EP1393059A1 - Device for analysing a chemical or biological sample, comparative analysis assembly, and related analysis method - Google Patents

Device for analysing a chemical or biological sample, comparative analysis assembly, and related analysis method

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Publication number
EP1393059A1
EP1393059A1 EP02745510A EP02745510A EP1393059A1 EP 1393059 A1 EP1393059 A1 EP 1393059A1 EP 02745510 A EP02745510 A EP 02745510A EP 02745510 A EP02745510 A EP 02745510A EP 1393059 A1 EP1393059 A1 EP 1393059A1
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EP
European Patent Office
Prior art keywords
micro
fractionation
columns
capture
column
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Application number
EP02745510A
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German (de)
French (fr)
Inventor
François Geli
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Individual
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Individual
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Publication of EP1393059A1 publication Critical patent/EP1393059A1/en
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/466—Flow patterns using more than one column with separation columns in parallel
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6095—Micromachined or nanomachined, e.g. micro- or nanosize
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/38—Flow patterns
    • G01N30/46—Flow patterns using more than one column
    • G01N30/461—Flow patterns using more than one column with serial coupling of separation columns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/60—Construction of the column
    • G01N30/6034—Construction of the column joining multiple columns
    • G01N30/6043—Construction of the column joining multiple columns in parallel
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/76—Acoustical detectors
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25375—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
    • Y10T436/255—Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.] including use of a solid sorbent, semipermeable membrane, or liquid extraction

Definitions

  • the subject of the present invention is a device for chemical or biochemical analysis of samples making it possible to overcome these drawbacks.
  • the subject of the invention is also a device for chemical or biochemical analysis of samples allowing improved separation of the constituents of a sample, and in particular separations of the constituents of a sample according to different selectivity criteria.
  • the device comprises fluid means for capturing fractionation products at the level of a terminal element of each fractionation micro-column situated upstream from its discharge orifice, micro-channels of ' capture intended to recover the captured fractionation products, and sets of selective micro-levers associated with the fractionation micro-columns and located downstream of the capture micro-channels, a micro-lever comprising detection means connected to means of 'analysis.
  • the fractionation micro-column designates a portion of micro-channel provided with separation means.
  • the micro-channel portion forming the fractionation micro-column can be preceded by downstream or upstream micro-channel portions devoid of separation means.
  • the end of the micro-channel portion configured as a fractionation micro-column is designated by the orifice for introducing and removing the fractionation micro-column.
  • Separation means can be a phase called stationary phase, of the type used in chromatography, or electrochromatography, or an electrophoresis gel, or electrical means.
  • micro-columns or micro-column groups of different constituents we will find in the terminal elements.
  • the terminal elements of the longest micro-columns we will find the most rapidly migrating molecules.
  • the terminal elements of the shortest micro-columns we will find the slowly migrating molecules, the rapidly migrating molecules having then been entrained towards the evacuation orifice of the micro-column thanks to the circulation of the enriched mobile phase.
  • each fractionation micro-column differs from an immediately longer fractionation micro-column by an element of given length.
  • a gradient of micro-columns is thus obtained allowing differential separation of the samples.
  • the device comprises secondary fractionation micro-columns situated downstream of the fluidic capture means and upstream of the set of micro-levers associated with a fractionation microcolumn, and intended for the secondary fractionation of fractionation products captured.
  • the constituents captured in a terminal element, or portion of the length of a micro-column are again separated before being analyzed using micro-levers, for more precise detection.
  • the device comprises several batches of fractionation micro-columns, each batch of fractionation micro-columns having a selectivity determined by the means for separating its fractionation micro-columns comprising a stationary phase coated or not and / or electrical separation means.
  • a separation of a sample in batches of microcolumns of different selectivities makes it possible to highlight, in each batch, different constituents of the sample. The comprehensiveness of the sample analysis is improved.
  • the device comprises a support carrying several batches of fractionation micro-columns, capture means and sets of associated micro-levers, and a supply channel for all of the batches of micro-columns fractionation.
  • the selective micro-levers comprise means of detection as a function of their surface state or of the surface state of a coating, of their chemical nature or of the chemical nature of a coating. Separate constituents, such as proteins, may or may not react with one or more specific micro-levers, which will indicate their presence in the sample.
  • the micro-levers comprise means for detection by adsorption of molecules.
  • micro-levers are intended for the detection of molecules attached to it. Antibodies grafted onto microlevers will make it possible to detect the presence of a specific molecule, such as a specific protein, the nature of which is already known. Micro-levers intended to detect molecules which are adsorbed there allow detection of molecules and in particular of proteins whose nature is not known a priori.
  • the selectivity of preliminary fractionation micro-columns is adapted as a function of the selectivity of fractionation micro-columns to promote the prior extraction of extracts containing constituents which will be well separated in the associated fractionation micro-columns, given their own selectivity.
  • a pre-fractionation stage comprises a plurality of pre-fractionation micro-columns, each intersected by a capture micro-channel, the capture micro-channels being connected to a recovery channel.
  • a prior fractionation stage comprises a plurality of micro-columns of prior fractionation, and a capture microchannel successively intersecting the micro-columns of prior fractionation, and opening into a recovery channel.
  • a terminal portion of the fractionation micro-column may include terminal separation means different from the intermediate separation means .
  • the constituents arriving substantially at the same instant downstream of a fractionation micro-column have the same characteristics in relation to the selectivity of the intermediate separation means. A change in selectivity of a terminal portion makes it possible to separate these constituents.
  • fluidic capture means associated with a fractionation micro-column comprise a capture micro-channel comprising an upstream portion opening at a downstream end of a capture segment of the fractionation micro-column and a downstream portion opening at an upstream end of the capture segment.
  • the offset of the upstream and downstream portions of a capture micro-channel allows the capture of constituents of a sample lying on a length segment of a fractionation micro-column.
  • Such capture ico-channels with offset portions can be provided for a main fractionation micro-column or a prior fractionation micro-column.
  • a capture on a preliminary fractionation micro-column a greater variety of constituents is captured.
  • a capture on a preliminary extraction micro-column During a capture on a fractionation micro-column, the counter-current circulation in the capture segment of a capture eluent, different from a mobile phase circulating in the micro-column, can allow a secondary fractionation of the fractionation product present in the capture segment at the time of capture.
  • the analysis device comprises a micro-washing channel for selective micro-levers opening into capture micro-channels directly upstream of selective micro-levers.
  • a micro-washing pipe allows to bring a washing buffer or an eluent directly on the micro-levers.
  • the micro-levers retain certain molecules according to their surface properties.
  • the washing eluent is chosen for its affinity with the molecules retained on certain micro-levers, in order to entrain the molecules previously retained by these microlevers.
  • a washing buffer removes any molecule attached to the microlevers.
  • the invention also relates to a set of chemical or biochemical comparative analysis of at least two biological or chemical samples comprising at least two devices comprising a plurality of micro-columns for fractionation of constituents of a sample, each micro-column for fractionation comprising a micro-channel provided with an orifice for introducing a mobile phase enriched in the sample, an evacuation orifice situated at a terminal end and intermediate separation means.
  • a device further comprises fluid means for capturing fractionation products at a terminal element of each fractionation micro-column situated upstream from its discharge orifice, capture micro-channels intended for recovering the products of fractionation captured, and sets of selective micro-levers associated with the fractionation microcolumns and located downstream of the capture micro-channels, a micro- lever comprising detection means connected to analysis means.
  • a set comprising separation and analysis devices allows rapid comparison of the samples to determine the differences in composition, for example as a function of differences in the physiological or pathological state of cells making up the samples.
  • a captured fractionation product is fractionated before analyzing it.
  • the fractionation product includes certain fractional components of the sample.
  • constituents of a fractionation product are detected using micro-levers, according to characteristics of polarity, solvophobicity or porosity of the material which constitutes them or of a coating of the microlevers, or according to characteristics of polarity, solvophobicity, ion exchange or affinity with functional groups grafted onto the micro-levers.
  • the sample can be fractionated by chromatography, by micro-electrophoresis, or by interaction with nano-electrodes.
  • the deviation or the frequency of vibration of the micro-levers is analyzed.
  • a molecule such as a protein or a peptide can be fixed on a micro-lever, according to a selectivity due for example to a surface state or a coating.
  • micro-lever can also be excited in vibration at a certain frequency, for example its resonant frequency.
  • a protein binds to the micro-lever, a change in vibration frequency is measured.
  • the fractionation elements are analyzed by mass spectrometry, before or after the analysis using micro-levers.
  • a first sample is analyzed
  • a second sample is analyzed, and the analysis results of the two samples are compared.
  • the samples are analyzed using suitable micro-levers, which will be able to detect differences in the composition of the samples, taking into account the difference in state of the latter, and the difference in composition that l 'we presuppose.
  • a preliminary extraction is carried out on a sample before a differential fractionation of the sample.
  • Figure 1 is a partial schematic view of an analysis device comprising micro-columns according to one aspect of the invention
  • Figure 2 is a schematic view of a first variant of the analysis device according to Figure 1;
  • Figures 4 and 5 are partial schematic views of an analysis device showing a particular arrangement of supports
  • Figure 8 is a schematic overview of an analysis device according to one aspect of the invention.
  • Figure 9 is a partial schematic view of a stage of prior extraction of a support;
  • FIGS 10 and 11 are partial schematic views of alternative pre-extraction stages according to Figure 9;
  • FIG. 12 is a view of a circuit for washing selective micro-levers
  • FIG. 13 is a partial view of an analysis support with mobile phase supply and separate sample supply
  • the length L2 of the shortest fractionation micro-columns 2 varies by way of example in no way limiting in a range 1 to 20 centimeters.
  • the length L1 of the longest fractionation micro-columns 2 varies by way of example in no way limiting in a range 5 to 40 centimeters.
  • Said fractionation micro-columns 2 have a diameter of around 1 to 100 microns, and in particular 10 to 100 microns.
  • the difference in length between a fractionation micro-column 2 and an immediately longer fractionation micro-column 2 is understood, by way of example in no way limiting, between 1 to 100 microns.
  • the fluidic capture means 7 comprise capture micro-channels 8 intersecting the fractionation micro-channels 3 at a terminal element or terminal portion 9 of each fractionation micro-channel 3, at a determined distance from the end end of the fractionation micro-channel 3, that is to say at a determined distance from its discharge orifice 3b.
  • Each fractionation micro-channel 3 is associated with a capture micro-channel 8.
  • the input ends of the capture micro-channels 8, located upstream of the intersection with the micro-channels 3, are connected to a channel secondary supply 15, intended for supply with secondary eluent.
  • the support 1 also comprises secondary fractionation micro-columns 10 located in downstream portions of the capture micro-channels 8, being located upstream of detection zones 11.
  • a secondary fractionation micro-column 10 is associated with a microphone -capture channel 8 and a detection zone 11.
  • a secondary fractionation micro-column 10 comprises fractionation means similar to those of the fractionation micro-column 2 with which it is associated.
  • a detection zone 11 comprises a circulation channel 12 passing through one or more selective micro-levers 13.
  • a mobile phase preferably in the form of an eluent, enriched in the sample, is brought in by the feed channel 4 and enters the fractionation micro-columns 2 through the introduction orifices. 3a.
  • the mobile phase enriched in sample circulates from the introduction orifice 3a to the evacuation orifice 3b while being separated in the micro-channel 3.
  • the terminal elements 9, where the catches are made, are located at distances different from the introduction orifices 3a of the fractionation micro-columns 2.
  • the migration rates of the constituents of a sample are different. Therefore, at a given time after the start of migration, we will find in the various terminal elements 9 of the microcolumns fractionation 2 sets of different constituents.
  • a micro or nano-flow of secondary eluent is circulated simultaneously in all of the capture microchannels 8.
  • a nano-flow of secondary eluent flowing in a capture micro-channel 8 crosses the terminal element of length Delta L contained in the associated micro-channel 3.
  • fractionation product includes a plurality of components of the sample.
  • the fractionation products are in particular separate molecules, non-separated molecular complexes and non-disaggregated molecular aggregates.
  • the means for separating the secondary fractionation micro-columns 10 are similar to those for the fractionation micro-columns 2. However, provision may be made for the means for separating a secondary fractionation micro-column 10 to be different from those of the associated fractionation micro-column 2.
  • the selectivity of the separation means may be different, to promote separation of the constituents present in the fractionation products. These constituents, which were captured at the same time in a fractionation product after a first separation, have similar migration characteristics in relation to the selectivity of the fractionation micro-column 2 from which they are derived. A second separation with different selectivity allows effective additional separation.
  • the secondary eluent is chosen to favor this secondary separation.
  • the fractionation micro-columns 2 can be fed from a collective enrichment column, that is to say a feed channel for enriched mobile phase, or from a plurality of enrichment columns, a specific batch being associated with a specific enrichment column, for example circulating a specific eluent, corresponding to the particular means of separation of the fractionation micro-columns 2 of the lot. Indeed, depending on the selectivity of the fractionation micro-columns and the nature of the eluent, the migration speeds of the molecules may be different.
  • fractionation micro-columns of each batch can have length gradients between micro-columns or groups of microcolumns.
  • a fractionation of a sample followed by a capture of fractionation products, and the detection of their constituents makes it possible to recover an "imprint" of the sample. You can make a series of imprints.
  • To allow successive captures and detections using the same micro-levers detection it is possible to provide for successive washing steps of the micro-levers 13, in particular by passing a particular eluent capable of entraining molecules retained in on the micro-levers.
  • the molecules entrained by a mobile phase in the fractionation micro-columns 2 are retained, according to the selectivity of the means of separation of the fractionation microcolumns.
  • a variation in the composition of an eluent allows for different entrainment of the constituents and improved separation.
  • the variation in composition can be carried out in successive steps, or continuously. In this case, we speak of an eluent gradient.
  • the capture of the fractionation products is carried out in step-by-step mode, each capture step being based on a precise physical or chemical or hydrodynamic condition prevailing at the intersection of a fractionation micro-column 2 with said micro-channel of capture 10.
  • the series of successive fingerprints of a first sample is compared to the series of successive fingerprints of a second sample using analysis means of the computer type, the series of detection fingerprints then being archived in a computer database.
  • (n) is a number between 1 and 5
  • (m) is a number between 1 and 5
  • (x) is a number between 5 and 50
  • (z) is a number between 1 and 5
  • (t) is a number between 10 and 10 000, in particular between 10 and 1000.
  • a set of supports is used for the analysis of a sample, another set of supports being used for the analysis of another sample.
  • the fingerprint made up of (n.m ⁇ .z.x) detections of the first sample is compared to the footprint made up of (n.m.t.z.x) detections of the second sample.
  • the primary and secondary elutions are made by elution gradient.
  • several fingerprints on micro-levers are made, at fixed or varied time intervals. Between each impression, micro-lever washes can take place.
  • the method of analysis of the sample gives rise, for each set of supports, to a passage of (p.t.z.x) different fractionation products over detection zones.
  • the separation in the fractionation micro-columns 2 or the secondary fractionation micro-columns 10 can be carried out by way of nonlimiting example by electrophoresis, chromatography or electrochromatography.
  • organic solvents can be used such as a mixture of dichloromethane-hexafluoro-2-propanol containing traces of pyridine with a linear gradient formic acid-2-propanol and formic acid-water on a stationary phase.
  • non-polar such as Vydac C4 (Cf. Bollhagen R, Schmiedberger M, Grell E. High performance liquid chromatography purification of extremely hydrophobic peptides: transmembrane segments Journal of Chromatography A, 1995, 711, 181-186).
  • nonaqueous solvents it is also possible to use nonaqueous solvents, as mentioned in combined use with methods of separation by non-capillary electrophoresis.
  • aqueous Cf. Cottet H, Struijk MP, Nan Dongen JLJ, Claessens HA, Cramers CA.
  • Electrophoresis 20 (1999) 111-117; Jussila, M., Sinervo, K., Porras, SP and Riekkola, M.-L. Modified liquid junction interface for nonaqueous capillary electrophoresis-mass spectrometry. Electrophoresis 21 (2000) 3311-3317; Koch, JT, Beam, B., Phillips, KS and Wheeler, JF Hydrophobic interaction electrokinetic chromatography for the separation of polycyclic àromatic hydrocarbons using non-aqueous matrices. J. Chromatogr. A 914 (2001) 223-231; Li, S.
  • C10-APSO4 respectively 3 (nonyl-dimethyl-ammonio) propyl sulfate and 3 (decyl - dimethyl-ammonio) propyl sulfate, for the extraction-preconcentration of hydrophobic molecular species (Cf. Saitoh T, Hinze WL. Concentration of hydrophobic organic compounds and extraction of proteins using alkylammoniosulfate zwitterionic surfactant mediated phase separations. Anal. Chem 1991, 63 (21): 2520-5.)
  • Basic analytes can also be separated in combination with nonaqueous capillary electrophoresis (Cf. Karbaum, A. and Jira, Th. Nonaqueous capillary electrophoresis: Application possibilities and suitability of various solvents for the separation of basic analytes. Electrophoresis, 1999 , 20, 3396-3401).
  • size exclusion chromatography can also be used with stationary apolar phases and elution with a ternary mixture such as (chloroform-methanol-trifluoroacetic acid) (Cf. Bunger H, Kaufner L, Pison U. Quantitative analysis of hydrophobic pulmonary surfactant proteins by high performance liquid chromatography with light-scattering detection. J. Chromatogr A, 2000, 18, 870 (1-2), 363-9.)
  • micellar retro-extraction in which the proteins encapsulated inside micelles are recovered after destruction of the micelles by a surfactant having a counter-electrostatic action (Cf. Jarudilokkul S,
  • Extraction and chromatography are based in particular on the concept of polarity, which comes from an asymmetric distribution of electronic clouds within molecules.
  • Polarity scales are designed in several independent ways which refer to the various consequences of polarity phenomena:
  • this method gives, by measurement of adsorption on alumina, the following polarity orders: water> methanol> ethanol> 2-propanol> dimethyl sulfoxide> acetonitrile> methyl ethyl ketone.
  • solubility parameters Hildebrand and Scott
  • solubility parameters Hildebrand and Scott
  • this method gives the following polarity orders: water> methanol> ethanol> dimethyl sulfoxide> acetonitrile> 2-propanol> methyl ethyl ketone.
  • the partition chromatography is based on the differential solubilities of the solutes between two liquid phases, more precisely between a mobile liquid phase and another liquid phase, called stationary, matching the meshes of a porous solid phase of fine particle size.
  • the solid phase may be polar, such as for example consisting of silica gel grains grafted with aminopropyl or paranitrobenzyl, or alkylnitrile, or glyceropropyl groups.
  • the weakly polar mobile phase as can be a mixture (95% hexane, 5% dichloromethane) will receive a "polar modifier" to give a mixture with increased polarity, such as (80% hexane, 20% dichloromethane ), until moving polar solutes interacting with the stationary polar liquid phase. This is normal phase sharing chromatography.
  • the solid phase can also be apolar, such as matrices such as a styrene-divinybenzene copolymer matrix, or else a pyrocarbon matrix, or silica gels grafted with apolar functional groups, such as, for example, alkyl or phenyl groups.
  • the mobile, polar phase as a mixture can be (40% methanol or acetonitrile, 60% water), will receive a "polar modifier" to give a mixture with less polarity (60% methanol or acetonitrile, 40 % water), until displacing apolar solutes interacting with the stationary apolar liquid phase. This is reverse phase partition chromatography.
  • the other chromatographic separation techniques are based on a differential retention of solutes contained in a mobile, liquid or gaseous phase, which passes through a solid stationary phase.
  • the retention mode is size, adsorption or affinity.
  • Size exclusion chromatography is based on a stationary phase consisting of porous beads forming a gel. The distribution of the pore diameters inside the porous beads corresponds to a fairly wide range. Depending on their steric hindrance, the molecules may or may not pass inside a greater or lesser number of pores of the porous beads. Those which pass most easily inside the pores of the porous beads are the most delayed. In practice, the phenomenon is biased by ionic or hydrophobic interactions between the solutes and the stationary phase.
  • Size exclusion chromatography in denaturing or non-denaturing conditions can also help, while minimizing the use of detergents, in the characterization of molecular aggregates in which membrane proteins are involved (see Lôster K, Baum O, Hofman W, Reutter W. Characterization of molecular aggregates of alpha 1 betal integrin and other rat liver membrane pro teins by combination of size exclusion chromatography and chemical cross-linking. Journal of Chromatography A, 1995, 711, 187-199.) Techniques Particularly preferred chromatography are those based on a differential adsorption of solutes contained in a mobile phase, liquid or gas, which passes through a solid stationary phase.
  • the selectivity in adsorption chromatography is based on a complete process for each of the solutes: entrainment by the mobile phase and interaction of specific energy with the stationary phase.
  • the polarity of the solute is intermediate between that of the mobile phase and that of the stationary phase. If the polarity of the solute is too far from that of the mobile phase, there is not sufficient solubility of the solute in the mobile phase to prevent irreversible retention on the stationary phase. If the polarity of the solute is too far from that of the stationary phase, there is no interaction with the stationary phase.
  • a solvent is all the more eluting as its polarity approaches that of the solute, and finally that of the stationary phase, since the latter is supposed to be close to that of the solute.
  • the selectivity is all the better when a small variation in the polarity of the solvent results in a selective change in the adsorption equilibrium of solutes of similar polarities.
  • matrices comprising a stationary phase, the grafts of which consist of polymers of molecules having an apolar side and a polar side, such as a macroporous copolymer produced from an equilibrium between the two monomers, divinylbenzene, apolar, and polar N-vinylpyrrolidone.
  • Normal phase adsorption chromatography is based on the differential adsorption of solutes on a solid and polar stationary phase, such as in particular based on alumina but especially on silicates, or hydrophilic polymers, such as agarose or dextran gels , the mobile phase being, it, apolar.
  • a solid and polar stationary phase such as in particular based on alumina but especially on silicates, or hydrophilic polymers, such as agarose or dextran gels , the mobile phase being, it, apolar.
  • micro-beads are obtained by a hot emulsion-gelling process first using a water-immiscible solvent, then a stabilizer, said process ending with the removal of the solvent by suction filtration.
  • Agarose gels can be crosslinked with crosslinking agents such as epichlorohydrin, 2,3 dibromopropanol or divinylsulfone.
  • crosslinking agents such as epichlorohydrin, 2,3 dibromopropanol or divinylsulfone.
  • 2B, 4B, 6B are examples of the names 2B, 4B, 6B respectively.
  • Sepharose CL is crosslinked with 2,3-dibromopropanol under strong alkaline conditions, operation followed by alkaline hydrolysis of its sulfate groups under very reducing conditions, so as to make it non-ionic or very weakly ionic.
  • Sephadex is a dextran gel crosslinked with epichlorohydrin which is stable under alkaline, saline and weakly acidic conditions, but which is hydrolyzed under marked acid or oxidizing conditions.
  • Sephadex (LH-20) and (LH-60) gels can be grafted with hydroxypropyl groups which bind by ether linkages to the glucose units of the dextran chains, with the effect of modulating their polarity.
  • Silica is insoluble in water for a pH varying from 2 to 8. Its polarity is provided by the presence on its surface of silanol groups (SiOH), 4.6 in number per nm 2 , of which the OH group is polar and proton donor in hydrogen bonds.
  • a silanol group can remain free (free silanol), or else initiate a hydrogen bond with a neighboring silanol group (bound silanol), or else initiate a hydrogen bond with a water molecule.
  • the OH group of a free silanol can also be a proton donor to a water molecule (free silanol hydrated by a layer of monomolecular water), or to another polar molecule.
  • bound silanols can attract water molecules: we then silanols hydrated by a layer of pluri-molecular water, these are highly hydrated silicon gels. Sihce gels are very porous. Depending on whether their specific surface is more or less large (it varies from 200 to 600 m 2 per gram), they comprise pores of a more or less large diameter and consequently a more or less large masking of the free silanols.
  • the free silanol groups are “strong” adsorption sites, completely available for hydrogen bonding. Hydrated free silanol groups and linked silanol groups are still adsorption sites.
  • silanol groups hydrated by a layer of pluri-molecular water are rather sites of partition chromatography.
  • highly hydrated silica gels whose specific surface exceeds 550 m 2 per gram and whose water content exceeds 5%, it is considered that partition chromatography becomes preponderant compared to adsorption chromatography.
  • the silica gels sold have a varied particle size and mention the number of silanols remaining free per unit area (for example Lichosorb Si 100 has 2.95 free silanol groups per nm 2 for a specific surface of 309 m 2 per gram, while that Lichosorb Si 80 contains 2.20 for a specific surface of 482 m 2 per gram).
  • adsorption chromatography In adsorption chromatography, an attempt is made to keep the adsorption capacity of the absorbent constant, whatever the mobile phase. To do this, we adjusts the water content of a solvent to a level called "isoactivating water content" so that the adsorption energy of the adsorbent is equivalent to what it can have with a reference solvent having a content of given water (for example, the reference solvent could be ethyl acetate at 0.06% water when we want a reference for the absorbency of a silica with a specific surface of 550 m_ per gram .)
  • the polarity of the silicas can be modified by grafting.
  • the polar grafts can be of the aminopropyl, paranitrobenzyl, alkylnitrile (nitro), glyceropropyl (diol) type.
  • the grafts can be carried out by silanization, that is to say by reactivity of mono, di- or tri-functional alkoxysilanes or chlorosilanes. For this reaction to take place, the silane molecules must penetrate into the pores of the silica, which supposes a pore diameter greater than 10 nm.
  • the non-polymerized grafted commercial phases have a grafting rate of 3.5 to 3.7 micromoles per nm 2 .
  • Reverse phase adsorption chromatography is based on the differential adsorption of solutes on a stationary solid and nonpolar phase, such as in particular silicas grafted with nonpolar groups, the mobile phase being more or less polar, according for example to various proportions of more or less polar solvents, for example water and methanol or alternatively water and acetonitrile.
  • a stationary solid and nonpolar phase such as in particular silicas grafted with nonpolar groups
  • the mobile phase being more or less polar, according for example to various proportions of more or less polar solvents, for example water and methanol or alternatively water and acetonitrile.
  • apolar functional groups grafted onto the stationary phases such as sihce or Sepharose can be alkyl groups Cl 8 or C8 or C4, or phenyl groups .
  • the grafting procedure for grafted silicas with nonpolar groups is obtained by silanization, as is the grafting procedure for grafted silicas with polar groups. It should be noted, as in the case of virgin (polar) silicas or polar grafted silicas, the presence of residual silanols originating from the hydrolysis of reactive groups of trifunctional silanes which have not reacted during the synthesis.
  • the organic solvent molecules of a mixture are preferentially fix on the surface of the apolar grafts
  • the solute molecules interact with the stationary liquid phase.
  • the interaction mechanism is either a solute sharing mechanism between the mobile phase and the adsorbed liquid phase, or based on a hydrophobic interaction between the solute molecules and the stationary apolar phase.
  • the solute molecules can displace molecules from the stationary polar liquid phase.
  • the accessible residual silanol groups can be eliminated (this process is called "end-capping") by a treatment with trimethylchlorosilane (TMCS).
  • TMCS trimethylchlorosilane
  • Apolar matrices other than silica grafted with C18 or C8 or C4 or phenyl alkyl groups can be used.
  • Phenyl and Octyl-Sepharose can be used in chromatography of hydrophobic interactions, and are obtained when the crosslinking of Sepharose CL is coupled with a derivation with phenyl or octyl groups.
  • Styrene-divinylbenzene or pyrocarbon copolymer matrices can be used, which have the advantage over grafted silicas of being stable in a much wider pH range (1 to 13) against (2 to 7.5), because silica is attackable by OH ions. This silica defect can be resolved by applying a silicone coating to the surface of the pores found in stationary commercial phases such as Capcell Pak.
  • a weak point of the copolymer matrices can be improved by using macroporous copolymer matrices.
  • macroporous copolymer matrices comprise both a very highly crosslinked part, impermeable to solvents, and a weakly crosslinked part favorable to exchanges between the stationary phase and the mobile phase, and macro-pores, without the presence of polymers.
  • Other stationary phases such as porous zirconium oxide or porous graphite naturally offer the qualities of stability (pH from 1 to 14) and mechanical resistance.
  • copolymeric matrices Another characteristic of the copolymeric matrices mentioned is the presence of aromatic groups, capable of interacting in the formation of donor-acceptor complexes of electrons with the solutes.
  • Other copolymer matrices can be used, such as for example those based on vinyl alcohol or polymethylmethacrylates.
  • the stationary phase which comprises a matrix to which are grafted fixed, ionized functional groups capable of fixing counterions.
  • the microparticles of the ionized stationary phase accept ions of opposite charge within them, and exclude ions of the same charge.
  • the matrices of the stationary phase can be grafted silicas or copolymeric matrices.
  • the matrices By their role of exclusion, as well as by their own composition, such as for example the presence of aromatic nuclei in the polystyrene divinyl benzene copolymer matrices giving rise to interactions by pi electrons, the matrices contribute to the process which is added to those of their ionized functional groups.
  • a competition for binding to the stationary phase is established between the solute ions of the mobile phase and counterions releasable by said stationary phase and therefore exchangeable.
  • the mobile phase is a buffer solution whose pH makes it possible to control the electrostatic attractions of the solutes, insofar as a certain value of the pH will correspond to a certain charge of the solutes.
  • the amino acids of proteins can be present in the solution in the form of zwitterionic molecules or in the form of anions or in the form of cations.
  • the grafted matrices are porous (they are micro-particles of porous layer or organic copolymers of microporous or macro-porous structure of poly (styrene / divinylbenzene) or polyacrylate type), which gives rise at the same time to a non-ionic separation mechanism, for example mechanisms for sharing molecules with a given polarity. For example, nonionic solutes do not undergo electrostatic repulsion to penetrate inside the pores of the matrix.
  • a matrix can be a cation exchanger or anion exchanger, weak or strong.
  • Strong cation exchangers (SCX for Strong Cation Exchangers), of the strong acid type, can be sulfonic, that is to say grafted with sulfonate SO3- functional groups.
  • the weak cation exchangers, of the weak acid type can be carboxylic, that is to say grafted with functional groups CO2- carboxylates.
  • the strong anion exchangers (SAX for Strong Anion Exchangers), of the strong base type, can be quaternary ammonium, that is to say grafted with NR3 + functional groups, such as for example trimethylammonium.
  • the weak anion exchangers of the weak base type can be non-quaternary ammonium, that is to say grafted with protonated forms of primary, secondary or tertiary amines (NHR2 + functional groups, such as for example diethylaminoethylammonium).
  • NHR2 + functional groups such as for example diethylaminoethylammonium.
  • the abbreviations of everyday language relate, for cation exchangers, the CM, weakly acid, for Carboxymethyl, as well as SP and S, strongly acid, for respectively Sulphopropyl and methylSulfonate.
  • the eluting force partly depends on the nature of the developer ion conveyed by the mobile phase.
  • a second mode of ion pair chromatography or ion interaction chromatography advantage is taken of the presence in the mobile phase of large ions (they are called counterions) comprising an apolar part and a charge opposite to that of the solute. Electro-neutrality is ensured by the presence of co-ions of the same sign as the ions of the solute.
  • each counterion can form a pair with a solute molecule by hydrophobic interaction.
  • a nonpolar stationary phase such as silica grafted with alkyl groups
  • the counterion can be adsorbed on the nonpolar grafts of the stationary phase, while having to leave free 60 to 70% of them.
  • the ions of the solute are sufficiently hydrophobic, it is then also possible to assist in the sharing of pairs of ions (solute / counterion) which are fixed on the alkyl groups which remain free from the stationary phase and the solubilization of these same ions (solute and storytelling) in the mobile phase.
  • solutes The retention of solutes depends on their degree of ionization, the content of organic solvent, and the concentration of counterions in the mobile phase.
  • solutes When the solutes are capable of forming complexes with a cation (Cu_ +, Zn_ +, Cd2 +, Ni_ +) or a donor or acceptor complex, it is possible to carry out chromatography by ligand exchange respectively, or chromatography with transfer of loads.
  • the metal cation for example copper
  • the stationary phase for example a virgin silica
  • a covalent bond of copper with silica is obtained in the presence of ammonia, giving rise to silicas covered with cupri-diamine silicates.
  • These cupri-diamine silicates attached to the stationary phase are capable of exchanging ammonia with a doubling-giving solute, which becomes the new ligand by forming a hedge with the copper of the stationary phase.
  • cupri-diamine silicates can solvate water molecules, which makes them very hydrophilic.
  • solute The retention of a solute will depend on its donor character (complexing character) and on its hydrophilic character, as well as on the ammonia content of the mobile phase, generally a ternary water-acetonitrile-ammonia mixture whose water content does not exceed step 50% in order to preserve the stability of the stationary phase.
  • the mobile phase contains a complex of a transition metal with a ligand comprising a hydrophobic chain
  • the stationary phase for example a silica grafted with nonpolar groups, such as groups C18 alkyl
  • the transition metal is in excess with respect to the hydrophobic ligand, so as to be free to also be able to preserve weak hedge sites with solvent molecules.
  • solutes are capable of forming complexes with the transition metal, they are divided between bonds with the metal in the mobile phase and bonds with the metal engaged in complexes with the hydrophobic ligand, itself adsorbed on the stationary phase hydrophobic.
  • the solvent molecules can also solvate the grafts of the stationary phase. Consequently, there can be, to receive electrons from the stationary phase, a two-way competition (solute, polar modifier) with a bias which is that the solute can also interact with the grafts solvated by the polar modifier. Finally, there is competition between the solutes and the polar modifier to give (or accept) electrons to (or from) free, unsolvated grafts from the stationary phase.
  • the retention of solutes is all the stronger as the number of free grafts of the stationary phase is high, and the number of aromatic nuclei per graft and the spatial density of these nuclei are high. All things being equal, the competition for binding to the stationary phase will be decided on the content of the polar modifier of the mobile phase.
  • the hydrophilicity of a protein or peptide depends on its amino acid composition.
  • the proportion of hydrohile or polar amino acids is high, the hydrophobic or apolar amino acids (isoleucine, valine, leucine, phenylalanine) are pushed back inside the molecule.
  • the proportion of non-polar or hydrophobic amino acids is high, there is a more direct interaction between certain hydrophobic amino acids and the aqueous medium.
  • a first means of increasing selectivity is to use changes in the composition of binary or ternary mixtures of solvents of different polarities to obtain a variation in polarity of the mobile phase, in order to completely modify the spatial conformation of the peptide or protein.
  • a second means of increasing selectivity is to use solvation variators of functional groups, such as salts.
  • functional groups such as salts.
  • hydrophobic or apolar functional groups are surrounded by self-organizing water molecules.
  • a strong ionic force unmasks the hydrophobic or apolar functional groups, by disorganizing the water molecules which surround them.
  • Chromatography of hydrophobic interactions consists of starting with a strong ionic strength and then decreasing it until the hydrophobic or apolar functional groups of the peptides or proteins find their mask in an aqueous medium of weak ionic strength.
  • a stationary apolar phase such as grafted with alkyl groups Cl 8, C8 or C4, or phenyl
  • the proteins which have the most hydrophobic functional groups are those which are the most delayed.
  • Phenyl and Octyl-Sepharose are often used for this type of chromatography.
  • Several models have attempted to describe the laws of separation in chromatography, in particular by attempting to configure the theoretical plateau height in the micro-column.
  • Nan Deemter equation is of the form:
  • A is a term which accounts for axial diffusion
  • B is a term which accounts for incomplete mass transfers between mobile phase and stationary phase
  • C is a term which accounts for, on the one hand, paths of unequal length to cross the column on the other hand difficulty for the mobile phase and the solutes to access the meshes formed by the stationary phase: it is optimal that the mobile phase and the solutes reach said meshes by convection rather than by diffusion
  • d is the flow rate of the mobile phase through the column.
  • Altria KD Overview of capillary electrophoresis and electrochromatography, Journal of Chromatography A, 1999, 856, 443-463; Quirino JP, Terabe S. Electrokinetic chromatography, Journal of Chromatography A, 1999, 465-482; Smith NW, Carter-Finch AS, Electrochromatography, Journal of Chromatography A, 2000, 892, 219-255; Bartle KD, Carney RA, Cavazza A , Cikalo MG, Myers P, Robson MM, Roulin SCP, Sealey K. Capillary electrochromatography on silica columns: factors inflencing performance. Journal of Chromatography A, 2000, 892, 279-299; Pyell U.
  • Micellar electrochromatography can be performed on miniaturized supports as described in the following document: (Cf. Culbertson CT, Jacobson SC, Ramsey JR. Micro-chip device for high efficiency separations. Anal. Chem, 2000, 72, 5814 -5819).
  • Elution gradients can be used, and in particular micro-gradients of elution (cf. AH, Kahle N, ⁇ ovotny MN. A micro-gradient elution system for capillary electrochromatography. J. Microcolumn separations, 2000, 12 (1 ), 1-5.).
  • the separation and in particular the separation of the peptides or the proteins can be carried out using separation methods by micro-chromatography, micro-electrochromatography or micro-electrophoresis.
  • a separation of peptides and proteins can be used by reverse phase chromatography on 1.5 micron non-porous non-porous silicas (Cf. Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Retention and selectivity of proteins and peptides in gradient elution of non-porous monodisperse 1,5 micron reversed phase silica. Journal of Chromatography A. 1987, 397, 71-80; Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Mobile phase and surface mediated effects on recovery of native proteins in gradient elution on non-porous monodisperse 1,5 micron reversed phase silica. Journal of Chromatography A. 1987, 397, 80-89.)
  • Peptides and proteins can be separated by anion exchange chromatography on a 3-micron non-porous polymeric phase of 3 microns of poly (styrene-divinylbenzene) (Cf. Régnier FE, Rounds MA. Synthesis of a non-porous, polystyrene-based anion-exchange packing material and its application to fast high-performance liquid chromatography of proteins. Journal of Chromatography A. 1988. 443. 73-83).
  • Peptides and proteins can be separated by hydrophobic interaction chromatography on 1.5 micron non-porous non-porous silicas (Cf. Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Performance of non -porous monodisperse 1.5 micron bonded silicas in the separation of proteins by hydrophobic interaction chromatography Journal of Chromatography A. 1987, 397, 91-97).
  • Proteins can be separated in solutions of pH 5, 7 and 9 by affinity chromatography for cupric ions immobilized on stationary Sepharose CL-4B phases thanks to an epoxy coupling procedure and using the tridentate chelating ligand N- (2- pyridylmethyl) aminoacetate (Cf. Chaouk H, Hearn MTW. New ligand, N- (2-pyridylmethyl) aminoacetate, for use in the immobiled metal iion affinity chromatography separation of pro teins. Journal of Chromatography A, 1999, 852, 105-115 ).
  • Synthetic peptides having a greater or lesser number of histidine residues can be retained in affinity chromatography for metal ions immobilized on stationary phases Sepharose CL-4B and using the iminodiacetic acid ion as tridentate chelating ligand or the acid ion.
  • nitrilotriacétqiue as tetradenté chelating ligand Cf Kronina NN, Wirth HJ, Hearn MTW. Characterization by immobiled metal ion affinity chromatography procedures of the binding behavior of several synthetic peptides designed to have high affinity for Cu (U) ions. Journal of Chromatography A, 1999, 852, 261-272.
  • micellar chromatography One can use a peptide chromatography by micellar chromatography as that was presented in the following article: (Cf. Kord AS, Khaledi MG. Selectivity of organic solvents in micellar liquid chromatography of amino-acids and peptides. Journal of Chromatography A. 1993, 631, 1255-132).
  • the separation of biological samples can be carried out by micro-electrophoresis with a prior enrichment phase (Cf. Lichtenberg J, Nerpoorte E, de Rooij Sample. Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis. Electrophoresis 2001, 22, 258-271; Wu XZ, Hosaka A, Hobo T. An on-line electrophoretic concentration method for capillary electrophoresis of proteins. Anal. Chem, 1998, 70, 2081-2084; Tragas C, Pawliszyn J. On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface.
  • Isoelectric focusing that is to say a pH gradient over the length of the separation capillary
  • This gradient can be obtained by a set of small ampholyte molecules charged according to their pi, such as those synthesized based on acrylic acid and polyamines or obtained by coupling with epichlorohydrin.
  • the pH gradient can generally vary from 3 to 10.
  • the gradient can also be obtained by grafting, in a polyacrylamide gel, modified acrylamide monomers carrying ionizable chemical groups of acidic or basic PK.
  • the pH gradient can vary from 1 to 12.5 (Cf Kawano Y, Ito Y, Yamakawa Y, Yamashino T, Horii T, Hasegawa T, Ohta M.
  • a free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry. Rapid Commun Mass Spectrom. 2000; 14 (14): 1269-74; Wen J, Lin Y, Xiang F, Maison DW, Udseth HR, Smith RD. Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry. Electrophoresis. 2000 Jan; 21 (l): 191; Rossier JS, Schwarz A, Reymond F, Ferrigno R, Blanchi F, Girault HH. Microchannel networks for electrophoretic separations. Electrophoresis.
  • the fractionation micro-columns comprise means for separation by chromatography.
  • the particular selectivity of a batch is determined by the nature of the stationary phase of all the chromatographic fractionation micro-columns 2 forming the batch.
  • the selectivity of the fractionation micro-columns 2 of a batch is determined by the intrinsic polarity and the solvophobicity of a stationary phase and by the polarity, the amphipaticity and the solvophobicity of the functional groups which therein. are grafted.
  • the selectivity of the fractionation micro-columns 2 is determined secondarily by other criteria such as the micro- porosity, macro-porosity, the ability to exchange ions or to the interaction of pairs of ions or to exchange ligands or to charge transfer or to the affinity reactions of said stationary phase or the granting of a pH gradient over the length over the entire length of said fractionation micro-columns 2, said pH gradient extending over a range all the wider as said fractionation micro-columns 2 are long.
  • Said selectivity is determined thirdly by an electric field applied to stationary phases of said fractionation micro-columns 2 (Cf. Method of electric field flow fractionation which the polarity of the electric field is periodically reversed. US Patent N ° 6113819).
  • each batch of fractionation micro-columns 2 receives a mobile phase, of the eluent type, which is specific to it and in relation to the nature of the stationary phase of its fractionation micro-columns 2 .
  • the separation in fractionation micro-columns 2 is carried out by a first method of chromatography, and that the separation in the corresponding secondary fractionation micro-columns 10 is carried out by a second chromatography method different from the first.
  • a second chromatography method is therefore preferably chosen according to a different selectivity allowing effective separation of the molecules of the fractionation product obtained with the first method.
  • the first chromatography method is an ion exchange method, the second method being a hydrophobic interaction chromatography method.
  • the detection is carried out on the detection zones 10 by detection by micro-levers, followed or optionally preceded by additional detection by one of the methods known to those skilled in the art for the detection of eluates from chromatography, directly or after hyphenation, for example the mass spectrometry mentioned previously.
  • the fractionation micro-columns comprise means of separation by micro-electrophoresis, of the zone micro-electrophoresis type, or of the micro-isochatophoresis type, or of the micellar micro-electrophoresis type, or of the micro-electrophoresis type with isoelectric focusing obtained by the granting of a pH gradient over the length over the entire length of said micro-channels, said pH gradient extending over a range that is wider the longer said micro-channels.
  • the detection is carried out using micro-levers and possibly by spectrometry.
  • the separation in the fractionation micro-columns is carried out by a method called Field Flow Fractionation (cf. Suslov SA, Roberts AJ., Modeling os sample dynamics in rectangular asymetrical flow field flow fractionation channels. Anal Chem 2000, 72 (18), 4331-45).
  • micro-channels of micro-columns are provided along their length with nano-electrodes (cf. US 6,123,819).
  • the molecules entrained by an eluent are all the more braked as their charges interact with an electromagnetic field created by the nano-electrodes. Rectilinear and parallel fractionation micro-columns have been described.
  • fractionation micro-columns 2 can for example be rectilinear, curved or sinuous. They are manufactured in part or in whole thanks to the techniques used in micro-manufacturing on silicon or glass or ceramic or plastic. In one embodiment, the fractionation micro-columns are monoliths.
  • micro-column beds that is to say grooves formed on a support and intended to be covered and closed using another support having a flat surface where corresponding grooves can be directly etched when said supports are based on silicon or glass or ceramic.
  • the fractionation micro-columns 2 and said secondary microcolumns 10 can be manufactured in part or in whole thanks to the techniques used in micro-manufacturing such as photoengraving, micromolding, micro-stamping, photolymerization, thermopolymerization.
  • micro-manufacturing such as photoengraving, micromolding, micro-stamping, photolymerization, thermopolymerization.
  • the microparticulate network can be obtained by micro-molding or micro-stamping or photopolymerization or thermopolymerization in situ stamping when said supports are plastic-based, or else can be made up of micro or nano-rods inserted in the ht of said micro-columns (Cf. Gusev I, Huang X, Horvath C. Capillary columns with in situ formed porous monolithic packing for micro- high performance liquid chromatography and capillary electrochromatography, Journal of Chromatography A, 1999, 855, 273-290; Yu C, Svec F., Fréchet J.
  • Micro-particles can also be immobilized in a continuous bed (Adam T., Unger KK, Dittmann MM, Rozing GP. Towards the column bed stabilization of columns in capillary electroendosmotic Chromatography. Immobilization of microparticulate silica columns to a continuous bed. J.
  • micro-particulate network of micro-columns which constitutes the stationary phase of a thin film of hydrophobic or hydrophilic nature and can be subjected to coupling chemistries known to those skilled in the art to graft characterized molecules. mainly by their polarity and their amphipathicity. Chemical etching increases the retention properties, as indicated in the following document: (Cf. Pesek, Protein and peptides separations on high surface area capillaries, Electrophoresis, 1999, 20, 2343-2348).
  • micro-channels can be provided with micro-rods of polymer monoliths suitable for the separation of proteins both by electrochromatography and by micro-HPLC (Cf. Hjerten, Electroosmosis and Pressure-driven chromatography in chips using continuous beds. Anal. Chem, 2000 , 72, 81-87).
  • Stationary chromatography phases can be obtained by molding using silicon molds.
  • the very high resolution of micro-plastic molding techniques is directly linked to that of silicon molds. It is therefore at the level required to consider the manufacture of stationary chromatography phases directly molded in the plastic using silicon molds dimensioned to manufacture stationary phases comprising a very fine microparticulate plastic network, such as for example by being made of cubes. 5 micron polymeric edges separated by spaces of 500 nanometers.
  • the conduits and components that guide or receive the fluids are miniaturized, on the other hand that the components which manage the flow of fluids and reagents (micro-valves, micropumps, micro-sensors, micro-heaters, etc.) are also miniaturized, and finally that connections can be made set up inside and outside the device.
  • components which manage the flow of fluids and reagents micro-valves, micropumps, micro-sensors, micro-heaters, etc.
  • connections can be made set up inside and outside the device.
  • the desired micro-system can hold in a more or less flattened volume represented by the superposition of subcomponents themselves clearly flattened.
  • wet chemical etching techniques of photolithography dry etching with various photonic or particulate radiation, micro-shaping with micro-tools or lasers, cutting, ablation, fusion assembly or anodic assembly , gluing, welding, molding, hot stamping (hot-embossing in English), punching, drilling, electroplating, chemical vapor deposition, production by progression by successive sheets (lamination in English ).
  • wet etching is applied in a known manner to silicon and its derivatives in the microelectronics industry. It can be isotropic. It can also be anisotropic when one seeks to take advantage of the orientation of the crystals and the properties of the gravants to control its direction. (S ato K., Shikida M, Yamashiro M, Tsunekawa M, Ito S. Characterization of anisotropic etching properties of single crystal silicon: surface roughening as asolution of crystallographic orientation, the 1 lth IEEE International Workshop on MEMS, Heidelberg, Germany, 1998, 201-206).
  • the wet etching techniques both isotropic and anisotropic, have many variants.
  • Knowledge in materials physics, orbital chemistry, radiation physics, doping of materials allow to take advantage of the atomic structure of different materials used, help to design methods for controlling the direction, depth and stopping of engravings on different layers.
  • the techniques cited have many variations.
  • Knowledge of surface treatment makes it possible to improve the qualities required of materials during manufacture or the qualities required of the finished product.
  • thermophysics and differential thermochemistry between two materials makes it possible to envisage new techniques of fusion, molding, stamping, punching, in particular plastics.
  • a technique of micro-manufacturing of polymers by stereolithography can be used, in particular for rapid 3D prototyping.
  • micro-manufacturing techniques are applicable not only to the manufacture of the finished products, but also to those of the tools used to carry out these micro-manufacturing, as well as to the micro-molds and to the hot stamping micro-matrices used. to micro-replicate en masse a micro-object.
  • the other criteria which will help to select a manufacturing method and material are the intrinsic qualities of the materials making up the finished object, and the prospects for controlling manufacturing costs.
  • Some techniques assume a manufacturing method that is less suitable for mass production: dry etching by photonic or particulate radiation (Bean. Anisotropic etching of silicon. 1978. vol ED-25 (10), pp 1185-1193. IEEE Transactions of Electron devices.), laser ablation, etching with micro-tip.
  • Microfluidic devices for ⁇ -TAS applications fabricated by polymer hot embossing. Proceedings of SPLE. Microfluidic Devices and Systems. 21-22 Sept 1998, Santa Clara, ppl77-182 _ Grzybowski BA, Haag R, Bowden N, Whitesides GM. Generation of micrometer-sized patterns for microanalytical applications using a laser direct-write method and microcontact printing. Anal. Chem, 1998, 70, 4645-4652 _ Martynova L,
  • micro-manufacture replication masters for example micro-molds for injection molding or for reactive molding, or hot stamping micro-dies
  • two qualities are combined. : a high aspect ratio and a surface compatible with the requirements of the replication process. Indeed, certain steps in replication are crucial, in particular the separation of the replication matrix from the newly replicated object.
  • the complexity of the method chosen for manufacturing a replication matrix is taken into account.
  • new techniques of dry etching and especially of wet etching with increased performances can prove to be more flexible with aspect ratios which approach more and more the LIGA technique.
  • anisotropic wet etching has progressed a lot (Hôlke A., Henderson HT. Ultra-deep anisotropic etching of (110) silicon; J. Micromech. Microeng. 1999, 9, 51-57).
  • Other results also show progress in the performance of wet chemical isotropic etching procedures of silicon - a possibility for the production of deep structured microcomponents. Schwesinger N, Albrecht A .. SPLE vol 3223, p 72- 79 ).
  • Certain unitary techniques can be adapted to mass production when the manufacturing instruments themselves used to implement them are miniaturized and can be used in a massively parallel manner. This is a close prospect for laser ablation (thanks to the manufacture of micro-lasers) and microtip etching, more distant for certain dry etching techniques.
  • Manufacturing in large quantities is possible with certain techniques such as: wet etching on silicon and derivatives, and on glasses, UN photolithography on photoresists, production by progression by successive layers of polymers with the use of sacrificial layers according to Webster and Mastrangelo cited below in reference, molding of poly (dimethylsiloxane) (PDMS), molding of plastics by injection with micro-mold, molding of ceramics and metals, hot stamping of polymers with micro-matrix of stamping.
  • PDMS poly (dimethylsiloxane)
  • wet etching can be applied to all types of silicon and quartz derivatives, as well as to different types of glass (for example pyrex, borophospho-silicate glasses, etc.).
  • micro-fluidics an important criterion is compatibility with the use of micro-electrophoresis, 2D micro-electrophoresis and micro-electro-chromatography to separate the molecules.
  • Compatibility with electro-osmosis to move fluids is also and above all important, this technique having the advantage of avoiding components such as micro-valves and micro-pumps.
  • electro-osmosis as well as micro-electro-chromatography combined with electro-osmosis require the application of significant potential differences. Consequently, they are not very compatible with the use of silicon. However, they are compatible with glasses and plastics.
  • thermo-capillary force Bosset MA, Mastrangelo CH, Sammarco T, Man FP, Webster JR, Johnson BN, Foerster B, Jones D, Fields Y, Kaiser AR, Burke DT .
  • Microfabricated structures for integrated DNA analysis. PNAS 1996, vol. 93, pp5556-5561 or the forces coupled to alternating surfaces or hydrophobic-surface lines or hydrophilic lines (Jones DK, Mastrangelo CH, Burns MA, Burke DT. Selective hydrophobic and hydrophhilic texturing of surfaces using photolithographic photodeposition of polymers.
  • Device for fluid supply of a micro-metering device US Patent N ° 5805189 _ Beckton Dickinson. DNA microwell device and method. US Patent No. 5795748).
  • Transparency a quality sought after in biological analysis, is a quality shared between glasses (Kricka L, Wilding P, et al. Micromachined Glass-Glass Microchips for In Nitro Fertilization, Clinical Chemistry, 1995, 41, 9, 1358-1359) and some plastics.
  • the glasses offer, among other things for biochemical analysis, compatibility with fluorescence detection and a good heat exchange coefficient. They are however etched only in an isotropic mode, which for example today limits the shape of the micro-channels on glass to a circular shape.
  • Plastics even if they have less compatibility with fluorescence detection and a lower heat exchange coefficient than glasses, have many other qualities, including the low cost price.
  • the very low cost of manufacturing micro-manufactured plastic objects comes from the low price of the raw material, the simplicity of the production processes which can be envisaged, and the ability to replicate by molding or by hot stamping, even for plastics photoresists to photolithography.
  • metal can be deposited once the product is finished.
  • the support can also be marked with a conductive ink.
  • - photoresists which can be machined among other things by photolithography, including for example PMMA for X-ray lithography, SU-8 (negative photoresist) and Novolac de Hoescht and AZ 9260 (positive photoresists ) for UN photolithography (Lorenz H, Despont M, Fahrni ⁇ , LaBianca ⁇ , Renaud P, SU-8: a low-cost negative resist for MEMS, J. Micromech. Microeng, 1997, 7, 121-124. _.
  • - silicone elastomers including poly (dimethylsiloxane) (PDMS), usable among others by simple molding, (Mac Donald JC, Duffy DC, Anderson JR, Chiu DT, Hongkai Wu, Schueller O, Whitesides GM, Fabrication of microfluidic Systems in poly (di ethylsiloxane), Electrophoresis 2000, 21, 27-40.
  • PDMS poly (dimethylsiloxane)
  • PA polycarbonates
  • PC polyoxymethylenes
  • POM polyoxymethylenes
  • COC cyclopentadienenorbomen copolymer
  • PMMA polymethylmethacrylates
  • PE-ld low density polyethylene
  • PE-hd high density polyethylene
  • PP polypropylene
  • PS polystyrenes
  • COC polyetheretherketone
  • Still other plastics can be micro-manufactured: polybutyleneterphthalate (PBT), polyphenylene ether (PPE), polysulfone (PSU), liquid crystal polymer (LCD), polyetherimide (PEI).
  • PBT polybutyleneterphthalate
  • PPE polyphenylene ether
  • PSU polysulfone
  • LCD liquid crystal polymer
  • PEI polyetherimide
  • Biodegradable polyactide can also be microfabricated.
  • PMMA and PC are widely used in injection molding and hot stamping. COC is commonly cited in hot stamping.
  • photohthography of photoresists including for example X-ray lithography for PMMA, UN photohthography for the Epson SU-8 photopolymer.
  • the "fill process” This is done by filling with a sacrificial layer, such as, for example, the Ciba-Geigy Araldite GT6063 between the second and the third photoresist layer. At the end of the process, the sacrificial layer is dissolved. - the "mask process”. A layer of metal is interposed on the second layer of photoresist that is not developed. This second layer of metal masks the microchannel. A third layer of photoresist is deposited and then illuminated. Then the photoresist is developed inside and outside of said micro-channel.
  • a sacrificial layer such as, for example, the Ciba-Geigy Araldite GT6063 between the second and the third photoresist layer.
  • the sacrificial layer is dissolved.
  • the "mask process” A layer of metal is interposed on the second layer of photoresist that is not developed. This second layer of metal masks the microchannel.
  • a third layer of photoresist
  • the "lamination process” a process without dissolution, where a layer of dry film of SU-8 is unwound on the construction made from the first layer of photoresist to seal it.
  • Plastic surface treatments depend on the application and the material used. For example, a hydrophobic surface must often be made hydrophilic.
  • Several methods exist for assembling and sealing plastic micro-products with a cover We can cite among others:
  • Microcolumn Separations 2000, 12 (7), 407-11; Alarie JP, Jacobson SC, Ramsey JM. Electrophoretic injection bias in a microchip valving scheme. Electrophoresis. 2001. Jan; 22 (2 ): 312-7; Rocklin RD, Ramsey RS, Ramsey JM. A microfabricated fluidic device for performing two-dimensional liquid-phase separations.
  • Electrophoresis 2001 Mar; 22 (5): 864-73; Colyer C. Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection. Cell Biochem Biophys. 2000; 33 (3): 323-37; Bonneil E, Waldron KC. On-line solid-phase preconcentration for sensitivity enhancement in capillary electrophoresis. J Capillary Electrophor. 1999 May-Aug; 6 (3-4): 61-73; Horvath J, Dolnik N. Polymer wall coatings for capillary electrophoresis.Electrophoresis.
  • Beds of fractionation micro-columns 2 can be etched on a support based on silicon or glass or ceramic. Beds of fractionation micro-columns 2 can be micro-molded or micro-stamped using silicon matrices when the support is plastic-based. Fractionation micro-column beds 2 can be coated with a thin film of hydrophobic or hydrophilic nature.
  • the micro-particulate network can be obtained by micro-molding or micro-stamping or photopolymerization or thermopolymerization in situ, or can be made up of micro or nano-rods fitting into said ht of said micro -colonnes.
  • a micro-particulate network of fractionation micro-columns 2 which constitutes the stationary phases can for example be obtained by photoengraving when the support is based on silicon or glass or ceramic.
  • a micro-particulate network of fractionation micro-columns 2 which constitutes the stationary phases can for example be obtained by micro-molding, micor-stamping, photopolymerization or thermopolymerization in situ, or may consist of micro or nano-rods fitting into the bed of said micro-columns.
  • a microparticulate network can be coated with a thin film of hydrophobic or hydrophilic nature.
  • a microparticulate network can be subjected to coupling chemistries known to those skilled in the art to graft molecules characterized by their polarity and their amphipathicity.
  • the stationary phases can be coated with peptides by grafting, using direct coupling chemistries or with spacer arms known to those skilled in the art, such as via cyanogen bromide, or carbodiimide or carbonyldiimidazole, or oxirane or azlactone.
  • An increasingly used method is the immobilization on tentacular gels of peptides via fixation by activation of epoxy gel and azlactone derivative (Pribl M. Beêt der Epoxyend phenomenon in mod en chromatographischen Sorbentien un Gelen. Anal. Chem. 1980. 303. 113 -116.).
  • the stationary phases can be coated with peptides by peptide synthesis in solid phase, the solid phase serving for the synthesis being also said stationary phase (Kumar KS, Rajasekharan Pillai NN, Das MR. Syntheses of four peptides from the immunodominant region of hepatitis C viral pathogens using PS TTEGDA support fot the investigation of HCN infection in human blood J. Peptide Res., 2000, 56, 88-96)
  • Stationary phases can be co-grafted into microcolumns for fractionation 2 of lipid monolayers of cell membranes such as, for example, phosphatidylcholines.
  • lipid monolayers of cell membranes such as, for example, phosphatidylcholines.
  • micro-levers In general, the detection of specific interactions is possible thanks to the measurement of the variation of mechanical properties of microstructures. In most cases, these microstructures are in the form of micro-levers.
  • Detection using micro-levers can be performed in static mode or in dynamic mode.
  • static mode the formation of a layer on the surface of the lever during a specific interaction generates a mechanical stress effect which results in a curvature of the lever.
  • the sensitivity is dependent on the stiffness of the micro-lever. It is of the order of 0.1 N / m in general or even lower.
  • the addition of a mass following a specific interaction on a resonant micro-lever causes a reduction in its resonant frequency.
  • the stiffnesses are greater between 1 and 100 N / m and the quality factors are between 10 and 500 in air, between 1 and 10 in liquids.
  • the sensitivity of such a detection is greatly increased if the measurements are carried out under vacuum (the quality factor can reach values greater than 104).
  • a first approach is based on the principle of laser optical deflection which is used as a detection system in commercial atomic force microscopes. It is an external detection. This technique is very sensitive and allows access to variations in deflection less than the angstrom or variations in resonant frequency of a few hertz. It is used in most cases (cf. patents WO 00/14539 or US 5,445,008 or J. Fritz et al., Science 288, 316, 2000).
  • the second approach consists in integrating the function of detection. They are generally of the piezoresistive type (cf.
  • the micro-levers can be coated with a particular molecule which will give it adsorption or affinity properties.
  • the positioning of the active part (specifically treated for molecular recognition particuhere) on the surface of the micro-lever, it can extend over the entire surface of the micro-lever in the case of a static measurement ( constraint effect) .
  • constraint effect being maximum when the micro-lever is embedded, an active surface reduced to the micro-lever mounting area may be sufficient.
  • the active part In the case of a dynamic measurement, if it is considered that the added mass does not modify the stiffness properties of the micro-lever, the active part must be positioned at the end of the micro-lever. However, an active part covering the entire surface of the micro-lever is conceivable.
  • micro-levers in the case where they are used with external optical detection.
  • Surface and volume micro-machining associated with deposits of thin layers make it possible to develop levers in silicon, in silicon oxide, in silicon nitride.
  • These micro-levers can also be metallized (gold, platinum ).
  • the dimensions of these micro-levers are typically a few hundred microns for the length, a few tens of microns for the width and a few tenths of a micron (for a static detection) or a few microns (for a dynamic detection) for the thickness.
  • the mechanical properties of the materials used as well as the dimensions of the micro-levers will modify their stiffness and their natural resonance frequencies.
  • the number of connections is equal to 2 per micro-lever, the first for the upper electrode and the second for the lower electrode.
  • the lower electrode is generally grounded and all of the lower electrodes are connected together to form a common ground. There are therefore (n + 1) electrical connections for n piezoelectric lever arms, which significantly reduces the number of electrical connections.
  • the other advantage of a piezoelectric detection is that it ensures not only the detection function but also the actuation function (resonance in the case of a dynamic measurement) thanks to the piezoelectric effect direct and reverse.
  • the second drawback concerns the stability of the ferroelectric and piezoelectric properties which are subject to thermal drifts, hysteresis effects and especially aging and fatigue which very strongly limits their lifespan for dynamic uses.
  • the number of connections is at least 2 for each micro-lever, i.e. 2n electrical connections for n micro-levers . This number can be equal to 4 if the piezoresistors are connected in Wheatstone bridge which gives 4n electrical connections.
  • the integration of a Wheatstone bridge makes it possible to reduce the compactness of the complete system compared to an assembly with an external Wheatstone bridge. In addition, it eliminates the effects of thermal drifts.
  • the disadvantage of a piezoresistive detection in the case of a dynamic measurement is that it supposes that there is an external mechanical excitation or that the quality factors of the microlevers are sufficiently important (> 100) to that the resonant frequency is detectable in white noise (thermomechanical excitation due to Brownian motion).
  • the selectivity of the micro-levers 13 depends on the intrinsic polarity, the solvophobicity and the porosity of the material which constitutes the micro-lever or a thin film coated on the micro-lever , and according to the polarity and the solvophobicity of the functional groups grafted onto the micro-lever.
  • the selectivity of the micro-levers 13 also depends on criteria such as the ion exchange and the affinity of the functional groups, the successive conditions of secondary micro-elution in the fractionation micro-columns 2 as well as the successive conditions of micro- extraction and micro-digestion carried out upstream of said micro-levers 13.
  • the capture of a molecule, and in particular of a protein, by a microlever 13 can be carried out by affinity. This is the case, for example, when the microlever has been coated with an antibody. In this case, the micro-lever captures a precise, known protein, and indicates its presence.
  • the capture of a molecule, and in particular of a protein, by a microlever 13 can be carried out by adsorption.
  • the same micro-lever will be able to detect a category of proteins having similar adsorption properties. Proteins not known or researched a priori can be detected.
  • fingerprint comparisons By carrying out fingerprint comparisons on different samples, it will be possible to identify differences in fingerprints, in particular differences in detection on adsorption micro-levers 13. Subsequently, by reproducing the capture with the same steps of selection, we can isolate the different proteins thus obtained to analyze them more specifically.
  • a stepwise or gradient elution allows the passage of different secondary eluents entraining different molecules according to their affinity with these molecules and the affinity of micro-levers with these same molecules. Successive fingerprints are recorded, at each elution step for a step-by-step elution, or at different times for an elution with an elution gradient. As already indicated, successive washes on the micro-levers 13 can be carried out, the retention of secondary micro-eluates or secondary micro-extracts or secondary digestion products of the fractionation products on the micro-levers 13 being measured by the deviation or by the vibration frequency of said micro-levers 13, several successive fingerprints being recorded.
  • the series of successive fingerprints for detecting the first sample on micro-levers 13 is for example then compared to the series of successive fingerprints for detection on micro-levers 13 of a second sample.
  • the passage of washing eluent into the detection zones can be carried out using micro-capture channels 8, into which a washing eluent is sent.
  • an additional washing circuit is provided, making it possible to bring a washing eluent directly upstream of a detection zone.
  • the analysis device comprises secondary fractionation micro-columns.
  • a fractionation micro-column 2 is intersected at the level of a terminal element by a capture micro-channel 8, connected upstream of the intersection with a secondary eluent supply conduit 15, and comprising downstream of the intersection a secondary fractionation micro-column 10.
  • a detection zone 11 comprising micro-levers 13 is located on the capture micro-channel 8 downstream of the secondary fractionation micro-column 10.
  • a washing micro-duct 70 comprises an inlet orifice 71 for supplying washing eluent, and an outlet orifice 72 opening into the micro- capture channel 8, downstream of the secondary fractionation micro-column 10, and upstream of the detection zone 11.
  • washing eluent If a washing eluent is brought in by the capture micro-channel 8 passing through the secondary fractionation micro-column 10, the washing eluent will entrain molecules retained in the secondary fractionation micro-column 10.
  • the micro-duct of washing 70 makes it possible to bring the washing eluent directly upstream of the detection zone for washing the micro-levers 13, without washing the secondary fractionation micro-column 10.
  • additional detection can be provided downstream of the detection zones li using micro-levers 13, for example by mass spectrometry.
  • mass spectrometry methods are discussed below.
  • a second variant after a comparison of successive fingerprints of two samples, provision is made for detection of secondary micro-eluates or secondary micro-extracts or secondary digestion products of the fractionation products to be carried out by mass spectrometry, but essentially on the detection zones 11 where the series of fingerprints by micro-levers 13 of the first sample differs from the series of fingerprints by micro-levers 13 of the second sample.
  • Mass spectrometry detection can be carried out in a known manner, as described in the following documents: (Cf. Dongré AR, Eng JK, Yates JR LU. Emerging tandem-mass spectrometry techniques for the rapid identification of proteins.
  • Detection by mass spectrometry can also be coupled in a known manner to a separation of peptides and proteins in capillaries or on miniaturized supports with micro-channels or micro-columns by micro-chromatography, micro-electrochromatography or micro-electrophoresis.
  • Documents describe more particularly the detection of peptides, proteins and carbohydrates by mass spectrometry.
  • polypeptides are analyzed by mass spectrometry before or after enzymatic digestion (Cf. Roepstroff P. Mass spectrometry in protein studies from genome to function. Current Opinion in Biotechnology, 1997, 8, 6-13), by mass spectrometry techniques using ionization in nebulization or desorption.
  • Post-translational modifications of proteins can be studied by subjecting the analytes to phosphatases or glycosylases (Cf. Qin J, Chait BT. Identifications and characterization of posttranslational modifications of proteins by MALDI Ion Trap mass spectrometry. Anal Chem, 1997, 69 , 4002-4009.) When the analysis by mass spectrometry takes place after digestion with a given endopeptidase, one can compare the spectra of the masses observed with the banks of the spectra of theoretical masses of the digestion residues with said endopeptidase.
  • the samples can be deposited on poly (vinylidene difluoride) or polyurethane membranes (Cf. Me Comb ME, Oleschuk RD, Manley DM, Donald L, Chow A, O'neil JD, Ens W , Stabding KG, Perreault H.
  • polyurethane membranes Cf. Me Comb ME, Oleschuk RD, Manley DM, Donald L, Chow A, O'neil JD, Ens W , Stabding KG, Perreault H.
  • non-porous polyurethane membrane as a sample support for matrix-assisted laser désorption ionisation time-of-flight mass spectrometry of peptides and proteins. Rapid Commun Mass Spectrom, 1997, 11 (15), 1716 -22).
  • we can plan to analyze glycoproteins by mass spectrometry Cf.
  • a first support 20 in the form of a flat plate, comprises a supply channel 4 intended for supply in the mobile phase and comprising an enrichment zone 21 of the mobile phase in the sample.
  • the first support 20 comprises a fractionation micro-column 2 comprising a micro-channel portion 3 provided with an introduction orifice 3a in fluid communication with a supply channel 4 downstream of an enrichment zone 18 d 'a mobile phase in sample, and an evacuation orifice 3b.
  • the first support 20 includes a capture micro-channel 8 intersecting the fractionation micro-column 2 at a terminal element 9, and a secondary eluent supply channel 15 in fluid communication with an inlet of the micro-channel of capture 8 upstream of the intersection with the fractionation micro-column 2.
  • a second support 22, in the form of a flat plate, comprises a micro-conduit 23 opening at one end into a secondary fractionation micro-column in fluid communication on the opposite side with a detection zone 11 provided with micro-levers 13.
  • a third support 24, also in the form of a flat plate, comprises a discharge channel 6.
  • the second and third supports 22, 24 are arranged on either side of the first support 20, parallel to the first support, being attached to the latter, so that the discharge channel 6 of the third support 24 is in fluid communication with the discharge orifice 3b of the fractionation micro-column 2, and the micro-conduit 23 of the second support is in fluid communication with the capture micro-channel 8, downstream of the intersection with the micro-column fractionation 2.
  • a first support 20 comprises a plurality of fractionation microcolumns 2, and a plurality of associated capture micro-channels 8.
  • the second support 22 comprises a plurality of micro-conduits in fluid communication with the capture micro-channels 8, and a plurality of detection zones 11.
  • the third support 24 comprises a discharge channel 6 in fluid communication with all of the discharge orifices 3b of the micro-channels 3.
  • the first, second and third supports 20, 22, 24 are arranged in parallel.
  • the second and third supports 22, 24 are arranged perpendicular to the first support 20.
  • the first support 20 comprises a single fractionation micro-column 2.
  • the first support 20 comprises a plurality of fractionation micro-columns 2, the second and third supports 22, 24 being adapted accordingly.
  • a support comprises four lots of fractionation micro-columns 2 arranged substantially in a star.
  • the fractionation micro-columns 2 of the first batch fall within a range of small lengths.
  • the fractionation microcolumns 2 of the second batch fall within a range of longer lengths, those of the third batch fall within a range of even longer lengths, those of the fourth batch fall within a range of even longer lengths.
  • the support comprises a central enrichment micro-column 25 of square shape, in fluid communication from which each of the batch fractionation micro-columns comes, at the center of which opens a channel for introducing the sample located in a vertical plane, and not shown in FIG. 8.
  • it is possible to provide for preliminary extractions of the constituents of the sample before introducing the sample into the fractionating microcolumns.
  • a preliminary extraction stage 30 comprises a support 31 shown partially and provided with a plurality of prior fractionation mico-columns 32 rectilinear parallel and of the same length, with a supply channel 33 in fluid communication with the fractionation micro-columns 32, and a discharge channel 34 in fluid communication with the fractionation micro-columns 32 on the side opposite to the feed channel 33.
  • the fractionation micro-columns 32 are formed by portions of micro-channels provided with intermediate separation means.
  • the support 31 comprises fluid capture means 35 in the form of capture micro-channels 36, supplied by eluting on one side from a supply line 37, and in fluid communication on the opposite side with a supply line recovery 38 common.
  • Each capture micro-channel transversely passes through a fractionation micro-column 32 at the level of a terminal element situated near an evacuation orifice of the fractionation micro-column 32 on the side of the evacuation channel 34.
  • the sample transported by a mobile phase is brought through the feed channel 33, circulates in the fractionation micro-columns 32, where it undergoes separation according to a selectivity of the separation means of the fractionation micro-columns 32
  • the association of a plurality of fractionation micro-columns 32 allows a significant separation in fractionation micro-columns 32 of small diameter, without limiting a flow rate of a mobile phase enriched in sample.
  • the capture means 35 allow successive captures of the constituents of the sample present at a given capture time at the terminal element of intersection of the capture micro-channels 36 with the fractionation micro-columns 32.
  • a portion of the constituents of the sample is recovered in the recovery conduit 38, which is conveyed to fractionation micro-columns and detection zones as described above.
  • the constituents separated during the prior extraction in a batch of preliminary fractionation micro-columns 32 having a particular selectivity can be better separated thereafter by adapting the selectivity of one or more batches of analysis fractionation micro-columns to which the captured portion is routed.
  • the micro-columns are of equal length in order to recover at the end of each micro-column substantially the same constituents.
  • the products captured on different portions are collectively evacuated in a separation conduit.
  • the fractionation micro-columns can be of different length to allow differential fractionation, and each capture micro-channel communicates with an associated detection zone.
  • an analysis device it is possible to provide a plurality of batches of preliminary fractionation microcolumns each having a different separation selectivity, and each communicating with a plurality of batches of analysis fractionation micro-columns, each batch of micro- analysis fractionation columns having a particular selectivity, preferably adapted as a function of the selectivity of the batch of prior extraction.
  • a support 31 comprises a single capture micro-channel 36 successively passing through the prior fractionation micro-columns 32 of the same batch, and finally opening into the recovery duct 38.
  • each capture micro-channel 36 comprises an upstream portion 36a situated between the supply duct 37 and the intersection with the preliminary fractionation micro-column 32, and a downstream portion 36b situated between the preliminary fractionation micro-column 32 and the recovery duct 38.
  • the upstream portion 36a and the downstream portion 36b open onto the preliminary fractionation micro-column 32 at offset points, at the ends of a capture segment 40.
  • the upstream portion 36a opens at the downstream end of the capture segment 40, the downstream portion 36b opening at the upstream end of the capture segment 40.
  • the separate components of the sample located at the capture segment 40 will be captured.
  • a larger number of constituents is captured during the same capture.
  • a support 1 comprises fractionation micro-columns 2, associated capture micro-channels 8 provided with upstream portions 8a and downstream portion 8b offset, opening into each fractionation micro-column 2 respectively downstream and upstream of a capture segment 40.
  • the downstream portion 8b of a capture micro-channel 8 leads downstream to a detection zone 11 provided with micro-levers 13.
  • An outlet channel 45 connects all of the capture micro-channels 8 downstream of the zones of detection 11, for evacuation of the mobile phases and of the constituents not retained by the selective micro-levers.
  • the support 1 comprises a washing micro-duct 46 in fluid communication with each of the capture micro-channels 8, directly upstream of the detection zones 11.
  • a fractionation micro-column to comprise a terminal portion. provided with a selectivity different from the selectivity of the upstream portion of the micro- column. Given the selectivity of the upstream portion, it is known that constituents having certain characteristics will migrate more quickly and reach the terminal portion first. The selectivity of the terminal portion is then adapted for additional separation of the constituents arriving at substantially the same time at the end of the fractionation micro-column.
  • This difference in selectivity of a portion of a fractionation micro-column can be applied to a fractionation micro-column, a secondary fractionation microcolumn, a prior fractionation micro-column.
  • terminal portion is meant a portion located just upstream of an outlet or of capture means.
  • an analysis support 1 comprises means for supplying the sample and the mobile phase separately.
  • a support 1 partially shown comprises a sample supply channel 4 comprising an introduction orifice 4a and a discharge orifice 4b.
  • the support 1 also includes a mobile phase supply channel
  • mobile phase supply micro-conduits 42 comprising an inlet orifice 43 opening into the mobile phase supply channel 41, and an outlet orifice 44 opening into the sample supply channel 4.
  • Each discharge orifice of a mobile phase supply micro-duct 42 opens into the sample supply channel 4 opposite an introduction orifice 3a of a fractionation micro-column 2.
  • Each mobile phase supply micro-duct 42 extended by a fractionation micro-column 2 forms a micro-channel intersecting the sample-raising channel 4.
  • a phase supply micro-duct mobile 42 can be envisaged as a portion of a micro-channel devoid of separation means, and located upstream of a portion of micro-channel provided with means of separation and thus forming a fractionation micro-column 2, the sample supply micro-channel 4 intersecting all of the micro-channels at the level of the introduction orifice 3a of the fractionation micro-columns 2.
  • a sample circulates in the sample supply channel 4, from the introduction orifice 4a to the discharge orifice 4b.
  • a circulation of mobile phase is caused in the mobile phase supply micro-conduits 42
  • the mobile phase crosses the sample supply channel 4 transversely, enriching itself in the sample, then is recovered downstream by the fractionation micro-columns 2.
  • the mobile phase supply micro-conduits 42 allow at a given instant an injection simultaneously into all of the fractionation micro-columns 2 of the same quantity of mobile phase enriched in sample.
  • a difference in mobile phase flow rate enriched from one fractionation micro-column 3 to another could lead to detection variations upstream of the fractionation micro-columns 3, in particular if provision is made for a simultaneous capture of fractionation products at the level of terminal elements of the fractionation micro-columns.
  • an analysis device comprising a preliminary extraction stage, it is of course possible to provide, upstream of the preliminary extraction stage, a sample capture zone as described above.
  • a detection of proteins or peptides has been described indifferently.
  • a biological cell contains a large number of proteins, which can generate an even greater number of peptides after digestion.
  • one or more sorting micro-columns can be provided upstream of micro-columns, and in particular a micro-column for sorting by size exclusion chromatography, already mentioned previously.
  • Example 1 examples of possible analyzes using an analysis device according to one aspect of the invention are provided.
  • Two biological samples can be analyzed, each using 8 supports of the type shown in FIG. 8. On the supports, the samples are separated by electrochromatography supplemented by additional pressure.
  • each of the supports contains 4 lots of 1000 fractionation microcolumns having a length gradient, with a minimum length difference of 20 microns between two micro-columns, so that between the earliest and the last of the 1000 micro-columns columns, there is a length difference of 20 mm.
  • the 1000 microcolumns in the first batch have lengths between 12 and 14 cm.
  • the 1000 microcolumns in the second batch have lengths between 14 and 16 cm.
  • the 1000 microcolumns in the third batch have lengths between 16 and 18 cm.
  • the 1000 microcolumns in the fourth batch have lengths between 18 and 20 cm.
  • FC fractionation microcolumns having a length gradient, with a minimum length difference of 20 microns between two micro-columns, so that between the earliest and the last of the 1000 micro-columns columns, there is a length difference of 20 mm.
  • the 1000 microcolumns in the first batch have lengths between 12 and 14 cm.
  • the media used include micro-capture channels.
  • the fractionation products adsorbed at time t on a fractionation micro-column at the point of intersection with said corresponding capture micro-channel are captured simultaneously, and undergo secondary, orthogonal, parallel micro or nano-elutions, terminal, simultaneous.
  • the capture micro-channels lead to detection zones with microlevers with optical detection.
  • the stationary phases of the fractionation micro-columns of the first support (FC, 4, 1000, 20, 12-20) are grafted with C30 alkyl chain molecules.
  • Such a support can be called (FC, 4, 1000, 20, 12-20) -C30.
  • the stationary phases of the fractionation micro-columns of the second support (FC, 4, 1000, 20, 12-20) are grafted with butyl molecules. (FC, 4, 1000, 20, 12-20) -butyl.
  • the stationary phases of the fractionation micro-columns of the third support (FC, 4, 1000, 20, 12-20) are grafted with cyclo-hexyl molecules.
  • Such a support can be called (FC, 4, 1000, 20, 12-20) -cyclohexyl.
  • the stationary phases of the fractionation micro-columns of the fourth support (FC, 4, 1000, 20, 12-20) are grafted with phenyl molecules.
  • Such a support can be called (FC, 4, 1000, 20, 12-20) - phenyl.
  • the stationary phases of the fractionation micro-columns of the fifth support (FC, 4, 1000, 20, 12-20) are grafted with ethyl molecules.
  • Such a support can be called (FC, 4, 1000, 20, 12-20) -ethyl.
  • the stationary phases of the fractionation micro-columns of the sixth support (FC, 4, 1000, 20, 12-20) are grafted with amino-propyl molecules.
  • Such a support can be called (FC, 4, 1000, 20, 12-20) -amino-propyl.
  • the stationary phases of the fractionation micro-columns of the seventh support (FC, 4, 1000, 20, 12-20) are grafted with dihydroxypropyl molecules.
  • Such a support can be called (FC, 4, 1000, 20, 12-20) -dihydroxypropyl
  • the stationary phases of the fractionation micro-columns of the eighth support (FC, 4, 1000, 20, 12-20) are grafted with cyanopropyl molecules.
  • Such a support can be called (FC, 4, 1000, 20, 12-20) - cyanopropyl.
  • Each detection zone associated with a capture micro-channel comprises eight micro-levers each provided with a specific coating.
  • the first may have a coating based on C30 alkyl chains, the second a coating based on octadecyl chains, the third a coating based on octyl chains, the fourth based on butyl chains, the fourth based on chains phenyl, the fifth based on cyclo-hexyl chains, the fifth based on ethyl chains, the sixth based on amino-propyl chains, the seventh based on dihyroxypropyl chains, the eighth based on cyanopropyl chains.
  • the basic solvents for the primary elutions of the fractionation chromatographies carried out on format supports can be ternary mixtures of water, trifluoroacetic acid (TFA), acetonitrile.
  • TFA trifluoroacetic acid
  • Six primary elution steps can be provided, the eluent used in each step having successively the following composition variants: (water, 10% acetonitrile, 0.1% TFA). (water, 15% acetonitrile, 0.1% TFA), (water, 20% acetonitrile, 0.1% TFA), (water, 25% acetonitrile, 0.1% TFA), (water, 30% acetonitrile, TFA 0.1%), (water, 35% acetonitrile, 0.1% TFA).
  • the successive series of imprints mentioned above are produced for each sample.
  • the series of successive fingerprints of the first sample is then compared to the series of successive fingerprints of the second sample, the series detection fingerprints then being archived in a computer database.
  • the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art.
  • the method is applicable to any search for differential expression of proteins for a given tissue, in particular for the comparison of a healthy individual and an individual suffering from a pathology. It is also applicable to the comparison of protein expression in two different physiological situations. It is also applicable to the comparison of protein expression in two strains of microorganisms (viruses, bacteria, yeasts), or applicable to the detection of differential expression of proteins on microorganisms (viruses, bacteria, yeasts) subjected to specific stimuli.
  • the device can be used to compare fingerprints of basic proteins between two samples. Each sample is analyzed on a support of the type shown in FIG. 8, and of format (FC, 4, 1000, 20, 12-20), in accordance with the name used in Example 1.
  • fractionation micro-columns are strongly zwitterionic in nature and contain copolymers based on sulfoalkylbetaine (N, N-dimethyl-N-methacryloyloxyethyl-N- (3-sulfopropyl) ammonium betaine (Cf. Niklund C, Sjorgen A, Irgum K , ⁇ es I. Anal. Chem. 2001. Feb 1, 73, (3), 444-52).
  • sulfoalkylbetaine N-dimethyl-N-methacryloyloxyethyl-N- (3-sulfopropyl) ammonium betaine
  • the basic proteins are separated in the fractionation micro-columns according to different methods with primary eluent (eluent A: water; eluent B: water, 10 mM sodium phosphate).
  • the fractionation products are separated in secondary fractionation micro-columns located downstream of capture means. Secondary elutions are modulated by thiocyanate ions (primary eluent + 10 mM thiocyanate) or by perchlorate ions (primary eluent + 10 mMperchlorate).
  • the series of successive fingerprints of the first sample is compared with the series of successive fingerprints of the second sample, the series of fingerprints then being archived in a computer database. Where differences are detected, the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art.
  • the fingerprints of peptides and membrane proteins from two samples can be compared specifically.
  • each sample is analyzed on two supports of the type shown in FIG. 8, having a format of the type (FC, 4, 1000, 20, 12-20).
  • the integrated supports have fractionation micro-columns grafted with C4 alkyl chains.
  • the membrane peptides are dissolved in dichloromethane (CH2C12) -hexafluoro-2-propanol (HFIP) (4: 1) containing traces of pyridine, then separated in micro-columns of fractionation using different primary elutions in different successive fingerprints.
  • CH2C12 dichloromethane
  • HFIP hexafluoro-2-propanol
  • the primary elutions are based on mixtures of eluent A (formic acid-water (2: 3)) on the one hand and eluent B (formic acid -2-propanol (4: 1)) on the other go.
  • the primary elutions successively present the following composition variants: (A 100%, B 0%); (A 80%, B 20%); (A 60%, B 40%); (At 40%,
  • the membrane proteins after extraction with Triton X-114, precipitation with 90% ethanol and redissolution in 65% formic acid, are separated in the micro-columns fractionation using different primary elutions in different successive fingerprints.
  • the primary elutions are based on mixtures of eluent A (formic acid-water (65:35)) and eluent B (acetonitrile-water (65:35)).
  • the primary elutions successively present the following composition variants: (A 100%, B 0%); (A 80%, B 20%); (A 60%, B 40%); (A 40%, B 60%); (A 20%, B 80%); (A 0%, B 100%).
  • the series of successive fingerprints of the first sample is then compared to the series of successive fingerprints of the second sample, the series of detection fingerprints then being archived in a computer database. Where differences are detected, the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art.
  • the data acquired during analyzes carried out using a device according to examples 1 to 3 described above can be used to configure a rapid test on miniaturized consumable. For such a test, it is possible to use a device provided with supports provided only with fractionating microcolumns where numerous comparisons have shown differences in reproducible fingerprints for a given pathology.
  • a healthy individual shows an A imprint, the individual suffering from pathology showing a B imprint.
  • a first support (sA) is dedicated to the recognition of the A imprint, a second support (sB) being dedicated to the recognition of the 'imprint B.
  • microcolumns In this test targeted at said pathology, the number of microcolumns, microchannels and microlevers is very small.
  • the fractionation micro-columns chosen, more specifically the selectivity of the separation methods in relation to the composition of the solid and liquid phases used, are adapted to demonstrate the presence in a sample of proteins of which the presence or absence is characteristic. of the targeted pathology.
  • An analysis device is suitable for the comparative chemical or biochemical analysis of two samples of a chemical nature, or else two samples of a biochemical nature such as cell extracts which are raw or derived from a prior extraction or which have undergone enzymatic digestion.
  • a biological sample is characterized in particular by its composition in each of the proteins, glycoproteins, phosphoproteins, lipoproteins, lipids, polysaccharides, hormones, vitamins synthesized permanently or occasionally depending on the tissue or the physiological or pathological condition envisaged.
  • An analysis device allows the detection of these constituents by separation and by obtaining fingerprints.
  • An analysis device can rely on a length gradient of a large number of micro-channels or micro-separation columns, for example by micro-electrophoresis, micro-chromatography or micro-electrochromatography.
  • a set of fractionation micro-columns can be associated with a second or even a third set of micro-channels or micro-columns, each or each of the micro-channels or microcolumns of separation of the first set being individually coupled to each or each of the micro-channels or micro-columns for separating said second set of micro-channels or micro-columns.
  • a detection by selective micro-levers has been described which makes it possible to adapt the selectivity of the micro-levers to the selectivity of the fractionation micro-columns with which they are associated. Additional detection can be performed by mass spectrometry.
  • the analysis device can also use other detection means known to those skilled in the art such as fluorescence, surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), electrochemistry, spectrophotometry, this list not being not limiting. Additional detection can be performed by mass spectrometry.
  • the analysis device can also use other means of detection known to those skilled in the art such as fluorescence, surface plasmon resonance.
  • an analysis device is obtained allowing separation of the constituents of a sample, according to different selectivities, and detection of the constituents.
  • An analysis device allows an exhaustive and rapid analysis of a sample and its comparison with another sample.
  • Detection by micro-levers linked to analysis means allows storage and comparison of recorded data.
  • a device according to the invention is suitable for miniaturization.
  • the invention is not limited to the embodiments and to the variants described above. Modifications can be made without departing from the scope of the invention.

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Abstract

The invention concerns a device for biological or chemical analysis of biological or chemical samples, in particular for a comparative analysis of at least two samples, comprising a plurality of fractionating micro-columns (2) for constituents of a sample, each micro-column (2) including at least a micro-channel portion (3) provided with intermediate separating means, the micro-channel portion (3) comprising an inlet (3a) for a sample-enriched mobile phase and an outlet (3b) located at a terminal end. The device further comprises fluidic means for capturing (7) fractionated products at a terminal element (9) of each fractionating micro-column (2) located upstream of its outlet (3b), capture micro-channels (8) for recovering the captured fractionated products, and selective micro-lever assemblies (13) associated with the fractionating micro-columns (2) located downstream of the capture micro-channels (8), a micro-lever (13) including detection means connected to analysing means.

Description

Dispositif d'analyse d'échantillon chimique ou biochimique, ensemble d'analyse comparative, et procédé d'analyse associé. Chemical or biochemical sample analysis device, comparative analysis set, and associated analysis method.
La présente invention concerne un dispositif d'analyse chimique ou biochimique d'échantillons chimiques ou biologiques, notamment pour une analyse comparative d'au moins deux échantillons. L'invention concerne également un ensemble d'analyse comparative, ainsi qu'un procédé d'analyse.The present invention relates to a device for chemical or biochemical analysis of chemical or biological samples, in particular for a comparative analysis of at least two samples. The invention also relates to a comparative analysis assembly, as well as to an analysis method.
L'analyse et la comparaison d'échantillons chimiques et biochimiques, et notamment l'analyse des protéines contenues dans des échantillons, complètent l'étude des gênes par l'étude de l'expression fonctionnelle de ces gênes sous forme de protéines.The analysis and comparison of chemical and biochemical samples, and in particular the analysis of proteins contained in samples, complete the study of genes by studying the functional expression of these genes in the form of proteins.
Chez les Eucaryotes, la génomique fonctionnelle (étude de la fonction des gènes déchiffrés) et la protéomique (étude de la fonction des protéines) font apparaître une diversité bien plus grande que celle du strict déchiffrage du code génétique. Ainsi, chez l'Homme, certaines estimations conduisent à penser que 25 000 gènes sont potentiellement capables d'exprimer un million de protéines différentes. Ces approches fonctionnelles se déclinent tant sur une recherche de voies intracellulaires et intercellulaires comprenant la notion de cascades d'interactions cellulaires que sur une recherche des combinaisons d'expression de gènes intervenant dans les cascades d'interactions. Que l'on considère les cascades d'interaction cellulaire ou un mode combiné d'expression de plusieurs gènes, l'état des modifications post-traductionnelles des protéines exprimées est essentiel à leur fonction et doit donc être connu.In eukaryotes, functional genomics (study of the function of deciphered genes) and proteomics (study of the function of proteins) reveal a much greater diversity than that of the strict deciphering of the genetic code. Thus, in humans, some estimates suggest that 25,000 genes are potentially capable of expressing a million different proteins. These functional approaches are available both for a search for intracellular and intercellular pathways including the concept of cascades of cellular interactions as for a search for combinations of expression of genes intervening in cascades of interactions. Whether we consider cell interaction cascades or a combined mode of expression of several genes, the state of post-translational modifications of the expressed proteins is essential to their function and must therefore be known.
Il existe un besoin de bénéficier d'outils d'analyse et de comparaison d'échantillons chimiques et biochimiques permettant une séparation des constituants d'un échantillons en vue de leur analyse et éventuellement de la comparaison des constituants de deux échantillons. Notamment, on désire souvent comparer les protéines exprimées par plusieurs groupes de cellules différents dans des états physiologiques ou pathologiques sensiblement différents. On connaît une méthode consistant à séparer les constituants d'un échantillon par migration dans un gel. Cependant, ses limites sont un manque d'exhaustivité, un manque de discrimination, une reproductibilité insuffisante et une inaptitude à l'étude des protéines hydrophobes.There is a need to benefit from tools for analysis and comparison of chemical and biochemical samples allowing a separation of the constituents of a sample with a view to their analysis and possibly of the comparison of the constituents of two samples. In particular, it is often desired to compare the proteins expressed by several different groups of cells in significantly different physiological or pathological states. A method is known which consists in separating the constituents of a sample by migration in a gel. However, its limits are a lack of completeness, a lack of discrimination, insufficient reproducibility and an inability to study hydrophobic proteins.
La présente invention a pour objet un dispositif d'analyse chimique ou biochimique d'échantillons permettant d'obvier ces inconvénients.The subject of the present invention is a device for chemical or biochemical analysis of samples making it possible to overcome these drawbacks.
L'invention a pour objet un dispositif d'analyse chimique ou biochimique d'échantillons permettant une séparation rapide des constituants d'un échantillon et une analyse rapide des constituants séparés, ainsi que la comparaison d'analyses de différents échantillons.The subject of the invention is a device for chemical or biochemical analysis of samples allowing rapid separation of the constituents of a sample and a rapid analysis of the separate constituents, as well as the comparison of analyzes of different samples.
L'invention a également pour objet un dispositif d'analyse chimique ou biochimique d'échantillons permettant une séparation améliorée des constituants d'un échantillon, et notamment des séparations des constituants d'un échantillon selon différents critères de sélectivité.The subject of the invention is also a device for chemical or biochemical analysis of samples allowing improved separation of the constituents of a sample, and in particular separations of the constituents of a sample according to different selectivity criteria.
Un tel dispositif d'analyse chimique ou biochimique d'échantillons biologique ou chimique, notamment pour une analyse comparative d'au moins deux échantillons, comprend une pluralité de micro-colonnes de fractionnement de constituants d'un échantillon, chaque micro-colonne de fractionnement comprenant au moins une portion d'un micro-canal munie de moyens de séparation intermédiaires, la portion de micro-canal comprenant un orifice d'introduction d'une phase mobile enrichie en échantillon et un orifice d'évacuation situé à une extrémité terminale. Selon un aspect de l'invention, le dispositif comprend des moyens fluidiques de capture de produits de fractionnement au niveau d'un élément terminal de chaque micro-colonne de fractionnement situé en amont de son orifice d'évacuation, des micro-canaux de ' capture destinés à récupérer les produits de fractionnement capturés, et des ensembles de micro-leviers sélectifs associés aux micro-colonnes de fractionnement et situés en aval des micro-canaux de capture, un micro-levier comportant des moyens de détection reliés à des moyens d'analyse.Such a device for chemical or biochemical analysis of biological or chemical samples, in particular for a comparative analysis of at least two samples, comprises a plurality of micro-columns for fractionation of constituents of a sample, each micro-column for fractionation comprising at least a portion of a micro-channel provided with intermediate separation means, the micro-channel portion comprising an orifice for introducing a mobile phase enriched in sample and an evacuation orifice located at a terminal end. According to one aspect of the invention, the device comprises fluid means for capturing fractionation products at the level of a terminal element of each fractionation micro-column situated upstream from its discharge orifice, micro-channels of ' capture intended to recover the captured fractionation products, and sets of selective micro-levers associated with the fractionation micro-columns and located downstream of the capture micro-channels, a micro-lever comprising detection means connected to means of 'analysis.
On désigne par micro-colonne de fractionnement une portion de micro-canal munie de moyens de séparation. La portion de micro-canal formant la micro-colonne de fractionnement peut être précédée de portions de micro-canal aval ou amont dénuées de moyens de séparation. Dans la suite, on désigne par orifice d'introduction et d'évacuation de la micro-colonne de fractionnement les extrémités de la portion de micro-canal configurée en micro-colonne de fractionnement. Des moyens de séparations peuvent être une phase appelée phase stationnaire, du type utilisée en chromatographie, ou électrochromatographie, ou un gel d'électrophorèse, ou des moyens électriques.The fractionation micro-column designates a portion of micro-channel provided with separation means. The micro-channel portion forming the fractionation micro-column can be preceded by downstream or upstream micro-channel portions devoid of separation means. In the following, the end of the micro-channel portion configured as a fractionation micro-column is designated by the orifice for introducing and removing the fractionation micro-column. Separation means can be a phase called stationary phase, of the type used in chromatography, or electrochromatography, or an electrophoresis gel, or electrical means.
En chromatographie, on parle de phase stationnaire par opposition à la phase mobile circulant dans la portion de micro-canal. Une phase mobile peut être de façon avantageuse un éluant présentant selon sa composition une affinité plus ou moins importante avec des constituants de l'échantillon, du type molécules ou protéines, et par conséquent plus ou moins apte à entraîner les constituants, une phase stationnaire ayant tendance à freiner la migration des constituants, plus ou moins selon leurs caractéristiques.In chromatography, we speak of the stationary phase as opposed to the mobile phase circulating in the micro-channel portion. A mobile phase can advantageously be an eluent which, depending on its composition, has a greater or lesser affinity with constituents of the sample, of the molecule or protein type, and therefore more or less able to entrain the constituents, a stationary phase having tendency to slow down the migration of constituents, more or less according to their characteristics.
Une phase mobile circule dans un micro-canal en entraînant un échantillon. Les moyens de séparation prévus dans la portion de micro-canal formant une micro- colonne de fractionnement provoquent un fractionnement ou une séparation des constituants de l'échantillon.A mobile phase circulates in a micro-channel carrying a sample. The separation means provided in the micro-channel portion forming a micro- fractionation column causes fractionation or separation of the components of the sample.
La séparation est effectuée par migrations différentielles de chaque constituant de l'échantillon dans la micro-colonne, en fonction d'une sélectivité de la micro-colonne et de la phase mobile. La phase mobile entraîne les constituants de l'échantillon vers l'orifice d'évacuation du micro-canal.The separation is carried out by differential migrations of each constituent of the sample in the micro-column, as a function of a selectivity of the micro-column and of the mobile phase. The mobile phase drives the components of the sample to the micro-channel discharge orifice.
Les moyens fluidiques de capture permettent de prélever des constituants séparés de l'échantillon se situant à l'instant du prélèvement dans une portion terminale déterminée de faible longueur de la micro-colonne de fractionnement, ladite portion terminale étant située en amont de l'orifice d'évacuation du micro-canal.The fluidic capture means make it possible to take separate constituents from the sample located at the time of sampling in a determined terminal portion of short length of the fractionation micro-column, said terminal portion being located upstream of the orifice. micro-channel discharge.
Les constituants capturés sont entraînés dans des micro-canaux de capture vers des micro-leviers sélectifs, en vue de leur détection et de leur analyse.The captured constituents are entrained in micro-capture channels towards selective micro-levers, with a view to their detection and analysis.
L' association de moyens fluidiques de capture et de micro-leviers permet une analyse rapide des constituants capturés pour déterminer la composition de l'échantillon. Les moyens d' analyse reliés aux micro-leviers permettent l' obtention rapide de résultats.The combination of fluidic capture means and micro-levers allows a rapid analysis of the captured components to determine the composition of the sample. The means of analysis linked to the micro-levers allow rapid results to be obtained.
Pour une séparation rapide et améliorée des constituants d'un échantillon, on peut prévoir que chaque micro-colonne de fractionnement ou un groupe de microcolonnes de fractionnement de même longueur diffère par sa longueur des autres microcolonnes de fractionnement ou groupes de micro-colonnes de fractionnement, les éléments terminaux étant situés sur chaque micro-colonne de fractionnement à une distance donnée de l'extrémité terminale de la micro-colonne de fractionnement. La phase mobile enrichie en échantillon circule de l'orifice d'introduction des microcanaux vers l'orifice d'évacuation. Les éléments terminaux, où s' effectuent les captures, sont situés à des distances différentes de l'entrée des micro-colonnes. Les vitesses de migration des constituants sont différentes. Dès lors, à un instant donné après le début de la migration, on retrouvera dans les éléments terminaux des micro-colonnes ou de groupes de micro-colonne des constituants différents. Dans les éléments terminaux des micro-colonnes les plus longues, on retrouvera les molécules migrant le plus rapidement. Dans les éléments terminaux des micro-colonnes les plus courtes, on retrouvera les molécules migrant lentement, les molécules migrant rapidement ayant alors été entraînées vers l'orifice d'évacuation de la micro-colonne grâce à la circulation de la phase mobile enrichie.For rapid and improved separation of the constituents of a sample, provision may be made for each fractionation micro-column or a group of fractionation microcolumns of the same length to differ in length from the other fractionation microcolumns or groups of fractionation micro-column , the terminal elements being located on each fractionation micro-column at a given distance from the terminal end of the fractionation micro-column. The mobile phase enriched in sample flows from the micro-channel introduction opening towards the evacuation opening. The terminal elements, where the catches are made, are located at different distances from the entry of the micro-columns. The migration rates of the constituents are different. Therefore, at a given time after the start of migration, we will find in the terminal elements micro-columns or micro-column groups of different constituents. In the terminal elements of the longest micro-columns, we will find the most rapidly migrating molecules. In the terminal elements of the shortest micro-columns, we will find the slowly migrating molecules, the rapidly migrating molecules having then been entrained towards the evacuation orifice of the micro-column thanks to the circulation of the enriched mobile phase.
De préférence, chaque micro-colonne de fractionnement diffère d'une micro-colonne de fractionnement immédiatement plus longue par un élément de longueur donné. On obtient ainsi un gradient de micro-colonnes permettant une séparation différentielle des échantillons. Pour améliorer une discrimination des constituants capturés, le dispositif comprend des micro-colonnes de fractionnement secondaire situées en aval des moyens fluidiques de capture et en amont de l'ensemble de micro-leviers associé à une microcolonne de fractionnement, et destinées au fractionnement secondaire des produits de fractionnement capturés. Ainsi, les constituants capturés dans un élément terminal, ou portion de longueur d'une micro-colonne, sont à nouveau séparés avant d'être analysés à l'aide des micro-leviers, pour une détection plus précise.Preferably, each fractionation micro-column differs from an immediately longer fractionation micro-column by an element of given length. A gradient of micro-columns is thus obtained allowing differential separation of the samples. To improve discrimination of the captured constituents, the device comprises secondary fractionation micro-columns situated downstream of the fluidic capture means and upstream of the set of micro-levers associated with a fractionation microcolumn, and intended for the secondary fractionation of fractionation products captured. Thus, the constituents captured in a terminal element, or portion of the length of a micro-column, are again separated before being analyzed using micro-levers, for more precise detection.
Dans un mode de réalisation, le dispositif comprend plusieurs lots de micro- colonnes de fractionnement, chaque lot de micro-colonnes de fractionnement possédant une sélectivité déterminée par les moyens de séparation de ses micro-colonnes de fractionnement comprenant une phase stationnaire revêtue ou non et/ou des moyens électriques de séparation. Une séparation d'un échantillon dans des lots de microcolonnes de sélectivités différentes permet de mettre en évidence, dans chaque lot, des constituants différents de l'échantillon. L'exhaustivité de l'analyse de l'échantillon est améliorée.In one embodiment, the device comprises several batches of fractionation micro-columns, each batch of fractionation micro-columns having a selectivity determined by the means for separating its fractionation micro-columns comprising a stationary phase coated or not and / or electrical separation means. A separation of a sample in batches of microcolumns of different selectivities makes it possible to highlight, in each batch, different constituents of the sample. The comprehensiveness of the sample analysis is improved.
Dans un mode de réalisation, le dispositif comprend un support portant plusieurs lots de micro-colonnes de fractionnement, des moyens de captures et des ensembles de micro-leviers associés, et un canal d'alimentation de l'ensemble des lots de micro-colonnes de fractionnement. Les micro-leviers sélectifs comprennent des moyens de détection en fonction de leur état de surface ou de l'état de surface d'un revêtement, de leur nature chimique ou de la nature chimique d'un revêtement. Des constituants séparés, tels que des protéines, pourront réagir ou non avec un ou plusieurs mico-leviers spécifiques, ce qui indiquera leur présence dans l'échantillon. De préférence, les micro-leviers comprennent des moyens de détection par adsorption de molécules.In one embodiment, the device comprises a support carrying several batches of fractionation micro-columns, capture means and sets of associated micro-levers, and a supply channel for all of the batches of micro-columns fractionation. The selective micro-levers comprise means of detection as a function of their surface state or of the surface state of a coating, of their chemical nature or of the chemical nature of a coating. Separate constituents, such as proteins, may or may not react with one or more specific micro-levers, which will indicate their presence in the sample. Preferably, the micro-levers comprise means for detection by adsorption of molecules.
Les micro-leviers sont prévus pour la détection de molécules qui y sont fix ées. Des anticorps greffés sur des microleviers permettront de détecter la présence d'une molécule spécifique, telle qu'une protéine spécifique, dont on connaît déjà la nature. Des micro-leviers prévus pour détecter des molécules qui y sont adsorbees permettent une détection de molécules et notamment de protéines dont on ne connaît pas la nature à priori.The micro-levers are intended for the detection of molecules attached to it. Antibodies grafted onto microlevers will make it possible to detect the presence of a specific molecule, such as a specific protein, the nature of which is already known. Micro-levers intended to detect molecules which are adsorbed there allow detection of molecules and in particular of proteins whose nature is not known a priori.
La comparaison des empreintes d' adsorption sur les micro-leviers de protéines de plusieurs échantillons pourra permettre de mettre en évidence les protéines exprimées différentiellementThe comparison of the adsorption fingerprints on the micro-levers of proteins of several samples may make it possible to highlight the proteins expressed differently.
De préférence, les micro-colonnes présentent un diamètre compris entre 1 micron (μm) et 100 microns (μ ). Dans un mode de réalisation, le dispositif comprend un support de fractionnement portant les micro-colonnes de fractionnement et un support de détection portant les micro-leviers, les supports étant sensiblement plans, et les supports étant sensiblement parallèles ou perpendiculaires entre eux. Afin d'améliorer une discrimination de constituants d'un échantillon, on peut prévoir en amont des micro-colonnes de fractionnement au moins un étage de fractionnement préalable comprenant au moins une micro-colonne de fractionnement préalable, des moyens de capture fluidiques au niveau d'un élément terminal de la micro-colonne de fractionnement préalable, et un canal de récupération des extraits préalables prévu pour amener les extraits préalables vers les micro-colonnes de fractionnement. L'étage d'extraction préalable permet une analyse d'une portion de l'échantillon comprenant un nombre réduit de constituants à détecter.Preferably, the micro-columns have a diameter of between 1 micron (μm) and 100 microns (μ). In one embodiment, the device comprises a fractionation support carrying the fractionation micro-columns and a detection support carrying the micro-levers, the supports being substantially planar, and the supports being substantially parallel or perpendicular to each other. In order to improve discrimination of the constituents of a sample, it is possible to provide upstream of the fractionation micro-columns at least one preliminary fractionation stage comprising at least one prior fractionation micro-column, fluid capture means at the level a terminal element of the preliminary fractionation micro-column, and a recovery channel for the preliminary extracts provided for bringing the preliminary extracts to the fractionation micro-columns. The preliminary extraction stage allows an analysis of a portion of the sample comprising a reduced number of constituents to be detected.
De préférence, la sélectivité de micro-colonnes de fractionnement préalable est adaptée en fonction de la sélectivité de micro-colonnes de fractionnement pour favoriser l'extraction préalable d'extraits contenant des constituants qui seront bien séparés dans les micro-colonnes de fractionnement associées, compte tenu de leur propre sélectivité.Preferably, the selectivity of preliminary fractionation micro-columns is adapted as a function of the selectivity of fractionation micro-columns to promote the prior extraction of extracts containing constituents which will be well separated in the associated fractionation micro-columns, given their own selectivity.
On prévoira éventuellement une pluralité d'étages d'extraction préalable de sélectivités différentes, chacun associé à des lots de micro-colonnes de fractionnement. Dans un étage d'extraction préalable, on peut prévoir des captures successives régulières.A plurality of preliminary extraction stages of different selectivities may be provided, each associated with batches of fractionation micro-columns. In a preliminary extraction stage, regular successive catches can be provided.
Dans un mode de réalisation, un étage de fractionnement préalable comprend une pluralité de micro-colonnes de fractionnement préalable, chacune intersectée par un micro-canal de capture, les micro-canaux de capture étant reliés à un canal de récupération.In one embodiment, a pre-fractionation stage comprises a plurality of pre-fractionation micro-columns, each intersected by a capture micro-channel, the capture micro-channels being connected to a recovery channel.
Dans un mode de réalisation, un étage de fractionnement préalable comprend une pluralité de micro-colonnes de fractionnement préalable, et un microcanal de capture intersectant successivement les micro-colonnes de fractionnement préalable, et débouchant dans un canal de récupération. Pour améliorer une séparation de constituant d'un échantillon dans une micro-colonne de fractionnement principal, secondaire ou préalable, on peut prévoir qu'une portion terminale de la micro-colonne de fractionnement comprend des moyens de séparation terminaux différents des moyens de séparation intermédiaires. Les constituants parvenant sensiblement à un même instant en aval d'une micro-colonne de fractionnement présentent les mêmes caractéristiques en rapport avec la sélectivité des moyens de séparation intermédiaires. Un changement de sélectivité d'une portion terminale permet de séparer ces constituants. Dans un mode de réalisation, des moyens fluidiques de capture associés à une micro-colonne de fractionnement comprennent un micro-canal de capture comprenant une portion amont débouchant à une extrémité aval d'un segment de capture de la micro-colonne de fractionnement et une portion aval débouchant à une extrémité amont du segment de capture. Le décalage des portions amont et aval d'un micro-canal de capture permet une capture de constituants d'un échantillon se situant sur un segment de longueur d'une micro-colonne de fractionnement.In one embodiment, a prior fractionation stage comprises a plurality of micro-columns of prior fractionation, and a capture microchannel successively intersecting the micro-columns of prior fractionation, and opening into a recovery channel. To improve a separation of the constituent of a sample in a main, secondary or prior fractionation micro-column, provision may be made for a terminal portion of the fractionation micro-column to include terminal separation means different from the intermediate separation means . The constituents arriving substantially at the same instant downstream of a fractionation micro-column have the same characteristics in relation to the selectivity of the intermediate separation means. A change in selectivity of a terminal portion makes it possible to separate these constituents. In one embodiment, fluidic capture means associated with a fractionation micro-column comprise a capture micro-channel comprising an upstream portion opening at a downstream end of a capture segment of the fractionation micro-column and a downstream portion opening at an upstream end of the capture segment. The offset of the upstream and downstream portions of a capture micro-channel allows the capture of constituents of a sample lying on a length segment of a fractionation micro-column.
De tels ico-canaux de capture avec portions décalées peuvent être prévus pour une micro-colonne de fractionnement principal ou une micro-colonne de fractionnement préalable. Lors d'une capture sur une micro-colonne de fractionnement préalable, une plus grande variété de constituants est capturée. Lors d'une capture sur une micro-colonne d'extraction préalable. Lors d'une capture sur une micro-colonne de fractionnement, la circulation à contre-courant dans le segment de capture d'un éluant de capture, différent d'une phase mobile circulant dans la micro-colonne, peut permettre un fractionnement secondaire du produit de fractionnement présent dans le segment de capture à l'instant de la capture.Such capture ico-channels with offset portions can be provided for a main fractionation micro-column or a prior fractionation micro-column. During a capture on a preliminary fractionation micro-column, a greater variety of constituents is captured. During a capture on a preliminary extraction micro-column. During a capture on a fractionation micro-column, the counter-current circulation in the capture segment of a capture eluent, different from a mobile phase circulating in the micro-column, can allow a secondary fractionation of the fractionation product present in the capture segment at the time of capture.
Dans un mode de réalisation, le dispositif d'analyse comprend un microconduit de lavage de micro-leviers sélectifs débouchant dans des micro-canaux de capture directement en amont de micro-leviers sélectifs. Un micro-conduit de lavage permet d' amener un tampon de lavage ou un éluant directement sur les micro-leviers. Les micro-leviers retiennent certaines molécules selon leurs propriétés de surfaces. L' éluant de lavage est choisi pour son affinité avec les molécules retenues sur certains micro-leviers, afin d'entraîner les molécules préalablement retenues par ces microleviers. Un tampon de lavage permet d'enlever toute molécule fixée sur les microleviers.In one embodiment, the analysis device comprises a micro-washing channel for selective micro-levers opening into capture micro-channels directly upstream of selective micro-levers. A micro-washing pipe allows to bring a washing buffer or an eluent directly on the micro-levers. The micro-levers retain certain molecules according to their surface properties. The washing eluent is chosen for its affinity with the molecules retained on certain micro-levers, in order to entrain the molecules previously retained by these microlevers. A washing buffer removes any molecule attached to the microlevers.
L'invention concerne également un ensemble d'analyse comparative chimique ou biochimique d'au moins deux échantillons biologiques ou chimiques comprenant au moins deux dispositifs comprenant une pluralité de micro-colonnes de fractionnement de constituants d'un échantillon, chaque micro-colonne de fractionnement comprenant un micro-canal muni d'un orifice d'introduction d'une phase mobile enrichie en échantillon, un orifice d'évacuation situé à une extrémité terminale et de moyens de séparation intermédiaires. Un dispositif comprend en outre des moyens fluidiques de capture de produits de fractionnement au niveau d'un élément terminal de chaque micro-colonne de fractionnement situé en amont de son orifice d'évacuation, des micro-canaux de capture destinés à récupérer les produits de fractionnement capturés, et des ensembles de micro-leviers sélectifs associés aux microcolonnes de fractionnement et situés en aval des micro-canaux de capture, un micro- levier comportant des moyens de détection reliés à des moyens d'analyse. Un ensemble comprenant des dispositifs de séparation et d'analyse permet une comparaison rapide des échantillons pour déterminer les différences de composition, par exemple en fonction de différences d'état physiologique ou pathologique de cellules composant les échantillons.The invention also relates to a set of chemical or biochemical comparative analysis of at least two biological or chemical samples comprising at least two devices comprising a plurality of micro-columns for fractionation of constituents of a sample, each micro-column for fractionation comprising a micro-channel provided with an orifice for introducing a mobile phase enriched in the sample, an evacuation orifice situated at a terminal end and intermediate separation means. A device further comprises fluid means for capturing fractionation products at a terminal element of each fractionation micro-column situated upstream from its discharge orifice, capture micro-channels intended for recovering the products of fractionation captured, and sets of selective micro-levers associated with the fractionation microcolumns and located downstream of the capture micro-channels, a micro- lever comprising detection means connected to analysis means. A set comprising separation and analysis devices allows rapid comparison of the samples to determine the differences in composition, for example as a function of differences in the physiological or pathological state of cells making up the samples.
L'invention concerne également un procédé d'analyse chimique ou biochimique d'échantillons biologique ou chimique, dans lequel on réalise des fractionnements différentiels d'une phase mobile enrichie en échantillon, on capture simultanément différents produits de fractionnement obtenus, et on analyse chacun des produits de fractionnement à l'aide d'un ensemble de micro-leviers sélectifs. Le fractionnement différentiel permet une séparation rapide des constituants de l'échantillon, et une capture simultanée des produits de fractionnement.The invention also relates to a method of chemical or biochemical analysis of biological or chemical samples, in which differential fractionations of a mobile phase enriched in sample are carried out, different fractionation products obtained are captured simultaneously, and each of the fractionation products using a set of selective micro-levers. Differential fractionation allows rapid separation of the components of the sample, and simultaneous capture of the fractionation products.
Pour améliorer une différenciation de constituants lors de l'analyse, on fractionne un produit de fractionnement capturé avant de l'analyser. Le produit de fractionnement comprend certains constituants fractionnés de l'échantillon. Une séparation ou un fractionnement supplémentaire permet d'obtenir des constituants plus séparés, qui pourront être analysés successivement avec plus de précision.To improve the differentiation of constituents during the analysis, a captured fractionation product is fractionated before analyzing it. The fractionation product includes certain fractional components of the sample. An additional separation or fractionation makes it possible to obtain more separate constituents, which can be analyzed successively with more precision.
Dans un mode de mise en œuvre, on détecte des constituants d'un produit de fractionnement à l'aide de micro-leviers, selon des caractéristiques de polarité, solvophobicité ou porosité du matériau qui les constitue ou d'un revêtement des microleviers, ou selon des caractéristiques de polarité, solvophobicité, échange d'ion ou affinité avec des groupements fonctionnels greffés sur les micro-leviers.In one embodiment, constituents of a fractionation product are detected using micro-levers, according to characteristics of polarity, solvophobicity or porosity of the material which constitutes them or of a coating of the microlevers, or according to characteristics of polarity, solvophobicity, ion exchange or affinity with functional groups grafted onto the micro-levers.
On peut fractionner l'échantillon par chromatographie, par micro- électrophorèse, ou par interaction avec des nano-électrodes. Dans un mode de mise en œuvre, on analyse la déviation ou la fréquence de vibration des micro-leviers. Une molécule telle qu'une protéine ou un peptide peut se fixer sur un micro-levier, selon une sélectivité due par exemple à un état de surface ou un revêtement.The sample can be fractionated by chromatography, by micro-electrophoresis, or by interaction with nano-electrodes. In one embodiment, the deviation or the frequency of vibration of the micro-levers is analyzed. A molecule such as a protein or a peptide can be fixed on a micro-lever, according to a selectivity due for example to a surface state or a coating.
On peut mesurer une déviation du micro-levier provoquée par la fixation d'une molécule. On peut également exciter en vibration le micro-levier à une certaine fréquence, par exemple sa fréquence de résonance. Lorsqu'une protéine se fixe sur le micro-levier, on mesure une modification de fréquence de vibration.We can measure a deviation of the micro-lever caused by the fixation of a molecule. The micro-lever can also be excited in vibration at a certain frequency, for example its resonant frequency. When a protein binds to the micro-lever, a change in vibration frequency is measured.
Dans un mode de mise en œuvre, on analyse les éléments de fractionnement par spectrométrie de masse, avant ou après l'analyse à l'aide des micro-leviers. Pour comparer un échantillon à un échantillon de référence, on analyse un premier échantillon, on analyse un second échantillon, et on compare les résultats d'analyse des deux échantillons. Dans ce cas, on analyse les premier et second échantillons en vue de la comparaison d'empreintes de protéines des échantillons, à l'aide de micro-leviers sélectifs aptes à montrer une empreinte de protéines différentielle. En d'autres termes, on analyse les échantillons à l'aide de micro-leviers adaptés, qui pourront détecter des différences de composition des échantillons, compte tenu de la différence d'état de ce dernier, et de la différence de composition que l'on présuppose.In one embodiment, the fractionation elements are analyzed by mass spectrometry, before or after the analysis using micro-levers. To compare a sample to a reference sample, a first sample is analyzed, a second sample is analyzed, and the analysis results of the two samples are compared. In this case, we analyze the first and second samples for comparison of protein fingerprints of the samples, using selective micro-levers capable of showing a differential protein imprint. In other words, the samples are analyzed using suitable micro-levers, which will be able to detect differences in the composition of the samples, taking into account the difference in state of the latter, and the difference in composition that l 'we presuppose.
Avantageusement, on effectue une extraction préalable sur un échantillon avant un fractionnement différentiel de l'échantillon.Advantageously, a preliminary extraction is carried out on a sample before a differential fractionation of the sample.
La présente invention et ses avantages seront mieux compris à l'étude de la description détaillée de modes de réalisation pris à titre d'exemple nullement limitatif et illustrée par les dessins annexés sur lesquels :The present invention and its advantages will be better understood on studying the detailed description of embodiments taken by way of nonlimiting example and illustrated by the appended drawings in which:
La figure 1 est une vue schématique partielle d'un dispositif d'analyse comprenant des micro-colonnes selon un aspect de l'invention ;Figure 1 is a partial schematic view of an analysis device comprising micro-columns according to one aspect of the invention;
La figure 2 est une vue schématique d'une première variante du dispositif d'analyse selon la figure 1 ;Figure 2 is a schematic view of a first variant of the analysis device according to Figure 1;
La figure 3 est une vue schématique d'une seconde variante du dispositif selon la figure 1, où des moyens fluidiques de capture sont représentés, selon un aspect de l'invention ;Figure 3 is a schematic view of a second variant of the device according to Figure 1, where fluid capture means are shown, according to one aspect of the invention;
Les figures 4 et 5 sont des vues schématiques partielles d'un dispositif d'analyse montrant un agencement particulier de supports ;Figures 4 and 5 are partial schematic views of an analysis device showing a particular arrangement of supports;
Les figures 6 et 7 sont des vues schématiques partielles d'un dispositif d'analyse montrant un autre agencement de supports ;Figures 6 and 7 are partial schematic views of an analysis device showing another arrangement of supports;
La figure 8 est une vue schématique d'ensemble d'un dispositif d'analyse selon un aspect de l'invention ; La figure 9 est une vue schématique partielle d'un étage d'extraction préalable d'un support ;Figure 8 is a schematic overview of an analysis device according to one aspect of the invention; Figure 9 is a partial schematic view of a stage of prior extraction of a support;
Les figures 10 et 11 sont des vues schématiques partielles de variantes d'étages d'extraction préalable selon la figure 9 ;Figures 10 and 11 are partial schematic views of alternative pre-extraction stages according to Figure 9;
La figure 12 est une vue d'un circuit de lavage de micro-leviers sélectifs ; La figure 13 est une vue partielle d'un support d'analyse à alimentation en phase mobile et alimentation en échantillon séparées ; etFIG. 12 is a view of a circuit for washing selective micro-levers; FIG. 13 is a partial view of an analysis support with mobile phase supply and separate sample supply; and
La figure 14 est une variante d'un dispositif selon la figure 3. Sur la figure 1, un support 1 comprend une pluralité de micro-colonnes 2 arrangées en parallèle et présentant un gradient de longueur. Les micro-colonnes 2 sont représentées en traits épais.Figure 14 is a variant of a device according to Figure 3. In Figure 1, a support 1 comprises a plurality of micro-columns 2 arranged in parallel and having a length gradient. The micro-columns 2 are shown in thick lines.
Chaque micro-colonne 2 comprend une portion de micro-canal 3 munie d'un orifice d'introduction 3a et d'un orifice d'évacuation 3b. Chaque portion de micro- canal 3 est pourvu de moyens de séparation intermédiaires. Un canal d'alimentation 4 relie l'ensemble des orifices d'introduction 3a des micro-colonnes fractionnement 2. Le canal d'alimentation 4 est relié aux orifices d'introduction 3a des micro-colonnes de fractionnement 2 par des mico-canaux intermédiaires 5 représentés en traits fins pour les différencier des micro-colonnes de fractionnement 2. Les micro-canaux intermédiaires 5 sont en fait des portions de micro-canaux dénuées de moyens de séparation et situées en amont des portions de micro-canaux 3 formant les micro-colonnes de fractionnemnet 2. Un canal d'évacuation 6 relie l'ensemble des orifices d'évacuation 3b des microcolonnes 2. Les portions de micro-canaux 3 formant les micro-colonnes de fractionnement 2 possèdent des longueurs différentes, chaque micro-canal 3 différant du micro-canal suivant 3 par un élément de longueur déterminée delta 1. Les micro-canaux 3 présentent donc un gradient de longueur. La micro-colonne de fractionnement 2 la plus courte possède une longueur Ll. La micro-colonne de fractionnement 2 la plus longue possède une longueur L2.Each micro-column 2 comprises a micro-channel portion 3 provided with an introduction orifice 3a and with an evacuation orifice 3b. Each portion of micro- channel 3 is provided with intermediate separation means. A feed channel 4 connects all of the introduction orifices 3a of the fractionation micro-columns 2. The feed channel 4 is connected to the introduction orifices 3a of the fractionation micro-columns 2 by intermediate micro-channels 5 shown in fine lines to differentiate them from the fractionation micro-columns 2. The intermediate micro-channels 5 are in fact portions of micro-channels devoid of separation means and located upstream of the portions of micro-channels 3 forming the micro - fractionation columns 2. An evacuation channel 6 connects all of the evacuation orifices 3b of the microcolumns 2. The portions of micro-channels 3 forming the fractionation micro-columns 2 have different lengths, each micro-channel 3 differing from the following micro-channel 3 by an element of determined length delta 1. The micro-channels 3 therefore have a length gradient. The shortest fractionation micro-column 2 has a length L1. The longest fractionation micro-column 2 has a length L2.
La longueur L2 des micro-colonnes de fractionnement 2 les plus courtes varie à titre d'exemple nullement limitatif dans une fourchette 1 à 20 centimètres. La longueur Ll des micro-colonnes de fractionnement 2 les plus longues varie à titre d'exemple nullement limitatif dans une fourchette 5 à 40 centimètres. Lesdites micro- colonnes de fractionnement 2 présentent un diamètre avoisinant 1 à 100 microns, et notamment 10 à 100 microns. La différence de longueur entre une micro-colonne fractionnement 2 et une micro-colonne fractionnement 2 immédiatement plus longue est comprise, à titre d'exemple nullement limitatif entre 1 à 100 microns.The length L2 of the shortest fractionation micro-columns 2 varies by way of example in no way limiting in a range 1 to 20 centimeters. The length L1 of the longest fractionation micro-columns 2 varies by way of example in no way limiting in a range 5 to 40 centimeters. Said fractionation micro-columns 2 have a diameter of around 1 to 100 microns, and in particular 10 to 100 microns. The difference in length between a fractionation micro-column 2 and an immediately longer fractionation micro-column 2 is understood, by way of example in no way limiting, between 1 to 100 microns.
Sur la figure 2, les références aux éléments semblables à ceux de la figure 1 ont été reprises. Un support intégré 1 est pourvu de groupes de micro-colonnes de fractionnement 2. Les micro-colonnes de fractionnement 2 d'un même groupe possèdent la même longueur. Les micro-colonnes de fractionnement 2 d'un groupe diffèrent des micro-colonnes de fractionnement 2 d'un autre groupe par leur longueur. Plus précisément, les micro-colonnes de fractionnement 2 d'un groupe diffèrent des micro-colonnes de fractionnement 2 du groupe suivant par un très petit élément de longueur. En d'autres termes, on retrouve un gradient de longueur entre les groupes de micro-colonnes de fractionnement 2.In FIG. 2, the references to elements similar to those of FIG. 1 have been repeated. An integrated support 1 is provided with groups of fractionation micro-columns 2. The fractionation micro-columns 2 of the same group have the same length. The fractionation micro-columns 2 of a group differ from the fractionation micro-columns 2 of another group by their length. More precisely, the fractionation micro-columns 2 of a group differ from the fractionation micro-columns 2 of the following group by a very small element of length. In other words, there is a length gradient between the groups of fractionation micro-columns 2.
Tel que représenté sur la figure 2, un canal d'alimentation 4 relie directement les orifices d'introduction 3a des micro-colonnes de fractionnement 2, en étant dépourvu de micro-canaux intermédiaires.As shown in FIG. 2, a supply channel 4 directly connects the introduction orifices 3a of the fractionation micro-columns 2, being devoid of intermediate micro-channels.
Les micro-canaux 3 sont entièrement configurés en micro-colonnes de fractionnement 2 en étant pourvus sur toute leur longueur de moyens de séparation. Sur la figure 3, où les références aux éléments semblables à ceux de la figure 1 ont été reprises, un support 1 comprend des micro-colonnes de fractionnementThe micro-channels 3 are entirely configured as fractionating micro-columns 2, being provided over their entire length with separation means. In FIG. 3, where the references to elements similar to those of FIG. 1 have been repeated, a support 1 comprises micro-columns for fractionation
2 et des moyens de capture fluidique 7. Les orifices d'introduction 3a sont reliés directement au canal d'alimentation 4. Les micro-colonnes de fractionnement 2 présentent un gradient de longueur.2 and fluid capture means 7. The introduction orifices 3a are connected directly to the supply channel 4. The fractionation micro-columns 2 have a length gradient.
Les moyens de capture fluidiques 7 comprennent des micro-canaux de capture 8 intersectant les micro-canaux de fractionnement 3 au niveau d'un élément terminal ou portion terminale 9 de chaque micro-canal de fractionnement 3, à une distance déterminée de l'extrémité terminale du micro-canal de fractionnement 3, c'est- à-dire à une distance déterminée de son orifice d'évacuation 3b. Chaque micro-canal de fractionnement 3 est associé a un micro-canal de capture 8. Les extrémités d'entrée des micro-canaux de capture 8, situées en amont de l'intersection avec les micro-canaux 3, sont reliées à un canal d'alimentation secondaire 15, prévu pour l'alimentation en éluant secondaire. Le support 1 comprend également des micro-colonnes de fractionnement secondaire 10 situées dans des portions avals des micro-canaux de capture 8, en se situant en amont de zones de détection 11. Une micro-colonne de fractionnement secondaire 10 est associée à un micro-canal de capture 8 et une zone de détection 11. Une micro-colonne de fractionnement secondaire 10 comprend des moyens de fractionnement similaires à ceux de la micro-colonne de fractionnement 2 à laquelle elle est associée. Une zone de détection 11 comprend un canal de circulation 12 passant par un ou plusieurs micro-leviers sélectifs 13.The fluidic capture means 7 comprise capture micro-channels 8 intersecting the fractionation micro-channels 3 at a terminal element or terminal portion 9 of each fractionation micro-channel 3, at a determined distance from the end end of the fractionation micro-channel 3, that is to say at a determined distance from its discharge orifice 3b. Each fractionation micro-channel 3 is associated with a capture micro-channel 8. The input ends of the capture micro-channels 8, located upstream of the intersection with the micro-channels 3, are connected to a channel secondary supply 15, intended for supply with secondary eluent. The support 1 also comprises secondary fractionation micro-columns 10 located in downstream portions of the capture micro-channels 8, being located upstream of detection zones 11. A secondary fractionation micro-column 10 is associated with a microphone -capture channel 8 and a detection zone 11. A secondary fractionation micro-column 10 comprises fractionation means similar to those of the fractionation micro-column 2 with which it is associated. A detection zone 11 comprises a circulation channel 12 passing through one or more selective micro-levers 13.
Pour une analyse d'un échantillon, une phase mobile, de préférence sous la forme d'un éluant, enrichie en échantillon est amenée par le canal d'alimentation 4 et entre dans les micro-colonnes de fractionnement 2 par les orifices d'introduction 3a. La phase mobile enrichie en échantillon circule de l'orifice d'introduction 3a vers l'orifice d'évacuation 3b en étant séparé dans le micro-canal 3. Les éléments terminaux 9, où s'effectuent les captures, sont situés à des distances différentes des orifices d'introduction 3a des micro-colonnes de fractionnement 2. Les vitesses de migration des constituants d'un échantillon sont différentes. Dès lors, à un instant donné après le début de la migration, on retrouvera dans les différents éléments terminaux 9 des microcolonnes de fractionnement 2 des ensembles de constituants différents. Dans les éléments terminaux 9 des micro-colonnes les plus longues, on retrouvera les constituants migrant rapidement. Au même instant, dans les éléments terminaux des micro-colonnes les plus courtes, on retrouvera des constituants migrant plus lentement. Les molécules dépassant les éléments terminaux 9 sont évacuées par les orifices d'évacuation 3b et le canal d'évacuation 6. On notera que les vitesses de migration des constituants et donc les séparations, dépendent de la sélectivité des moyens de séparation d'une micro-colonne et de la nature de la phase mobile ou éluant entraînant les constituants. Les moyens de séparation tendent à retenir les constituants, plus ou moins selon leurs caractéristiques, alors que l'éluant tend à entraîner les constituants, également selon leurs caractéristiques.For an analysis of a sample, a mobile phase, preferably in the form of an eluent, enriched in the sample, is brought in by the feed channel 4 and enters the fractionation micro-columns 2 through the introduction orifices. 3a. The mobile phase enriched in sample circulates from the introduction orifice 3a to the evacuation orifice 3b while being separated in the micro-channel 3. The terminal elements 9, where the catches are made, are located at distances different from the introduction orifices 3a of the fractionation micro-columns 2. The migration rates of the constituents of a sample are different. Therefore, at a given time after the start of migration, we will find in the various terminal elements 9 of the microcolumns fractionation 2 sets of different constituents. In the terminal elements 9 of the longest micro-columns, the rapidly migrating constituents will be found. At the same time, in the terminal elements of the shortest micro-columns, one will find constituents migrating more slowly. The molecules protruding from the terminal elements 9 are evacuated through the evacuation orifices 3b and the evacuation channel 6. It will be noted that the migration speeds of the constituents and therefore the separations depend on the selectivity of the means of separation of a micro-column and on the nature of the mobile phase or eluent entraining the constituents. The separation means tend to retain the constituents, more or less according to their characteristics, while the eluent tends to entrain the constituents, also according to their characteristics.
Pour capturer les constituants séparés de l' échantillon, un micro ou nano- flux d' éluant secondaire est mis en circulation simultanément dans tous les microcanaux de capture 8. Un nano-flux d' éluant secondaire circulant dans un micro-canal de capture 8 traverse l'élément terminal de longueur Delta L contenu dans le micro-canal 3 associé.To capture the separate constituents of the sample, a micro or nano-flow of secondary eluent is circulated simultaneously in all of the capture microchannels 8. A nano-flow of secondary eluent flowing in a capture micro-channel 8 crosses the terminal element of length Delta L contained in the associated micro-channel 3.
Le nano-flux d'éluant secondaire est récupéré par une portion aval du micro-canal de capture 8 après son passage à travers l'élément terminal. Les constituants de l'échantillon présents dans l'élément terminal à l'instant de la capture sont entraînés dans le micro-canal de capture 8. On choisit de préférence un éluant secondaire apte à entraîner les constituants retenus dans la micro-colonne de fractionnement 2.The nano-flow of secondary eluent is recovered by a downstream portion of the capture micro-channel 8 after it has passed through the terminal element. The components of the sample present in the terminal element at the time of capture are entrained in the capture micro-channel 8. A secondary eluent is preferably chosen which is capable of entraining the constituents retained in the fractionation micro-column. 2.
Les constituants capturés seront désignés par la suite par produit de fractionnement. Un produit de fractionnement comprend une pluralité de constituants de l'échantillon. Les produits de fractionnement sont notamment des molécules séparées, des complexes moléculaires non séparés et des agrégats moléculaires non désagrégés.The constituents captured will be designated subsequently by fractionation product. A fractionation product includes a plurality of components of the sample. The fractionation products are in particular separate molecules, non-separated molecular complexes and non-disaggregated molecular aggregates.
Les produits de fractionnement sont amenés par les micro-canaux de capture 8 vers les micro-colonnes de fractionnement secondaire 10. En traversant ces microcolonnes de fractionnement secondaire 10, les produits de fractionnement subissent une nouvelle séparation. Dans les micro-colonnes de fractionnement secondaires 10 les produits de fractionnement peuvent subir des micro- ou nano-extractions ou séparations secondaires, terminales, parallèles, simultanées, ou/et de micro- ou nano- digestions secondaires, enzymatiques, terminales, parallèles, simultanées.The fractionation products are brought by the capture micro-channels 8 to the secondary fractionation micro-columns 10. By passing through these secondary fractionation microcolumns 10, the fractionation products undergo a new separation. In the secondary fractionation micro-columns, the fractionation products can undergo secondary, terminal, parallel, simultaneous micro- or nano-extractions or separations, and / or secondary, enzymatic, terminal, parallel micro- or nano-digestions, concurrent.
Les produits de micro ou nano-élution secondaire, de micro ou nano- digestion secondaire et de micro ou nano-extraction secondaire, appelés par la suite produits de fractionnement secondaire, circulent dans les micro-canaux de capture 8 en aval des micro-colonnes de fractionnement secondaire 10, vers les zones de détection 11. La détection des constituants présents dans les produits de fractionnement secondaire s'effectue sur les micro-leviers sélectifs 13. La rétention des constituants présents dans les produits de fractionnement secondaire sur les micro-leviers 13 est mesurée par l'intermédiaire d'une mesure de la déviation des micro-leviers 13 ou par la mesure de variation de fréquence de vibration des micro-leviers 13.The products of secondary micro or nano-elution, of secondary micro or nanodigestion and of secondary micro or nano-extraction, hereinafter called secondary fractionation products, circulate in the capture micro-channels 8 downstream of the micro-columns secondary fractionation 10, towards the detection zones 11. The components present in the secondary fractionation products are detected on the selective micro-levers 13. The retention of the constituents present in the secondary fractionation products on the micro-levers 13 is measured by measuring the deviation of the micro-levers 13 or by measuring the variation in the frequency of vibration of the micro-levers 13.
Les moyens de séparation des micro-colonnes de fractionnement secondaire 10 sont similaires à ceux des micro-colonnes de fractionnement 2. Cependant, on peut prévoir que les moyens de séparation d'une micro-colonne de fractionnement secondaire 10 soient différents de ceux de la micro-colonne de fractionnement 2 associée. Notamment, la sélectivité des moyens de séparation peut être différente, pour favoriser une séparation des constituants présents dans les produits de fractionnement. Ces constituants, qui ont été capturés au même instant dans un produit de fractionnement après une première séparation, possèdent des caractéristiques de migration similaires en rapport la sélectivité de la micro-colonne de fractionnement 2 dont ils sont issus. Une seconde séparation avec une sélectivité différente permet une séparation supplémentaire efficace. Bien entendu, l' éluant secondaire est choisi pour favoriser cette séparation secondaire. On peut prévoir un lot de micro-colonnes de fractionnement 2 comprenant des moyens de séparation identiques. On peut également prévoir différents lots de micro-colonnes de fractionnement 2, chaque lot comprenant des micro-colonnes de fractionnement munies de moyens de fractionnement particuliers, présentant par exemple une sélectivité différente. Ainsi, selon la sélectivité d'un lot, un constituant particulier est mieux séparé dans ce lot et peut être détecté plus facilement en aval de ce lots.The means for separating the secondary fractionation micro-columns 10 are similar to those for the fractionation micro-columns 2. However, provision may be made for the means for separating a secondary fractionation micro-column 10 to be different from those of the associated fractionation micro-column 2. In particular, the selectivity of the separation means may be different, to promote separation of the constituents present in the fractionation products. These constituents, which were captured at the same time in a fractionation product after a first separation, have similar migration characteristics in relation to the selectivity of the fractionation micro-column 2 from which they are derived. A second separation with different selectivity allows effective additional separation. Of course, the secondary eluent is chosen to favor this secondary separation. It is possible to provide a batch of fractionation micro-columns 2 comprising identical separation means. It is also possible to provide different batches of fractionation micro-columns 2, each batch comprising fractionation micro-columns provided with particular fractionation means, for example having a different selectivity. Thus, according to the selectivity of a batch, a particular constituent is better separated in this batch and can be more easily detected downstream of this batch.
Dans le cas d'une pluralité de lots de micro-colonnes fractionnement 2, les micro-colonnes de fractionnement 2 peuvent être alimentées à partir d'une colonne d'enrichissement collective, c'est-à-dire un canal d'alimentation en phase mobile enrichie, ou à partir d'une pluralité de colonnes d'enrichissement, un lot spécifique étant associé à une colonne d'enrichissement spécifique, où circule par exemple un éluant spécifique, correspondant aux moyens de séparation particuliers des micro-colonnes de fractionnement 2 du lot. En effet, selon la sélectivité des micro-colonnes de fractionnement et la nature de l' éluant, les vitesses de migration des molécules pourront être différentes.In the case of a plurality of batches of fractionation micro-columns 2, the fractionation micro-columns 2 can be fed from a collective enrichment column, that is to say a feed channel for enriched mobile phase, or from a plurality of enrichment columns, a specific batch being associated with a specific enrichment column, for example circulating a specific eluent, corresponding to the particular means of separation of the fractionation micro-columns 2 of the lot. Indeed, depending on the selectivity of the fractionation micro-columns and the nature of the eluent, the migration speeds of the molecules may be different.
Bien entendu, les micro-colonnes de fractionnement de chaque lot peuvent présenter des gradients de longueur entre micro-colonnes ou groupes de microcolonnes.Of course, the fractionation micro-columns of each batch can have length gradients between micro-columns or groups of microcolumns.
Un fractionnement d'un échantillon suivi d'une capture de produits de fractionnement, et de la détection de leurs constituants permet de récupérer une "empreinte" de l'échantillon. On peut réaliser des séries d'empreintes. Pour permettre des captures et des détections successives à l'aide des mêmes micro-leviers de détection, on peut prévoir des étapes de lavages successifs des micro-leviers 13, notamment par passage d'un éluant particulier apte à entraîner des molécules retenues dans sur les micro-leviers.A fractionation of a sample followed by a capture of fractionation products, and the detection of their constituents makes it possible to recover an "imprint" of the sample. You can make a series of imprints. To allow successive captures and detections using the same micro-levers detection, it is possible to provide for successive washing steps of the micro-levers 13, in particular by passing a particular eluent capable of entraining molecules retained in on the micro-levers.
On peut effectuer une séparation des constituants d'un échantillon en mode isocratique, en mode d'élution pas à pas, ou encore par gradient d'élution, c'est-à-dire par variation continue progressive de la composition d'un éluant.It is possible to separate the constituents of a sample in isocratic mode, in step-by-step elution mode, or even by elution gradient, that is to say by continuous progressive variation of the composition of an eluent. .
Les molécules entraînées par une phase mobile dans les micro-colonnes de fractionnement 2 sont retenues, selon la sélectivité des moyens de séparation des microcolonnes de fractionnement 2. Le passage d'un éluant présentant une composition particulière et donc une affinité particulière avec certaines molécules, permet d'entraîner préférentiellement ces molécules, les autres étant retenues par une phase stationnaire des moyens de séparation.The molecules entrained by a mobile phase in the fractionation micro-columns 2 are retained, according to the selectivity of the means of separation of the fractionation microcolumns. The passage of an eluent having a particular composition and therefore a particular affinity with certain molecules, preferentially entrain these molecules, the others being retained by a stationary phase of the separation means.
Les constituants de l'échantillon sont séparés en fonction de leur vitesse de migration, fonction de leur caractéristique, de la sélectivité des moyens de séparation, et de leur affinité avec une phase mobile du type éluant.The constituents of the sample are separated as a function of their speed of migration, a function of their characteristic, of the selectivity of the separation means, and of their affinity with a mobile phase of the eluent type.
Une variation de la composition d'un éluant permet en entraînement différents des constituants et une séparation améliorée. La variation de composition peut être réalisée par pas successifs, ou de façon continue. Dans ce cas, on parle de gradient d' éluant. La capture des produits de fractionnement est effectuée en mode pas à pas, chaque pas de capture s' appuyant sur une condition physique ou chimique ou hydrodynamique précise régnant à l'intersection d'une micro-colonne de fractionnement 2 avec ledit micro-canal de capture 10.A variation in the composition of an eluent allows for different entrainment of the constituents and improved separation. The variation in composition can be carried out in successive steps, or continuously. In this case, we speak of an eluent gradient. The capture of the fractionation products is carried out in step-by-step mode, each capture step being based on a precise physical or chemical or hydrodynamic condition prevailing at the intersection of a fractionation micro-column 2 with said micro-channel of capture 10.
On peut comparer des séries d'empreintes de deux échantillons. La série d'empreintes successives d'un premier échantillon est comparée à la série d'empreintes successives d'un deuxième échantillon à l'aide de moyens d'analyse du type informatique, les séries d'empreintes de détection étant ensuite archivées dans une base de données informatiques.We can compare sets of fingerprints from two samples. The series of successive fingerprints of a first sample is compared to the series of successive fingerprints of a second sample using analysis means of the computer type, the series of detection fingerprints then being archived in a computer database.
Considérons un dispositif d'analyse selon un aspect de l'invention, comprenant deux ensembles de (x) supports chacun muni de (z) lots de (t) microcolonnes de fractionnement.Consider an analysis device according to one aspect of the invention, comprising two sets of (x) supports each provided with (z) lots of (t) fractionating microcolumns.
Pour chaque échantillon, on peut procéder à une élution pas à pas à (n) pas d'élution primaire, dans (t) micro-colonnes de fractionnement 2. Pour chaque pas d'élution primaire, on peut procéder à des micro ou nano-élutions secondaires pas à pas comportant (m) pas d'élutions. Au total, le procédé d'analyse de l'échantillon donne lieu, pour chaque ensemble de supports à un passage de (n.m.Lz.x) produits de fractionnement différents sur des zones de détection.For each sample, it is possible to carry out a stepwise elution with (n) primary elution step, in (t) fractionation micro-columns 2. For each primary elution step, it is possible to carry out micro or nano - secondary stepwise elutions comprising (m) no elutions. In total, the sample analysis process gives rise, for each set of supports, to the passage of (nmLz.x) different fractionation products over detection zones.
À titre d'exemple nullement limitatif, (n) est un nombre compris entre 1 et 5, (m) est un nombre compris entre 1 et 5, (x) est un nombre compris entre 5 et 50 (z) est un nombre compris entre 1 et 5, et (t) est un nombre compris entre 10 et 10 000, notamment entre 10 et 1000.By way of nonlimiting example, (n) is a number between 1 and 5, (m) is a number between 1 and 5, (x) is a number between 5 and 50 (z) is a number between 1 and 5, and (t) is a number between 10 and 10 000, in particular between 10 and 1000.
Un ensemble de supports est utilisé pour l'analyse d'un échantillon, un autre ensemble de support étant utilisé pour l'analyse d'un autre échantillon. L'empreinte constituée des (n.mΛ.z.x) détections du premier échantillon est comparée à l'empreinte constituée de (n.m.t.z.x) détections du deuxième échantillon.A set of supports is used for the analysis of a sample, another set of supports being used for the analysis of another sample. The fingerprint made up of (n.mΛ.z.x) detections of the first sample is compared to the footprint made up of (n.m.t.z.x) detections of the second sample.
Alternativement, les élutions primaires et secondaires se font par gradient d'élution. Dans ce cas, plusieurs empreintes sur micro-leviers sont effectuées, à des intervalles de temps fixes, ou variés. Entre chaque empreinte, des lavages de micro- leviers peuvent avoir heu.Alternatively, the primary and secondary elutions are made by elution gradient. In this case, several fingerprints on micro-levers are made, at fixed or varied time intervals. Between each impression, micro-lever washes can take place.
Si le nombre d'empreintes observées est (p), alors au total, le procédé d'analyse de l'échantillon donne lieu, pour chaque ensemble de supports à un passage de (p.t.z.x) produits de fractionnement différents sur des zones de détection.If the number of fingerprints observed is (p), then in total, the method of analysis of the sample gives rise, for each set of supports, to a passage of (p.t.z.x) different fractionation products over detection zones.
La séparation dans les micro-colonnes de fractionnement 2 ou les micro- colonnes de fractionnement secondaire 10 peut être effecutee à titre d'exemple nullement limitatif par électrophorèse, chromatographie ou électrochromatographie.The separation in the fractionation micro-columns 2 or the secondary fractionation micro-columns 10 can be carried out by way of nonlimiting example by electrophoresis, chromatography or electrochromatography.
Des méthodes de séparation par chromatographique ou électrophorèse utilisables sont celles présentées dans l'article (Neraart JR, Lingeman H, Brin man UA T. Coupling of biological sample handling and capillary electrophoresis. Journal of Chromatography A, 1999, 856, 483-514).Methods of separation by chromatography or electrophoresis that can be used are those presented in the article (Neraart JR, Lingeman H, Brin man UA T. Coupling of biological sample handling and capillary electrophoresis. Journal of Chromatography A, 1999, 856, 483-514) .
On peut utiliser des méthodes de séparation des peptides et des protéines, y compris les protéines hydrophobes, qui ont été présentées dans les articles suivants : (Herraiz T, Casai N. Evaluation of solid-phase extraction procédures in peptide analysis. Journal of Chromatography A, 1995, 708, 209, 221 ; Schweitz L, Petersson M, Johansson T, Νilsson S. Alternative methods providing enhanced sensitivity ansd selectivity in capillary electro-separation experiments. Journal of Chromatography A, 2000, 892, 203-217; Bosserhoff A, Wallach J, Frank RW. Micropreparative séparation of peptides derived from sodium dodecyl sulphate-solubilized proteins. J. Chromatogr, 1989, 473(1). 71-77; Huang JX, Guiochon G. Applications of preparative high- performance liquid chromatography to the séparation and purification of peptides and proteins. J chromatography 1989, 492, 431-69; Rivasseau C, Nanhoenacker G, Sandra P, Hennion MC. On-line solid-phase extraction in Microcolumn- Liquid Chromatography coupled to UN or MS détection: application to the analysis of cyanobacterial toxins. J. Microcolumn séparations, 2000, 12(5), 323-332; Kutter JP, Jacobson SC, Ramsey JM. Solid phase extraction on micro-fluidic devices. J. Microcolumn Séparations, 2000, 12(2), 93-97.). On peut utiliser des méthodes de séparation de protéines membranaires telles qu'évoquées dans les articles suivants : (Cf. Santoni N, Kieffer S, Desclaux D, Masson F, Rabilloud T. Membrane Proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 2000. 21 (16). 3329-44; Santoni N, Doumas P, Rouquie D, Mansion M, Rabilloud T, Rossignol M. large scale characterization of plant plasma membrane proteins. Biochimie 1999. 81(6). 655-61; Thomas TC, Mac Νamee MG. Purification of membrane proteins. Methods in Enzymology. Nol 182, 499-520; Power SD, Lochrie MA, Poyton RO. Reversed-phase high performance liquid chromatographie purification of subunits of oligomeric membrane proteins. The nuclear coded subunits of yeast cytochrome c oxidase. J Chromatogr, 1983, 266, 585-98 ; Josic D, Hofmann W, Habermann R, Becker A, Reuter W. High performance liquid affinity chromatography of liver plasma membrane proteins. J. of Chromatography A, 1987, 397, 39-46.; Lee RP, Doughty SW, Ashman K, Walker J. Purification of hydrophobic intégral membrane proteins from mycoplasma hyopneumoniae by reversed-phase high performance liquid chromatography. Journal of Chromatography A, 1996, 737, 273-279; Sivars U, Tjerneld F. . Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase Systems for purification of intégral membrane proteins . Biochimica et Biophysica Acta, 2000, 1474, 133-146; Ferro M, Seigneurin-Berny D, Rolland Ν, Chapel A, Salvi D, Garin J, Joyard J. Organic solvent extraction as a versatile procédure to identify hydrophobic chloroplast membrane proteins. Electrophoresis 2000, 21, 3517-3526; Stark M, Jornvall H, Johansson J. Isolation and characterization of hydrophobic polypeptides in human bile. Eur J Biochem 1999. 266(1). 209-14. ).Methods of separating peptides and proteins, including hydrophobic proteins, which have been presented in the following articles can be used: (Herraiz T, Casai N. Evaluation of solid-phase extraction procedures in peptide analysis. Journal of Chromatography A , 1995, 708, 209, 221; Schweitz L, Petersson M, Johansson T, Νilsson S. Alternative methods providing enhanced sensitivity ansd selectivity in capillary electro-separation experiments. Journal of Chromatography A, 2000, 892, 203-217; Bosserhoff A , Wallach J, Frank RW. Micropreparative separation of peptides derived from sodium dodecyl sulphate-solubilized proteins. J. Chromatogr, 1989, 473 (1). 71-77; Huang JX, Guiochon G. Applications of preparative high-performance liquid chromatography to the separation and purification of peptides and proteins. J chromatography 1989, 492, 431-69; Rivasseau C, Nanhoenacker G, Sandra P, Hennion MC. On-line solid-phase extraction in Microcolumn- Liquid Chromatography coupled to UN or MS detection: application to the analysis of cyanobacterial toxins. J. Microcolumn separations, 2000, 12 (5), 323-332; Kutter JP, Jacobson SC, Ramsey JM. Solid phase extraction on micro-fluidic devices. J. Microcolumn Séparations, 2000, 12 (2), 93-97.). We can use membrane protein separation methods as mentioned in the following articles: (Cf. Santoni N, Kieffer S, Desclaux D, Masson F, Rabilloud T. Membrane Proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 2000. 21 (16). 3329-44; Santoni N, Doumas P, Rouquie D, Mansion M, Rabilloud T, Rossignol M. large scale characterization of plant plasma membrane proteins . Biochemistry 1999. 81 (6). 655-61; Thomas TC, Mac Νamee MG. Purification of membrane proteins. Methods in Enzymology. Nol 182, 499-520; Power SD, Lochrie MA, Poyton RO. Reversed-phase high performance liquid chromatography purification of subunits of oligomeric membrane proteins. The nuclear coded subunits of yeast cytochrome c oxidase. J Chromatogr, 1983, 266, 585-98; Josic D, Hofmann W, Habermann R, Becker A, Reuter W. High performance liquid affinit y chromatography of liver plasma membrane proteins. J. of Chromatography A, 1987, 397, 39-46 .; Lee RP, Doughty SW, Ashman K, Walker J. Purification of hydrophobic integral membrane proteins from mycoplasma hyopneumoniae by reversed-phase high performance liquid chromatography. Journal of Chromatography A, 1996, 737, 273-279; Sivars U, Tjerneld F.. Mechanisms of phase behavior and protein partitioning in detergent / polymer aqueous two-phase Systems for purification of integral membrane proteins. Biochimica and Biophysica Acta, 2000, 1474, 133-146; Ferro M, Seigneurin-Berny D, Rolland Ν, Chapel A, Salvi D, Garin J, Joyard J. Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins. Electrophoresis 2000, 21, 3517-3526; Stark M, Jornvall H, Johansson J. Isolation and characterization of hydrophobic polypeptides in human bile. Eur J Biochem 1999. 266 (1). 209-14. ).
Pour la purification des peptides très hydrophobes on peut utiliser des solvants organiques tels qu'un mélange de dichlorométhane-hexafluoro-2-propanol contenant des trace de pyridine avec un gradient linéaire acide formique-2-propanol et acide formique-eau sur une phase stationnaire non polaire telle que Vydac C4 (Cf. Bollhagen R, Schmiedberger M, Grell E. High performance liquid chromatographie purification of extremely hydrophobic peptides : transmembrane segments Journal of Chromatography A, 1995, 711, 181-186).For the purification of very hydrophobic peptides, organic solvents can be used such as a mixture of dichloromethane-hexafluoro-2-propanol containing traces of pyridine with a linear gradient formic acid-2-propanol and formic acid-water on a stationary phase. non-polar such as Vydac C4 (Cf. Bollhagen R, Schmiedberger M, Grell E. High performance liquid chromatography purification of extremely hydrophobic peptides: transmembrane segments Journal of Chromatography A, 1995, 711, 181-186).
On peut également utiliser des solvants non aqueux, comme évoqué en utilisation combinée avec des méthodes de séparation par l' électrophorèse capillaire non aqueuse (Cf. Cottet H, Struijk MP, Nan Dongen JLJ, Claessens HA, Cramers CA . Nonaqueous capillary electrophoresis using non-dissociating solvents. Application to the séparation of highly hydrophobic oligomers. Journal of Chromatography A, 2001, 915, 241-252; Veraart, J.R., Reinders, M.C., Lingeman, H. and Brinkman U.A. Non-aqueous capillary electrophoresis of biological samples after at-line solid-phase extraction. J. Chromatogr. A 811 (1998) 211-217; Yang, Q., Benson, L.M., Johnson, K.L. and Naylor, S. Analysis of lipophilic peptides and therapeutic drugs: on-line-nonaqueous capillary electrophoresis-mass spectrometry. J. Biochem. Biophys. Methods 38 (1999) 103-121 ;Belder, D., Elke, K. and Husmann, H. Use of coated capillaries for nonaqueous capillary electrophoresis. J. Microcol. Sep. 11 (1999) 209-213; Lister, A.S., Dorsey, J.G. and Burton, D.E. Current measurement of nonaqueous solvents: applications to capillary electrophoresis and electrochromatography. J. High Res. Chromatogr. 20 (1997) 523-528; Belder, D., Husmann, H. and Warnke, Directed control of electroosmotic flow in nonaqueous electrolytes using poly ethylene glycol coated capillaries. J. Electrophoresis 22 (2001) 666-672 ; Bj0rnsdottir, I. and Hansen, S.H.Comparison of séparation selectivity in aqueous and non-aqueous capillary electrophoresis. J. Chromatogr. A 711 (1995) 313-322; Walbroehl, Y. and Jorgenson, J.W. Capillary zone electrophoresis of neutral organic molécules by solvophobic association with tetraalkylammonium ion. Anal. Chem. 58 (1986) 479-481 ; Wei, H. and Li, S. F. Y. Nonaqueous capillary zone electrophoresis for séparation of free fatty acids with indirect fluorescence détection. Electrophoresis 19 (1998) 2187-2192 ; Raith, K., Wolf, R., Wagner, J. and Neubert, R.H.H.Separation of phospholipids by nonaqueous capillary electrophoresis with electrospray ionization mass spectrometry. J. Microcol. Sep. 10 (1998) 681-685 ; Drange, E. and Lundanes, E. Détermination of long-chained fatty acids using non-aqueous capillary electrophoresis and indirect UN détection. J. Chromatogr. A 771 (1997) 301-309; Esaka, Y., Yoshimura, K., Goto, M. and Kano, K. Νon-aqueous capillary zone electrophoresis using polyethylene glycol as a matrix agent. J. Chromatogr. A 822 (1998) 107-115; Jansson, M. and Roeraade. [Ν- Methylformamide as a séparation médium in capillary electrophoresis. J. Chromatographia 40 (1995) 163-169 ; Esaka, Y., Inagaki, S., Uchida, D., Goto, M. and Kano, K.Polyacrylamides as hydrophilic selectors in non-aqueous capillary electrophoresis. J. Chromatogr. A 905 (2001) 291-297; Hansen, S.H., Tj0rnelund, J. and Bj0rnsdottir, I. Selectivity enhancement in capillary electrophoresis using nonaqueous média. Trends Anal. Chem. 15 (1996) 175-180; Jussila, M., Sundberg, S., Hopia, A., Mâkinen, M. and Riekkola, M.-L. Séparation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis. Electrophoresis 20 (1999) 111-117 ; Jussila, M., Sinervo, K., Porras, S. P. and Riekkola, M.-L. Modified liquid junction interface for nonaqueous capillary electrophoresis-mass spectrometry. Electrophoresis 21 (2000) 3311-3317; Koch, J.T., Beam, B., Phillips, K.S. and Wheeler, J.F. Hydrophobic interaction electrokinetic chromatography for the séparation of polycyclic àromatic hydrocarbons using non- aqueous matrices. J. Chromatogr. A 914 (2001) 223-231 ; Li, S. and Weber, S.G.Separation of neutral compounds in nonaqueous solvents by capillary zone electrophoresis. J. Am. Chem. Soc. 122 (2000) 3787-3788 ; Miller, J.L., Khaledi, M.G. and Shea, D. Séparation of hydrophobic solutés by nonaqueous capillary electrophoresis through dipolar and charge-transfer interactions with pyrylium salts. J. Microcol. Sep. 10 (1998) 681-685; Riekkola, M.-L., Wiedmer, S.K., Nalkό, LE. and Sirén, H. Selectivity in capillary electrophoresis in the présence of micelles, chiral selectors and non-aqueous média. . J. Chromatogr. A 792 (1997) 13-35 ; Riekkola, M.-L., Jussila, M., Porras, S.P. and Nalkό, LE. Νon-aqueous capillary electrophoresis. J. Chromatogr. A 892 (2000) 155-170 ; Sahota, R.S. and Khaledi, M.G. Nonaqueous capillary electrophoresis. Anal. Chem. 66 (1994) 1141-1146 ; Steiner, F. and Hassel, M. Nonaqueous capillary electrophoresis: A versatile completion of electrophoretic séparation techniques. Electrophoresis 21 (2000) 3994-4016; Tj0rnelund, J., Bazzanella, A., Lochmann, H. and Bâchmann, K.Coelectroosmotic séparations of anions in non-aqueous capillary electrophoresis. J. Chromatogr. A 811 (1998) 211-217; Wang, T.; Ward, N.L. and Khaledi, M.G. Efficiency studies in nonaqueous capillary electrophoresis. J. Chromatogr. A 859 (1999) 203-219 ; Wright, P.B., Lister, A.S. and Dorsey, J.G. Behavior and use of nonaqueous média without supporting electrolyte in capillary electrophoresis and capillary electrochromatography. Anal. Chem. 69 (1997) 3251-3259). On peut utiliser des surfactants zwittterioniques, comme C9-APSO4 ouIt is also possible to use nonaqueous solvents, as mentioned in combined use with methods of separation by non-capillary electrophoresis. aqueous (Cf. Cottet H, Struijk MP, Nan Dongen JLJ, Claessens HA, Cramers CA. Nonaqueous capillary electrophoresis using non-dissociating solvents. Application to the separation of highly hydrophobic oligomers. Journal of Chromatography A, 2001, 915, 241-252 ; Veraart, JR, Reinders, MC, Lingeman, H. and Brinkman UA Non-aqueous capillary electrophoresis of biological samples after at-line solid-phase extraction. J. Chromatogr. A 811 (1998) 211-217; Yang, Q. , Benson, LM, Johnson, KL and Naylor, S. Analysis of lipophilic peptides and therapeutic drugs: on-line-nonaqueous capillary electrophoresis-mass spectrometry. J. Biochem. Biophys. Methods 38 (1999) 103-121; Belder, D ., Elke, K. and Husmann, H. Use of coated capillaries for nonaqueous capillary electrophoresis. J. Microcol. Sep. 11 (1999) 209-213; Lister, AS, Dorsey, JG and Burton, DE Current measurement of nonaqueous solvents : applications to capillary electrophoresis and electrochromatography. J. High Res. Chromatogr. 20 (1997) 523- 528; Belder, D., Husmann, H. and Warnke, Directed control of electroosmotic flow in nonaqueous electrolytes using poly ethylene glycol coated capillaries. J. Electrophoresis 22 (2001) 666-672; Bj0rnsdottir, I. and Hansen, SHComparison of separation selectivity in aqueous and non-aqueous capillary electrophoresis. J. Chromatogr. A 711 (1995) 313-322; Walbroehl, Y. and Jorgenson, JW Capillary zone electrophoresis of neutral organic molecules by solvophobic association with tetraalkylammonium ion. Anal. Chem. 58 (1986) 479-481; Wei, H. and Li, SFY Nonaqueous capillary zone electrophoresis for separation of free fatty acids with indirect fluorescence detection. Electrophoresis 19 (1998) 2187-2192; Raith, K., Wolf, R., Wagner, J. and Neubert, RHHSeparation of phospholipids by nonaqueous capillary electrophoresis with electrospray ionization mass spectrometry. J. Microcol. Sep. 10 (1998) 681-685; Drange, E. and Lundanes, E. Determination of long-chained fatty acids using non-aqueous capillary electrophoresis and indirect UN detection. J. Chromatogr. A 771 (1997) 301-309; Esaka, Y., Yoshimura, K., Goto, M. and Kano, K. Νon-aqueous capillary zone electrophoresis using polyethylene glycol as a matrix agent. J. Chromatogr. A 822 (1998) 107-115; Jansson, M. and Roeraade. [Ν- Methylformamide as a medium separation in capillary electrophoresis. J. Chromatographia 40 (1995) 163-169; Esaka, Y., Inagaki, S., Uchida, D., Goto, M. and Kano, K. Polyacrylamides as hydrophilic selectors in non-aqueous capillary electrophoresis. J. Chromatogr. A 905 (2001) 291-297; Hansen, SH, Tj0rnelund, J. and Bj0rnsdottir, I. Selectivity enhancement in capillary electrophoresis using nonaqueous media. Trends Anal. Chem. 15 (1996) 175-180; Jussila, M., Sundberg, S., Hopia, A., Mâkinen, M. and Riekkola, M.-L. Séparation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis. Electrophoresis 20 (1999) 111-117; Jussila, M., Sinervo, K., Porras, SP and Riekkola, M.-L. Modified liquid junction interface for nonaqueous capillary electrophoresis-mass spectrometry. Electrophoresis 21 (2000) 3311-3317; Koch, JT, Beam, B., Phillips, KS and Wheeler, JF Hydrophobic interaction electrokinetic chromatography for the separation of polycyclic àromatic hydrocarbons using non-aqueous matrices. J. Chromatogr. A 914 (2001) 223-231; Li, S. and Weber, SG Separation of neutral compounds in nonaqueous solvents by capillary zone electrophoresis. J. Am. Chem. Soc. 122 (2000) 3787-3788; Miller, JL, Khaledi, MG and Shea, D. Separation of hydrophobic solutes by nonaqueous capillary electrophoresis through dipolar and charge-transfer interactions with pyrylium salts. J. Microcol. Sep. 10 (1998) 681-685; Riekkola, M.-L., Wiedmer, SK, Nalkό, LE. and Sirén, H. Selectivity in capillary electrophoresis in the presence of micelles, chiral selectors and non-aqueous media. . J. Chromatogr. A 792 (1997) 13-35; Riekkola, M.-L., Jussila, M., Porras, SP and Nalkό, LE. Νon-aqueous capillary electrophoresis. J. Chromatogr. A 892 (2000) 155-170; Sahota, RS and Khaledi, MG Nonaqueous capillary electrophoresis. Anal. Chem. 66 (1994) 1141-1146; Steiner, F. and Hassel, M. Nonaqueous capillary electrophoresis: A versatile completion of electrophoretic separation techniques. Electrophoresis 21 (2000) 3994-4016; Tj0rnelund, J., Bazzanella, A., Lochmann, H. and Bâchmann, K. Coelectroosmotic separations of anions in non-aqueous capillary electrophoresis. J. Chromatogr. A 811 (1998) 211-217; Wang, T .; Ward, NL and Khaledi, MG Efficiency studies in nonaqueous capillary electrophoresis. J. Chromatogr. A 859 (1999) 203-219; Wright, PB, Lister, AS and Dorsey, JG Behavior and use of nonaqueous media without supporting electrolyte in capillary electrophoresis and capillary electrochromatography. Anal. Chem. 69 (1997) 3251-3259). Zwitterionic surfactants, such as C9-APSO4 or
C10-APSO4, respectivement 3 ( nonyl-dimethyl-ammonio) propyl sulfate et 3 ( decyl - dimethyl-ammonio) propyl sulfate, pour l'extraction-préconcentration d'espèces moléculaires hydrophobes (Cf. Saitoh T, Hinze WL. Concentration of hydrophobic organic compounds and extraction of proteins using alkylammoniosulfate zwitterionic surfactant mediated phase séparations. Anal. Chem 1991, 63(21):2520-5.)C10-APSO4, respectively 3 (nonyl-dimethyl-ammonio) propyl sulfate and 3 (decyl - dimethyl-ammonio) propyl sulfate, for the extraction-preconcentration of hydrophobic molecular species (Cf. Saitoh T, Hinze WL. Concentration of hydrophobic organic compounds and extraction of proteins using alkylammoniosulfate zwitterionic surfactant mediated phase separations. Anal. Chem 1991, 63 (21): 2520-5.)
En jouant sur un équilibre entre les atomes de soufre et d'azote garantissant le caractère fortement zwitterionique d'une phase stationnaire constituée de monolithes macroporeux synthétisés in situ par photopolymérisation et contenant des copolymères à base de sulfoalkylbetaine (Ν,Ν-dimethyl-Ν-methacryloyloxyethyl-Ν-(3-sulfopropyl) ammonium betaine), des protéines basiques peuvent être extraites par interactions chromatographiques et élution avec des solutions aqueuses de faible force ionique modulée par des ions chaotropiques tel que perchlorate et thiocyanate. (Cf. Niklund C, Sjorgen A, Irgum K, Νes I. Anal. Chem. 2001. Feb 1, 73,(3) , 444-52).By playing on an equilibrium between the sulfur and nitrogen atoms guaranteeing the strongly zwitterionic character of a stationary phase made up of macroporous monoliths synthesized in situ by photopolymerization and containing copolymers based on sulfoalkylbetaine (Ν, Ν-dimethyl-Ν- methacryloyloxyethyl-Ν- (3-sulfopropyl) ammonium betaine), basic proteins can be extracted by chromatographic interactions and elution with aqueous solutions of low ionic strength modulated by chaotropic ions such as perchlorate and thiocyanate. (Cf. Niklund C, Sjorgen A, Irgum K, Νes I. Anal. Chem. 2001. Feb 1, 73, (3), 444-52).
Les analytes basiques peuvent eux aussi être séparés en combinaison avec l' électrophorèse capillaire non aqueuse (Cf. Karbaum, A. and Jira, Th. Nonaqueous capillary electrophoresis: Application possibilities and suitability of various solvents for the séparation of basic analytes. Electrophoresis, 1999, 20, 3396-3401).Basic analytes can also be separated in combination with nonaqueous capillary electrophoresis (Cf. Karbaum, A. and Jira, Th. Nonaqueous capillary electrophoresis: Application possibilities and suitability of various solvents for the separation of basic analytes. Electrophoresis, 1999 , 20, 3396-3401).
Pour la séparation de protéines hydrophobes, la chromatographie par exclusion de taille peut aussi être utilisée avec des phases stationnaires apolaires et élution avec un mélange ternaire tel que (chloroforme-méthanol-acide trifluoro- acétique) (Cf. Bunger H, Kaufner L, Pison U. Quantitative analysis of hydrophobic pulmonary surfactant proteins by high performance liquid chromatography with light- scattering détection. J. Chromatogr A, 2000, 18, 870 (1-2), 363-9.)For the separation of hydrophobic proteins, size exclusion chromatography can also be used with stationary apolar phases and elution with a ternary mixture such as (chloroform-methanol-trifluoroacetic acid) (Cf. Bunger H, Kaufner L, Pison U. Quantitative analysis of hydrophobic pulmonary surfactant proteins by high performance liquid chromatography with light-scattering detection. J. Chromatogr A, 2000, 18, 870 (1-2), 363-9.)
On peut utiliser une méthode de rétro-extraction micellaire dans laquelle les protéines encapsulées à l'intérieur de micelles sont récupérées après destruction des micelles par un surfactant ayant une action contre-électrostatique (Cf. Jarudilokkul S,One can use a method of micellar retro-extraction in which the proteins encapsulated inside micelles are recovered after destruction of the micelles by a surfactant having a counter-electrostatic action (Cf. Jarudilokkul S,
Poppenborg LH, Stuckey DC. Backward extraction of reverse micellar encapsulated proteins using a counterionic surfactant. Biotechnol. Bioeng. 1999. 62 (5). 593-601.)Poppenborg LH, Stuckey DC. Backward extraction of reverse micellar encapsulated proteins using a counterionic surfactant. Biotechnol. Bioeng. 1999. 62 (5). 593-601.)
On peut envisager de purifier des glycoprotéines par chromatographie d'affinité aux lectines comme cela est présenté dans l'article suivant : (Cf. Gérard G, Purification of glycoproteins. Methods in Enzymology, vol 182, 529-539).One can consider purifying glycoproteins by lectin affinity chromatography as presented in the following article: (Cf. Gérard G, Purification of glycoproteins. Methods in Enzymology, vol 182, 529-539).
On peut encore purifier des complexes multi-enzymatiques de la façon décrite dans l'article suivant (Cf. Srere PA, Matthews CK. Purification of Multienzyme Complexes. Methods in Enzymology, vol 182, 539-551).It is also possible to purify multi-enzymatic complexes as described in the following article (Cf. Srere PA, Matthews CK. Purification of Multienzyme Complexes. Methods in Enzymology, vol 182, 539-551).
L'extraction et la chromatographie reposent en particulier sur la notion de polarité, qui provient d'une répartition asymétrique des nuages électroniques au sein des molécules.Extraction and chromatography are based in particular on the concept of polarity, which comes from an asymmetric distribution of electronic clouds within molecules.
Les échelles de polarité sont conçues de plusieurs manières indépendantes qui renvoient à des conséquences diverses des phénomènes de polarité:Polarity scales are designed in several independent ways which refer to the various consequences of polarity phenomena:
- la première s'attache à mesurer l'énergie libre d' adsorption par unité de surface d'un solvant (avec une certaine polarité) à une phase solide (avec une certaine autre polarité). A titre d'exemple, cette méthode donne, par mesure d'adsorption sur l'alumine, les ordres de polarité suivants : eau > méthanol > éthanol > 2-propanol > diméthylsulfoxyde > acétonitrile > méthyléthylcétone.- the first focuses on measuring the free energy of adsorption per unit area of a solvent (with a certain polarity) to a solid phase (with a certain other polarity). By way of example, this method gives, by measurement of adsorption on alumina, the following polarity orders: water> methanol> ethanol> 2-propanol> dimethyl sulfoxide> acetonitrile> methyl ethyl ketone.
- la deuxième s'attache à mesurer expérimentalement (Rohrschneider) des coefficients de distribution de solutés tests entre plusieurs phases. A titre d'exemple, cette méthode donne les ordres de polarité suivants: eau> diméthylsulfoxyde > acétonitrile > méthanol > méthyléthylcétone > éthanol > 2-proρanol. On peut déterminer dans la polarité globale de Rohrschneider la part des polarités partielles, c'est-à-dire respectivement les "pouvoir à accepter des protons", "pouvoir à donner des protons", "pouvoir à créer des interactions dipôle-dipôle". Il apparaît que les alcools sont surtout "accepteurs de protons", que l'acétonitrile et le méthyléthylcétone ont un "pouvoir à créer des interactions dipôles-dipôles", et que le diméthylsulfoxyde a autant un pouvoir à "accepter des protons" qu'à "créer des interactions dipôles-dipôles".- the second attempts to measure experimentally (Rohrschneider) the distribution coefficients of test solutes between several phases. As an example, this method gives the following polarity orders: water> dimethyl sulfoxide>acetonitrile>methanol> methyl ethyl ketone>ethanol> 2-proρanol. We can determine in the global polarity of Rohrschneider the share of partial polarities, that is to say respectively "power to accept protons", "power to give protons", "power to create dipole-dipole interactions" . It appears that alcohols are above all "proton acceptors", that acetonitrile and methyl ethyl ketone have a "power to create dipole-dipole interactions", and that dimethyl sulfoxide has as much a power to "accept protons" as "create dipole-dipole interactions".
- la troisième s'attache à définir des paramètres de solubilité (Hildebrand et Scott), calculés à partir de l'énergie moléculaire de cohésion faisant le bilan de toutes les interactions inter-moléculaires d'un solvant, elle même calculée à partir de l'enthalpie molaire de vaporisation. A titre d'exemple, cette méthode donne les ordres de polarité suivants: eau > méthanol > éthanol > diméthylsulfoxyde > acétonitrile > 2-propanol > méthyléthylcétone.- the third endeavors to define solubility parameters (Hildebrand and Scott), calculated from the molecular energy of cohesion taking stock of all the inter-molecular interactions of a solvent, itself calculated from l molar enthalpy of vaporization. By way of example, this method gives the following polarity orders: water> methanol> ethanol> dimethyl sulfoxide> acetonitrile> 2-propanol> methyl ethyl ketone.
La chromatographie de partage repose sur les solubilités différentielles des solutés entre deux phases liquides, plus précisément entre une phase liquide mobile et une autre phase liquide, dite stationnaire, épousant les mailles d'une phase solide poreuse de fine granulométrie.The partition chromatography is based on the differential solubilities of the solutes between two liquid phases, more precisely between a mobile liquid phase and another liquid phase, called stationary, matching the meshes of a porous solid phase of fine particle size.
La phase solide peut être polaire, telle que par exemple constituée de grains de gel de silice greffée avec des groupements aminopropyle ou paranitrobenzyle, ou alkylnitrile, ou glycéropropyle. En ce cas, la phase mobile, faiblement polaire, comme peut l'être un mélange (95 % hexane, 5% dichlorométhane) recevra un "modificateur polaire" pour donner un mélange à polarité accrue, comme (80 % hexane, 20% dichlorométhane), jusqu'à déplacer des solutés polaires interagissant avec la phase liquide polaire stationnaire. Il s'agit là de chromatographie de partage en phase normale. La phase solide peut aussi être apolaire, comme des matrices telles que matrice copolymère styrène-divinybenzène, ou bien matrice pyrocarbone, ou gels de silice greffés avec des groupements fonctionnels apolaires, tels que par exemple des groupements alkyle ou phenyl. En ce cas, la phase mobile, polaire, comme peut l'être un mélange (40% méthanol ou acétonitrile, 60% eau), recevra un "modificateur polaire" pour donner un mélange à polarité moindre (60 % méthanol ou acétonitrile,40 % eau), jusqu'à déplacer des solutés apolaires interagissant avec la phase liquide apolaire stationnaire. H s'agit là de chromatographie de partage en phase inversée.The solid phase may be polar, such as for example consisting of silica gel grains grafted with aminopropyl or paranitrobenzyl, or alkylnitrile, or glyceropropyl groups. In this case, the weakly polar mobile phase, as can be a mixture (95% hexane, 5% dichloromethane) will receive a "polar modifier" to give a mixture with increased polarity, such as (80% hexane, 20% dichloromethane ), until moving polar solutes interacting with the stationary polar liquid phase. This is normal phase sharing chromatography. The solid phase can also be apolar, such as matrices such as a styrene-divinybenzene copolymer matrix, or else a pyrocarbon matrix, or silica gels grafted with apolar functional groups, such as, for example, alkyl or phenyl groups. In this case, the mobile, polar phase, as a mixture can be (40% methanol or acetonitrile, 60% water), will receive a "polar modifier" to give a mixture with less polarity (60% methanol or acetonitrile, 40 % water), until displacing apolar solutes interacting with the stationary apolar liquid phase. This is reverse phase partition chromatography.
Les autres techniques chromatographiques de séparation reposent sur une rétention différentielle de solutés contenus dans une phase mobile, liquide ou gazeuse, qui traverse une phase stationnaire solide. Selon la technique utilisée, le mode de rétention est la taille, l' adsorption ou l'affinité. La chromatographie par exclusion de taille repose sur une phase stationnaire constituée de billes poreuses formant un gel. La distribution des diamètres des pores à l'intérieur des billes poreuses correspond à une fourchette assez large. Selon leur encombrement stérique, les molécules peuvent ou non passer à l'intérieur d'un plus ou moins grand nombre de pores des billes poreuses. Celles qui passent le plus facilement à l'intérieur des pores des billes poreuses sont les plus retardées. Dans la pratique, le phénomène est biaisé par des interactions ioniques ou hydrophobes entre les solutés et la phase stationnaire. Par ailleurs des effets parasites - comme ceux causés par un écoulement turbulent de la phase mobile, ou bien comme ceux d'un effet gravitationnel dû à des différences de densité entre la phase mobile et les solutés - ont amené à conduire ce type de chromatographie avec des billes de très petite taille et à moyenne ou haute pression. La chromatographie par exclusion de taille est une technique douce, où les molécules peuvent rester dans tout milieu de toute force ionique ou de tout pH, avec toute teneur en détergents ou agents chaotropes, ou toute solution appropriée au maintien de leur intégrité. Par exemple, les protéines séparées par ce moyen peuvent garder une stabilité fonctionnelle ou structurale, car la phase mobile peut accepter les ions et les co-facteurs qui vont favoriser celles-ci.The other chromatographic separation techniques are based on a differential retention of solutes contained in a mobile, liquid or gaseous phase, which passes through a solid stationary phase. Depending on the technique used, the retention mode is size, adsorption or affinity. Size exclusion chromatography is based on a stationary phase consisting of porous beads forming a gel. The distribution of the pore diameters inside the porous beads corresponds to a fairly wide range. Depending on their steric hindrance, the molecules may or may not pass inside a greater or lesser number of pores of the porous beads. Those which pass most easily inside the pores of the porous beads are the most delayed. In practice, the phenomenon is biased by ionic or hydrophobic interactions between the solutes and the stationary phase. Furthermore, parasitic effects - such as those caused by a turbulent flow of the mobile phase, or else those of a gravitational effect due to differences in density between the mobile phase and the solutes - have led to conduct this type of chromatography with very small balls at medium or high pressure. Size exclusion chromatography is a gentle technique, where the molecules can remain in any medium of any ionic strength or any pH, with any content of detergents or chaotropic agents, or any solution suitable for maintaining their integrity. For example, proteins separated by this means can keep a functional or structural stability, because the mobile phase can accept ions and the co-factors which will favor them.
La chromatographie d'exclusion de taille en conditions dénaturantes ou non dénaturantes peut aussi aider, tout en minimisant l'emploi de détergents, à la caracterisation d'agrégats moléculaires où des protéines de membranes sont partie prenante (Cf. Lôster K, Baum O, Hofman W, Reutter W. Characterization of molecular aggregates of alpha 1 betal integrin and other rat liver membrane pro teins by combination of size exclusion chromatography and chemical cross-linking. Journal of Chromatography A, 1995, 711, 187-199.) Des techniques chromatographiques particulièrement préférées sont celles qui reposent sur une adsorption différentielle de solutés contenus dans une phase mobile, liquide ou gazeuse, qui traverse un phase stationnaire solide. La sélectivité en chromatographie d' adsorption, comme dans d'autres techniques de chromatographie, repose sur un process complet pour chacun des solutés : entraînement par la phase mobile et interaction d'énergie spécifique avec la phase stationnaire. La polarité du soluté est intermédiaire entre celle de la phase mobile et celle de la phase stationnaire. Si la polarité du soluté est trop éloignée de celle de la phase mobile, il n' y a pas une solubilité suffisante du soluté dans la phase mobile pour empêcher une rétention irréversible sur la phase stationnaire. Si la polarité du soluté est trop éloignée de celle de la phase stationnaire, il n'y a pas d'interaction avec la phase stationnaire. Cette contradiction peut être partiellement résolue par l'emploi de mélanges binaires ou ternaires avec des polarités variées et des process successifs avec différentes phases mobiles de forces éluantes croissantes, c'est-à-dire des phases mobiles où l'on fait varier la composition des mélanges binaires ou ternaires de solvants à polarité différente pendant la séparation. L'apparition d'un mélange binaire ou ternaire suppose que le micro-environnement de la phase stationnaire va être le siège de gradient de concentrations entre différents solvants, selon que leur polarité est plus ou moins éloignée de celle de ladite phase stationnaire, ce qui va pouvoir donner lieu à des phénomènes de chromatographie de partage.Size exclusion chromatography in denaturing or non-denaturing conditions can also help, while minimizing the use of detergents, in the characterization of molecular aggregates in which membrane proteins are involved (see Lôster K, Baum O, Hofman W, Reutter W. Characterization of molecular aggregates of alpha 1 betal integrin and other rat liver membrane pro teins by combination of size exclusion chromatography and chemical cross-linking. Journal of Chromatography A, 1995, 711, 187-199.) Techniques Particularly preferred chromatography are those based on a differential adsorption of solutes contained in a mobile phase, liquid or gas, which passes through a solid stationary phase. The selectivity in adsorption chromatography, as in other chromatography techniques, is based on a complete process for each of the solutes: entrainment by the mobile phase and interaction of specific energy with the stationary phase. The polarity of the solute is intermediate between that of the mobile phase and that of the stationary phase. If the polarity of the solute is too far from that of the mobile phase, there is not sufficient solubility of the solute in the mobile phase to prevent irreversible retention on the stationary phase. If the polarity of the solute is too far from that of the stationary phase, there is no interaction with the stationary phase. This contradiction can be partially resolved by the use of binary or ternary mixtures with varied polarities and successive processes with different phases mobiles of increasing eluting forces, that is to say mobile phases in which the composition of the binary or ternary mixtures of solvents of different polarity is varied during separation. The appearance of a binary or ternary mixture supposes that the micro-environment of the stationary phase will be the seat of concentration gradient between different solvents, according to whether their polarity is more or less distant from that of said stationary phase, which will be able to give rise to sharing chromatography phenomena.
Un solvant est d'autant plus éluant que sa polarité se rapproche de celle du soluté, et finalement de celle de la phase stationnaire, puisque cette dernière est censée être proche de celle du soluté. Ceci rend difficile l'obtention d'un système chromatographique exhaustif : l'exhaustivité d'un système où la phase mobile peut contenir des solutés de polarité assez éloignées supposerait que la phase stationnaire ait une large gamme d' adsorption, c'est à dire qu'elle soit où très polaire ou très apolaire, ce qui va rendre difficile respectivement l' adsorption de solutés apolaires ou très polaires. A l'intérieur d'une échelle limitée de polarité des solutés, la sélectivité est d'autant meilleure qu'une faible variation de polarité du solvant entraîne un changement sélectif de l'équilibre d'adsorption de solutés de polarités voisines.A solvent is all the more eluting as its polarity approaches that of the solute, and finally that of the stationary phase, since the latter is supposed to be close to that of the solute. This makes it difficult to obtain an exhaustive chromatographic system: the exhaustiveness of a system where the mobile phase can contain solutes of fairly distant polarity would suppose that the stationary phase has a wide range of adsorption, ie whether it is very polar or very non-polar, which will make it difficult, respectively, to adsorb non-polar or very polar solutions. Within a limited scale of polarity of the solutes, the selectivity is all the better when a small variation in the polarity of the solvent results in a selective change in the adsorption equilibrium of solutes of similar polarities.
Pour une phase stationnaire donnée, on peut prévoir, outre la possibilité de faire varier la polarité de la phase mobile, d'autres moyens pour augmenter la sélectivité du processus chromatographique : on peut amener un compétiteur dans l' adsorption des solutés comme par exemple un cation ou un anion, ou bien modifier sélectivement les propriétés des solutés, comme par exemple en faisant varier le pH ou la force ionique.For a given stationary phase, it is possible to provide, in addition to the possibility of varying the polarity of the mobile phase, other means for increasing the selectivity of the chromatographic process: it is possible to bring a competitor into the adsorption of solutes such as for example a cation or anion, or selectively modify the properties of the solutes, such as by varying the pH or the ionic strength.
On peut utiliser des matrices comportant une phase stationnaire dont les greffons sont constitués de polymères de molécules ayant un côté apolaire et un coté polaire, comme un copolymère macroporeux réalisé à partir d'un équilibre entre les deux monomères que sont le divinylbenzène, apolaire, et le N-vinylpyrrolidone, polaire.It is possible to use matrices comprising a stationary phase, the grafts of which consist of polymers of molecules having an apolar side and a polar side, such as a macroporous copolymer produced from an equilibrium between the two monomers, divinylbenzene, apolar, and polar N-vinylpyrrolidone.
La chromatographie par adsorption en phase normale repose sur l' adsorption différentielle des solutés sur une phase stationnaire solide et polaire, telle que notamment à base d'alumine mais surtout de silicates, ou de polymères hydrophiles, comme des gels d'agarose ou de dextran, la phase mobile étant, elle, apolaire.Normal phase adsorption chromatography is based on the differential adsorption of solutes on a solid and polar stationary phase, such as in particular based on alumina but especially on silicates, or hydrophilic polymers, such as agarose or dextran gels , the mobile phase being, it, apolar.
A partir d'agarose, on obtient des micro-billes par un procédé d'émulsion- gélification à chaud utilisant d'abord un solvant non miscible à l'eau, puis un stabilisant, ledit procédé se terminant par l'éviction du solvant par succion filtration. Les gels d'agarose peuvent être réticulés par des agents réticulants tels que l'épichlorohydrine, le 2,3 dibromopropanol ou le divinylsulfone. Selon le pourcentage en agarose, (2, 4, 6%) les gels commerciaux Sepharose ont respectivement les appellations 2B, 4B, 6B. Le Sepharose CL, plus stable thermiquement et chimiquement, est réticulé avec du 2,3 -dibromopropanol en fortes conditions alcalines, opération suivie hydrolyse alcaline de ses groupements sulfates en conditions très réductrices, de manière à le rendre non-ionique ou très faiblement ionique. Le Sephadex est un gel de dextran réticulé avec l'épichlorohydrine qui est stable en conditions alcalines, salines, faiblement acides, mais qui est hydrolyse en conditions acides ou oxydantes marquées. Les gels Sephadex (LH-20) et (LH-60) peuvent être greffés avec des groupements hydroxypropyl qui se lient par des laisons ethers aux unités glucose des chaînes de dextranes, avec pour effet de moduler leur polaritéFrom agarose, micro-beads are obtained by a hot emulsion-gelling process first using a water-immiscible solvent, then a stabilizer, said process ending with the removal of the solvent by suction filtration. Agarose gels can be crosslinked with crosslinking agents such as epichlorohydrin, 2,3 dibromopropanol or divinylsulfone. Depending on the percentage of agarose, (2, 4, 6%) Sepharose commercial gels have the names 2B, 4B, 6B respectively. Sepharose CL, more thermally and chemically stable, is crosslinked with 2,3-dibromopropanol under strong alkaline conditions, operation followed by alkaline hydrolysis of its sulfate groups under very reducing conditions, so as to make it non-ionic or very weakly ionic. Sephadex is a dextran gel crosslinked with epichlorohydrin which is stable under alkaline, saline and weakly acidic conditions, but which is hydrolyzed under marked acid or oxidizing conditions. Sephadex (LH-20) and (LH-60) gels can be grafted with hydroxypropyl groups which bind by ether linkages to the glucose units of the dextran chains, with the effect of modulating their polarity.
La silice est insoluble dans l'eau pour un pH variant de 2 à 8. Sa polarité est apportée par la présence à sa surface de groupements silanols (SiOH), au nombre de 4,6 par nm2, dont le groupement OH est polaire et donneur de protons dans des liaisons hydrogène. Un groupement silanol peut rester libre (silanol libre), ou bien engager une liaison hydrogène avec un groupement silanol voisin (silanol lié), ou encore engager une liaison hydrogène avec une molécule d'eau. Le groupement OH d'un silanol libre peut aussi être donneur de protons à une molécule d'eau (silanol libre hydraté par une couche d'eau monomoléculaire), ou à une autre molécule polaire. Par ailleurs, des silanols liés peuvent attirer des molécules d'eau : on alors des silanols hydratés par une couche d'eau pluri-moléculaire, ce sont des gels de sihce fortement hydratés. Les gels de sihce sont très poreux. Selon que leur surface spécifique est plus ou moins grande (elle varie de 200 à 600 m2 par gramme), il comportent des pores d'un plus ou moins grand diamètre et par suite un masquage plus ou moins grand des silanols libres. Les groupements silanols libres sont des sites d'adsorption "forts", totalement disponibles pour une liaison hydrogène. Les groupements silanols libres hydratés et les groupements silanols liés sont encore des sites d'adsorption. Par contre, les groupements silanols hydratés par une couche d'eau pluri-moléculaire sont plutôt des sites de chromatographie de partage. Dans les gels de silice fortement hydratés dont la surface spécifique dépasse 550 m2 par gramme et dont la teneur en eau dépasse 5%, on considère que la chromatographie de partage devient prépondérante par rapport à la chromatographie d'adsorption. Les gels de silice commercialisés ont une granulométrie variée et mentionnent le nombre de silanols restés libres par unité de surface (par exemple le Lichosorb Si 100 comporte 2,95 groupements silanols libres par nm2 pour une surface spécifique de 309 m2 par gramme, tandis que le Lichosorb Si 80 en comporte 2,20 pour une surface spécifique de 482 m2 par gramme).Silica is insoluble in water for a pH varying from 2 to 8. Its polarity is provided by the presence on its surface of silanol groups (SiOH), 4.6 in number per nm 2 , of which the OH group is polar and proton donor in hydrogen bonds. A silanol group can remain free (free silanol), or else initiate a hydrogen bond with a neighboring silanol group (bound silanol), or else initiate a hydrogen bond with a water molecule. The OH group of a free silanol can also be a proton donor to a water molecule (free silanol hydrated by a layer of monomolecular water), or to another polar molecule. In addition, bound silanols can attract water molecules: we then silanols hydrated by a layer of pluri-molecular water, these are highly hydrated silicon gels. Sihce gels are very porous. Depending on whether their specific surface is more or less large (it varies from 200 to 600 m 2 per gram), they comprise pores of a more or less large diameter and consequently a more or less large masking of the free silanols. The free silanol groups are “strong” adsorption sites, completely available for hydrogen bonding. Hydrated free silanol groups and linked silanol groups are still adsorption sites. On the other hand, the silanol groups hydrated by a layer of pluri-molecular water are rather sites of partition chromatography. In highly hydrated silica gels whose specific surface exceeds 550 m 2 per gram and whose water content exceeds 5%, it is considered that partition chromatography becomes preponderant compared to adsorption chromatography. The silica gels sold have a varied particle size and mention the number of silanols remaining free per unit area (for example Lichosorb Si 100 has 2.95 free silanol groups per nm 2 for a specific surface of 309 m 2 per gram, while that Lichosorb Si 80 contains 2.20 for a specific surface of 482 m 2 per gram).
En chromatographie d'adsorption, on cherche à garder constant le pouvoir d'adsorption de l'absorbant, et ce quelle que soit la phase mobile. Pour ce faire, on ajuste la teneur en eau d'un solvant à un niveau dit de "teneur en eau isoactivante" pour que l'énergie d'adsorption de l'adsorbant soit équivalente à ce qu'elle peut avoir avec un solvant de référence ayant une teneur en eau donnée (par exemple, le solvant de référence pourra être l'acétate d'éthyle à 0,06% d'eau lorsqu'on veut une référence pour le pouvoir absorbant d'une silice d'une surface spécifique de 550 m_ par gramme.)In adsorption chromatography, an attempt is made to keep the adsorption capacity of the absorbent constant, whatever the mobile phase. To do this, we adjusts the water content of a solvent to a level called "isoactivating water content" so that the adsorption energy of the adsorbent is equivalent to what it can have with a reference solvent having a content of given water (for example, the reference solvent could be ethyl acetate at 0.06% water when we want a reference for the absorbency of a silica with a specific surface of 550 m_ per gram .)
On peut modifier la polarité des silices par greffage. Les greffons polaires peuvent être du type aminopropyle, paranitrobenzyle, alkylnitrile(nitro), glycéropropyle (diol). Les greffages peuvent être effectués par silanisation, c'est à dire par réactivité d'alkoxysilanes ou chlorosilanes mono, di- ou tri fonctionnels. Pour que cette réaction puisse avoir lieu, il faut que les molécules de silanes pénétrent dans les pores de la silice, ce qui suppose un diamètre de pores supérieur à 10 nm. De plus, une molécule de silane pouvant occuper deux fois la surface d'une molécule de silanol (0,4 nm2 contre 0,2 nm2), le rendement maximal de greffage est de 50%, c'est à dire 4 micromoles/nm_. Dans la pratique, les phases commerciales greffées non polymérisées ont un taux de greffage de 3,5 à 3,7 micromoles par nm2. Dans une réaction de silanisation avec des chlorosilanes trifonctionnels, seuls deux atomes de chlore peuvent réagir pour des raisons stériques, la liaison Si-Ci avec le troisième atome de chlore dudit silane tri- fonctionnel pouvant être hydrolysée par des traces d'eau, et la liaison Si-OH obtenue donnant lieu à une nouvelle réaction avec des silanes résiduels contenus dans le milieu reactionnel et conduisant à une réaction en chaîne de polymérisation. Les phases stationnaires polymérisées ont alors une grande capacité, mais une résistance importante au transfert de masse.The polarity of the silicas can be modified by grafting. The polar grafts can be of the aminopropyl, paranitrobenzyl, alkylnitrile (nitro), glyceropropyl (diol) type. The grafts can be carried out by silanization, that is to say by reactivity of mono, di- or tri-functional alkoxysilanes or chlorosilanes. For this reaction to take place, the silane molecules must penetrate into the pores of the silica, which supposes a pore diameter greater than 10 nm. In addition, a silane molecule being able to occupy twice the surface of a silanol molecule (0.4 nm 2 versus 0.2 nm 2 ), the maximum grafting yield is 50%, i.e. 4 micromoles / nm_. In practice, the non-polymerized grafted commercial phases have a grafting rate of 3.5 to 3.7 micromoles per nm 2 . In a silanization reaction with trifunctional chlorosilanes, only two chlorine atoms can react for steric reasons, the Si-Ci bond with the third chlorine atom of said tri-functional silane being able to be hydrolyzed by traces of water, and the Si-OH bond obtained giving rise to a new reaction with residual silanes contained in the reaction medium and leading to a reaction in the polymerization chain. The stationary polymerized phases then have a large capacity, but a significant resistance to mass transfer.
La chromatographie par adsorption en phase inversée repose sur l' adsorption différentielle des solutés sur une phase stationnaire solide et apolaire, telle que notamment des silices greffées avec des groupements apolaires, la phase mobile étant, elle, plus ou moins polaire, selon par exemple des proportions variées de solvants plus ou moins polaires, par exemple d'eau et de méthanol ou bien encore d'eau et d'acétonitrile.Reverse phase adsorption chromatography is based on the differential adsorption of solutes on a stationary solid and nonpolar phase, such as in particular silicas grafted with nonpolar groups, the mobile phase being more or less polar, according for example to various proportions of more or less polar solvents, for example water and methanol or alternatively water and acetonitrile.
Si certaines matrices telles que copolymère styrène-divinylbenzène ou pyrocarbone peuvent être apolaires "ex-abrupto", les groupements fonctionnels apolaires greffés sur les phases stationnaires telles que sihce ou Sepharose peuvent être des groupements alkyles Cl 8 ou C8 ou C4 , ou des groupements phényl. La procédure de greffage des silices greffées avec des groupements apolaires s'obtient par silanisation, tout comme la procédure de greffage des silices greffées avec des groupements polaires. Il faut noter, comme dans le cas de silices vierges (polaires) ou des silices greffées polaires, la présence de silanols résiduels provenant de l'hydrolyse des groupements réactifs de silanes trifonctionnels n'ayant pas réagi au cours de la synthèse. Ces silanols résiduels sont recouverts par des molécules d'eau et ceux qui sont accessibles créent un environnement propice à une chromatographie de partage sur phases stationnaires polaires: d'une part les molécules de solvant organique d'un mélange (eau-solvant organique) se fixent préférentiellement à la surface des greffons apolaires, d'autre part les molécules de soluté interagissent avec la phase liquide stationnaire. Le mécanisme d'interaction est soit un mécanisme de partage des solutés entre la phase mobile et la phase liquide adsorbée, soit basé sur une interaction hydrophobe entre les molécules de soluté et la phase stationnaire apolaire. De plus, si leur polarité est suffisante, les molécules de soluté peuvent déplacer des molécules de la phase liquide polaire stationnaire.If certain matrices such as styrene-divinylbenzene or pyrocarbon copolymer can be apolar "ex-abrupto", the apolar functional groups grafted onto the stationary phases such as sihce or Sepharose can be alkyl groups Cl 8 or C8 or C4, or phenyl groups . The grafting procedure for grafted silicas with nonpolar groups is obtained by silanization, as is the grafting procedure for grafted silicas with polar groups. It should be noted, as in the case of virgin (polar) silicas or polar grafted silicas, the presence of residual silanols originating from the hydrolysis of reactive groups of trifunctional silanes which have not reacted during the synthesis. These residual silanols are covered by water molecules and those which are accessible create an environment suitable for partition chromatography on polar stationary phases: on the one hand, the organic solvent molecules of a mixture (water-organic solvent) are preferentially fix on the surface of the apolar grafts, on the other hand the solute molecules interact with the stationary liquid phase. The interaction mechanism is either a solute sharing mechanism between the mobile phase and the adsorbed liquid phase, or based on a hydrophobic interaction between the solute molecules and the stationary apolar phase. In addition, if their polarity is sufficient, the solute molecules can displace molecules from the stationary polar liquid phase.
Les groupements silanols résiduels accessibles peuvent être éliminés (c'est le process appelé "end-capping") par un traitement au triméthylchlorosilane (TMCS).The accessible residual silanol groups can be eliminated (this process is called "end-capping") by a treatment with trimethylchlorosilane (TMCS).
On peut utiliser des matrices apolaires différentes des silices greffées avec des groupements alkyles C18 ou C8 ou C4 ou phényl. Par exemple, les Phenyl et Octyl- Sepharose peuvent être utilisés en chromatographie d'interactions hydrophobes, et sont obtenus lorsqu'on couple la réticulation du Sepharose CL avec une dérivation avec des groupements phenyl ou octyl.Apolar matrices other than silica grafted with C18 or C8 or C4 or phenyl alkyl groups can be used. For example, Phenyl and Octyl-Sepharose can be used in chromatography of hydrophobic interactions, and are obtained when the crosslinking of Sepharose CL is coupled with a derivation with phenyl or octyl groups.
On peut utiliser des matrices copolymère styrène-divinylbenzène ou pyrocarbone présentant l'avantage par rapport aux silices greffées d'être stables dans une fourchette de pH beaucoup plus larges (1 à 13) contre (2 à 7,5), car la silice est attaquable par les ions OH. Ce défaut des silices peut être résolu par l'application d'un revêtement siliconé sur la surface des pores que l'on trouve dans des phases stationnaires commerciales comme Capcell Pak.Styrene-divinylbenzene or pyrocarbon copolymer matrices can be used, which have the advantage over grafted silicas of being stable in a much wider pH range (1 to 13) against (2 to 7.5), because silica is attackable by OH ions. This silica defect can be resolved by applying a silicone coating to the surface of the pores found in stationary commercial phases such as Capcell Pak.
On peut améliorer un point faible des matrices copolymeres, à savoir la résistance mécanique, en employant des matrices copolymeres macroporeuses. Celles-ci comportent à la fois une partie très fortement réticulée, imperméable aux solvants, et une partie peu réticulée propice aux échanges entre la phase stationnaire et la phase mobile, et des macro-pores, sans présence de polymères. D'autres phases stationnaires comme l'oxyde de zirconium poreux, ou le graphite poreux offrent naturellement les qualités de stabilité (pH de 1 à 14) et de résistance mécanique.A weak point of the copolymer matrices, namely mechanical strength, can be improved by using macroporous copolymer matrices. These comprise both a very highly crosslinked part, impermeable to solvents, and a weakly crosslinked part favorable to exchanges between the stationary phase and the mobile phase, and macro-pores, without the presence of polymers. Other stationary phases such as porous zirconium oxide or porous graphite naturally offer the qualities of stability (pH from 1 to 14) and mechanical resistance.
Une autre caractéristique des matrices copolymeres évoquées est la présence de groupements aromatiques, susceptibles d'interagir dans la formation de complexes donneurs-accepteurs d'électrons avec les solutés. On peut utiliser d'autres matrices copolymeres, telles que par exemple celles à base d'alcool vinylique ou de polyméthylméthacrylates.Another characteristic of the copolymeric matrices mentioned is the presence of aromatic groups, capable of interacting in the formation of donor-acceptor complexes of electrons with the solutes. Other copolymer matrices can be used, such as for example those based on vinyl alcohol or polymethylmethacrylates.
On peut dans la présente invention utiliser des méthodes connues de séparation par chromatographie sur résines échangeuses d'ions permettant de séparer des solutés ioniques suivant leur attraction électrostatique à la phase stationnaire, laquelle comporte une matrice à laquelle sont greffés des groupements fonctionnels fixes, ionisés, et capables de fixer des contre-ions. Les micro-particules de la phase stationnaire ionisée accueillent en leur sein les ions de charge opposée, et en excluent les ions de même charge. Les matrices de la phase stationnaire peuvent être des silices greffées ou des matrices copolymeres. Par leur rôle d'exclusion, ainsi que par leur composition propre, comme par exemple la présence de noyaux aromatiques dans les matrices copolymeres polystyrène divinyl benzène donnant lieu à des interactions par électrons pi, les matrices apportent une contribution au process qui se rajoute à celles de leurs groupements fonctionnels ionisés. Une compétition pour la liaison à la phase stationnaire s'établit entre les ions solutés de la phase mobile et des contre-ions libérables par ladite phase stationnaire et donc échangeables. La phase mobile est une solution tampon dont le pH permet de contrôler les attractions électrostatiques des solutés, dans la mesure où à une certaine valeur du pH va correspondre une certaine charge des solutés. Par exemple les acides aminés des protéines, selon le pH, peuvent être présents dans la solution sous forme de molécules zwitterioniques ou sous forme d'anions ou sous forme de cations. De plus, les matrices greffées sont poreuses (il s'agit de micro-particules de sihce poreuse ou de copolymeres organiques de structure microporeuse ou macro-poreuse de type poly(styrène/divinylbenzène) ou polyacrylate), ce qui donne lieu en même temps à un mécanisme de séparation non-ionique, par exemple des mécanismes de partage de molécules avec une polarité donnée. Par exemple, des solutés non ioniques ne subissent pas de répulsion électrostatique pour pénétrer à l'intérieur des pores de la matrice. Ce faisant, ils sont soumis à un mécanisme de partage gouverné par des interactions hydrophobes et/ou des interactions par transfert de charges. Une matrice peut être échangeur de cations ou échangeur d'anions, faible ou fort. Les échangeurs de cations forts (SCX pour Strong Cation Exchangers), du type acides forts, peuvent être sulfoniques, c'est à dire greffés avec des groupements fonctionnels sulfonates SO3-. Les échangeurs de cations faibles, du type acides faibles, peuvent être carboxyliques, c'est à dire greffés avec des groupements fonctionnels carboxylates CO2-. Les échangeurs d'anions forts (SAX pour Strong Anion Exchangers), du type bases fortes, peuvent être des ammonium quaternaires, c'est à dire greffés avec des groupements fonctionnels NR3+, tels que par exemple triméthylammonium . Les échangeurs d'anions faibles, du type bases faibles, peuvent être des ammonium non quaternaires , c'est à dire greffés avec des formes protonées d'aminés primaire, secondaire ou tertiaire (groupements fonctionnels NHR2+, tels que par exemple diéthylaminoéthylammonium). Les abréviations du langage courant concernent, pour des échangeurs de cations, le CM, faiblement acide, pour Carboxymethyl, ainsi que SP et S, fortement acides, pour respectivement Sulphopropyl et methylSulfonate.It is possible in the present invention to use known methods of separation by chromatography on ion exchange resins making it possible to separate ionic solutes according to their electrostatic attraction to the stationary phase, which comprises a matrix to which are grafted fixed, ionized functional groups capable of fixing counterions. The microparticles of the ionized stationary phase accept ions of opposite charge within them, and exclude ions of the same charge. The matrices of the stationary phase can be grafted silicas or copolymeric matrices. By their role of exclusion, as well as by their own composition, such as for example the presence of aromatic nuclei in the polystyrene divinyl benzene copolymer matrices giving rise to interactions by pi electrons, the matrices contribute to the process which is added to those of their ionized functional groups. A competition for binding to the stationary phase is established between the solute ions of the mobile phase and counterions releasable by said stationary phase and therefore exchangeable. The mobile phase is a buffer solution whose pH makes it possible to control the electrostatic attractions of the solutes, insofar as a certain value of the pH will correspond to a certain charge of the solutes. For example, the amino acids of proteins, depending on the pH, can be present in the solution in the form of zwitterionic molecules or in the form of anions or in the form of cations. In addition, the grafted matrices are porous (they are micro-particles of porous layer or organic copolymers of microporous or macro-porous structure of poly (styrene / divinylbenzene) or polyacrylate type), which gives rise at the same time to a non-ionic separation mechanism, for example mechanisms for sharing molecules with a given polarity. For example, nonionic solutes do not undergo electrostatic repulsion to penetrate inside the pores of the matrix. In doing so, they are subject to a sharing mechanism governed by hydrophobic interactions and / or charge transfer interactions. A matrix can be a cation exchanger or anion exchanger, weak or strong. Strong cation exchangers (SCX for Strong Cation Exchangers), of the strong acid type, can be sulfonic, that is to say grafted with sulfonate SO3- functional groups. The weak cation exchangers, of the weak acid type, can be carboxylic, that is to say grafted with functional groups CO2- carboxylates. The strong anion exchangers (SAX for Strong Anion Exchangers), of the strong base type, can be quaternary ammonium, that is to say grafted with NR3 + functional groups, such as for example trimethylammonium. The weak anion exchangers of the weak base type can be non-quaternary ammonium, that is to say grafted with protonated forms of primary, secondary or tertiary amines (NHR2 + functional groups, such as for example diethylaminoethylammonium). The abbreviations of everyday language relate, for cation exchangers, the CM, weakly acid, for Carboxymethyl, as well as SP and S, strongly acid, for respectively Sulphopropyl and methylSulfonate.
Egalement, les abréviations du langage courant concernent, pour des échangeurs d'anions, le DMAE et DEAE, faiblement basiques, pour respectivementAlso, the abbreviations of the current language concern, for anion exchangers, DMAE and DEAE, weakly basic, for respectively
Dimethylaminoethyl et Diethylaminoethyl, ainsi que TMA, Q, et QAE, fortement basiques, pour respectivement Trimethylaminoethyl, Quaternary Ammonium,Dimethylaminoethyl and Diethylaminoethyl, as well as TMA, Q, and QAE, strongly basic, for respectively Trimethylaminoethyl, Quaternary Ammonium,
Quaternary aminoethyl.Quaternary aminoethyl.
La force éluante dépend en partie de la nature de l'ion développeur véhiculé par la phase mobile.The eluting force partly depends on the nature of the developer ion conveyed by the mobile phase.
Dans un premier mode de chromatographie de paires d'ions ou chromatographie d'interactions d'ions, on tire profit de la présence dans la phase mobile d'ions avec une charge opposée à celle du soluté. En présence de soluté, dans une phase mobile à faible constante diélectrique, chaque contre-ion peut former une paire avec une molécule de soluté de charge opposée par attraction électrostatique coulombienne.In a first mode of ion pair chromatography or ion interaction chromatography, advantage is taken of the presence in the mobile phase of ions with a charge opposite to that of the solute. In the presence of solute, in a mobile phase with low dielectric constant, each counterion can form a pair with a solute molecule of opposite charge by Coulomb electrostatic attraction.
Dans un deuxième mode de chromatographie de paires d'ions ou chromatographie d'interactions d'ions, on tire profit de la présence dans la phase mobile d'ions de grande taille (on les appelle les contre-ions) comportant une partie apolaire et une charge opposée à celle du soluté. L'électro-neutralité est assurée par la présence de co-ions de même signe que les ions du soluté. Dans une phase mobile à forte polarité comme l'eau, en présence de soluté, chaque contre-ion peut former une paire avec une molécule de soluté par interaction hydrophobe. Si l'on utilise une phase stationnaire apolaire, telle qu'une silice greffée avec des groupements alkyles, le contre-ion peut s'adsorber sur les greffons apolaires de la phase stationnaire, tout en devant laisser libres 60 à 70% d'entre eux. En effet, eux-mêmes se repoussent l'un l'autre par répulsion coulombienne entre ions de même charge lorsqu'ils se lient à des chaînes alkyles trop proches. Par ailleurs, tout en se liant à la phase stationnaire par leur partie apolaire, les contre-ions vont attirer au niveau de leur partie ionique des ions de charge opposée, comme les co-ions assurant l'électro-neutralité. Il s'opère un échange entre l'ion soluté et le co-ion qui sont de même charge. La capacité de rétention du soluté par la phase stationnaire dépendra, toutes choses égales, de la concentration en contre-ions. Si les ions du soluté sont suffisamment hydrophobes, on peut alors assister aussi au partage de paires d'ions (soluté/contre-ion) se fixant sur les groupements alkyles restés libres de la phase stationnaire et la solubilisation de ces mêmes ions (soluté et conte-ion) dans la phase mobile.In a second mode of ion pair chromatography or ion interaction chromatography, advantage is taken of the presence in the mobile phase of large ions (they are called counterions) comprising an apolar part and a charge opposite to that of the solute. Electro-neutrality is ensured by the presence of co-ions of the same sign as the ions of the solute. In a mobile phase with strong polarity like water, in the presence of solute, each counterion can form a pair with a solute molecule by hydrophobic interaction. If a nonpolar stationary phase is used, such as silica grafted with alkyl groups, the counterion can be adsorbed on the nonpolar grafts of the stationary phase, while having to leave free 60 to 70% of them. In fact, they repel each other by Coulomb repulsion between ions of the same charge when they bind to too close alkyl chains. Furthermore, while binding to the stationary phase by their apolar part, the counterions will attract ions at their ionic part of opposite charge, such as the co-ions ensuring electro-neutrality. There is an exchange between the solute ion and the co-ion which have the same charge. The retention capacity of the solute by the stationary phase will depend, all other things being equal, on the concentration of counterions. If the ions of the solute are sufficiently hydrophobic, it is then also possible to assist in the sharing of pairs of ions (solute / counterion) which are fixed on the alkyl groups which remain free from the stationary phase and the solubilization of these same ions (solute and storytelling) in the mobile phase.
La rétention des solutés dépend de leur degré d'ionisation, de la teneur en solvant organique, et de la concentration en contre-ions dans la phase mobile. Lorsque les solutés sont capables de former des complexes avec un cation (Cu_+, Zn_+, Cd2+, Ni_+) ou un complexe donneur ou accepteur, on peut procéder respectivement à une chromatographie par échange de ligands, ou à la chromatographie avec transferts de charges. Dans la chromatographie par échange de ligands en mode statique, le cation métallique, par exemple le cuivre, est fixé dans la phase stationnaire, par exemple une silice vierge, par des liaisons ioniques ou covalentes, ce qui donne heu au "cuivrage" desdites phases stationnaires. Ainsi, une liaison covalente du cuivre avec la silice est obtenue en présence d'ammoniaque, donnant lieu à des silices recouvertes de silicates de cupri-diamines. Ces silicates de cupri-diamines fixés à la phase stationnaire sont capables d'échanger l'ammoniaque avec un soluté donneur de doublets, qui devient le nouveau ligand en formant une haison avec le cuivre de la phase stationnaire. Dans le même temps, les silicates de cupri-diamines peuvent solvater des molécules d'eau, ce qui les rend très hydrophiles. La rétention d'un soluté va dépendre de son caractère donneur (caractère complexant) et de son caractère hydrophile, ainsi que de la teneur en ammoniaque de la phase mobile, généralement un mélange ternaire eau-acétonitrile- ammoniaque dont la teneur en eau ne dépasse pas 50% afin de préserver la stabilité de la phase stationnaire.The retention of solutes depends on their degree of ionization, the content of organic solvent, and the concentration of counterions in the mobile phase. When the solutes are capable of forming complexes with a cation (Cu_ +, Zn_ +, Cd2 +, Ni_ +) or a donor or acceptor complex, it is possible to carry out chromatography by ligand exchange respectively, or chromatography with transfer of loads. In chromatography by static ligand exchange, the metal cation, for example copper, is fixed in the stationary phase, for example a virgin silica, by ionic or covalent bonds, which gives uh to the "copper plating" of said phases. stationary. Thus, a covalent bond of copper with silica is obtained in the presence of ammonia, giving rise to silicas covered with cupri-diamine silicates. These cupri-diamine silicates attached to the stationary phase are capable of exchanging ammonia with a doubling-giving solute, which becomes the new ligand by forming a hedge with the copper of the stationary phase. At the same time, cupri-diamine silicates can solvate water molecules, which makes them very hydrophilic. The retention of a solute will depend on its donor character (complexing character) and on its hydrophilic character, as well as on the ammonia content of the mobile phase, generally a ternary water-acetonitrile-ammonia mixture whose water content does not exceed step 50% in order to preserve the stability of the stationary phase.
Dans la chromatographie par échange de ligands en mode dynamique, la phase mobile contient un complexe d'un métal de transition avec un ligand comportant une chaîne hydrophobe, et la phase stationnaire, par exemple une silice greffée avec des groupements apolaires, tels que des groupements alkyle C18, est capable de fixer ce complexe hydrophobe. Dans la phase mobile, le métal de transition est en excès par rapport au ligand hydrophobe, de manière à être libre de pouvoir aussi conserver des sites de haison faibles avec des molécules de solvant. Lorsque des solutés sont capables de former des complexes avec le métal de transition, ils se partagent entre des liaisons avec le métal dans la phase mobile et des liaisons avec le métal engagé dans des complexes avec le ligand hydrophobe, lui même adsorbé sur la phase stationnaire hydrophobe. Dans la chromatographie avec transferts de charges, il y a compétition entre les solutés et le solvant pour donner (ou accepter) des électrons à (ou de) des greffons de la phase stationnaire respectivement accepteurs ou donneurs d'électrons et former les complexes correspondants. Lesdits complexes correspondants formés avec les greffons sont très spécifiques, car un greffon peut être ou non accepteur ou donneur selon un deuxième composé en présence potentiellement accepteur ou donneur. Ces complexes ont une enthalpie de formation très faible, de quelques kilojoules. Ainsi, des silices greffées avec des greffons tels que composés aromatiques peuvent donner des électrons à un soluté particulier ou un solvant particulier accepteurs d'électrons et former des complexes. Ce solvant particulier est appelé le "modificateur polaire" de la phase mobile. Dans un tel système, les molécules de solvant peuvent aussi solvater les greffons de la phase stationnaire. En conséquence, il peut y avoir, pour recevoir des électrons de la phase stationnaire, une compétition à deux (soluté, modificateur polaire) avec un biais qui est que le soluté peut interagir aussi avec les greffons solvatés par le modificateur polaire. Finalement, il y a compétition entre les solutés et le modificateur polaire pour donner (ou accepter) des électrons à (ou de) des greffons libres, non solvatés, de la phase stationnaire. La rétention des solutés est d'autant plus forte que le nombre de greffons libres de la phase stationnaire est élevé, et le nombre de noyaux aromatiques par greffon et la densité spatiale de ces noyaux sont élevés. Toutes choses égales, la compétition pour la liaison à la phase stationnaire va se jouer sur la teneur en modificateur polaire de la phase mobile.In chromatography by ligand exchange in dynamic mode, the mobile phase contains a complex of a transition metal with a ligand comprising a hydrophobic chain, and the stationary phase, for example a silica grafted with nonpolar groups, such as groups C18 alkyl, is capable of fixing this hydrophobic complex. In the mobile phase, the transition metal is in excess with respect to the hydrophobic ligand, so as to be free to also be able to preserve weak hedge sites with solvent molecules. When solutes are capable of forming complexes with the transition metal, they are divided between bonds with the metal in the mobile phase and bonds with the metal engaged in complexes with the hydrophobic ligand, itself adsorbed on the stationary phase hydrophobic. In charge transfer chromatography, there is competition between the solutes and the solvent to give (or accept) electrons to (or from) grafts of the stationary phase respectively acceptors or electron donors and form the corresponding complexes. Said corresponding complexes formed with grafts are very specific, because a graft may or may not be an acceptor or a donor according to a second compound in the presence of potentially acceptor or donor. These complexes have a very low enthalpy of formation, of a few kilojoules. Thus, silicas grafted with grafts such as aromatic compounds can give electrons to a particular solute or a particular electron acceptor solvent and form complexes. This particular solvent is called the "polar modifier" of the mobile phase. In such a system, the solvent molecules can also solvate the grafts of the stationary phase. Consequently, there can be, to receive electrons from the stationary phase, a two-way competition (solute, polar modifier) with a bias which is that the solute can also interact with the grafts solvated by the polar modifier. Finally, there is competition between the solutes and the polar modifier to give (or accept) electrons to (or from) free, unsolvated grafts from the stationary phase. The retention of solutes is all the stronger as the number of free grafts of the stationary phase is high, and the number of aromatic nuclei per graft and the spatial density of these nuclei are high. All things being equal, the competition for binding to the stationary phase will be decided on the content of the polar modifier of the mobile phase.
Les principes précédemment évoqués sont compliqués dans le cas de molécules comme des peptides ou des protéines, qui sont des polymères d'acides aminés possédant chacun leur polarité, qui possèdent aussi leur charge nette globale pour un pH donné, et qui possèdent enfin une conformation spatiale qui dépend de la polarité du solvant. La conformation spatiale s'organise de telle sorte que les groupements fonctionnels de même polarité que celle du solvant soient exposés en surface de la molécule, tandis que les groupements fonctionnels de polarité opposée sont repoussés à l'intérieur de la molécule. Une autre possibilité est que les molécules de solvant s'auto-organisent (c'est le phénomène de solvatation) autour des groupements fonctionnels du soluté de polarité opposée, créant une sorte de poche de masquage. Cette dernière a pour effet de masquer la polarité desdits groupements fonctionnels du soluté de polarité opposée à celle du solvant.The principles previously mentioned are complicated in the case of molecules such as peptides or proteins, which are polymers of amino acids each having their polarity, which also have their overall net charge for a given pH, and which finally have a spatial conformation which depends on the polarity of the solvent. The spatial conformation is organized in such a way that the functional groups of the same polarity as that of the solvent are exposed on the surface of the molecule, while the functional groups of opposite polarity are pushed back inside the molecule. Another possibility is that the solvent molecules self-organize (this is the phenomenon of solvation) around the functional groups of the solute of opposite polarity, creating a kind of masking pocket. The latter has the effect of masking the polarity of said functional groups of the solute of polarity opposite to that of the solvent.
En milieux aqueux, l'hydrophilicité d'une protéine ou d'un peptide dépend de sa composition en acides aminés. Lorsqu'à l'intérieur d'une séquence, la proportion d'acides aminés hydrohiles ou polaires est forte, les acides aminés hydrophobes ou apolaires (isoleucine, valine, leucine, phenylalanine) sont repoussés à l'intérieur de la molécule. Par contre, lorsqu'à l'intérieur d'une séquence, la proportion d'acides aminés apolaires ou hydrophobes est forte, il y a une interaction plus directe entre certains acides aminés hydrophobes et le milieu aqueux.In aqueous media, the hydrophilicity of a protein or peptide depends on its amino acid composition. When within a sequence, the proportion of hydrohile or polar amino acids is high, the hydrophobic or apolar amino acids (isoleucine, valine, leucine, phenylalanine) are pushed back inside the molecule. On the other hand, when within a sequence, the proportion of non-polar or hydrophobic amino acids is high, there is a more direct interaction between certain hydrophobic amino acids and the aqueous medium.
Ainsi, un premier moyen d'augmenter la sélectivité est d'utiliser des changements de composition de mélanges binaires ou ternaires de solvants de différentes polarités pour obtenir une variation de polarité de la phase mobile, afin de complètement modifier la conformation spatiale du peptide ou de la protéine. On a alors des conformations spatiales de peptides ou de protéines spécifiques de la nouvelle polarité obtenue, d'autant plus éloignées de la configuration initiale (dénaturées) que la nouvelle polarité du solvant binaire ou ternaire est éloignée de sa polarité initiale. En partant d'un solvant tel qu'un mélange d'eau et d'acétontrile, qui a pour effet de repousser vers l'intérieur de la molécule les groupements des acides aminés hydrophobes ou apolaires d'une protéine ou d'un peptide ayant une forte proportion d'acides aminés hydrophiles ou polaires, l'addition d'un solvant moins polaire va modifier la conformation de la protéine ou du peptide jusqu'à exposer les groupements fonctionnels moins polaires ou non polaires. Les peptides ou les protéines sont alors adsorbees sur une phase stationnaire greffée avec des groupements apolaires, tels que des groupements alkyles Cl 8, C8 ou C4. Les protéines ou le peptides comportant le plus d'acides aminés avec des groupements fonctionnels apolaires seront les plus retardées.Thus, a first means of increasing selectivity is to use changes in the composition of binary or ternary mixtures of solvents of different polarities to obtain a variation in polarity of the mobile phase, in order to completely modify the spatial conformation of the peptide or protein. We then have spatial conformations of specific peptides or proteins of the new polarity obtained, all the more distant from the initial configuration (denatured) that the new polarity of the binary or ternary solvent is distant from its initial polarity. Starting from a solvent such as a mixture of water and acetontrile, which has the effect of pushing towards the interior of the molecule the groups of hydrophobic or nonpolar amino acids of a protein or peptide having a high proportion of hydrophilic or polar amino acids, the addition of a less polar solvent will modify the conformation of the protein or peptide until exposing the less polar or non-polar functional groups. The peptides or proteins are then adsorbed on a stationary phase grafted with nonpolar groups, such as alkyl groups Cl 8, C8 or C4. The proteins or peptides containing the most amino acids with nonpolar functional groups will be the most delayed.
Un second moyen d'augmenter la sélectivité est d'utiliser des variateurs de solvatation des groupements fonctionnels, tels que des sels. A faible force ionique, les groupements fonctionnels hydrophobes ou apolaires sont entourés de molécules d'eau qui s'auto-organisent. Par contre, une forte force ionique démasque les groupements fonctionnels hydrophobes ou apolaires, en désorganisant les molécules d'eau qui les entourent. La chromatographie d'interactions hydrophobes consiste à partir d'une forte force ionique puis à la diminuer jusqu'à ce que les groupements fonctionnels hydrophobes ou apolaires des peptides ou protéines retrouvent leur masque en milieu aqueux de faible force ionique. Si l'on utilise une phase stationnaire apolaire, tels que greffées avec des groupements alkyles Cl 8, C8 ou C4, ou phenyl, les protéines qui ont le plus de groupements fonctionnels hydrophobes sont celles qui sont le plus retardées. Pour ce type de chromatographie, on utilisez souvent les Phenyl et Octyl-Sepharose. Plusieurs modèles ont tenté de décrire les lois de séparation en chromatographie, en particulier en s'attachant à paramétrer la hauteur de plateau théorique dans la micro-colonne.A second means of increasing selectivity is to use solvation variators of functional groups, such as salts. At low ionic strength, hydrophobic or apolar functional groups are surrounded by self-organizing water molecules. On the other hand, a strong ionic force unmasks the hydrophobic or apolar functional groups, by disorganizing the water molecules which surround them. Chromatography of hydrophobic interactions consists of starting with a strong ionic strength and then decreasing it until the hydrophobic or apolar functional groups of the peptides or proteins find their mask in an aqueous medium of weak ionic strength. If a stationary apolar phase, such as grafted with alkyl groups Cl 8, C8 or C4, or phenyl, is used, the proteins which have the most hydrophobic functional groups are those which are the most delayed. Phenyl and Octyl-Sepharose are often used for this type of chromatography. Several models have attempted to describe the laws of separation in chromatography, in particular by attempting to configure the theoretical plateau height in the micro-column.
Différents modèles (Nan Deemter, Giddings, Huber, Knox, Horvath) renvoient à différentes équations pour calculer la hauteur de plateau théorique H. Par exemple, l'équation de Nan Deemter est de la forme:Different models (Nan Deemter, Giddings, Huber, Knox, Horvath) refer to different equations to calculate the theoretical plateau height H. For example, the Nan Deemter equation is of the form:
H = A/d + Bd + CH = A / d + Bd + C
A est un terme qui rend compte de la diffusion axiale, B est un terme qui rend compte des transferts de masse incomplets entre phase mobile et phase stationnaire, C est un terme qui rend compte d'une part de trajets de longueur inégale pour traverser la colonne, d'autre part de la difficulté pour la phase mobile et les solutés d'accéder aux mailles formées par la phase stationnaire: il est optimal que la phase mobile et les solutés atteignent lesdites mailles par convection plutôt que par diffusion, d est le débit de la phase mobile au travers de la colonne. Tous les modèles rendent compte, entre autres, du fait que la diffusion axiale, un phénomène qui est d'autant plus marqué que les molécules sont petites et qui contrecarre la finesse de résolution des séparations, est d'autant plus limitée que la vitesse de la phase mobile est grande, mais que dans le même temps le transfert de masse entre les phases stationnaire et mobile est d'autant mieux effectué que la vitesse de la phase mobile est faible. Excepté pour la chromatographie par exclusion de taille, ils rendent aussi compte du fait que la qualité des séparations est d'autant meilleure que l'accès par convection de la phase mobile aux mailles formées par la phase stationnaire l'emporte sur l'accès par diffusion, c'est à dire que le diamètre des particules de le phase stationnaire est faible.A is a term which accounts for axial diffusion, B is a term which accounts for incomplete mass transfers between mobile phase and stationary phase, C is a term which accounts for, on the one hand, paths of unequal length to cross the column on the other hand difficulty for the mobile phase and the solutes to access the meshes formed by the stationary phase: it is optimal that the mobile phase and the solutes reach said meshes by convection rather than by diffusion, d is the flow rate of the mobile phase through the column. All the models account, inter alia, for the fact that the axial diffusion, a phenomenon which is all the more marked as the molecules are small and which counteracts the fineness of resolution of the separations, is all the more limited as the speed of the mobile phase is large, but at the same time the mass transfer between the stationary and mobile phases is all the better when the speed of the mobile phase is low. Except for size exclusion chromatography, they also account for the fact that the quality of the separations is all the better as the convection access of the mobile phase to the meshes formed by the stationary phase prevails over the access by diffusion, ie the particle diameter of the stationary phase is small.
On peut dans la présente invention utiliser des méthodes connues de séparation par électrochromatographie, c'est-à-dire par électrophorèse effectuée dans un capillaire qui contient une phase stationnaire et qui est soumis à ses deux extrémités à un champ électrique (Cf. Manz A, Effenhauser CS, Burggraf, Harrison DJ, Seiler K, Fluri K . Electroosmotic pumping and electrophoretic séparations for miniaturized chemical analysis Systems. J. Micromech. Microeng, 1994, 4, 257-265 ; Jacobson SC, Kutter JP, Culbertson CT, Ramsey JM. Rapid electrophoretic and chromatographie analysis on microchips). Les solutés sont séparés à la fois selon leur mobilité électrophorétique et selon leur coefficient de distribution entre phase mobile et phase stationnaire (Cf. Altria KD. Overview of capillary electrophoresis and electrochromatography, Journal of Chromatography A, 1999, 856, 443-463; Quirino JP, Terabe S. Electrokinetic chromatography, Journal of Chromatography A, 1999, 465- 482 ; Smith NW, Carter-Finch AS, Electrochromatography, Journal of Chromatography A, 2000, 892, 219-255 ; Bartle KD, Carney RA, Cavazza A, Cikalo MG, Myers P, Robson MM, Roulin SCP, Sealey K. Capillary electrochromatography on silica columns : factors inflencing performance. Journal of Chromatography A, 2000, 892, 279-299; Pyell U. Advances in column technology and instrumentation in capillary electrochromatography, Journal of Chromatography A, 2000, 892, 257-278; Angus PDA, Demarest CW, Catalano T, Stobaugh JF. Aspects of column fabrication for packed capillary electrochromatography. Journal of Chromatography A, 887, 2000, 347-345; Rapp E, Bayer E. Improved column préparation and performance in capillary electrochromatography. Journal of Chromatography A, 2000, 887, 367-378; Luedtke S, Adam Th, von Doehren N, Unger KK. Towards the ultimate minimum particle diameter of silica packings in capillary electrochromatography, Journal of Chromatography A, 2000, 887, 339-346 ; Liu CH, Stationary phases for capillary electrophoresis and capillary electrochromatography. Electrophoresis 2001, 22, 612-628 ; Hayes JD, Malik A. Sol-gel open tubular ODS columns with reversed electroosmotic flow for capillary electrochromatography. Anal. Chem. 2001, 73, 987-996; Roed L, Lundanes E, Greibrokk T. Non-aqueous electrochromatography on continuous bed columns of sol- gel bonded large-pore C30 material: séparation of retinyl esters. J. Microcolumns Séparations. 2000. 12(11). 561-567.).In the present invention, it is possible to use known methods of separation by electrochromatography, that is to say by electrophoresis carried out in a capillary which contains a stationary phase and which is subjected at both ends to an electric field (cf. Manz A , Effenhauser CS, Burggraf, Harrison DJ, Seiler K, Fluri K. Electroosmotic pumping and electrophoretic separations for miniaturized chemical analysis Systems. J. Micromech. Microeng, 1994, 4, 257-265; Jacobson SC, Kutter JP, Culbertson CT, Ramsey JM. Rapid electrophoretic and chromatography analysis on microchips). The solutes are separated both according to their electrophoretic mobility and according to their distribution coefficient between mobile phase and stationary phase (Cf. Altria KD. Overview of capillary electrophoresis and electrochromatography, Journal of Chromatography A, 1999, 856, 443-463; Quirino JP, Terabe S. Electrokinetic chromatography, Journal of Chromatography A, 1999, 465-482; Smith NW, Carter-Finch AS, Electrochromatography, Journal of Chromatography A, 2000, 892, 219-255; Bartle KD, Carney RA, Cavazza A , Cikalo MG, Myers P, Robson MM, Roulin SCP, Sealey K. Capillary electrochromatography on silica columns: factors inflencing performance. Journal of Chromatography A, 2000, 892, 279-299; Pyell U. Advances in column technology and instrumentation in capillary electrochromatography, Journal of Chromatography A, 2000, 892, 257-278; Angus PDA, Demarest CW, Catalano T, Stobaugh JF. Aspects of column fabrication for packed capillary electrochromatography. Journal of Chromatogra phy A, 887, 2000, 347-345; Rapp E, Bayer E. Improved column preparation and performance in capillary electrochromatography. Journal of Chromatography A, 2000, 887, 367-378; Luedtke S, Adam Th, von Doehren N, Unger KK. Towards the ultimate minimum particle diameter of silica packings in capillary electrochromatography, Journal of Chromatography A, 2000, 887, 339-346; Liu CH, Stationary phases for capillary electrophoresis and capillary electrochromatography. Electrophoresis 2001, 22, 612-628; Hayes JD, Malik A. Sol-gel open tubular ODS columns with reversed electroosmotic flow for capillary electrochromatography. Anal. Chem. 2001, 73, 987-996; Roed L, Lundanes E, Greibrokk T. Non-aqueous electrochromatography on continuous bed columns of sol- gel bonded large-pore C30 material: separation of retinyl esters. J. Microcolumns Separations. 2000. 12 (11). 561-567.).
On peut appliquer un champ électrique de manière externe aux canaux sur leur longueur, comme cela a été présenté dans le document (Hayes MA. Extension of external voltage control of electro-osmosis to high pH buffers. Anal Chem, 1999, 71, 3793-3798).An electric field can be applied externally to the channels along their length, as was presented in the document (Hayes MA. Extension of external voltage control of electro-osmosis to high pH buffers. Anal Chem, 1999, 71, 3793- 3798).
On peut procéder à une électrochromatographie micellaire sur supports miniaturisés comme cela a été décrit dans le document suivant : (Cf. Culbertson CT, Jacobson SC, Ramsey JR. Micro-chip device for high efficiency séparations. Anal. Chem, 2000, 72, 5814-5819).Micellar electrochromatography can be performed on miniaturized supports as described in the following document: (Cf. Culbertson CT, Jacobson SC, Ramsey JR. Micro-chip device for high efficiency separations. Anal. Chem, 2000, 72, 5814 -5819).
On peut utiliser des gradients d'élution, et notamment des micro-gradients d'élution ( Cf .Que AH, Kahle N, Νovotny MN. A micro-gradient elution system for capillary electrochromatography. J. Microcolumn séparations, 2000, 12(1), 1-5.).Elution gradients can be used, and in particular micro-gradients of elution (cf. AH, Kahle N, Νovotny MN. A micro-gradient elution system for capillary electrochromatography. J. Microcolumn separations, 2000, 12 (1 ), 1-5.).
La séparation et notamment la séparation des peptides ou des protéines peut être effectuée à l'aide de procédés de séparation par micro-chromatographie, micro- électrochromatographie ou micro-électrophorèse.The separation and in particular the separation of the peptides or the proteins can be carried out using separation methods by micro-chromatography, micro-electrochromatography or micro-electrophoresis.
Le recueil "Protein Liquid Chromatography". Journal of ChromatographyThe collection "Protein Liquid Chromatography". Journal of Chromatography
Library, 2000, vol 61, éd. M. Kastner, Elsevier décrit la séparation des peptides ou protéines, sous la forme d'articles passant en revue leur séparation par chromatographie en phase inversée (Schliiter H. Reversed-Phase Chromatography, p 147-223), par chromatographie par échange d'ions (Roos P. Ion Exchange Chromatography. p 3-88), par chromatographie par interactions hydrophobes (Jacob LR . Hydrophobic InteractionLibrary, 2000, vol 61, ed. M. Kastner, Elsevier describes the separation of peptides or proteins, in the form of articles reviewing their separation by reverse phase chromatography (Schliiter H. Reversed-Phase Chromatography, p 147-223), by exchange chromatography. ions (Roos P. Ion Exchange Chromatography. p 3-88), by hydrophobic interaction chromatography (Jacob LR. Hydrophobic Interaction
Chromatography. p 235-267), par chromatograhie sur hydroxyapatite (Deppert WR,Chromatography. p 235-267), by chromatography on hydroxyapatite (Deppert WR,
Lukacin R, Hydroxyapatite chromatography, p 271-297), par chromatographie d'affinité pour des ions métaux immobilisés (Kastner M, Immobilized Ion AffinityLukacin R, Hydroxyapatite chromatography, p 271-297), by affinity chromatography for immobilized metal ions (Kastner M, Immobilized Ion Affinity
Chromatography, p 301-377), par chromato-focalisation (Lukacin R, Deppert WR,Chromatography, p 301-377), by chromato-focusing (Lukacin R, Deppert WR,
Chromatofocusing, p 385-413), par chromatographie d'affinité pour des ligands moléculaires (Kirchberger J., Bôhme HJ, Dye-Affinity Chromatography, p 415-446), par chromatographie par déplacement (Schliiter H, Jankowski J, Displacement Chromatography, p 505-522), par chromatographie de partage liquide-hquide (HanssonChromatofocusing, p 385-413), by affinity chromatography for molecular ligands (Kirchberger J., Bôhme HJ, Dye-Affinity Chromatography, p 415-446), by displacement chromatography (Schliiter H, Jankowski J, Displacement Chromatography, p 505-522), by liquid-liquid partition chromatography (Hansson
UB, Wingren C. Liquid liquid partition chromatography, p 469-502), par chromatographie par liaison par les résidus cystéine (Whitney D. Covalent Chromatography, p 639-663).UB, Wingren C. Liquid liquid partition chromatography, p 469-502), by cysteine residue binding chromatography (Whitney D. Covalent Chromatography, p 639-663).
On peut utiliser une séparation de peptides et de protéines par chromatographie en phase inversée sur des silices non poreuses mono-dispersées de 1,5 micron (Cf. Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Rétention and selectivity of proteins and peptides in gradient elution of non-porous monodisperse 1,5 micron reversed phase silica. Journal of Chromatography A. 1987, 397, 71-80 ; Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Mobile phase and surface mediated effects on recovery of native proteins in gradient elution on non-porous monodisperse 1,5 micron reversed phase silica. Journal of Chromatography A. 1987, 397, 80-89.)A separation of peptides and proteins can be used by reverse phase chromatography on 1.5 micron non-porous non-porous silicas (Cf. Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Retention and selectivity of proteins and peptides in gradient elution of non-porous monodisperse 1,5 micron reversed phase silica. Journal of Chromatography A. 1987, 397, 71-80; Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Mobile phase and surface mediated effects on recovery of native proteins in gradient elution on non-porous monodisperse 1,5 micron reversed phase silica. Journal of Chromatography A. 1987, 397, 80-89.)
On peut séparer des peptides et des protéines par chromatographie par échange d'anions sur une phase polymérique non poreuse mono-dispersée de 3 microns de poly(styrène-divinylbenzène) (Cf. Régnier FE, Rounds MA. Synthesis of a non- porous, polystyrene-based anion-exchange packing material and its application to fast high-performance liquid chromatography of proteins. Journal of Chromatography A. 1988. 443. 73-83).Peptides and proteins can be separated by anion exchange chromatography on a 3-micron non-porous polymeric phase of 3 microns of poly (styrene-divinylbenzene) (Cf. Régnier FE, Rounds MA. Synthesis of a non-porous, polystyrene-based anion-exchange packing material and its application to fast high-performance liquid chromatography of proteins. Journal of Chromatography A. 1988. 443. 73-83).
On peut séparer des peptides et des protéines par chromatographie par interactions hydrophobes sur des silices non poreuses mono-dispersées de 1,5 micron (Cf. Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Performance of non-porous monodisperse 1,5 micron bonded silicas in the séparation of proteins by hydrophobic interaction chromatography Journal of Chromatography A. 1987, 397, 91- 97).Peptides and proteins can be separated by hydrophobic interaction chromatography on 1.5 micron non-porous non-porous silicas (Cf. Jilge G, Janzen R, Giesche H, Unger KK, Kinkel JN, Hearn MTW. Performance of non -porous monodisperse 1.5 micron bonded silicas in the separation of proteins by hydrophobic interaction chromatography Journal of Chromatography A. 1987, 397, 91-97).
On peut séparer des protéines dans des solutions de pH 5, 7 et 9 par chromatographie par affinité pour des ions cuivriques immobilisés sur des phases stationnaires Sepharose CL-4B grâce à une procédure de couplage epoxy et utilisant le ligand chélateur tridenté N-(2-pyridylmethyl) aminoacétate (Cf. Chaouk H, Hearn MTW. New ligand, N-(2-pyridylmethyl)aminoacetate, for use in the immobilised métal iion affinity chromatographie séparation of pro teins. Journal of Chromatography A, 1999, 852, 105-115).Proteins can be separated in solutions of pH 5, 7 and 9 by affinity chromatography for cupric ions immobilized on stationary Sepharose CL-4B phases thanks to an epoxy coupling procedure and using the tridentate chelating ligand N- (2- pyridylmethyl) aminoacetate (Cf. Chaouk H, Hearn MTW. New ligand, N- (2-pyridylmethyl) aminoacetate, for use in the immobiled metal iion affinity chromatography separation of pro teins. Journal of Chromatography A, 1999, 852, 105-115 ).
On peut retenir des peptides synthétiques ayant un un nombre plus ou moins grand de résidus histidine en chromatographie par affinité pour des ions métalliques immobilisés sur des phases stationnaires Sepharose CL-4B et utilisant l'ion acide iminodiacétique comme ligand chélateur tridenté ou l'ion acide nitrilotriacétqiue comme ligand chélateur tetradenté (Cf Kronina NN, Wirth HJ, Hearn MTW. Characterization by immobilised métal ion affinity chromatographie procédures of the binding behaviour of several synthetic peptides designed to hâve high affinity for Cu(U) ions. Journal of Chromatography A, 1999, 852, 261-272.)Synthetic peptides having a greater or lesser number of histidine residues can be retained in affinity chromatography for metal ions immobilized on stationary phases Sepharose CL-4B and using the iminodiacetic acid ion as tridentate chelating ligand or the acid ion. nitrilotriacétqiue as tetradenté chelating ligand (Cf Kronina NN, Wirth HJ, Hearn MTW. Characterization by immobiled metal ion affinity chromatography procedures of the binding behavior of several synthetic peptides designed to have high affinity for Cu (U) ions. Journal of Chromatography A, 1999, 852, 261-272.)
On peut utiliser une chromatographie de peptides par chromatographie micellaire comme cela a été présenté dans l'article suivant : (Cf. Kord AS, Khaledi MG. Selectivity of organic solvents in micellar liquid chromatography of amino-acids and peptides. Journal of Chromatography A. 1993. 631, 1255-132).One can use a peptide chromatography by micellar chromatography as that was presented in the following article: (Cf. Kord AS, Khaledi MG. Selectivity of organic solvents in micellar liquid chromatography of amino-acids and peptides. Journal of Chromatography A. 1993, 631, 1255-132).
La séparation des échantillons biologiques peut être effectuée par micro- électrophorèse avec une phase préalable d'enrichissement (Cf. Lichtenberg J, Nerpoorte E, de Rooij Ν. Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis. Electrophoresis 2001, 22, 258-271; Wu XZ, Hosaka A, Hobo T. An on-line electrophoretic concentration method for capillary electrophoresis of proteins. Anal. Chem, 1998, 70, 2081-2084; Tragas C, Pawliszyn J. On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface. Electrophoresis 2000, 21, 227-237; Cappiello A, Berloni A, Famiglini G, Mangani F, Palma P. Micro-SPE method for sample introduction in capillary HPLC/MS. Anal. Chem 2001, 73, 298-302; Timperman AT, Aebersold R. Peptide électro-extraction for direct coupling of in-gel digests with capillary LC-MS MS for protein identification and sequencing. Anal. Chem. 2000, 72, 4115-4121; Tong W, Link A, Eng JK, Yates JR UI. Identification of proteins in complexes by solid-phase micro-extraction / multistep elution / capillary electyrophoresis / tandem mass spectrometry. Anal Chem 1999, 71, 2270-2278; Figeys D, Ducret A, Yates JR III, Aebersold R. Protein identification by solid-phase micro extraction / multistep elution / capillary electyrophoresis / tandem mass spectrometry. Nature Biotechnology, 1999, 14, 1579-1583; Stegehuis DS, Irth H, Tjaden UR, van der Greef J. Isochatophoresis as on-line concentration pretreatment technique in capillary electrophoresis. J. Chromât, 1991, 538(2), 393-402.; Poison NA, Savin DP, Hayes MA. Electrophoretic focusing preconcentration technique using a continuous buffer System for capillary electrophoresis. J. Microcolumn Séparations, 2000, 12(2), 98-106.).The separation of biological samples can be carried out by micro-electrophoresis with a prior enrichment phase (Cf. Lichtenberg J, Nerpoorte E, de Rooij Sample. Sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis. Electrophoresis 2001, 22, 258-271; Wu XZ, Hosaka A, Hobo T. An on-line electrophoretic concentration method for capillary electrophoresis of proteins. Anal. Chem, 1998, 70, 2081-2084; Tragas C, Pawliszyn J. On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface. Electrophoresis 2000, 21, 227-237; Cappiello A, Berloni A, Famiglini G, Mangani F, Palma P. Micro-SPE method for sample introduction in capillary HPLC / MS Anal. Chem 2001, 73, 298-302; Timperman AT, Aebersold R. Peptide electro-extraction for direct coupling of in-gel digests with capillary LC-MS MS for protein identification and sequencing. Anal. Chem. 2000, 72, 4 115-4121; Tong W, Link A, Eng JK, Yates JR UI. Identification of proteins in complexes by solid-phase micro-extraction / multistep elution / capillary electyrophoresis / tandem mass spectrometry. Anal Chem 1999, 71, 2270-2278; Figeys D, Ducret A, Yates JR III, Aebersold R. Protein identification by solid-phase micro extraction / multistep elution / capillary electyrophoresis / tandem mass spectrometry. Nature Biotechnology, 1999, 14, 1579-1583; Stegehuis DS, Irth H, Tjaden UR, van der Greef J. Isochatophoresis as on-line concentration pretreatment technique in capillary electrophoresis. J. Chromât, 1991, 538 (2), 393-402 .; Poison NA, Savin DP, Hayes MA. Electrophoretic focusing preconcentration technique using a continuous buffer System for capillary electrophoresis. J. Microcolumn Separations, 2000, 12 (2), 98-106.).
On peut utiliser dans des séparations électrophorétiques la focalisation iso- électrique, c'est-à-dire un gradient de pH sur la longueur du micro-capillaire de séparation. Ce gradient peut être obtenu par un ensemble de petites molécules ampholytes chargées selon leur pi, telles que celles synthétisées à base d'acide acrylique et de polyamines ou obtenues par couplage par épichlorhydrine . Le gradient de pH peut varier généralement de 3 à 10. Le gradient peut aussi être obtenu par greffage, dans un gel de polyacrylamide, de monomères d'acrylamide modifiés portant des groupements chimiques ionisables de PK acides ou basiques. Dans ce cas, le gradient de pH peut varier de 1 à 12,5 (Cf Kawano Y, Ito Y, Yamakawa Y, Yamashino T, Horii T, Hasegawa T, Ohta M. Rapid isolation and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography-electrospray ionization mass spectrometry .FEMS Microbiol Lett. 2000 Aug l;189(l):103-8 ; Bean SR, Lookhart GL.Electrophoresis of cereal storage proteins. J Chromatogr A. 2000 Jun 9;881(l-2):23-36 ; Issaq HJ. A décade of capillary electrophoresis. Electrophoresis. 2000 Jun;21(10):1921-39 ; Herrero-Martinez JM, Simo-Alfonso EF, Ra is-Ramos G, Gelfi C, Righetti PG. Détermination of cow's milk in non-bovine and mixed cheeses by capillary electrophoresis of whey proteins in acidic isoelectric buffers. J Chromatogr A. 2000 May 12;878(2):261-71; Jensen PK, Pasa-Tolic L, Peden KK, Martinovic S, Lipton MS, Anderson GA, Tolic N, Wong KK, Smith RD. Mass spectrometric détection for capillary isoelectric focusing séparations of complex protein mixtures. Electrophoresis. 2000 Apr;21(7):1372-80; Shen Y, Berger SJ, Anderson GA, Smith RD. High-efficiency capillary isoelectric focusing of peptides. Anal Chem. 2000 May l;72(9):2154-9; Shimura K, Zhi W, Matsumoto H, Kasai K. Accuracy in the détermination of isoelectric points of some proteins and a peptide by capillary isoelectric focusing: utility of synthetic peptides as isoelectric point markers. Anal Chem. 2000 Oct 1;72(19):4747- 57 ; Yang L, Lee CS, Hofstadler SA, Pasa-Tolic L, Smith RD. Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron résonance mass spectrometry for protein characterization. Anal Chem. 1998 Aug 1;70(15):3235-41; Chartogne A, Tjaden UR, Van der Greef J. A free-flow electrophoresis chip device for interfacing . capillary isoelectric focusing on-line with electrospray mass spectrometry. Rapid Commun Mass Spectrom. 2000; 14(14): 1269-74 ; Wen J, Lin Y, Xiang F, Maison DW, Udseth HR, Smith RD. Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry. Electrophoresis. 2000 Jan;21(l):191 ; Rossier JS, Schwarz A, Reymond F, Ferrigno R, Blanchi F, Girault HH. Microchannel networks for electrophoretic séparations. Electrophoresis. 1999 Apr-May;20(4-5):727- 31; Hofmann O, Che D, Cruickshank KA, Muller UR. Adaptation of capillary isoelectric focusing to microchannels on a glass chip. Anal Chem. 1999 Feb l;71(3):678-86; Horka M, Willimann T, Blum M, Nording P, Friedl Z, Slais K. Capillary isoelectric focusing with UN-induced fluorescence détection. J Chromatogr A. 2001 May 4;916(l-2):65-71; Hu JP, Lanthier P, White TC, McHugh SG, Yaguchi M, Roy R, Thibault P. Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry. J Chromatogr B Biomed Sci Appl. 2001 Mar 10;752(2):349-68; Shihabi ZK. Stacking in capillary zone electrophoresis. J Chromatogr A. 2000 Dec 1;902(1):107-17; Gubitz G, Schrnid MG. Récent progress in chiral séparation principles in capillary electrophoresis. Electrophoresis. 2000 Dec;21(18):4112-35; Mao Q, Pawliszyn J, Thormann W. Dynamics of capillary isoelectric focusing in the absence of fluid flow: high-resolution computer simulation and expérimental vah dation with whole column optical imaging. Anal Chem. 2000 Nov l;72(21):5493-502 ; Martinovic S, Berger SJ, Pasa-Tolic L, Smith RD. Séparation and détection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry. Anal Chem. 2000 Nov l;72(21):5356-60; Huang T, Wu XZ, Pawliszyn J. Capillary isoelectric focusing without carrier ampholytes.Anal Chem. 2000 Oct l;72(19):4758-61;Barkemeyer BM, Hempe JM.. Effect of transfusion on hemoglobin variants in preterm infants. J Perinatol. 2000 Sep;20(6):355-8 ; Sugano M, Hidaka H, Yamauchi K, Nakabayashi T, Higuchi Y, Fujita K, Okumura N, Ushiyama Y, Tozuka M, Katsuyama T. Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focusing method. Electrophoresis. 2000 Aug;21(14):3016-9 ; Lupi A, Niglio S, Luisetti M, Gorrini M, Coni P, Faa G, Cetta G, Iadarola P. Alphal- antitrypsin in sérum determined by capillary isoelectric focusing. Electrophoresis. 2000 Sep;21(15):3318-26 ; Martinovic S, Pasa-Tolic, Masselon C, Jensen PK, Stone CL, Smith RD. Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry. Electrophoresis. 2000 Jul;21(12):2368-75 ; Chartogne A, Tjaden UR, Nan der Greef J. A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry. Rapid Commun Mass Spectrom. 2000;14(14): 1269-74).Isoelectric focusing, that is to say a pH gradient over the length of the separation capillary, can be used in electrophoretic separations. This gradient can be obtained by a set of small ampholyte molecules charged according to their pi, such as those synthesized based on acrylic acid and polyamines or obtained by coupling with epichlorohydrin. The pH gradient can generally vary from 3 to 10. The gradient can also be obtained by grafting, in a polyacrylamide gel, modified acrylamide monomers carrying ionizable chemical groups of acidic or basic PK. In this case, the pH gradient can vary from 1 to 12.5 (Cf Kawano Y, Ito Y, Yamakawa Y, Yamashino T, Horii T, Hasegawa T, Ohta M. Rapid isolation and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography-electrospray ionization mass spectrometry .FEMS Microbiol Lett. 2000 Aug l; 189 (l): 103-8; Bean SR, Lookhart GL.Electrophoresis of cereal storage proteins. J Chromatogr A. 2000 Jun 9; 881 (1-2): 23-36; Issaq HJ. A decade of capillary electrophoresis. Electrophoresis. 2000 Jun; 21 (10): 1921-39; Herrero-Martinez JM, Simo-Alfonso EF, Ra is-Ramos G, Gelfi C, Righetti PG. Determination of cow's milk in non-bovine and mixed cheeses by capillary electrophoresis of whey proteins in acidic isoelectric buffers. J Chromatogr A. 2000 May 12; 878 (2): 261-71; Jensen PK, Pasa-Tolic L, Peden KK, Martinovic S, Lipton MS, Anderson GA, Tolic N, Wong KK, Smith RD. Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures. Electrophoresis. 2000 Apr; 21 (7): 1372-80; Shen Y, Berger SJ, Anderson GA, Smith RD. High-efficiency capillary isoelectric focusing of peptides. Anal Chem. 2000 May 1; 72 (9): 2154-9; Shimura K, Zhi W, Matsumoto H, Kasai K. Accuracy in the determination of isoelectric points of some proteins and a peptide by capillary isoelectric focusing: utility of synthetic peptides as isoelectric point markers. Anal Chem. 2000 Oct 1; 72 (19): 4747-57; Yang L, Lee CS, Hofstadler SA, Pasa-Tolic L, Smith RD. Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization. Anal Chem. 1998 Aug 1; 70 (15): 3235-41; Chartogne A, Tjaden UR, Van der Greef J. A free-flow electrophoresis chip device for interfacing. capillary isoelectric focusing on-line with electrospray mass spectrometry. Rapid Commun Mass Spectrom. 2000; 14 (14): 1269-74; Wen J, Lin Y, Xiang F, Maison DW, Udseth HR, Smith RD. Microfabricated isoelectric focusing device for direct electrospray ionization-mass spectrometry. Electrophoresis. 2000 Jan; 21 (l): 191; Rossier JS, Schwarz A, Reymond F, Ferrigno R, Blanchi F, Girault HH. Microchannel networks for electrophoretic separations. Electrophoresis. 1999 Apr-May; 20 (4-5): 727-31; Hofmann O, Che D, Cruickshank KA, Muller UR. Adaptation of capillary isoelectric focusing to microchannels on a glass chip. Anal Chem. 1999 Feb 1; 71 (3): 678-86; Horka M, Willimann T, Blum M, Nording P, Friedl Z, Slais K. Capillary isoelectric focusing with UN-induced fluorescence detection. J Chromatogr A. 2001 May 4; 916 (1-2): 65-71; Hu JP, Lanthier P, White TC, McHugh SG, Yaguchi M, Roy R, Thibault P. Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry. J Chromatogr B Biomed Sci Appl. 2001 Mar 10; 752 (2): 349-68; Shihabi ZK. Stacking in capillary zone electrophoresis. J Chromatogr A. 2000 Dec 1; 902 (1): 107-17; Gubitz G, Schrnid MG. Recent progress in chiral separation principles in capillary electrophoresis. Electrophoresis. 2000 Dec; 21 (18): 4112-35; Mao Q, Pawliszyn J, Thormann W. Dynamics of capillary isoelectric focusing in the absence of fluid flow: high-resolution computer simulation and experimental vah dation with whole column optical imaging. Anal Chem. 2000 Nov 1; 72 (21): 5493-502; Martinovic S, Berger SJ, Pasa-Tolic L, Smith RD. Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry. Anal Chem. 2000 Nov 1; 72 (21): 5356-60; Huang T, Wu XZ, Pawliszyn J. Capillary isoelectric focusing without carrier ampholytes.Anal Chem. 2000 Oct l; 72 (19): 4758-61; Barkemeyer BM, Hempe JM .. Effect of transfusion on hemoglobin variants in preterm infants. J Perinatol. 2000 Sep; 20 (6): 355-8; Sugano M, Hidaka H, Yamauchi K, Nakabayashi T, Higuchi Y, Fujita K, Okumura N, Ushiyama Y, Tozuka M, Katsuyama T. Analysis of hemoglobin and globin chain variants by a commonly used capillary isoelectric focusing method. Electrophoresis. 2000 Aug; 21 (14): 3016-9; Lupi A, Niglio S, Luisetti M, Gorrini M, Coni P, Faa G, Cetta G, Iadarola P. Alphal- antitrypsin in serum determined by capillary isoelectric focusing. Electrophoresis. 2000 Sep; 21 (15): 3318-26; Martinovic S, Pasa-Tolic, Masselon C, Jensen PK, Stone CL, Smith RD. Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry. Electrophoresis. 2000 Jul; 21 (12): 2368-75; Chartogne A, Tjaden UR, Nan der Greef J. A free-flow electrophoresis chip device for interfacing capillary isoelectric focusing on-line with electrospray mass spectrometry. Rapid Commun Mass Spectrom. 2000; 14 (14): 1269-74).
On peut utiliser dans les séparations électrophorétiques des isochatophorèses , c'est à dire une électroséparation avec gradient de champ électrique (Cf. Chen S, Lee ML. Automated instrumentation for comprehensive isotachophoresis- capillary zone electrophoresis. Anal Chem. 2000 Feb 15;72(4):816-20) Les méthodes de séparation décrites ci-dessus peuvent être facilement appliquées dans les micro-colonnes de fractionnement 2 ou les micro-colonnes de fractionnement secondaire 10.It is possible to use in electrophoretic separations of isochatophoresis, that is to say an electro-separation with electric field gradient (Cf. Chen S, Lee ML. Automated instrumentation for comprehensive isotachophoresis- capillary zone electrophoresis. Anal Chem. 2000 Feb 15; 72 ( 4): 816-20) The separation methods described above can be easily applied in the fractionation micro-columns 2 or the secondary fractionation micro-columns 10.
Dans un mode de réalisation préféré, les micro-colonnes de fractionnement comprennent des moyens de séparation par chromatographie. Dans le cas de plusieurs lots de micro-colonnes de fractionnement 2, la sélectivité particulière d'un lot est déterminée par la nature de la phase stationnaire de l'ensemble des micro-colonnes chromatographiques de fractionnement 2 formant le lot.In a preferred embodiment, the fractionation micro-columns comprise means for separation by chromatography. In the case of several batches of fractionation micro-columns 2, the particular selectivity of a batch is determined by the nature of the stationary phase of all the chromatographic fractionation micro-columns 2 forming the batch.
Selon la méthode de séparation utilisée, la sélectivité des micro-colonnes de fractionnement 2 d'un lot est déterminée par la polarité intrinsèque et la solvophobicité d'une phase stationnaire et par la polarité, l'amphipaticite et la solvophobicité des groupements fonctionnels qui y sont greffés. La sélectivité des micro-colonnes de fractionnement 2 est déterminée secondairement par d'autres critères comme la micro- porosité, la macro-porosité, l'aptitude à l'échange d'ions ou à l'interaction de paires d'ions ou à l'échange de ligands ou au transfert de charges ou aux réactions d'affinité de ladite phase stationnaire ou l'octroi d'un gradient de pH sur la longueur sur toute la longueur desdites micro-colonnes de fractionnement 2, ledit gradient de pH s'étendant sur une fourchette d'autant plus large que lesdites micro-colonnes de fractionnement 2 sont longues. Ladite sélectivité est déterminée tertiairement par un champ électrique appliqué à des phases stationnaires desdites micro-colonnes de fractionnement 2 (Cf. Method of electric field flow fractionation wherein the polarity of the electric field is periodically reversed. US Patent N°6113819). Pendant une séparation des constituants d'un échantillon, chaque lot de micro-colonnes de fractionnement 2 reçoit une phase mobile, du type éluant, qui lui est propre et en rapport avec la nature de la phase stationnaire de ses micro-colonnes de fractionnement 2.According to the separation method used, the selectivity of the fractionation micro-columns 2 of a batch is determined by the intrinsic polarity and the solvophobicity of a stationary phase and by the polarity, the amphipaticity and the solvophobicity of the functional groups which therein. are grafted. The selectivity of the fractionation micro-columns 2 is determined secondarily by other criteria such as the micro- porosity, macro-porosity, the ability to exchange ions or to the interaction of pairs of ions or to exchange ligands or to charge transfer or to the affinity reactions of said stationary phase or the granting of a pH gradient over the length over the entire length of said fractionation micro-columns 2, said pH gradient extending over a range all the wider as said fractionation micro-columns 2 are long. Said selectivity is determined thirdly by an electric field applied to stationary phases of said fractionation micro-columns 2 (Cf. Method of electric field flow fractionation which the polarity of the electric field is periodically reversed. US Patent N ° 6113819). During a separation of the constituents of a sample, each batch of fractionation micro-columns 2 receives a mobile phase, of the eluent type, which is specific to it and in relation to the nature of the stationary phase of its fractionation micro-columns 2 .
Dans une variante adaptée à un mode de réalisation dans lequel le dispositif comprend des micro-colonnes de fractionnement secondaire 10, on prévoit que la séparation dans des micro-colonnes de fractionnement 2 s'effectue par une première méthode de chromatographie, et que la séparation dans les micro-colonnes de fractionnement secondaire 10 correspondantes s'effectue par une seconde méthode de chromatographie différente de la première. Compte tenu de la première méthode de séparation employée et de la sélectivité d'une micro-colonne de fractionnement 2, on retrouvera regroupées dans un produit de fractionnement capturé des molécules possédant une vitesse de migration similaire. On choisit donc de préférence une seconde méthode de chromatographie selon une sélectivité différente permettant une séparation efficace des molécules du produit de fractionnement obtenu avec la première méthode. Par exemple, la première méthode de chromatographie est une méthode par échange d'ion, la deuxième méthode étant une méthode de chromatographie par interactions hydrophobes.In a variant adapted to an embodiment in which the device comprises secondary fractionation micro-columns 10, provision is made for the separation in fractionation micro-columns 2 to be carried out by a first method of chromatography, and that the separation in the corresponding secondary fractionation micro-columns 10 is carried out by a second chromatography method different from the first. Taking into account the first separation method used and the selectivity of a fractionation micro-column 2, we will find grouped in a captured fractionation product molecules having a similar migration speed. A second chromatography method is therefore preferably chosen according to a different selectivity allowing effective separation of the molecules of the fractionation product obtained with the first method. For example, the first chromatography method is an ion exchange method, the second method being a hydrophobic interaction chromatography method.
Dans le cas de séparation par chromatographie, la détection est effectuée sur les zones de détection 10 par une détection par micro-leviers, suivie ou éventuellement précédée d'une détection supplémentaire par une des méthodes connues de l'Homme de l'Art pour la détection des éluats de chromatographie, directement ou après hyphénation, par exemple la spectrométrie de masse évoquée auparavant.In the case of separation by chromatography, the detection is carried out on the detection zones 10 by detection by micro-levers, followed or optionally preceded by additional detection by one of the methods known to those skilled in the art for the detection of eluates from chromatography, directly or after hyphenation, for example the mass spectrometry mentioned previously.
On a décrit des micro-colonnes de séparation par chromatographie. On peut utiliser des séparations par des moyens différents dont certains ont déjà été évoqués, en obtenant des sélectivités encore différentes.Micro-columns for separation by chromatography have been described. Separations can be used by different means, some of which have already been mentioned, while obtaining still different selectivities.
Dans une variante, les micro-colonnes de fractionnement comprennent des moyens de séparation par micro-électrophorèse, de type micro-électrophorèse de zone, ou de type micro-isochatophorèse, ou de type micro-électrophorèse micellaire, ou de type micro-électrophorèse à focalisation iso-électrique obtenue par l'octroi d'un gradient de pH sur la longueur sur toute la longueur desdits micro-canaux, ledit gradient de pH s'étendant sur une fourchette d'autant plus large que lesdites micro-canaux sont longs. Dans ce cas également, la détection est effectuée à l'aide de micro-leviers et éventuellement par spectrométrie.In a variant, the fractionation micro-columns comprise means of separation by micro-electrophoresis, of the zone micro-electrophoresis type, or of the micro-isochatophoresis type, or of the micellar micro-electrophoresis type, or of the micro-electrophoresis type with isoelectric focusing obtained by the granting of a pH gradient over the length over the entire length of said micro-channels, said pH gradient extending over a range that is wider the longer said micro-channels. In this case also, the detection is carried out using micro-levers and possibly by spectrometry.
Dans une variante, la séparation dans les micro-colonnes de fractionnement est réalisée par une méthode dite de Field Flow Fractionation (cf. Suslov SA, Roberts AJ., Modeling os sample dynamics in rectangular asymetrical flow field flow fractionation channels. Anal Chem 2000, 72(18), 4331-45).In a variant, the separation in the fractionation micro-columns is carried out by a method called Field Flow Fractionation (cf. Suslov SA, Roberts AJ., Modeling os sample dynamics in rectangular asymetrical flow field flow fractionation channels. Anal Chem 2000, 72 (18), 4331-45).
Dans une variante, les micro-canaux de micro-colonnes sont pourvus sur leur longueur de nano-électrodes (cf. US 6 123 819). Les molécules entraînées par un éluant sont d'autant plus freinées que leurs charges interagissent avec un champ électromagnétique crée par les nano-électrodes. On a décrit des micro-colonnes de fractionnement rectilignes et parallèles.In a variant, the micro-channels of micro-columns are provided along their length with nano-electrodes (cf. US 6,123,819). The molecules entrained by an eluent are all the more braked as their charges interact with an electromagnetic field created by the nano-electrodes. Rectilinear and parallel fractionation micro-columns have been described.
On peut envisager des arrangements différents des micro-colonnes de fractionnement 2. Les micro-colonnes de fractionnement 2 peuvent par exemple être rectilignes, courbes ou sinueuses. Elles sont fabriquées en partie ou en totalité grâce aux techniques employées dans la micro-fabrication sur silicium ou verre ou céramique ou plastique. Dans un mode de réalisation, les micro-colonnes de fractionnement sont monolithes.One can envisage different arrangements of the fractionation micro-columns 2. The fractionation micro-columns 2 can for example be rectilinear, curved or sinuous. They are manufactured in part or in whole thanks to the techniques used in micro-manufacturing on silicon or glass or ceramic or plastic. In one embodiment, the fractionation micro-columns are monoliths.
Des méthodes de fabrication des supports sont décrites dans la suite de la description.Methods of manufacturing the supports are described in the following description.
De façon générale, des lits de micro-colonnes, c'est-à-dire des rainures formées sur un support et prévues pour être recouvertes et fermées à l'aide d'un autre support présentant une surface plane où des rainures correspondantes peuvent être directement gravées lorsque lesdits supports sont à base de silicium ou de verre ou de céramique.In general, micro-column beds, that is to say grooves formed on a support and intended to be covered and closed using another support having a flat surface where corresponding grooves can be directly etched when said supports are based on silicon or glass or ceramic.
Par exemple, les micro-colonnes de fractionnement 2 et lesdites microcolonnes secondaires 10 peuvent être fabriquées en partie ou en totalité grâce aux techniques employées dans la micro-fabrication telles que photogravure, micromoulage, micro-emboutissage, photolymérisation, thermopolymérisation. (Cf. He B, Tait N, Régnier F. Fabrication of Nanocolumns for liquid chromatography. Anal. Chem, 1998, 70, 3790-3797 ; Régnier FE, He B, Lin S, Busse J. Chromatography and electrophoresis on chips : critical éléments of future integrated, microfluidic analytical Systems for life science, TIBTECH, 1999, 17, 101-106 ; Pesek JJ, Matyska MT. Open tubular capillary electrokinetic chromatography in etched fused-silica tubes. Journal of Chromatography, 2000, 887, 31-41). On peut obtenir le réseau micro-particulaire des micro-colonnes qui constitue les phases stationnaires par photogravure lorsque lesdits supports intégrés sont à base de silicium ou de verre ou de céramique (Cf. He B., Régnier F. Microfabricated liquid chromatography columns based on collocated monolith support structures, 451- 455; He B., Tait N., Régnier F. Fabrication of nanocolumns for liquid chromatography. Anal. Chem, 1998, 70, 3790-3797).For example, the fractionation micro-columns 2 and said secondary microcolumns 10 can be manufactured in part or in whole thanks to the techniques used in micro-manufacturing such as photoengraving, micromolding, micro-stamping, photolymerization, thermopolymerization. (Cf. He B, Tait N, Régnier F. Fabrication of Nanocolumns for liquid chromatography. Anal. Chem, 1998, 70, 3790-3797; Régnier FE, He B, Lin S, Busse J. Chromatography and electrophoresis on chips: critical elements of future integrated, microfluidic analytical Systems for life science, TIBTECH, 1999, 17, 101-106; Pesek JJ, Matyska MT. Open tubular capillary electrokinetic chromatography in etched fused-silica tubes. Journal of Chromatography, 2000, 887, 31- 41). One can obtain the micro-particulate network of micro-columns which constitutes the stationary phases by photoengraving when said integrated supports are based on silicon or glass or ceramic (Cf. He B., Régnier F. Microfabricated liquid chromatography columns based on collocated monolith support structures, 451-455; He B., Tait N., Régnier F. Fabrication of nanocolumns for liquid chromatography. Anal. Chem, 1998, 70, 3790-3797).
On peut obtenir le réseau microparticulaire par micro-moulage ou microemboutissage ou photopolymérisation ou thermopolymérisation in situ emboutissage lorsque lesdits supports sont à base de plastiques, ou bien peut être constitué de micro ou nano-baguettes insérées dans le ht desdites micro-colonnes (Cf. Gusev I, Huang X, Horvath C. Capillary columns with in situ formed porous monolithic packing for micro- high performance liquid chromatography and capillary electrochromatography, Journal of Chromatography A, 1999, 855, 273-290;Yu C, Svec F., Fréchet J. Towards stationary phases for chromatography on a microchip: molded porous polymer monoliths prepared in capillaries by photo-initiated in situ polymerization as séparation média for electrochromatography. Electrophoresis 2000, 21, 120-127; Svec F., Peters E.C., Sykora D., Fréchet J. Design of the monolithic polymers used in capillary electrochromatography columns. J. of Chromatography A., 2000, 887, 3-29. Josic D., Buchacher A., Jungbauer A. Monoliths as stationary phases for séparation of proteins and polynucleotides and enzymatic conversion. Journal of Chromatography B, 2001, 752, 191-205; Ngola SM, Fintschenko Y., Choi WY, Shepodd TJ. Conduct-as-cast polymer monohths as séparation média for capillary electrochromatography ; Pursch M, Sander LC. Stationary phases for capillary electrochromatography. Journal of Chromatography A, 2000, 887, 313-326.) Des micro-particules peuvent aussi être immobilisées dans un lit continu (Adam T., Unger KK, Dittmann MM, Rozing GP. Towards the column bed stabilization of columns in capillary electroendosmotic chromatography. Immobilization of microparticulate silica columns to a continuous bed. J. of chromatography A, 2000, 327-337; Roed L, Lundanes E, Greibrokk T. Nonaqueous electrochromatography on continuous bed columns of sol-gel bonded large- pore C30 material: séparation of retinyl esters. J. Microcolumns Séparations. 2000. 12(11). 561-567)The microparticulate network can be obtained by micro-molding or micro-stamping or photopolymerization or thermopolymerization in situ stamping when said supports are plastic-based, or else can be made up of micro or nano-rods inserted in the ht of said micro-columns (Cf. Gusev I, Huang X, Horvath C. Capillary columns with in situ formed porous monolithic packing for micro- high performance liquid chromatography and capillary electrochromatography, Journal of Chromatography A, 1999, 855, 273-290; Yu C, Svec F., Fréchet J. Towards stationary phases for chromatography on a microchip: molded porous polymer monoliths prepared in capillaries by photo-initiated in situ polymerization as separation media for electrochromatography. Electrophoresis 2000, 21, 120-127; Svec F., Peters EC, Sykora D. , Fréchet J. Design of the monolithic polymers used in capillary electrochromatography columns. J. of Chromatography A., 2000, 887, 3-29. Josic D., Buchach er A., Jungbauer A. Monoliths as stationary phases for separation of proteins and polynucleotides and enzymatic conversion. Journal of Chromatography B, 2001, 752, 191-205; Ngola SM, Fintschenko Y., Choi WY, Shepodd TJ. Conduct-as-cast polymer monohths as media separation for capillary electrochromatography; Pursch M, Sander LC. Stationary phases for capillary electrochromatography. Journal of Chromatography A, 2000, 887, 313-326.) Micro-particles can also be immobilized in a continuous bed (Adam T., Unger KK, Dittmann MM, Rozing GP. Towards the column bed stabilization of columns in capillary electroendosmotic Chromatography. Immobilization of microparticulate silica columns to a continuous bed. J. of Chromatography A, 2000, 327-337; Roed L, Lundanes E, Greibrokk T. Nonaqueous electrochromatography on continuous bed columns of sol-gel bonded large- pore C30 material: separation of retinyl esters, J. Microcolumns Separations, 2000. 12 (11). 561-567)
On peut revêtir le réseau micro-particulaire des micro-colonnes qui constitue la phase stationnaire d'un film fin de nature hydrophobe ou hydrophile et peut être soumis à des chimies de couplage connues de l'Homme de l'Art pour greffer des molécules caractérisées par principalement par leur polarité et leur amphipathicité. La gravure chimique augmente les propriétés de rétention, comme cela est indiqué dans le document suivant : (Cf. Pesek, Protein and peptides séparations on high surface area capillaries, Electrophoresis, 1999, 20, 2343-2348).One can coat the micro-particulate network of micro-columns which constitutes the stationary phase of a thin film of hydrophobic or hydrophilic nature and can be subjected to coupling chemistries known to those skilled in the art to graft characterized molecules. mainly by their polarity and their amphipathicity. Chemical etching increases the retention properties, as indicated in the following document: (Cf. Pesek, Protein and peptides separations on high surface area capillaries, Electrophoresis, 1999, 20, 2343-2348).
On peut munir les micro-canaux de micro-baguettes de monolithes polymères adéquates pour la séparation des protéines tant par électrochromatographie que par micro-HPLC (Cf. Hjerten, Electroosmosis and Pressure-driven chromatography in chips using continuous beds. Anal. Chem, 2000, 72, 81-87).The micro-channels can be provided with micro-rods of polymer monoliths suitable for the separation of proteins both by electrochromatography and by micro-HPLC (Cf. Hjerten, Electroosmosis and Pressure-driven chromatography in chips using continuous beds. Anal. Chem, 2000 , 72, 81-87).
On peut obtenir des phases stationnaires de chromatographie par moulage à l'aide de moules en silicium. La très grande résolution des techniques de micro-moulage de plastique est directement liée à celle des moules en silicium. Elle se situe donc au niveau requis pour envisager la fabrication de phases stationnaires de chromatographie directement moulées dans le plastique grâce à des moules silicium dimensionnés pour fabriquer des phases stationnaires comportant un réseau plastique micro-particulaire très fin, comme par exemple en étant constitué de cubes polymériques de 5 microns d'arêtes séparés par des espaces de 500 nanomètres.Stationary chromatography phases can be obtained by molding using silicon molds. The very high resolution of micro-plastic molding techniques is directly linked to that of silicon molds. It is therefore at the level required to consider the manufacture of stationary chromatography phases directly molded in the plastic using silicon molds dimensioned to manufacture stationary phases comprising a very fine microparticulate plastic network, such as for example by being made of cubes. 5 micron polymeric edges separated by spaces of 500 nanometers.
On peut utiliser des techniques de "Molecular Imprinting" visant à faire mimer par les plastiques des surfaces de reconnaissance moléculaire de molécules A calquées sur celles de molécules B ayant une affinité pour lesdites molécules A (Cf. Rachkov A, Minoura N. Towards molecularly imprinted polymers sélective to peptides and proteins. The epitope approach. Biochim. Biophys Acta 2001. 1544(1-2). 255-266; Haupt K, Mosbach K. Plastic antibodies: developments and applications. Trends Biotech 1998. 16(11). 468-75; Ramstrom O, Mosbach K. Sybthesis and catalysis by molecularly imprinted materials. Current Opinion Chem. Biol. 1999, 3(6). 759-64; Heegaard NH, Nilsson S, Guzman NA. Affinity capillary electrophoreis: important application areas and some récent developments . J. chromatography B Biomed Sci Appl. 1998. 715, 29-54.; Schweitz L, Petersson M, Johansson T, Nilsson S. Alternative methods providing enhanced sensitivity ansd selectivity in capillary electro-separation experiments. Journal of Chromatography A, 2000, 892, 203-217)We can use "Molecular Imprinting" techniques aimed at mimicking by plastics molecular recognition surfaces of molecules A modeled on those of molecules B having an affinity for said molecules A (Cf. Rachkov A, Minoura N. Towards molecularly imprinted selective polymers to peptides and proteins. The epitope approach. Biochim. Biophys Acta 2001. 1544 (1-2). 255-266; Haupt K, Mosbach K. Plastic antibodies: developments and applications. Trends Biotech 1998. 16 (11). 468-75; Ramstrom O, Mosbach K. Sybthesis and catalysis by molecularly imprinted materials. Current Opinion Chem. Biol. 1999, 3 (6). 759-64; Heegaard NH, Nilsson S, Guzman NA. Affinity capillary electrophoreis: important application areas and some recent developments, J. chromatography B Biomed Sci Appl. 1998. 715, 29-54 .; Schweitz L, Petersson M, Johansson T, Nilsson S. Alternative methods providing enhanced sensitivity ansd selectivity in capillary electro-separation experiment s. Journal of Chromatography A, 2000, 892, 203-217)
Pour qu'un système d'analyse chimique ou biochimique puisse être miniaturisé, il faut d'une part que les conduits et composants qui guident ou reçoivent les fluides (micro-canaux, micro-réservoirs, micro-mixers, micro-colonnes, etc) soient miniaturisés, d'autre part que les composants qui gèrent le parcours des fluides et des réactifs (micro-vannes, micropompes, micro-capteurs, micro-chauffeurs, etc) soient eux aussi miniaturisés, et enfin que des connexions puissent s'établir à l'intérieur et vers l'extérieur du dispositif. (Cf. Elwenspoek M, Lammerink TS J, Miyaké R, Fluitman JHJ. Towards integrated microliquid handling Systems. J. Micromech. Microeng. 1994, 4, 227-245. _ Verpoorte EMJ, van der Schoot BH, Jeanneret S, Manz A, Widmer HM, de Rooij NF. Three-dimensional micro flow manifolds for miniaturized chemical anlysis Systems . J. Micromech. Microeng.1994, 4, 246-256, 1994 _ Schabmueller CGJ, Koch M, Evans AGR, Brunnschweiler A. . Design and fabrication of a microfluidic circuitboard. J. Micromech.Microeng. 1999, 9, 176-179._ Lammerink TSJ, Spiering VL, Elwenspoek M, van den Berg A. Modular concept for fluid handhng system. Proc. IEEE Micro Electro Mechanical Systems, 1996, San Diego pp389-384 _ Richter M, Prak A, Eberl M, Leeuwis H, Woias P, Steckenborn A. 1997. A chemical microanalysis system as a microfluid demonstrator. Proc. Transducers 97, LEEE Chicago, pp303-306._ Kovacs GTA, Petersen K, Albin M. Silicon micromachining: sensors to Systems. Analytical Chemistry, 1996, 407A - 412A _ Gravesen P, Branebjerg J, Jensen OS.. .Microfluidics. A review.J. Micromech. Microeng. 1993. 3. 168-182. _ Shoji S, Esahi M. Microflow devices and Systems. J. Micromech. Microeng. 1994. 4. 157-171. _ Bϋttgenbach S., Robohm C. Microflow devices for miniaturized chemical analysis Systems. SPIE 1998, vol 3539, 51-61 _ Urban G, Jobst G, Moser I. Chemo-and biosensor microsystems for clinical applications. SPLE 1998. Nol 3539, 46-50).For a chemical or biochemical analysis system to be miniaturized, on the one hand, the conduits and components that guide or receive the fluids (micro-channels, micro-reservoirs, micro-mixers, micro-columns, etc.) ) are miniaturized, on the other hand that the components which manage the flow of fluids and reagents (micro-valves, micropumps, micro-sensors, micro-heaters, etc.) are also miniaturized, and finally that connections can be made set up inside and outside the device. (Cf. Elwenspoek M, Lammerink TS J, Miyaké R, Fluitman JHJ. Towards integrated microliquid handling Systems. J. Micromech. Microeng. 1994, 4, 227-245. _ Verpoorte EMJ, van der Schoot BH, Jeanneret S, Manz A , Widmer HM, from Rooij NF. Three-dimensional micro flow manifolds for miniaturized chemical anlysis Systems. J. Micromech. Microeng. 1994, 4, 246-256, 1994 _ Schabmueller CGJ, Koch M, Evans AGR, Brunnschweiler A.. Design and fabrication of a microfluidic circuitboard. J. Micromech, Microeng. 1999, 9, 176-179._ Lammerink TSJ, Spiering VL, Elwenspoek M, van den Berg A. Modular concept for fluid handhng system. Proc. IEEE Micro Electro Mechanical Systems, 1996, San Diego pp389-384 _ Richter M, Prak A, Eberl M, Leeuwis H, Woias P, Steckenborn A. 1997. A chemical microanalysis system as a microfluid demonstrator. Proc. Transducers 97, LEEE Chicago, pp303-306._ Kovacs GTA, Petersen K, Albin M. Silicon micromachining: sensors to Systems. Analytical Chemistry, 1996, 407A - 412A _ Gravesen P, Branebjerg J, Jensen OS ... Microfluidics. A review.J. Micromech. Microeng. 1993. 3. 168-182. _ Shoji S, Esahi M. Microflow devices and Systems. J. Micromech. Microeng. 1994. 4. 157-171. _ Bϋttgenbach S., Robohm C. Microflow devices for miniaturized chemical analysis Systems. SPIE 1998, vol 3539, 51-61 _ Urban G, Jobst G, Moser I. Chemo-and biosensor microsystems for clinical applications. SPLE 1998. Nol 3539, 46-50).
On peut envisager des techniques et des fabrications séparées d'une part pour les supports avec micro-conduits et micro-canaux, d'autre part pour les microcomposants, quitte à procéder à un montage, de préférence automatisé, en fin de fabrication. Parmi les critères qui aident à orienter le choix d'un mode de fabrication pour une pièce donnée d'un dispositif miniaturisé d'analyse chimique ou biochimique figure le ratio d'aspect (aspect ratio en anglais) qui représente l'aptitude à respecter les côtes d'un plan en trois dimensions, en particulier à respecter un profil à partir de lignes brisées et non pas à partir de courbes Pour la fabrication des systèmes miniaturisés, au moins dans une première phase de fabrication, on peut partir part de supports plans et plats (parallèles à un plan et peu épais), dits en 2D, où l'essentiel des composants est fabriqué à partir de gravures, d'ablations et de dépôts sur surface plane.We can consider separate techniques and manufacturing on the one hand for supports with micro-conduits and micro-channels, on the other hand for microcomponents, even if it means mounting, preferably automated, at the end of manufacturing. Among the criteria that help guide the choice of a manufacturing method for a given part of a miniaturized chemical or biochemical analysis device is the aspect ratio which represents the ability to respect the ribs of a three-dimensional plane, in particular to respect a profile from broken lines and not from curves For the manufacture of miniaturized systems, at least in a first phase of manufacture, one can start from flat supports and flat (parallel to a plane and not very thick), called 2D, where most of the components are made from engravings, ablations and deposits on a flat surface.
On peut également faire des composants de moins en moins plans et plats tout en respectant de plus en plus finement un profil de fabrication, grâce à des techniques substractives du type gravure chimique, ablation physique, et des techniques additives du type dépôts tels qu' électrodéposition (electroplating), electroless plating, par vapeur chimique (CND et PENCD) et enfin des techniques de micro-moulage et de micro-emboutissage. Pour surmonter les limites de fabrication, notamment la profondeur limite obtenue par usinage, et les limites des techniques de dépôt ou de moulage pour sculpter des dispositifs où des formes en 3D avec un rapport élevé hauteur/ surface de base sont requises, on peut assembler plusieurs pièces, que l'on peut appeler «sous-composants», qui ont un degré de planéité et de platitude suffisant pour la mise en œuvre de techniques d'usinage d'une surface plane. On peut fabriquer des sous-composants juste assez plans et juste assez plats pour pouvoir être micro-fabriqués. Puis on les superpose et assemble par fusion ou collage après un éventuel emboîtement ou enclenchement, ce qui permet de reconstituer le micro-système voulu (US Patent N° 5 932 315: Microfluidic structure assembly with mating microfeatures _ US Patent N° 5 611 214. Microcomponent sheet architecture US Patent 5252294. Micromechanical structure).It is also possible to make components that are less and less planar and flat while respecting a more and more finely a manufacturing profile, by virtue of substractive techniques of the chemical etching type, physical ablation, and additive techniques of the deposit type such as electrodeposition. (electroplating), electroless plating, by chemical vapor (CND and PENCD) and finally micro-molding and micro-stamping techniques. To overcome the manufacturing limits, in particular the limit depth obtained by machining, and the limits of deposition or molding techniques for sculpting devices where 3D shapes with a high height / base surface ratio are required, several parts can be assembled, which can be called “sub-components”, which have a sufficient degree of flatness and flatness for the implementation of techniques for machining a flat surface. You can make subcomponents just flat enough and just flat enough to be micro-manufactured. Then they are superimposed and assembled by fusion or gluing after a possible interlocking or interlocking, which makes it possible to reconstitute the desired micro-system (US Patent N ° 5,932,315: Microfluidic structure assembly with mating microfeatures _ US Patent N ° 5,611,214 Microcomponent sheet architecture US Patent 5252294. Micromechanical structure).
Pour que cette solution soit applicable, il faut entre autres choses que le micro-système voulu puisse tenir dans volume plus ou moins aplati représenté par la superposition de sous-composants eux-mêmes nettement aplatis.For this solution to be applicable, it is necessary, among other things, that the desired micro-system can hold in a more or less flattened volume represented by the superposition of subcomponents themselves clearly flattened.
Il se peut que la fabrication de certains composants ne puisse pas faire appel à des techniques de micro-usinage des surfaces planes. Ce peut être le cas parce que les formes sont trop sophistiquées pour être techniquement réalisables ou pour être fabriquées par ces techniques à un coût raisonnable. Ce peut aussi être le cas parce que la fonction demandée à ces composants s'accommode mal de la miniaturisation elle- même ou des techniques de miniaturisation. La conséquence d'une telle situation est que ces composants vont rester à une macro-échelle, et que le packaging du microsystème devra être conçu pour assembler ou connecter des macro-pièces avec des micro-pièces. ( van der Schoot BH, Interfacing micro and macro mechanical worlds. J. Micromech. Microeng.1995, 5, 72-73 ).The manufacturing of certain components may not require micromachining techniques for flat surfaces. This may be the case because the shapes are too sophisticated to be technically feasible or to be manufactured by these techniques at a reasonable cost. This may also be the case because the function demanded of these components is ill-suited to the miniaturization itself or to the miniaturization techniques. The consequence of such a situation is that these components will remain on a macro-scale, and that the packaging of the microsystem will have to be designed to assemble or connect macro-parts with micro-parts. (van der Schoot BH, Interfacing micro and macro mechanical worlds. J. Micromech. Microeng. 1995, 5, 72-73).
On peut utiliser des techniques de gravure chimique humide de photolithographie, de gravure sèche avec divers rayonnements photoniques ou particulaires, de micro-façonnage avec micro-outillages ou lasers, de découpage, d'ablation, d'assemblage par fusion ou d'assemblage anodique, de collage, de soudure, de moulage, d'estampage à chaud (hot-embossing en anglais), de poinçonnage, de forage, d'électrodéposition, de dépôt de vapeur chimique, de fabrication par progression par feuilles successives (lamination en anglais).We can use wet chemical etching techniques of photolithography, dry etching with various photonic or particulate radiation, micro-shaping with micro-tools or lasers, cutting, ablation, fusion assembly or anodic assembly , gluing, welding, molding, hot stamping (hot-embossing in English), punching, drilling, electroplating, chemical vapor deposition, production by progression by successive sheets (lamination in English ).
La gravure humide est appliquée de façon connue au silicium et à ses dérivés dans l'industrie de la micro-électronique. Elle peut être isotrope. Elle peut aussi être anisotrope lorsqu'on cherche à profiter de l'orientation des cristaux et des propriétés des substances gravantes pour maîtriser sa direction. (S ato K., Shikida M, Yamashiro M, Tsunekawa M, Ito S. Characterization of anisotropic etching properties of single crystal silicon: surface roughening as a fonction of crystallographic orientation, the 1 lth IEEE International Workshop on MEMS, Heidelberg, Germany, 1998, 201-206).Wet etching is applied in a known manner to silicon and its derivatives in the microelectronics industry. It can be isotropic. It can also be anisotropic when one seeks to take advantage of the orientation of the crystals and the properties of the gravants to control its direction. (S ato K., Shikida M, Yamashiro M, Tsunekawa M, Ito S. Characterization of anisotropic etching properties of single crystal silicon: surface roughening as a fonction of crystallographic orientation, the 1 lth IEEE International Workshop on MEMS, Heidelberg, Germany, 1998, 201-206).
Les techniques de gravure humide tant isotropes qu'anisotropes possèdent de nombreuses variantes. Les connaissances en physique des matériaux, chimie orbitale, physique des rayonnements, dopage des matériaux, permettent de tirer profit de la structure atomique de différents matériaux utilisés, aident à concevoir des méthodes de contrôle de la direction, de la profondeur et de l'arrêt des gravures sur différentes couches. Les techniques citées possèdent de nombreuses variantes. Les connaissances en traitement de surface permettent d'améliorer les qualités demandées aux matériaux en cours de fabrication ou les qualités demandées au produit fini.The wet etching techniques, both isotropic and anisotropic, have many variants. Knowledge in materials physics, orbital chemistry, radiation physics, doping of materials, allow to take advantage of the atomic structure of different materials used, help to design methods for controlling the direction, depth and stopping of engravings on different layers. The techniques cited have many variations. Knowledge of surface treatment makes it possible to improve the qualities required of materials during manufacture or the qualities required of the finished product.
Les connaissances en thermophysique et thermochimie différentielle entre deux matériaux permettent d'envisager des nouvelles techniques de fusion, de moulage, d'estampillage, de poinçonnage, en particulier des plastiques.The knowledge in thermophysics and differential thermochemistry between two materials makes it possible to envisage new techniques of fusion, molding, stamping, punching, in particular plastics.
On peut utiliser une technique de micro-fabrication des polymères par stéréolithographie, en particulier pour le prototypage rapide en 3D.A technique of micro-manufacturing of polymers by stereolithography can be used, in particular for rapid 3D prototyping.
Selon que l'on grave un matériau dans sa masse ou que l'on grave seulement des couches superficielles, on parle respectivement de "bulk micromachining" et de "surface micromachining".Depending on whether one engraves a material in its mass or whether one engraves only surface layers, one speaks respectively of "bulk micromachining" and "surface micromachining".
Toutes ces techniques de micro-fabrication sont applicables non seulement à la fabrication des produits finis, mais aussi à celles des outils utilisés pour effectuer ces micro-fabrications, ainsi qu'aux micro-moules et aux micro-matrices d'estampage à chaud utilisés pour micro-répliquer en masse un micro-objet. Parmi les autres critères qui vont aider à sélectionner un mode et un matériau de fabrication figurent les qualités intrinsèques des matériaux composant l'objet fini, et les perspectives de maîtrise des coûts de fabrication.All these micro-manufacturing techniques are applicable not only to the manufacture of the finished products, but also to those of the tools used to carry out these micro-manufacturing, as well as to the micro-molds and to the hot stamping micro-matrices used. to micro-replicate en masse a micro-object. Among the other criteria which will help to select a manufacturing method and material are the intrinsic qualities of the materials making up the finished object, and the prospects for controlling manufacturing costs.
Certaines techniques supposent un mode de fabrication moins adapté à la fabrication en grande quantité : gravure sèche par rayonnements photoniques ou particulaires (Bean. Anisotropic etching of silicon. 1978. vol ED-25(10), pp 1185-1193. IEEE Transactions of Electron devices.), ablation lasers, gravure avec micro-pointe.Some techniques assume a manufacturing method that is less suitable for mass production: dry etching by photonic or particulate radiation (Bean. Anisotropic etching of silicon. 1978. vol ED-25 (10), pp 1185-1193. IEEE Transactions of Electron devices.), laser ablation, etching with micro-tip.
Mais on peut utiliser ces techniques comme première étape dans un procédé de fabrication en grande quantité d'objets en plastique ou en céramique ou en métal selon des procédés appelés "par réplication" (Niggemann M., Ehrfeld W., Weber L.. Fabrication of miniaturized biotechnical devices. SPLE Conférence on Micromachining and Microfabrication Process Technology IN, Santa Clara, California, Sept 1998, vol 3511, pp 204 - 213 _ Ruprecht R, Bâcher W, Hausselt JH, Piotter N. Injection Molding of LIGA and LIGA-similar microstructures using filled and unfilled thermoplastics. SPLE, vol 2639, ppl46-158 _ Fleming JG, Barron CC, Νovel silicon fabrication process for high aspect ratio micromachined parts, SPLE vol 2639, 185-190 _ Keller CG, Howe RT. Νickel-filled HEXSLL thermally actuated tweezers, 8th International Conférence on Solid-State Sensors and Actuators, Stockholm, Sweden, 1995, June 25-29, pp 376-379. _ Selvakumar A, Najafi K, High density vertical comb array microactuators fabricated using a novel bulk/polysilicon trench refill technology, Solid State Sensor and ActuatorHowever, these techniques can be used as the first step in a process for the mass production of plastic or ceramic or metal objects according to processes called "by replication" (Niggemann M., Ehrfeld W., Weber L .. Manufacturing of miniaturized biotechnical devices. SPLE Conference on Micromachining and Microfabrication Process Technology IN, Santa Clara, California, Sept 1998, vol 3511, pp 204 - 213 _ Ruprecht R, Bâcher W, Hausselt JH, Piotter N. Injection Molding of LIGA and LIGA- similar microstructures using filled and unfilled thermoplastics. SPLE, vol 2639, ppl46-158 _ Fleming JG, Barron CC, Νovel silicon fabrication process for high aspect ratio micromachined parts, SPLE vol 2639, 185-190 _ Keller CG, Howe RT. Νickel- filled HEXSLL thermally actuated tweezers, 8th International Conférence on Solid-State Sensors and Actuators, Stockholm, Sweden, 1995, June 25-29, pp 376-379. _ Selvakumar A, Najafi K, High density vertical comb array microactuators fabricated using a novel bulk / polysilicon trench refill technology, Solid State Sensor and Actuator
Workshop, Hilton head , 1994, SC June 13-16, pp 138-141 _ Becker H., Dietz W..Workshop, Hilton head, 1994, SC June 13-16, pp 138-141 _ Becker H., Dietz W ..
Microfluidic devices for μ-TAS applications fabricated by polymer hot embossing. Proceedings of SPLE. Microfluidic Devices and Systems. 21-22 sept 1998, Santa Clara, ppl77-182 _ Grzybowski BA, Haag R, Bowden N, Whitesides GM. Génération of micrometer-sized patterns for microanalytical applications using a laser direct-write method and microcontact printing. Anal. Chem, 1998, 70, 4645-4652 _ Martynova L,Microfluidic devices for μ-TAS applications fabricated by polymer hot embossing. Proceedings of SPLE. Microfluidic Devices and Systems. 21-22 Sept 1998, Santa Clara, ppl77-182 _ Grzybowski BA, Haag R, Bowden N, Whitesides GM. Generation of micrometer-sized patterns for microanalytical applications using a laser direct-write method and microcontact printing. Anal. Chem, 1998, 70, 4645-4652 _ Martynova L,
Locascio E, Gaitan G, Kramer W, Christensen RG, MacCrehan WA.. Fabrication of plastic microfluid channels by imprinting methods. Anal. Chem. 1997, 69, 4763-4789).Locascio E, Gaitan G, Kramer W, Christensen RG, MacCrehan WA .. Fabrication of plastic microfluid channels by imprinting methods. Anal. Chem. 1997, 69, 4763-4789).
On peut utiliser ces techniques à vocation unitaire pour micro-fabriquer des masters de réplication (par exemple des micro-moules pour moulage par injection ou pour moulage réactif, ou des micro-matrices d'estampage à chaud), à condition de réunir deux qualités : un ratio d'aspect élevé et une surface compatible avec les exigences du procédé de réplication. En effet, certaines étapes dans la réplication sont cruciales, en particulier la séparation de la matrice de réplication de l'objet nouvellement répliqué.These unitary techniques can be used to micro-manufacture replication masters (for example micro-molds for injection molding or for reactive molding, or hot stamping micro-dies), provided that two qualities are combined. : a high aspect ratio and a surface compatible with the requirements of the replication process. Indeed, certain steps in replication are crucial, in particular the separation of the replication matrix from the newly replicated object.
De préférence, on prend en compte la complexité du procédé choisi pour fabriquer une matrice de réplication. Par exemple, on peut fabriquer avec une très grande précision un micro-moule de moulage par injection ou une micro-matrice d'estampage à chaud avec la technique LIGA, où un rayonnement synchrotron issu d'une machinerie très onéreuse, très rare et très lourde, est utilisé dans les premières étapes. Mais de nouvelles techniques de gravure sèche et surtout de gravure humide avec des performances accrues peuvent se révéler plus souples avec des ratios d'aspect qui se rapprochent de plus en plus de la technique LIGA. Ainsi le gravure humide anisotrope a beaucoup progressé (Hôlke A., Henderson HT. Ultra-deep anisotropic etching of (110) silicon; J. Micromech. Microeng. 1999, 9, 51-57). D'autres résultats montrent aussi un progrès dans les performances de la gravure humide isotrope (Wet chemical isotropic etching procédures of silicon - a possibility for the production of deep structured microcomponents. Schwesinger N, Albrecht A.. SPLE vol 3223, p 72- 79).Preferably, the complexity of the method chosen for manufacturing a replication matrix is taken into account. For example, we can manufacture with very high precision an injection molding micro-mold or a hot stamping micro-matrix with the LIGA technique, where synchrotron radiation from very expensive machinery, very rare and very heavy, is used in the early stages. But new techniques of dry etching and especially of wet etching with increased performances can prove to be more flexible with aspect ratios which approach more and more the LIGA technique. Thus anisotropic wet etching has progressed a lot (Hôlke A., Henderson HT. Ultra-deep anisotropic etching of (110) silicon; J. Micromech. Microeng. 1999, 9, 51-57). Other results also show progress in the performance of wet chemical isotropic etching procedures of silicon - a possibility for the production of deep structured microcomponents. Schwesinger N, Albrecht A .. SPLE vol 3223, p 72- 79 ).
Certaines techniques à vocation unitaire, peuvent être adaptées à la fabrication en masse lorsque les instruments de fabrication eux-mêmes qui servent à les mettre en œuvre sont miniaturisés et peuvent être utilisés de manière massivement parallèles. C'est une perspective proche pour l'ablation laser (grâce à la fabrication de micro-lasers) et la gravure par micropointe, plus lointaine pour certaines techniques de gravure sèche. La fabrication en grande quantité est possible avec certaines techniques telles que : gravure humide sur silicium et dérivés, et sur verres, photolithographie UN sur photorésists, fabrication par progression par couches successives de polymères avec utilisation de couches sacrificielles selon Webster et Mastrangelo cités ci-après en référence, moulage de poly(dimethylsiloxane) (PDMS), moulage de plastiques par injection avec micro-moule, moulage de céramiques et de métaux, estampage à chaud de polymères avec micro-matrice d'estampage.Certain unitary techniques can be adapted to mass production when the manufacturing instruments themselves used to implement them are miniaturized and can be used in a massively parallel manner. This is a close prospect for laser ablation (thanks to the manufacture of micro-lasers) and microtip etching, more distant for certain dry etching techniques. Manufacturing in large quantities is possible with certain techniques such as: wet etching on silicon and derivatives, and on glasses, UN photolithography on photoresists, production by progression by successive layers of polymers with the use of sacrificial layers according to Webster and Mastrangelo cited below in reference, molding of poly (dimethylsiloxane) (PDMS), molding of plastics by injection with micro-mold, molding of ceramics and metals, hot stamping of polymers with micro-matrix of stamping.
La gravure humide peut être appliquée à tous types de dérivés du silicium et au quartz, ainsi qu'aux différents types de verre (par exemple pyrex, verres boro- phospho-silicatés, etc).Wet etching can be applied to all types of silicon and quartz derivatives, as well as to different types of glass (for example pyrex, borophospho-silicate glasses, etc.).
En matière de micro-fluidique, un critère important est la compatibilité avec l'utilisation de la micro-électrophorèse, de la micro-électrophorèse 2D et de la micro- électro-chromatographie pour séparer les molécules. Est importante aussi et surtout la compatibilité avec l'électro-osmose pour mouvoir des fluides, cette technique ayant l'avantage d'éviter des composants tels que micro-vannes et micro-pompes. Comme la micro-électrophorèse et la micro-électrophorèse 2D, l'électro-osmose ainsi que la micro-électro-chromatographie alliée à l'électro-osmose nécessitent l'application de différences de potentiel importantes. En conséquence, elles sont peu compatibles avec l'utilisation du silicium. Par contre, elles sont compatibles avec les verres et les plastiques. (ManzIn micro-fluidics, an important criterion is compatibility with the use of micro-electrophoresis, 2D micro-electrophoresis and micro-electro-chromatography to separate the molecules. Compatibility with electro-osmosis to move fluids is also and above all important, this technique having the advantage of avoiding components such as micro-valves and micro-pumps. Like micro-electrophoresis and 2D micro-electrophoresis, electro-osmosis as well as micro-electro-chromatography combined with electro-osmosis require the application of significant potential differences. Consequently, they are not very compatible with the use of silicon. However, they are compatible with glasses and plastics. (Manz
A., Effenhauser CS, Burggraf Ν, Harrison DJ, Seiler K, Fluri K. Electroosmotic pumping and electrophoretic séparations for miniaturized chemical analysis Systems. J. Micromech. Microeng., 1994, 4, 257-265. - Mac Cormick RM, Nelson JR, Alonso- Amigo MG, Benvegnu DJ, Hooper HH. Microchannel electrophoretic séparations of DNA in injection-molded plastic substrates. Anal. Chem., 1997, 69, 2626-2630 _ Jacobson SJ, Kutter JP, Culbertson CT, Ramsey JM. .Rapid electrophoretic and chromatographie analysis on microchips, μ-TAS 1998, Banff, Canada, 315-318._ Microfabricated liquid chromatography columns based on collocated monolith support structures, μ-TAS 1998, Banff, Canada, 451-455. _ Paulus A., Williams SJ, Sassi AP, Kao PH, Tan H, Hooper HH . Integrated capillary electrophoresis using glass and plastic chips for multiplexed DNA analysis, pp 94-103. SPIE Proceedings Nol 3515 #3515-08. _ PM Martin, DW Matson, Bennett WD, Hammerstrom DJ. Fabrication of plastic microfluidic components. Polymer-based microfluidic analytical devices. SPLE Proceedings Vol 3515 # 3515-19). On peut envisager d'utiliser d'autres forces que la force électro-osmotique pour mouvoir les liquides où l'utilisation de micro-vannes et micropompes peut être minimisée, comme la force centrifuge (Madou MJ, Kellogg GJ: The LabCD: a centrifuge-based microfluidic platform for diagnostics. SPLE vol 3259, pp 80-93).A., Effenhauser CS, Burggraf Ν, Harrison DJ, Seiler K, Fluri K. Electroosmotic pumping and electrophoretic separations for miniaturized chemical analysis Systems. J. Micromech. Microeng., 1994, 4, 257-265. - Mac Cormick RM, Nelson JR, Alonso- Amigo MG, Benvegnu DJ, Hooper HH. Microchannel electrophoretic separations of DNA in injection-molded plastic substrates. Anal. Chem., 1997, 69, 2626-2630 _ Jacobson SJ, Kutter JP, Culbertson CT, Ramsey JM. .Rapid electrophoretic and chromatographie analysis on microchips, μ-TAS 1998, Banff, Canada, 315-318._ Microfabricated liquid chromatography columns based on collocated monolith support structures, μ-TAS 1998, Banff, Canada, 451-455. _ Paulus A., Williams SJ, Sassi AP, Kao PH, Tan H, Hooper HH. Integrated capillary electrophoresis using glass and plastic chips for multiplexed DNA analysis, pp 94-103. SPIE Proceedings Nol 3515 # 3515-08. _ PM Martin, DW Matson, Bennett WD, Hammerstrom DJ. Fabrication of plastic microfluidic components. Polymer-based microfluidic analytical devices. SPLE Proceedings Vol 3515 # 3515-19). We can consider using other forces than electro-osmotic force to move liquids where the use of micro-valves and micropumps can be minimized, like centrifugal force (Madou MJ, Kellogg GJ: The LabCD: a centrifuge-based microfluidic platform for diagnostics. SPLE vol 3259, pp 80-93).
D'autres modes de propulsion de liquides peuvent être envisagés comme la force thermo-capillaire ( Burns MA, Mastrangelo CH, Sammarco T, Man FP, Webster JR, Johnson BN, Foerster B, Jones D, Fields Y, Kaiser AR, Burke DT. Microfabricated structures for integrated DNA analysis. P.N.A.S. 1996, vol. 93, pp5556-5561), ou les forces couplées à des alternances surfaces ou raies hydrophobes-surfaces ou raies hydrophiles (Jones DK, Mastrangelo CH, Burns MA, Burke DT. Sélective hydrophobic and hydrophhilic texturing of surfaces using photolithographic photodeposition of polymers. SPLE vol 3515, 136- 143 _ Eastman Kodak.. Device for fluid supply of a micro-metering device; US Patent N° 5805189 _ Beckton Dickinson. DNA microwell device and method.US Patent N° 5795748).Other modes of liquid propulsion can be considered such as thermo-capillary force (Burns MA, Mastrangelo CH, Sammarco T, Man FP, Webster JR, Johnson BN, Foerster B, Jones D, Fields Y, Kaiser AR, Burke DT . Microfabricated structures for integrated DNA analysis. PNAS 1996, vol. 93, pp5556-5561), or the forces coupled to alternating surfaces or hydrophobic-surface lines or hydrophilic lines (Jones DK, Mastrangelo CH, Burns MA, Burke DT. Selective hydrophobic and hydrophhilic texturing of surfaces using photolithographic photodeposition of polymers. SPLE vol 3515, 136- 143 _ Eastman Kodak .. Device for fluid supply of a micro-metering device; US Patent N ° 5805189 _ Beckton Dickinson. DNA microwell device and method. US Patent No. 5795748).
On pourra également envisager de faire acquérir au silicium une compatibilité avec des différences de potentiel importantes. (Characterization of silicon- based insulated channels for capillary electrophoresis, Nan den Berg et al., μ-TAS 98, Canada, pp-327-330).We could also consider making silicon acquire compatibility with significant potential differences. (Characterization of silicon-based insulated channels for capillary electrophoresis, Nan den Berg et al., Μ-TAS 98, Canada, pp-327-330).
La transparence, qualité recherchée en analyse biologique, est une qualité partagée entre les verres (Kricka L, Wilding P, et al.. Micromachined Glass-Glass Microchips for In Nitro Fertilization, Clinical Chemistry, 1995, 41, 9, 1358-1359) et certains plastiques.Transparency, a quality sought after in biological analysis, is a quality shared between glasses (Kricka L, Wilding P, et al. Micromachined Glass-Glass Microchips for In Nitro Fertilization, Clinical Chemistry, 1995, 41, 9, 1358-1359) and some plastics.
Certains verres ayant les bons compromis de conditions de dopage et d'expansion thermique, ont la qualité de pouvoir facilement être assemblés au silicium (Albaugh KB, Rasmussen DH, "Mechanisms of anodic bonding of silicon to pyrex glass. Proc LEEE Solid State Sensors and Actuators Workshop. 1988. 109-110). La gravure humide sur verre, par nature isotrope, est parfaitement maîtriséeCertain glasses having the good compromises of conditions of doping and thermal expansion, have the quality of being able to be easily assembled with silicon (Albaugh KB, Rasmussen DH, "Mechanisms of anodic bonding of silicon to pyrex glass. Proc LEEE Solid State Sensors and Actuators Workshop, 1988. 109-110) Wet etching on glass, isotropic in nature, is perfectly mastered
(A new fabrication method for borosilicate glass capillary tubes with latéral inlets and outlets. Grétillat MA, Paoletti F, Thiébaud P, Roth S, Koudelka-Hep M, de Rooij ΝF. Sensors and Actuators A 60, 1997, 219-222. _ Corman T, Enoksson P, Stemme G. Deep wet etching of borosilicate glass using an anodically bonded silicon substrate as mask J. Micromech. Microeng., 1998, 8, 84-87.)(A new fabrication method for borosilicate glass capillary tubes with lateral inlets and outlets. Grétillat MA, Paoletti F, Thiébaud P, Roth S, Koudelka-Hep M, de Rooij ΝF. Sensors and Actuators A 60, 1997, 219-222. _ Corman T, Enoksson P, Stemme G. Deep wet etching of borosilicate glass using an anodically bonded silicon substrate as mask J. Micromech. Microeng., 1998, 8, 84-87.)
Comparés aux plastiques, les verres offrent entre autres pour l'analyse biochimique la compatibilité avec la détection par fluorescence et un bon coefficient d'échange thermique. Ils sont gravés cependant uniquement selon un mode isotrope, ce qui par exemple limite aujourd'hui la forme des micro-canaux sur verre à une forme circulaire.Compared to plastics, the glasses offer, among other things for biochemical analysis, compatibility with fluorescence detection and a good heat exchange coefficient. They are however etched only in an isotropic mode, which for example today limits the shape of the micro-channels on glass to a circular shape.
Les plastiques, même s'ils ont une moindre compatibilité avec la détection par fluorescence et un moindre coefficient d'échange thermique que les verres, ont de nombreuses autres qualités, dont le faible prix de revient. On peut envisager l'amélioration de la détection par fluorescence avec les plastiques et réhmination des bruits de fond (en modulant la vitesse de migration des analytes et en utilisant une source lumineuse LED), comme cela a été rapporté (Wang Shau-Chun et Michael D. Morris de l'Université du Michigan à la 10 ème "Frederick Conférence on Capillary Electrophoresis " en Octobre 1999).Plastics, even if they have less compatibility with fluorescence detection and a lower heat exchange coefficient than glasses, have many other qualities, including the low cost price. We can envisage the improvement of detection by fluorescence with plastics and rehmination of background noise (by modulating the migration speed of the analytes and using an LED light source), as has been reported (Wang Shau-Chun and Michael D. Morris of the University of Michigan at the 10th "Frederick Conference on Capillary Electrophoresis" in October 1999).
Le très faible coût de fabrication des objets micro-fabriqués en plastique vient du faible prix de la matière première, de la simplicité des procédés de production qui peuvent être envisagés, et de l'aptitude à la réplication par moulage ou par estampage à chaud, voire même pour les plastiques photoresists à la photolithographie. Pour la circuiterie électrique sur supports plastiques, on peut déposer du métal une fois le produit fini. On peut également marquer le support à l'aide d'une encre conductrice.The very low cost of manufacturing micro-manufactured plastic objects comes from the low price of the raw material, the simplicity of the production processes which can be envisaged, and the ability to replicate by molding or by hot stamping, even for plastics photoresists to photolithography. For electrical circuitry on plastic supports, metal can be deposited once the product is finished. The support can also be marked with a conductive ink.
Parmi les plastiques, on peut faire la classification suivante : - les photoresists, usinables entre autres par photolithographie, dont par exemple le PMMA pour la lithographie aux rayons X, SU-8 ( photorésist négatif) et Novolac de Hoescht et AZ 9260 (photoresists positif) pour la photolithographie UN (Lorenz H, Despont M, Fahrni Ν, LaBianca Ν, Renaud P, SU-8: a low-cost négative resist for MEMS, J. Micromech. Microeng, 1997, 7, 121-124. _ .Loechtel B, Maciossek A, Surface micro components fabricated by UN depth lithography and electroplating, SPIE vol 2639, 174- 184 _ Conédéra N, Le Goff B, Fabre Ν. Potentialities of a new positive photorésist for the realization of thick moulds, J. Micromech. Microeng, 1999, 9, 173-175. _ Guérin LJ, Bossel M, Demierre M, Calmes S, Renaud P. Simple and low cost fabrication of embedded microchannels by using a new thick-film photoplastic. Proceedings of Transducers, Chicago, USA, 1997, ppl419-1422.)Among plastics, the following classification can be made: - photoresists, which can be machined among other things by photolithography, including for example PMMA for X-ray lithography, SU-8 (negative photoresist) and Novolac de Hoescht and AZ 9260 (positive photoresists ) for UN photolithography (Lorenz H, Despont M, Fahrni Ν, LaBianca Ν, Renaud P, SU-8: a low-cost negative resist for MEMS, J. Micromech. Microeng, 1997, 7, 121-124. _. Loechtel B, Maciossek A, Surface micro components fabricated by UN depth lithography and electroplating, SPIE vol 2639, 174- 184 _ Conédéra N, Le Goff B, Fabre Pot. Potentialities of a new positive photoresist for the realization of thick molds, J. Micromech, Microeng, 1999, 9, 173-175. _ Guérin LJ, Bossel M, Demierre M, Calmes S, Renaud P. Simple and low cost fabrication of embedded microchannels by using a new thick-film photoplastic. Proceedings of Transducers, Chicago , USA, 1997, ppl419-1422.)
- les élasto mères siliconés, dont le poly(dimethylsiloxane) (PDMS), utilisables entre autres par moulage simple, (Mac Donald JC, Duffy DC, Anderson JR, Chiu DT, Hongkai Wu, Schueller O, Whitesides GM, Fabrication of microfluidic Systems in poly(di ethylsiloxane), Electrophoresis 2000, 21, 27-40. _ Ocvirk G, Munroe M; Tang T, Oleschuk R, Westra K, Harrison DJ, Electrokinetic control of fluid flow in native poly(dimethysiloxane) capillary electrophoretic devices, Electrophoresis 2000, 21, 107-115.)- silicone elastomers, including poly (dimethylsiloxane) (PDMS), usable among others by simple molding, (Mac Donald JC, Duffy DC, Anderson JR, Chiu DT, Hongkai Wu, Schueller O, Whitesides GM, Fabrication of microfluidic Systems in poly (di ethylsiloxane), Electrophoresis 2000, 21, 27-40. _ Ocvirk G, Munroe M; Tang T, Oleschuk R, Westra K, Harrison DJ, Electrokinetic control of fluid flow in native poly (dimethysiloxane) capillary electrophoretic devices, Electrophoresis 2000, 21, 107-115.)
- un ensemble de plus en plus vaste usinable entre autres par moulage par injection et par emboutissage à chaud. Parmi ces derniers, on peut citer des polyamides- an increasingly large assembly which can be machined, inter alia, by injection molding and hot stamping. Among these, mention may be made of polyamides
(PA), des polycarbonates (PC), des polyoxyméthylènes (POM), le cyclopentadienenorbomen copolymer (COC), des polyméthylmethacrylates (PMMA), le polyéthylène basse densité (PE-ld), le polyéthylène haute densité (PE-hd), le polypropylène (PP), des polystyrènes (PS), le cycloolefin copolymère (COC), le polyetheretherketone (PEEK). (Niggemann M., Ehrfeld W., Weber L.; Fabrication of miniaturized biotechnical devices, SPLE , vol 3511, pp 204 - 213 _ Becker H, Gartner C, Polymer microfabrication methods for microfluidic analytical applications, Electrophoresis 2000, 21, 12-26).(PA), polycarbonates (PC), polyoxymethylenes (POM), cyclopentadienenorbomen copolymer (COC), polymethylmethacrylates (PMMA), low density polyethylene (PE-ld), high density polyethylene (PE-hd), polypropylene (PP), polystyrenes (PS), cycloolefin copolymer (COC), polyetheretherketone (PEEK). (Niggemann M., Ehrfeld W., Weber L .; Fabrication of miniaturized biotechnical devices, SPLE, vol 3511, pp 204 - 213 _ Becker H, Gartner C, Polymer microfabrication methods for microfluidic analytical applications, Electrophoresis 2000, 21, 12- 26).
D'autres plastiques encore peuvent être micro-fabriqués: le polybutylèneterphtalate (PBT), le polyphenylène ether (PPE), le polysulfone (PSU), le liquid crystal polymer (LCD), le polyetherimide (PEI).Le polyactide biodégradable peut aussi être micro-fabriqué.Still other plastics can be micro-manufactured: polybutyleneterphthalate (PBT), polyphenylene ether (PPE), polysulfone (PSU), liquid crystal polymer (LCD), polyetherimide (PEI). Biodegradable polyactide can also be microfabricated.
Le PMMA et le PC sont couramment employés dans le moulage par injection et l'estampage à chaud. Le COC est couramment cité dans l'estampage à chaud.PMMA and PC are widely used in injection molding and hot stamping. COC is commonly cited in hot stamping.
Les procédés de fabrication de masse des plastiques sont très variés. On peut envisager comme procédés :The mass production processes for plastics are very varied. We can consider as processes:
- l'impression par matrices filiformes (wire imprinting) ( Locascio LF, Gitan M, Hong J, Eldefrawi M, Plastic microfluidic devices for clinical measurements, μ-TAS 1998, 367- 370 _ Chen YH, Chen SH, Analysis of DNA fragments by microchip electrophoresis fabricated on poly(methyl methacrylate) substrates using a wire- imprinting method, Electrophoresis 2000, 21, 165-170)- printing using wire imprinting (Locascio LF, Gitan M, Hong J, Eldefrawi M, Plastic microfluidic devices for clinical measurements, μ-TAS 1998, 367- 370 _ Chen YH, Chen SH, Analysis of DNA fragments by microchip electrophoresis fabricated on poly (methyl methacrylate) substrates using a wire- imprinting method, Electrophoresis 2000, 21, 165-170)
- l'estampage à chaud (Hot embossing) (Becker H., Dietz W, Dannberg P. Microfluidic manifolds by polymer hot embossing for μ-TAS applications. μ-TAS 1998, Banff, Canada, 253-256. _ Kempen LU, Kunz RE, Gale MT. Micromolded structures for integrated optical sensors. SPLE vol 2639, 278-285.). - le moulage par injection (Hagmann P, Ehrfeld W. Fabrication of microstructures of extrême structural heights by reaction injection molding, International Polymer Processing, 1989, Vol IN, Ν°3, pp 188-195. _ Weber L, Ehrfeld W, Freimuth H, Lâcher M, Lehr H, Pech B. . Micro-moulding - a powerful tool for the large scale production of précise microstructures. Proc. SPIE Symp. Micromachining and Microfabrication, 1996, vol 2879, pp 156- 167.).- hot stamping (Hot embossing) (Becker H., Dietz W, Dannberg P. Microfluidic manifolds by polymer hot embossing for μ-TAS applications. μ-TAS 1998, Banff, Canada, 253-256. _ Kempen LU, Kunz RE, Gale MT. Micromolded structures for integrated optical sensors. SPLE vol 2639, 278-285.). - injection molding (Hagmann P, Ehrfeld W. Fabrication of microstructures of extreme structural heights by reaction injection molding, International Polymer Processing, 1989, Vol IN, Ν ° 3, pp 188-195. _ Weber L, Ehrfeld W, Freimuth H, Lâcher M, Lehr H, Pech B.. Micro-molding - a powerful tool for the large scale production of precise microstructures. Proc. SPIE Symp. Micromachining and Microfabrication, 1996, vol 2879, pp 156- 167.).
- le moulage simple pour les élastomères siliconés (Kumar A, Whitesides GM. Appl. Phys. Lett, 1993, 63, 2002-2004 _ Wilbur JL, Kumar A, kim E, Whitesides GM, Adv. Mat. 1994, 7, 600-604.).- simple molding for silicone elastomers (Kumar A, Whitesides GM. Appl. Phys. Lett, 1993, 63, 2002-2004 _ Wilbur JL, Kumar A, kim E, Whitesides GM, Adv. Mat. 1994, 7, 600 -604.).
- la photohthographie des photoresists, dont par exemple la lithographie aux rayons X pour le PMMA, la photohthographie UN pour le photopolymère Epson SU-8.- photohthography of photoresists, including for example X-ray lithography for PMMA, UN photohthography for the Epson SU-8 photopolymer.
Dans cette dernière, trois procédés sont couramment utilisés (Renaud P., Nan Lintel H, Heusckel M, Guérin L.. Photo-polymer microchannel technologies and applications. μ-TAS 1998, Banff. Canada, ppl7-22.). Ils commencent toutes trois par le dépôt d'une première couche de SU-8 qu'on expose aux UN. Pour la fabrication d'un microcanal, la première couche de photorésist fait le fond dudit micro-canal à section rectangulaire. La deuxième couche de photorésist fait les parois verticales dudit micro- canal. La troisième couche de photorésist termine le capillaire en constituant la partie couvercle.In the latter, three methods are commonly used (Renaud P., Nan Lintel H, Heusckel M, Guérin L .. Photo-polymer microchannel technologies and applications. μ-TAS 1998, Banff. Canada, ppl7-22.). They all start with the deposition of a first layer of SU-8 which is exposed to the UN. For the manufacture of a microchannel, the first photoresist layer forms the bottom of said micro-channel with rectangular section. The second photoresist layer forms the vertical walls of said micro-channel. The third layer of photoresist terminates the capillary by constituting the cover part.
- le "fill process" . On procède par remplissage avec une couche sacrificielle, comme par exemple l'Araldite GT6063 de Ciba-Geigy entre la deuxième et la troisième couche de photorésist. En fin de process, la couche sacrificielle est dissoute. - le "mask process" . On interpose une couche de métal sur la deuxième couche de photorésist qu'on ne développe pas. Cette deuxième couche de métal masque le microcanal. Une troisième couche de photorésist est déposée puis illuminée. Puis le photorésist est développé à l'intérieur et à l'extérieur dudit micro-canal.- the "fill process". This is done by filling with a sacrificial layer, such as, for example, the Ciba-Geigy Araldite GT6063 between the second and the third photoresist layer. At the end of the process, the sacrificial layer is dissolved. - the "mask process". A layer of metal is interposed on the second layer of photoresist that is not developed. This second layer of metal masks the microchannel. A third layer of photoresist is deposited and then illuminated. Then the photoresist is developed inside and outside of said micro-channel.
- le "lamination process", un procédé sans dissolution, où l'on déroule une couche de film sec de SU-8 sur la construction faite à partir de la première couche de photorésist pour la sceller.- the "lamination process", a process without dissolution, where a layer of dry film of SU-8 is unwound on the construction made from the first layer of photoresist to seal it.
- la fabrication par progression par couches successives de polymères, avec utilisation de couches sacrificielles, dont par exemple le process utilisé par Webster JR, Burns MA, Mastrangelo CH, Man PF, Jones DK, Burke DT., (Webster JR, Burns MA, Burke DT., Mastrangelo CH , An inexpensive plastic technology for microfabricated capillary electrophoresis chips, μ-TAS 1998, 249-252), une technique qui part de parylène déposé sur du polycarbonate ou du silicium avec utilisation ultérieure de photorésist sacrificiel. L'avantage de cette technique réside dans le scellement des microcanaux naturellement inclus dans la méthode. La micro-fabrication par laser des plastiques est aussi possible, tout en étant réservée à une production unitaire. Ce peut être par exemple l'ablation directe dans la masse ou la découpe d'un joint que l'on glissera en sandwich entre deux couvercles.- the production by progression by successive layers of polymers, with the use of sacrificial layers, including for example the process used by Webster JR, Burns MA, Mastrangelo CH, Man PF, Jones DK, Burke DT., (Webster JR, Burns MA, Burke DT., Mastrangelo CH, An inexpensive plastic technology for microfabricated capillary electrophoresis chips, μ-TAS 1998, 249-252), a technique which starts from parylene deposited on polycarbonate or silicon with subsequent use of sacrificial photoresist. The advantage of this technique lies in the sealing of the microchannels naturally included in the method. Laser micro-manufacturing of plastics is also possible, while being reserved for unit production. It can be for example direct ablation in the mass or the cutting of a joint which one will slide in sandwich between two lids.
Les traitements de surface de plastiques dépendent de l'application et du matériau utilisé. Par exemple, il faut souvent rendre hydrophile une surface hydrophobe. Pour assembler et sceller d'un couvercle des micro-fabrications en plastique, plusieurs procédés existent. On peut citer entre autres :Plastic surface treatments depend on the application and the material used. For example, a hydrophobic surface must often be made hydrophilic. Several methods exist for assembling and sealing plastic micro-products with a cover. We can cite among others:
- le scellement par déroulement à chaud d'une feuille recouvrante de PET d'environ 30 microns coatée avec une couche d'un matériau, le plus souvent un polymère que l'on porte à son point de fusion pour qu'elle se mélange avec le substrat, - le scellement d'un couvercle ou l'assemblage d'une partie complémentaire par collage, ou par pression à chaud, ou par soudure au laser, ou par l'emploi d'ultrasons, ou par l'emploi de plasmas, etc. En ce qui concerne en particulier les séparations électrophorétiques par micro-électrophorèse d'échantillons biologiques, on peut effectuer ces séparations sur des supports gravés, moulés ou emboutis ( Cf. Liu Y, Foote RS, Culbertson CT, Jacobson SC, Ramsey RS, Ramsey JR. Electrophoretic séparations on microchips. J. Microcolumn Séparations, 2000, 12(7), 407-11; Alarie JP, Jacobson SC, Ramsey JM. Electrophoretic injection bias in a microchip valving scheme. Electrophoresis. 2001. Jan;22(2):312-7; Rocklin RD, Ramsey RS, Ramsey JM. A microfabricated fluidic device for performing two-dimensional liquid-phase séparations. Anal Chem. 2000 Nov l;72(21):5244-9; Liu Y, Foote RS, Jacobson SC, Ramsey RS, Ramsey JM. Electrophoretic séparation of proteins on a microchip with noncovalent, postcolumn labeling. Anal Chem. 2000 Oct 1;72(19):4608-13; Khandurina J, McKnight TE, Jacobson SC, Waters LC, Foote RS, Ramsey JM. Integrated system for rapid PCR- based DNA analysis in microfluidic devices. Anal Chem. 2000 Jul l;72(13):2995-3000; Alarie JP, Jacobson SC, Culbertson CT, Ramsey JM. Effects of the electric field distribution on microchip valving performance. Electrophoresis. 2000 Jan;21(l): 100-6.; Khandurina J, Jacobson SC, Waters LC, Foote RS, Ramsey JM. Microfabricated porous membrane structure for sample concentration and electrophoretic analysis. Anal Chem. 1999 May 1;71(9): 1815 ; Waters LC, Jacobson SC, Kroutchinina N, Khandurina J, Foote RS, Ramsey JM. Multiple sample PCR amplification and electrophoretic analysis on a microchip. Anal Chem. 1998 Dec 15;70(24):5172-6. ; Waters LC, Jacobson SC, Kroutchinina N, Khandurina J, Foote RS, Ramsey JM. Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing.Anal Chem. 1998 Jan l;70(l):158-62 ; von Brocke A, Nicholson G, Bayer E. Récent advances in capillary electrophoresis/electrospray-mass spectrometry. Electrophoresis. 2001 Apr;22(7):1251- 66; Schmid MG, Grobuschek N, Lecnik O, Gubitz G. Chiral hgand-exchange capillary electrophoresis. J Biochem Biophys Methods. 2001 Apr 24;48(2): 143-54 ; Nishi H, Kuwahara Y. Enantiomer séparation by capillary electrophoresis utilizing noncyclic mono-, oligo- and polysaccharides as chiral selectors. J Biochem Biophys Methods. 2001 Apr 24;48(2):89-102; Castellanos-Serra L, Hardy E. Détection of biomolecules in electrophoresis gels with salts of imidazole and zinc II: a décade of research. Electrophoresis. 2001 Mar;22(5): 864-73; Colyer C. Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence détection. Cell Biochem Biophys. 2000;33(3):323-37; Bonneil E, Waldron KC. On-line solid-phase preconcentration for sensitivity enhancement in capillary electrophoresis. J Capillary Electrophor. 1999 May-Aug;6(3-4):61-73; Horvath J, Dolnik N. Polymer wall coatings for capillary electrophoresis.Electrophoresis. 2001 ;22(4): 644-55 ; Kricka LJ. Microchips, microarrays, biochips and nanochips: personal laboratories for the 21st century. Clin Chim Acta. 2001 May;307(l-2):219-23. Wang J, Chatrathi MP, Tian B ; Brahmasandra SN, Ugaz NM, Burke DT, Mastroangelo CH, Bums MA. Electrophoresis in microfabricated devices using photopolymerized polyacrylamide gels and electrode- defined sample injection. Electrophoresis. 2001 Jan;22(2):300-ll; Dutta D, Leighton DT Jr. Dispersion réduction in pressure-driven flow through microetched channels. Anal Chem. 2001 Feb 1;73(3):504-13; Baldwin RP. Récent advances in electrochemical détection in capillary electrophoresis. Electrophoresis. 2000 Dec;21(18):4017-28 ; Bruin GJ. Récent developments in electrokinetically driven analysis on microfabricated devices. Electrophoresis. 2000 Dec;21(18):3931-51; Krishnan M, Namasivayam N, Lin R, Pal R, Burns MA. Microfabricated reaction and séparation Systems. Curr Opin Biotechnol. 2001 Feb;12(l):92-8; Hutt LD, Glavin DP, Bada JL, Mathies RA. Microfabricated capillary electrophoresis amino acid chirality analyzer for extraterrestrial exploration. Anal Chem. 1999 Sep 15;71(18):4000-6; Chiem ΝH, Harrison DJ. Microchip Systems for immunoassay: an integrated immunoreactor with electrophoretic séparation for sérum theophylline détermination. Clin Chem. 1998 Mar;44(3):591-8; Woolley AT, Lao K, Glazer AN, Mathies RA. Capillary electrophoresis chips with integrated electrochemical détection. Anal Chem. 1998 Feb 15;70(4):684-8; Colyer CL, Tang T, Chiem N, Harrison DJ. Clinical potential of microchip capillary electrophoresis Systems. Electrophoresis. 1997 Sep;l 8(10): 1733- 41).- the sealing by hot unwinding of a PET covering sheet of about 30 microns coated with a layer of a material, most often a polymer which is brought to its melting point so that it mixes with the substrate, - the sealing of a cover or the assembly of a complementary part by gluing, or by hot pressing, or by laser welding, or by the use of ultrasound, or by the use of plasmas , etc. As regards in particular the electrophoretic separations by micro-electrophoresis of biological samples, these separations can be carried out on engraved, molded or stamped supports (Cf. Liu Y, Foote RS, Culbertson CT, Jacobson SC, Ramsey RS, Ramsey JR. Electrophoretic separations on microchips. J. Microcolumn Separations, 2000, 12 (7), 407-11; Alarie JP, Jacobson SC, Ramsey JM. Electrophoretic injection bias in a microchip valving scheme. Electrophoresis. 2001. Jan; 22 (2 ): 312-7; Rocklin RD, Ramsey RS, Ramsey JM. A microfabricated fluidic device for performing two-dimensional liquid-phase separations. Anal Chem. 2000 Nov l; 72 (21): 5244-9; Liu Y, Foote RS , Jacobson SC, Ramsey RS, Ramsey JM. Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling. Anal Chem. 2000 Oct 1; 72 (19): 4608-13; Khandurina J, McKnight TE, Jacobson SC, Waters LC , Foote RS, Ramsey JM. Integrated system for rapid PCR- based DNA analysis in microfluidic d evices Anal Chem. 2000 Jul 1; 72 (13): 2995-3000; Alarie JP, Jacobson SC, Culbertson CT, Ramsey JM. Effects of the electric field distribution on microchip valving performance. Electrophoresis. 2000 Jan; 21 (l): 100-6 .; Khandurina J, Jacobson SC, Waters LC, Foote RS, Ramsey JM. Microfabricated porous membrane structure for sample concentration and electrophoretic analysis. Anal Chem. 1999 May 1; 71 (9): 1815; Waters LC, Jacobson SC, Kroutchinina N, Khandurina J, Foote RS, Ramsey JM. Multiple sample PCR amplification and electrophoretic analysis on a microchip. Anal Chem. 1998 Dec 15; 70 (24): 5172-6. ; Waters LC, Jacobson SC, Kroutchinina N, Khandurina J, Foote RS, Ramsey JM. Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing.Anal Chem. 1998 Jan l; 70 (l): 158-62; von Brocke A, Nicholson G, Bayer E. Recent advances in capillary electrophoresis / electrospray-mass spectrometry. Electrophoresis. 2001 Apr; 22 (7): 1251-66; Schmid MG, Grobuschek N, Lecnik O, Gubitz G. Chiral hgand-exchange capillary electrophoresis. J Biochem Biophys Methods. 2001 Apr 24; 48 (2): 143-54; Nishi H, Kuwahara Y. Enantiomer separation by capillary electrophoresis utilizing noncyclic mono-, oligo- and polysaccharides as chiral selectors. J Biochem Biophys Methods. 2001 Apr 24; 48 (2): 89-102; Castellanos-Serra L, Hardy E. Detection of biomolecules in electrophoresis gels with salts of imidazole and zinc II: a decade of research. Electrophoresis. 2001 Mar; 22 (5): 864-73; Colyer C. Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection. Cell Biochem Biophys. 2000; 33 (3): 323-37; Bonneil E, Waldron KC. On-line solid-phase preconcentration for sensitivity enhancement in capillary electrophoresis. J Capillary Electrophor. 1999 May-Aug; 6 (3-4): 61-73; Horvath J, Dolnik N. Polymer wall coatings for capillary electrophoresis.Electrophoresis. 2001; 22 (4): 644-55; Kricka LJ. Microchips, microarrays, biochips and nanochips: personal laboratories for the 21st century. Clin Chim Acta. 2001 May; 307 (1-2): 219-23. Wang J, Chatrathi MP, Tian B; Brahmasandra SN, Ugaz NM, Burke DT, Mastroangelo CH, Bums MA. Electrophoresis in microfabricated devices using photopolymerized polyacrylamide gels and electrode- defined sample injection. Electrophoresis. 2001 Jan; 22 (2): 300-ll; Dutta D, Leighton DT Jr. Dispersion reduction in pressure-driven flow through microetched channels. Anal Chem. 2001 Feb 1; 73 (3): 504-13; Baldwin RP. Recent advances in electrochemical detection in capillary electrophoresis. Electrophoresis. 2000 Dec; 21 (18): 4017-28; Bruin GJ. Recent developments in electrokinetically driven analysis on microfabricated devices. Electrophoresis. 2000 Dec; 21 (18): 3931-51; Krishnan M, Namasivayam N, Lin R, Pal R, Burns MA. Microfabricated reaction and separation Systems. Curr Opin Biotechnol. 2001 Feb; 12 (l): 92-8; Hutt LD, Glavin DP, Bada JL, Mathies RA. Microfabricated capillary electrophoresis amino acid chirality analyzer for extraterrestrial exploration. Anal Chem. 1999 Sep 15; 71 (18): 4000-6; Chiem ΝH, Harrison DJ. Microchip Systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination. Clin Chem. 1998 Mar; 44 (3): 591-8; Woolley AT, Lao K, Glazer AN, Mathies RA. Capillary electrophoresis chips with integrated electrochemical detection. Anal Chem. 1998 Feb 15; 70 (4): 684-8; Colyer CL, Tang T, Chiem N, Harrison DJ. Clinical potential of microchip capillary electrophoresis Systems. Electrophoresis. 1997 Sep; l 8 (10): 1733-41).
En résumé, pour la fabrication des supports, on peut choisir du silicium, du verre ou en céramique ou en plastique.In summary, for the manufacture of the supports, one can choose silicon, glass or ceramic or plastic.
Des lits de micro-colonnes de fractionnement 2 peuvent être gravés sur un support à base de silicium ou de verre ou de céramique. Des lits de micro-colonnes de fractionnement 2 peuvent être micro-moulés ou micro-emboutis à l'aide de matrices en silicium lorsque le support est à base de plastiques. Des lits de micro-colonnes de fractionnement 2 peuvent être revêtus d'un film fin de nature hydrophobe ou hydrophile.Beds of fractionation micro-columns 2 can be etched on a support based on silicon or glass or ceramic. Beds of fractionation micro-columns 2 can be micro-molded or micro-stamped using silicon matrices when the support is plastic-based. Fractionation micro-column beds 2 can be coated with a thin film of hydrophobic or hydrophilic nature.
Lorsque le support est à base de plastiques, le réseau micro-particulaire peut être obtenu par micro-moulage ou micro-emboutissage ou photopolymérisation ou thermopolymérisation in situ, ou peut être constitué de micro ou nano-baguettes s'insérant dans ledit ht desdites micro-colonnes.When the support is based on plastics, the micro-particulate network can be obtained by micro-molding or micro-stamping or photopolymerization or thermopolymerization in situ, or can be made up of micro or nano-rods fitting into said ht of said micro -colonnes.
Un réseau micro-particulaire des micro-colonnes de fractionnement 2 qui constitue les phases stationnaires peut par exemple être obtenu par photogravure lorsque le support est à base de silicium ou de verre ou de céramique. Un réseau micro- particulaire des micro-colonnes de fractionnement 2 qui constitue les phases stationnaires peut par exemple être obtenu par micro-moulage, micor-emboutissage, photopolymérisation ou thermopolymérisation in situ, ou peut être constitué de micro ou nano-baguettes s'insérant dans le lit desdites micro-colonnes. Un réseau micro- particulaire peut être revêtus d'un film fin de nature hydrophobe ou hydrophile. Un réseau micro-particulaire peut être soumis à des chimies de couplage connues de l'Homme de l'Art pour greffer des molécules caractérisées par leur polarité et leur amphipathicité.A micro-particulate network of fractionation micro-columns 2 which constitutes the stationary phases can for example be obtained by photoengraving when the support is based on silicon or glass or ceramic. A micro-particulate network of fractionation micro-columns 2 which constitutes the stationary phases can for example be obtained by micro-molding, micor-stamping, photopolymerization or thermopolymerization in situ, or may consist of micro or nano-rods fitting into the bed of said micro-columns. A microparticulate network can be coated with a thin film of hydrophobic or hydrophilic nature. A microparticulate network can be subjected to coupling chemistries known to those skilled in the art to graft molecules characterized by their polarity and their amphipathicity.
Des méthodes d'obtention de revêtement des phases stationnaires sont évoquées par la suite.Methods for obtaining a coating of the stationary phases are discussed below.
On peut effectuer le revêtement des phases stationnaires avec des peptides par greffage, grâce aux chimies de couplage directes ou avec bras espaceurs connues de l'Homme de l'Art, telles que via le bromure de cyanogène, ou le carbodiimide ou le carbonyldiimidazole, ou oxirane ou l'azlactone. Une méthode de plus en plus utilisé est l'immobihsation sur gels tentaculaires de peptides via une fixation par activation de gel epoxy et dérivé azlactone (Pribl M. Bestimmung der Epoxyendgruppen in modifizierten chromatographischen Sorbentien un Gelen. Anal. Chem. 1980. 303. 113-116.).The stationary phases can be coated with peptides by grafting, using direct coupling chemistries or with spacer arms known to those skilled in the art, such as via cyanogen bromide, or carbodiimide or carbonyldiimidazole, or oxirane or azlactone. An increasingly used method is the immobilization on tentacular gels of peptides via fixation by activation of epoxy gel and azlactone derivative (Pribl M. Bestimmung der Epoxyendgruppen in modifizierten chromatographischen Sorbentien un Gelen. Anal. Chem. 1980. 303. 113 -116.).
On peut effectuer le revêtement des phases stationnaires avec des peptides par synthèse peptidique en phase solide, la phase solide servant à la synthèse étant aussi ladite phase stationnaire (Kumar KS, Rajasekharan Pillai NN, Das MR. Synthèses of four peptides from the immunodominant région of hepatitis C viral pathogens using PS TTEGDA support fot the investigation of HCN infection in human blood J. Peptide Res., 2000, 56, 88-96)The stationary phases can be coated with peptides by peptide synthesis in solid phase, the solid phase serving for the synthesis being also said stationary phase (Kumar KS, Rajasekharan Pillai NN, Das MR. Syntheses of four peptides from the immunodominant region of hepatitis C viral pathogens using PS TTEGDA support fot the investigation of HCN infection in human blood J. Peptide Res., 2000, 56, 88-96)
On peut greffer de façon coonue des phases stationnaires des microcolonnes de fractionnements 2 de monocouches de lipides de membranes cellulaires tels que par exemple des phosphatidylcholines. (Maget-Dana R. The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes. Biochim Biophys Acta. 1999 Dec 15;1462(l-2): 109-40; Mozsolits H, Lee TH, Wirth HJ, Perlmutter P, Aguilar MI. The interaction of bioactive peptides with an immobilized phosphatidylchoiline monolayer. Biophys. J, 1999, 1428-1444, 77, 3.). La détection de molécules à l'aide de micro-leviers est décrite plus en détail dans la suite de la description.Stationary phases can be co-grafted into microcolumns for fractionation 2 of lipid monolayers of cell membranes such as, for example, phosphatidylcholines. (Maget-Dana R. The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes. Biochim Biophys Acta. 1999 Dec 15; 1462 (l-2): 109-40 ; Mozsolits H, Lee TH, Wirth HJ, Perlmutter P, Aguilar MI. The interaction of bioactive peptides with an immobilized phosphatidylchoiline monolayer. Biophys. J, 1999, 1428-1444, 77, 3.). The detection of molecules using micro-levers is described in more detail in the following description.
De façon générale, la détection d'interactions spécifiques est possible grâce à la mesure de la variation de propriétés mécaniques de microstructures. Dans la plupart des cas, ces microstructures se présentent sous la forme de micro-leviers. ( Betts TA, Tipple CA, Sepaniak MJ, Datskos PG. Selectivity of chemical sensors based on micro- cantilevers coated with thin polymer films. Anal. Chimica Acta, 2000, 422, 89; Fagan B, Xue B, Datkos P, Sepaniak M.. Modification of micro-cantilevers sensors with sol- gels to enhance performance and immobilize chemically sélective phases. Talanta, 2000, 53, 599; Wadu-Mesthrige K, Amro NA, Garno JC, Xu S, Liu G- Y. Fabrication of nanometer-sized proteins patterns using atomic force microscopy and sélective immobilization. Biophysical Journal. 2001, 80, 1891-1899; Viani MB, Pietrasanta LI, Thompson JB, Chand A, Gebeshuber IC, Kindt JH, Richter M, Hansma HG, Hansma PK. Probing protein-protein interactions in real time. Nat Struct Biol. 2000 Aug;7(8):644-7; Luckham PF, Smith K. Direct measurement of récognition forces between proteins and membrane receptors. Faraday Discuss. 1998;(lll):307-20; discussion 331-43; Micic M, Chen A, Leblanc RM, Moy NT. Scanning électron microscopy studies of protein-functionalized atomic force microscopy cantilever tips. Scanning. 1999 Νov-Dec;21(6):394-7; Bryant Z, Pande VS, Rokshar DS. Mechanical unfolding of a beta-hairpin using molecular dynamics. Biophys J. 2000 Feb ;78 (2): 584- 9; Willemsen OH, Snel MM, van Noort SJ, van der Werf KO, de Grooth BG, Figdor CG, Grève J. Optimization of adhésion mode atomic force microscopy résolves individual molécules in topography and adhésion. Ultramicroscopy. 1999 Oct;80(2): 133-44; Willemsen OH, Snel MM, Kuipers L, Figdor CG, Grève J, de Grooth BG.A physical approach to reduce nonspecific adhésion in molecular récognition atomic force microscopy. Biophys J. 1999 Feb;76(2):716-24; Heinz WF, Hoh JH. Relative surface charge density mapping with the atomic force microscope. Biophys J. 1999 Jan;76(l Pt l):528-38; Eckert R, Jeney S, Horber JK. Understanding intercellular interactions and cell adhésion: lessons from studies on protein-metal interactions. Cell Biol Int. 1997.21(11):707-13; Oberlheithner H, Schneider SW, Henderson RM. Structural activity of a cloned potassium channel (ROMK) monitored with the atomic force microscope : the « molecular sandwich » technique PNAS, 1997, 94(25), 14144-9; You H, Yu L. Investigation of the image contrast of tapping-mode atomic force microscopy using protein-modified cantilever tips. Biophys J. 1997 Dec;73(6):3299-308; Tokunaga M, Aoki T, Hiroshima M, Kitamura K, Yanagida T. Subpiconewton intermolecular force microscopy. Biochem Biophys Res Commun. 1997 Feb 24;231(3):566-9; Mitsui K, Hara M, Ikai A. Mechanical unfolding of alpha2- macroglobulin molécules with atomic force microscope. FEBS Lett. 1996 Apr 29;385(l-2):29-33; Florin EL, Moy NT, Gaub HE. Adhésion forces between individual ligand-receptor pairs. Science. 1994 Apr 15;264(5157):415-7; Fritz J, Baller MK, Lang HP, Rothuizen H, Nettiger P, Meyer E, Guntherodt H, Gerber C, Gimzewski JK. Translating biomolecular récognition into nanomechanics. Science. 2000 Apr 14;288(5464):316-8 ; Baller MK, Lang HP, Fritz J, Gerber C, Gimzewski JK, Drechsler U, Rothuizen H, Despont M, Nettiger P, Battiston FM, Ramseyer JP, Fornaro P, Meyer E, Guntherodt HJ.A cantilever array-based artificial nose. Ultramicroscopy. 2000 Feb;82(l-4):l-9).In general, the detection of specific interactions is possible thanks to the measurement of the variation of mechanical properties of microstructures. In most cases, these microstructures are in the form of micro-levers. (Betts TA, Tipple CA, Sepaniak MJ, Datskos PG. Selectivity of chemical sensors based on micro-cantilevers coated with thin polymer films. Anal. Chimica Acta, 2000, 422, 89; Fagan B, Xue B, Datkos P, Sepaniak M .. Modification of micro-cantilevers sensors with sol- gels to enhance performance and immobilize chemically selective phases. Talanta, 2000, 53, 599; Wadu-Mesthrige K, Amro NA, Garno JC, Xu S, Liu G- Y. Fabrication of nanometer-sized proteins patterns using atomic force microscopy and selective immobilization. Biophysical Journal. 2001, 80, 1891-1899; Viani MB, Pietrasanta LI, Thompson JB, Chand A, Gebeshuber IC, Kindt JH, Richter M, Hansma HG, Hansma PK. Probing protein-protein interactions in real time. Nat Struct Biol. 2000 Aug; 7 (8): 644-7; Luckham PF, Smith K. Direct measurement of recognition forces between proteins and membrane receptors. Faraday Discuss. 1998; (III): 307-20; discussion 331-43; Micic M, Chen A, Leblanc RM, Moy NT. Scanning electron microscopy studies of protein-functionalized atomic force microscopy cantilever tips. Scanning. 1999 Νov-Dec; 21 (6): 394-7; Bryant Z, Pande VS, Rokshar DS. Mechanical unfolding of a beta-hairpin using molecular dynamics. Biophys J. 2000 Feb; 78 (2): 584-9; Willemsen OH, Snel MM, van Noort SJ, van der Werf KO, de Grooth BG, Figdor CG, Grève J. Optimization of adhesion mode atomic force microscopy resolved individual molecules in topography and adhesion. Ultramicroscopy. 1999 Oct; 80 (2): 133-44; Willemsen OH, Snel MM, Kuipers L, Figdor CG, Grève J, de Grooth BG.A physical approach to reduce nonspecific adhesion in molecular recognition atomic force microscopy. Biophys J. 1999 Feb; 76 (2): 716-24; Heinz WF, Hoh JH. Relative surface charge density mapping with the atomic force microscope. Biophys J. 1999 Jan; 76 (l Pt l): 528-38; Eckert R, Jeney S, Horber JK. Understanding intercellular interactions and cell adhesion: lessons from studies on protein-metal interactions. Cell Biol Int. 1997.21 (11): 707-13; Oberlheithner H, Schneider SW, Henderson RM. Structural activity of a cloned potassium channel (ROMK) monitored with the atomic force microscope: the "molecular sandwich" technique PNAS, 1997, 94 (25), 14144-9; You H, Yu L. Investigation of the image contrast of tapping-mode atomic force microscopy using protein-modified cantilever tips. Biophys J. 1997 Dec; 73 (6): 3299-308; Tokunaga M, Aoki T, Hiroshima M, Kitamura K, Yanagida T. Subpiconewton intermolecular force microscopy. Biochem Biophys Res Commun. 1997 Feb 24; 231 (3): 566-9; Mitsui K, Hara M, Ikai A. Mechanical unfolding of alpha2- macroglobulin molecules with atomic force microscope. FEBS Lett. 1996 Apr 29; 385 (1-2): 29-33; Florin EL, Moy NT, Gaub HE. Membership forces between individual ligand-receptor pairs. Science. 1994 Apr 15; 264 (5157): 415-7; Fritz J, Baller MK, Lang HP, Rothuizen H, Nettiger P, Meyer E, Guntherodt H, Gerber C, Gimzewski JK. Translating biomolecular recognition into nanomechanics. Science. 2000 Apr 14; 288 (5464): 316-8; Baller MK, Lang HP, Fritz J, Gerber C, Gimzewski JK, Drechsler U, Rothuizen H, Despont M, Nettiger P, Battiston FM, Ramseyer JP, Fornaro P, Meyer E, Guntherodt HJ.A cantilever array-based artificial nose. Ultramicroscopy. 2000 Feb; 82 (l-4): l-9).
Une détection à l'aide de micro-leviers peut être effectuée en mode statique ou en mode dynamique. En mode statique, la formation d'une couche à la surface du levier lors d'une interaction spécifique engendre un effet de contrainte mécanique qui se traduit par une courbure du levier. La sensibilité est dépendante de la raideur du micro-levier. Elle est de l'ordre de 0.1 N/m en général voire inférieure.Detection using micro-levers can be performed in static mode or in dynamic mode. In static mode, the formation of a layer on the surface of the lever during a specific interaction generates a mechanical stress effect which results in a curvature of the lever. The sensitivity is dependent on the stiffness of the micro-lever. It is of the order of 0.1 N / m in general or even lower.
En mode dynamique, l'ajout d'une masse consécutive à une interaction spécifique sur un micro-levier résonnant engendre une diminution de sa fréquence de résonance. La sensibilité sera d'autant plus importante que la fréquence de résonance et le facteur de qualité seront élevés. Dans ce cas, les raideurs sont plus importantes entre 1 et 100 N/m et les facteurs de qualité sont compris entre 10 et 500 dans l'air, entre 1 et 10 dans les liquides. La sensibilité d'une telle détection est fortement augmentée si on effectue les mesures sous vide (le facteur de qualité peut atteindre des valeurs supérieures à 104).In dynamic mode, the addition of a mass following a specific interaction on a resonant micro-lever causes a reduction in its resonant frequency. The higher the resonant frequency and the higher the quality factor, the higher the sensitivity. In this case, the stiffnesses are greater between 1 and 100 N / m and the quality factors are between 10 and 500 in air, between 1 and 10 in liquids. The sensitivity of such a detection is greatly increased if the measurements are carried out under vacuum (the quality factor can reach values greater than 104).
Une telle approche suppose que l'on peut mesurer la déflexion dans le cas d'une mesure en statique ou la fréquence de résonance dans le cas d'une mesure en dynamique. On peut effectuer deux types de mesures. Une première approche est basée sur le principe de la déflexion optique laser qui est utilisé comme système de détection dans les microscopes à force atomique commerciaux. Il s'agit d'une détection externe. Cette technique est très sensible et permet d'accéder à des variations de déflexion inférieures à l'angstrom ou à des variations de fréquence de résonance de quelques hertz. Elle est utilisée dans la majeure partie des cas (cf. brevets WO 00/14539 ou US 5,445,008 ou J.Fritz et al., Science 288, 316, 2000).La seconde approche consiste à intégrer sur le micro-levier la fonction de détection. Elles sont en général de type piézorésistive (cf. brevet US 5,807,758 ou J. Thaysen et al., MEMS, 401, Interlaken, Janvier 2001) ou piézoélectrique (cf. Brevets US 5,719,324 ou US 6,054,277). L'avantage de cette deuxième approche est qu'elle permet, même si les sensibilités sont moindres, d'augmenter la compacité du système et surtout d'avoir une transduction électrique directe ce qui le rend facilement intégrable dans un système plus complexe.Such an approach supposes that one can measure the deflection in the case of a static measurement or the resonant frequency in the case of a dynamic measurement. Two types of measurements can be made. A first approach is based on the principle of laser optical deflection which is used as a detection system in commercial atomic force microscopes. It is an external detection. This technique is very sensitive and allows access to variations in deflection less than the angstrom or variations in resonant frequency of a few hertz. It is used in most cases (cf. patents WO 00/14539 or US 5,445,008 or J. Fritz et al., Science 288, 316, 2000). The second approach consists in integrating the function of detection. They are generally of the piezoresistive type (cf. US patent 5,807,758 or J. Thaysen et al., MEMS, 401, Interlaken, January 2001) or piezoelectric (cf. US Patents 5,719,324 or US 6,054,277). The advantage of this second approach is that it allows, even if the sensitivities are lower, to increase the compactness of the system and above all to have a direct electrical transduction which makes it easy to integrate into a more complex system.
On peut revêtir les micro-leviers d'une molécule particulière qui va lui conférer des propriétés d'adsorption ou d'affinité. En ce qui concerne le positionnement de la partie active (traitée spécifiquement pour une reconnaissance moléculaire particuhere) sur la surface du micro-levier, elle peut s'étendre sur toute la surface du micro-levier dans le cas d'une mesure en statique (effet de contrainte).L' effet de contrainte étant maximal à l'encastrement du micro-levier, une surface active réduite à la zone d'encastrement du micro-levier peut être suffisante. Dans le cas d'une mesure en dynamique, si on considère que la masse ajoutée ne modifie pas les propriétés de raideur du micro-levier, la partie active doit être positionnée à l' extrémité du microlevier. Cependant, une partie active couvrant la totalité de la surface du micro-levier est envisageable.The micro-levers can be coated with a particular molecule which will give it adsorption or affinity properties. Regarding the positioning of the active part (specifically treated for molecular recognition particuhere) on the surface of the micro-lever, it can extend over the entire surface of the micro-lever in the case of a static measurement ( constraint effect) .The constraint effect being maximum when the micro-lever is embedded, an active surface reduced to the micro-lever mounting area may be sufficient. In the case of a dynamic measurement, if it is considered that the added mass does not modify the stiffness properties of the micro-lever, the active part must be positioned at the end of the micro-lever. However, an active part covering the entire surface of the micro-lever is conceivable.
On connaît des techniques de fabrication de micro-leviers simples dans le cas où ils sont utilisés avec une détection optique externe. Des micro-usinages de surface et de volume associés à des dépôts de couches minces permettent d'élaborer des leviers en silicium, en oxyde de silicium, en nitrure de silicium. Ces micro-leviers peuvent être également métallisés (or, platine...). Les dimensions de ces micro-leviers sont typiquement de quelques centaines de microns pour la longueur, quelques dizaines de microns pour la largeur et quelques dixièmes de microns (pour une détection en statique) ou quelques microns (pour une détection en dynamique) pour l'épaisseur. Bien évidemment, les propriétés mécaniques des matériaux utilisés ainsi que les dimensions des micro-leviers vont modifier leurs raideurs et leurs fréquences propres de résonance. La réalisation de micro-leviers intégrant la fonction de détection est plus complexe et nécessite un nombre d'étapes de fabrication plus important. D'autre part, il faut prévoir les connexions électriques et ce nombre de connexions va évidemment croissant avec le nombre de micro-leviers. Dans le cas d'une détection piézoélectrique, le nombre de connexions est égale à 2 par micro-levier, la première pour l'électrode supérieure et la seconde pour l'électrode inférieure. L'électrode inférieure est en général mise à la masse et toutes les électrodes inférieures sont connectées ensemble pour former une masse commune. Il y a donc (n+1) connexions électriques pour n bras de leviers piézoélectriques, ce qui réduit sensiblement le nombre de connexions électriques. L'autre avantage d'une détection piézoélectrique est qu'elle permet d'assurer non seulement la fonction de détection mais également la fonction d'actionnement (mise en résonance dans le cas d'une mesure en dynamique) grâce à l'effet piézoélectrique direct et inverse. Il y a cependant deux inconvénients à utiliser une détection piézoélectrique. Le premier est lié au fait que les technologies d'élaboration de couches minces piézoélectriques (sol-gel ou evaporation radio fréquence) sont complexes et leur compatibilité avec les technologies silicium problématiques (effets d'interface notamment). Le deuxième inconvénient concerne la stabilité des propriétés ferroélectriques et piézoélectriques qui sont sujettes à des dérives thermiques, des effets d'hystérésis et surtout de vieillissement et de fatigue ce qui limite très fortement leur durée de vie pour des utilisations en dynamique.Techniques are known for manufacturing simple micro-levers in the case where they are used with external optical detection. Surface and volume micro-machining associated with deposits of thin layers make it possible to develop levers in silicon, in silicon oxide, in silicon nitride. These micro-levers can also be metallized (gold, platinum ...). The dimensions of these micro-levers are typically a few hundred microns for the length, a few tens of microns for the width and a few tenths of a micron (for a static detection) or a few microns (for a dynamic detection) for the thickness. Obviously, the mechanical properties of the materials used as well as the dimensions of the micro-levers will modify their stiffness and their natural resonance frequencies. The realization of micro-levers integrating the detection function is more complex and requires a larger number of manufacturing steps. On the other hand, it is necessary to provide the electrical connections and this number of connections obviously increases with the number of micro-levers. In the case of piezoelectric detection, the number of connections is equal to 2 per micro-lever, the first for the upper electrode and the second for the lower electrode. The lower electrode is generally grounded and all of the lower electrodes are connected together to form a common ground. There are therefore (n + 1) electrical connections for n piezoelectric lever arms, which significantly reduces the number of electrical connections. The other advantage of a piezoelectric detection is that it ensures not only the detection function but also the actuation function (resonance in the case of a dynamic measurement) thanks to the piezoelectric effect direct and reverse. There are, however, two drawbacks to using piezoelectric detection. The first is linked to the fact that the technologies for developing thin piezoelectric layers (sol-gel or radio frequency evaporation) are complex and their compatibility with problematic silicon technologies (interface effects in particular). The second drawback concerns the stability of the ferroelectric and piezoelectric properties which are subject to thermal drifts, hysteresis effects and especially aging and fatigue which very strongly limits their lifespan for dynamic uses.
Dans le cas d'une détection piézorésistive (cf. E. Cocheteau, C. Bergaud, B. Bélier, L. Bary, R. Plana « Formation of ultra-shallow p+/n junctions with BF2 implantation for the fabrication of improved piezoresistive cantilevers », Transducers' 2001/Eurosensors XN, Munich, 10-14 juin 2001), le nombre de connexions est égal à 2 au minimum pour chaque micro-levier soit 2n connexions électriques pour n micro-leviers. Ce nombre peut être égal à 4 si les piézorésistances sont connectées en pont de Wheatstone ce qui donne 4n connexions électriques. L'intégration d'un pont de Wheatstone permet de réduire la compacité du système complet par rapport à un montage avec un pont de Wheatstone externe. En outre, il permet de s'affranchir des effets de dérives thermiques. L'inconvénient d'une détection piezoresistive dans le cas d'une mesure en dynamique est qu'elle suppose que l'on dispose d'une excitation mécanique externe ou bien que les facteurs de qualité des microleviers sont suffisamment importants (>100) pour que la fréquence de résonance soit détectable en bruit blanc (excitation thermomécanique due au mouvement brownien). Il y a deux avantages à utiliser une détection piezoresistive. Le premier est que la réalisation de microleviers piézorésistifs est relativement simple et parfaitement compatible avec les technologies de la microélectronique silicium. Le second avantage est la sensibilité plus importante d'une détection piezoresistive par rapport à une détection piézoélectrique.In the case of piezoresistive detection (cf. E. Cocheteau, C. Bergaud, B. Bélier, L. Bary, R. Plana “Formation of ultra-shallow p + / n junctions with BF2 implantation for the fabrication of improved piezoresistive cantilevers ", Transducers' 2001 / Eurosensors XN, Munich, 10-14 June 2001), the number of connections is at least 2 for each micro-lever, i.e. 2n electrical connections for n micro-levers . This number can be equal to 4 if the piezoresistors are connected in Wheatstone bridge which gives 4n electrical connections. The integration of a Wheatstone bridge makes it possible to reduce the compactness of the complete system compared to an assembly with an external Wheatstone bridge. In addition, it eliminates the effects of thermal drifts. The disadvantage of a piezoresistive detection in the case of a dynamic measurement is that it supposes that there is an external mechanical excitation or that the quality factors of the microlevers are sufficiently important (> 100) to that the resonant frequency is detectable in white noise (thermomechanical excitation due to Brownian motion). There are two advantages to using piezoresistive detection. The first is that the production of piezoresistive microlevers is relatively simple and perfectly compatible with the technologies of silicon microelectronics. The second advantage is the greater sensitivity of a piezoresistive detection compared to a piezoelectric detection.
La sélectivité des micro-leviers 13, c'est-à-dire leur capacité à capturer des molécules particulières, dépend de la polarité intrinsèque, la solvophobicité et la porosité du matériau qui constitue le micro-levier ou un film fin revêtu sur le microlevier, et selon la polarité et la solvophobicité des groupements fonctionnels greffés sur le micro-levier. La sélectivité des micro-leviers 13 dépend également de critères comme l'échange d'ions et l'affinité des groupements fonctionnels, des conditions successives de micro-élution secondaire dans les micro-colonnes de fractionnement 2 ainsi que des conditions successives de micro-extraction et de micro-digestion conduites en amont desdits micro-leviers 13.The selectivity of the micro-levers 13, that is to say their capacity to capture particular molecules, depends on the intrinsic polarity, the solvophobicity and the porosity of the material which constitutes the micro-lever or a thin film coated on the micro-lever , and according to the polarity and the solvophobicity of the functional groups grafted onto the micro-lever. The selectivity of the micro-levers 13 also depends on criteria such as the ion exchange and the affinity of the functional groups, the successive conditions of secondary micro-elution in the fractionation micro-columns 2 as well as the successive conditions of micro- extraction and micro-digestion carried out upstream of said micro-levers 13.
La capture d'une molécule, et notamment d'une protéine, par un microlevier 13 peut être effectuée par affinité. C'est le cas par exemple lorsque le microlevier a été revêtu avec un anticorps. Dans ce cas, le micro-levier capture une protéine précise, connue, et en indique la présence.The capture of a molecule, and in particular of a protein, by a microlever 13 can be carried out by affinity. This is the case, for example, when the microlever has been coated with an antibody. In this case, the micro-lever captures a precise, known protein, and indicates its presence.
La capture d'une molécule, et notamment d'une protéine, par un microlevier 13 peut être effectué par adsorption. Dans ce cas, un même micro-levier sera apte à détecter une catégorie de protéines présentant des propriétés d'adsorption similaires. Des protéines non connues ou recherchées à priori pourront être détectées. En effectuant des comparaisons d'empreintes sur des échantillons différents, on pourra dégager des différences d'empreintes, notamment des différences de détections sur des micro-leviers 13 d'adsorption. Par la suite, en reproduisant la capture avec les mêmes étapes de sélection, on peut isoler les protéines différentes ainsi obtenues pour les analyser plus spécifiquement.The capture of a molecule, and in particular of a protein, by a microlever 13 can be carried out by adsorption. In this case, the same micro-lever will be able to detect a category of proteins having similar adsorption properties. Proteins not known or researched a priori can be detected. By carrying out fingerprint comparisons on different samples, it will be possible to identify differences in fingerprints, in particular differences in detection on adsorption micro-levers 13. Subsequently, by reproducing the capture with the same steps of selection, we can isolate the different proteins thus obtained to analyze them more specifically.
Dans un mode de réalisation dans lequel le dispositif d'analyse ne comprend pas de micro-colonnes de fractionnement secondaire, une elution pas à pas ou avec gradient permet le passage d' éluant secondaires différents entraînant différentes molécules selon leur affinité avec ces molécules et l'affinité des micro-leviers avec ces mêmes molécules. Des empreintes successives sont enregistrées, à chaque pas d'élution pour une elution pas à pas, ou à des instants différents pour une elution avec gradient d'élution. Comme on l'a déjà indiqué des lavages successifs sur les micro-leviers 13 peuvent être effectués, la rétention des micro-éluats secondaires ou des micro-extraits secondaires ou des produits de digestion secondaires des produits de fractionnements sur les micro-leviers 13 étant mesurée par la déviation ou par la fréquence de vibration desdits micro-leviers 13, plusieurs empreintes successives étant enregistrées. La série d'empreintes successives de détection du premier échantillon sur micro-leviers 13 est par exemple comparée ensuite à la série d'empreintes successives de détection sur micro-leviers 13 d'un deuxième échantillon. Pour un lavage des micro-leviers, on prévoit le passage dans les zones de détection d'un éluant apte à entraîner des molécules retenues sur les micro-leviers 13. Selon la sélectivité des micro-leviers 13 d'une zone de détection, on prévoit d'adapter le composition de l' éluant pour favoriser le décrochage des molécules retenues.In an embodiment in which the analysis device does not include secondary fractionation micro-columns, a stepwise or gradient elution allows the passage of different secondary eluents entraining different molecules according to their affinity with these molecules and the affinity of micro-levers with these same molecules. Successive fingerprints are recorded, at each elution step for a step-by-step elution, or at different times for an elution with an elution gradient. As already indicated, successive washes on the micro-levers 13 can be carried out, the retention of secondary micro-eluates or secondary micro-extracts or secondary digestion products of the fractionation products on the micro-levers 13 being measured by the deviation or by the vibration frequency of said micro-levers 13, several successive fingerprints being recorded. The series of successive fingerprints for detecting the first sample on micro-levers 13 is for example then compared to the series of successive fingerprints for detection on micro-levers 13 of a second sample. For washing the micro-levers, provision is made for passage into the detection zones of an eluent capable of entraining molecules retained on the micro-levers 13. Depending on the selectivity of the micro-levers 13 of a detection zone, plans to adapt the composition of the eluent to promote the detachment of the molecules retained.
Le passage d' éluant de lavage dans les zones de détection peut être effectuée à l'aide de micro-canaux de capture 8, dans lesquels on envoie un éluant de lavage.The passage of washing eluent into the detection zones can be carried out using micro-capture channels 8, into which a washing eluent is sent.
Dans une variante, on prévoit un circuit supplémentaire de lavage permettant d'amener un éluant de lavage directement en amont d'une zone de détection. Un tel mode de réalisation est préférable dans le cas où le dispositif d' analyse comprend des micro-colonnes de fractionnement secondaires.In a variant, an additional washing circuit is provided, making it possible to bring a washing eluent directly upstream of a detection zone. Such an embodiment is preferable in the case where the analysis device comprises secondary fractionation micro-columns.
Sur la figure 12, on a repris les références aux éléments semblables à ceux de la figure 3. Une micro-colonne de fractionnement 2 est intersectée au niveau d'un élément terminal par un micro-canal de capture 8, reliée en amont de l'intersection à un conduit d'alimentation en éluant secondaire 15, et comprenant en aval de l'intersection une micro-colonne de fractionnement secondaire 10. Une zone de détection 11 comprenant des micro-leviers 13 est située sur le micro-canal de capture 8 en aval de la micro-colonne de fractionnement secondaire 10. Un micro-conduit de lavage 70 comprend un orifice d'entrée 71 pour l'alimentation en éluant de lavage, et un orifice de sortie 72 débouchant dans le micro- canal de capture 8, en aval de la micro-colonne de fractionnement secondaire 10, et en amont de la zone de détection 11.In FIG. 12, the references to elements similar to those of FIG. 3 have been repeated. A fractionation micro-column 2 is intersected at the level of a terminal element by a capture micro-channel 8, connected upstream of the intersection with a secondary eluent supply conduit 15, and comprising downstream of the intersection a secondary fractionation micro-column 10. A detection zone 11 comprising micro-levers 13 is located on the capture micro-channel 8 downstream of the secondary fractionation micro-column 10. A washing micro-duct 70 comprises an inlet orifice 71 for supplying washing eluent, and an outlet orifice 72 opening into the micro- capture channel 8, downstream of the secondary fractionation micro-column 10, and upstream of the detection zone 11.
Si un éluant de lavage est amené par le micro-canal de capture 8 en traversant la micro-colonne de fractionnement secondaire 10, l' éluant de lavage entraînera des molécules retenue dans la micro-colonne de fractionnement secondaire 10. Le micro-conduit de lavage 70 permet d'amener l'éluant de lavage directement en amont de la zone de détection pour un lavage des micro-leviers 13, sans lavage de la micro-colonne de fractionnement secondaire 10.If a washing eluent is brought in by the capture micro-channel 8 passing through the secondary fractionation micro-column 10, the washing eluent will entrain molecules retained in the secondary fractionation micro-column 10. The micro-duct of washing 70 makes it possible to bring the washing eluent directly upstream of the detection zone for washing the micro-levers 13, without washing the secondary fractionation micro-column 10.
Dans une première variante du dispositif d'analyse, on peut prévoir une détection supplémentaire, en aval des zones de détection li a l'aide de micro-leviers 13, par exemple par spectrométrie de masse. Des méthodes de spectrométrie de masse sont évoquées par la suite.In a first variant of the analysis device, additional detection can be provided downstream of the detection zones li using micro-levers 13, for example by mass spectrometry. Mass spectrometry methods are discussed below.
Dans une seconde variante, après une comparaison d'empreintes successives de deux échantillons, on prévoit qu'une détection des micro-éluats secondaires ou des micro-extraits secondaires ou des produits de digestion secondaires des produits de fractionnement est effectuée par spectrométrie de masse, mais essentiellement sur les zones de détection 11 où la série d'empreintes par micro-leviers 13 du premier échantillon diffère de la série d'empreintes par micro-leviers 13 du deuxième échantillon. On peut effectuer des détection par spectrométrie de masse de façon connue, comme cela est exposé dans les documents suivants : (Cf. Dongré AR, Eng JK, Yates JR LU. Emerging tandem-mass spectrometry techniques for the rapid identification of proteins. TiBTECH, 1997, 15, 418-425; Anderegg RJ, Wagner DS, Blackburn RK, Opiteck GJ, Jorgenson JW. A multidimensional approach to protein characterization. Journal of Protein Chemistry, 1997, 16, 5 ; Huang P, Jin X, Chen Y, Srinivasan JR, Lubman DM. Use of a mixed mode packing and voltage tuning for peptide mixture séparation in pressurized capillary electrochromatography with an ion trap storage/reflectron Time-of-Flight mass spectrometer detector. Anal. Chem, 1999, 71, 1786-1791 ; Martin SE, Shabanowitz J, Hunt DF, Marto JA. Subfemtomole MS and MS/MS peptide séquence analysis using Nano-HPLC micro-ESI Fourier Transform Ion Cyclotron Résonance Mass Spectrometry; Anal. Chem 2000, 72, 4266-4274; Gafiin CL, Eng JK, Cross ST, Detter JC, Yates JR LU. Automated identification of amino-acid séquence variation in proteins by HPLC/Microspray tandem Mass Spectrometry. Anal. Chem, 2000, 72, 757-763; Ji J, Chakraborty A, geng M, Zhnag X, Amini A, Bina M, Régnier F. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. Journal of Chromatography B, 2000, 745, 197-210 ; Nan Pelt CK, Corso TΝ, Schultz GA, Lowes S, Henion J. A four-column parallel chromatography system for isocratic or gradient LC/MS analyses. Anal. Chem. 2001, 73, 582-5888; Patterson SD et al. Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. Proteomics 2001, 1, 93-107 et 2, 108-117). Des techniques de spectrométrie de masse, ainsi que des solutions d'interfaçage entre la chromatograhie en phase liquide et lesdites techniques de spectrométrie de masse ont été décrites dans l'ouvrage de Niessen, "Liquid Chromatography - Mass Spectrometry, vol 79, Chromatographie Science Séries, Ed Jack Cazes, et le couplage LC-MS appliqué à l'analyse des protéines a été plus particulièrement décrit au chapitre 15, pp 501 - 537. La détection par spectrométrie de masse peut notamment être couplée de façon connue à deux méthodes de séparation préalable par chromatographie en phase liquide (notation abrégée LC-MS (LC = liquid chromatography, MS = mass spectrometry)). ( Cf. Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JR III. Direct analysis of protein complexes using mass spectrometry. Nature Biotechnology, 1999, 17, 676-681; Washburn MP, Wolters D, Yates JR UI. Large-scale analysis of the yeast proteome by multi-dimensional protein identification technology. Nature Biotechnology. 2001, 19, 242-247; Davis MT, Beierle J, Bures ET, Mac Ginley MD, Mort J, Robinson JH, Saphr CS, YU W, Luethy R, Patterson SD. Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyses. Journal of Chromatography B, 2001, 752, 281-291 ; Zhou H, Watts JD, Aebersold R. A systematic approach to the analysis of protein phosphorylation, 2001, 375-382).In a second variant, after a comparison of successive fingerprints of two samples, provision is made for detection of secondary micro-eluates or secondary micro-extracts or secondary digestion products of the fractionation products to be carried out by mass spectrometry, but essentially on the detection zones 11 where the series of fingerprints by micro-levers 13 of the first sample differs from the series of fingerprints by micro-levers 13 of the second sample. Mass spectrometry detection can be carried out in a known manner, as described in the following documents: (Cf. Dongré AR, Eng JK, Yates JR LU. Emerging tandem-mass spectrometry techniques for the rapid identification of proteins. TiBTECH, 1997, 15, 418-425; Anderegg RJ, Wagner DS, Blackburn RK, Opiteck GJ, Jorgenson JW. A multidimensional approach to protein characterization. Journal of Protein Chemistry, 1997, 16, 5; Huang P, Jin X, Chen Y, Srinivasan JR, Lubman DM. Use of a mixed mode packing and voltage tuning for peptide mixture separation in pressurized capillary electrochromatography with an ion trap storage / reflectron Time-of-Flight mass spectrometer detector. Anal. Chem, 1999, 71, 1786-1791 ; Martin SE, Shabanowitz J, Hunt DF, Marto JA. Subfemtomole MS and MS / MS peptide sequence analysis using Nano-HPLC micro-ESI Fourier Transform Ion Cyclotron Résonance Mass Spectrometry; Anal. Chem 2000, 72, 4266-4274; Gafiin CL , Eng JK, Cross ST, Detter JC, Yat es JR LU. Automated identification of amino acid sequence variation in proteins by HPLC / Microspray tandem Mass Spectrometry. Anal. Chem, 2000, 72, 757-763; Ji J, Chakraborty A, geng M, Zhnag X, Amini A, Bina M, Régnier F. Strategy for qualitative and quantitative analysis in proteomics based on signature peptides. Journal of Chromatography B, 2000, 745, 197-210; Nan Pelt CK, Corso TΝ, Schultz GA, Lowes S, Henion J. A four-column parallel chromatography system for isocratic or gradient LC / MS analyzes. Anal. Chem. 2001, 73, 582-5888; Patterson SD et al. Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. Proteomics 2001, 1, 93-107 and 2, 108-117). Mass spectrometry techniques, as well as solutions for interfacing between liquid chromatography and said mass spectrometry techniques have been described in Niessen's book, "Liquid Chromatography - Mass Spectrometry, vol 79, Chromatographie Science Séries , Ed Jack Cazes, and the LC-MS coupling applied to protein analysis was more particularly described in chapter 15, pp 501 - 537. Detection by mass spectrometry can in particular be coupled in a known manner to two separation methods prior by liquid chromatography (abbreviated LC-MS notation (LC = liquid chromatography, MS = mass spectrometry)) (Cf. Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, Garvik BM, Yates JR III. Direct analysis of protein complexes using mass spectrometry. Nature Biotechnology, 1999, 17, 676-681; Washburn MP, Wolters D, Yates JR UI. Large-scale analysis of the yeast proteome by multi-dimensional protei n identification technology. Nature Biotechnology. 2001, 19, 242-247; Davis MT, Beierle J, Bures ET, Mac Ginley MD, Mort J, Robinson JH, Saphr CS, YU W, Luethy R, Patterson SD. Automated LC-LC-MS-MS platform using binary ion-exchange and gradient reversed-phase chromatography for improved proteomic analyzes. Journal of Chromatography B, 2001, 752, 281-291; Zhou H, Watts JD, Aebersold R. A systematic approach to the analysis of protein phosphorylation, 2001, 375-382).
Une détection par spectrométrie de masse peut également être couplée de façon connue à une séparation des peptides et des protéines en capillaires ou sur supports miniaturisés avec micro-canaux ou micro-colonnes par micro- chromatographie, micro-électrochromatographie ou micro-électrophorèse. (Cf. Xie S.,Detection by mass spectrometry can also be coupled in a known manner to a separation of peptides and proteins in capillaries or on miniaturized supports with micro-channels or micro-columns by micro-chromatography, micro-electrochromatography or micro-electrophoresis. (Cf. Xie S.,
Allington R.W., Svec F., Fréchet J. Rapid reverse-phase séparation of proteins and peptides using optimized moulded monolithic poly(styrene-co-divinylbenzene) columns; Josic D., Buchacher A., Jungbauer A. Monoliths as stationary phases for séparation of proteins and polynucleotides and enzymatic conversion. Journal ofAllington R.W., Svec F., Fréchet J. Rapid reverse-phase separation of proteins and peptides using optimized molded monolithic poly (styrene-co-divinylbenzene) columns; Josic D., Buchacher A., Jungbauer A. Monoliths as stationary phases for separation of proteins and polynucleotides and enzymatic conversion. Journal of
Chromatography B, 2001, 752, 191-205; Walhagen K, Unger KK, Hearn MTW,Chromatography B, 2001, 752, 191-205; Walhagen K, Unger KK, Hearn MTW,
Capillary electroendoosmotic chromatography of peptides, Journal of ChromatographyCapillary electroendoosmotic chromatography of peptides, Journal of Chromatography
A, 2000, 887, 165-185 ; Krull IS, Sebag A, Stevenson R, Spécifie applications of capillary electrochromatography to biopolymers, including proteins, nucleic acids, peptide mapping, antibodies, and so forth, Journal of chromatography A, 2000, 887,A, 2000, 887, 165-185; Krull IS, Sebag A, Stevenson R, Specifies applications of capillary electrochromatography to biopolymers, including proteins, nucleic acids, peptide mapping, antibodies, and so forth, Journal of chromatography A, 2000, 887,
137-136. ; He B, Ji J, Régnier FE. Capillary electrochromatography of peptides in a micro-fabricated system. Journal of Chromatography A, 1999, 853, 257-262).Pour une détection par spectrométrie de masse, on peut utiliser l'ionisation en nébulisation, comme par exemple la spectrométrie de masse en tandem avec ionisation en electrospray (ESI-MS, ou Electrospray Ionisation Mass Spectrometry) ou en désorption, comme par exemple la désorption sur matrice et assistée par laser dans la spectrométrie de masse MALDI (Martix Assisted Laser Désorption Ionization).137-136. ; He B, Ji J, Régnier FE. Capillary electrochromatography of peptides in a micro-fabricated system. Journal of Chromatography A, 1999, 853, 257-262). detection by mass spectrometry, one can use the ionization in nebulization, like for example mass spectrometry in tandem with ionization in electrospray (ESI-MS, or Electrospray Ionization Mass Spectrometry) or in desorption, like for example the desorption on matrix and laser assisted in mass spectrometry MALDI (Martix Assisted Laser Desorption Ionization).
On peut prévoir l'utilisation d'une détection par spectrométrie de masse par triple quadrupole, ou par piège à ions (Ion Trap) ou en tandem avec ionisation en electrospray (ESI-MS-MS, ou ou Electrospray Ionisation Tandem Mass Spectrometry), on peut tirer profit de la dissociation par collision (CID, ou Collision Induced Dissociation): chaque peptide est susceptible de posséder un spectre de masse de dissocation par collision archivé dans les banques de données (Cf. Figeys D, Ning Y, Aebersold R. A microfabricated device for rapid protein identification by microelectrospray Ion Trap mass Spectrometry. Anal Chem, 1997, 69, 3153-3160) .We can foresee the use of a detection by mass spectrometry by triple quadrupole, or by ion trap (Ion Trap) or in tandem with electrospray ionization (ESI-MS-MS, or or Electrospray Ionization Tandem Mass Spectrometry), we can take advantage of collision dissociation (CID, or Collision Induced Dissociation): each peptide is likely to have a mass spectrum of dissocation by collision archived in databases (Cf. Figeys D, Ning Y, Aebersold R. A microfabricated device for rapid protein identification by microelectrospray Ion Trap mass Spectrometry. Anal Chem, 1997, 69, 3153-3160).
Des documents décrivent plus particulièrement la détection des peptides, protéines et carbohydrates par spectrométrie de masse.Documents describe more particularly the detection of peptides, proteins and carbohydrates by mass spectrometry.
Les polypeptides sont analysés par spectrométie de masse avant ou après digestion enzymatique (Cf. Roepstroff P. Mass spectrometry in protein studies from génome to function. Current Opinion in Biotechnology, 1997, 8, 6-13), par des techniques de spectrométrie de masse utilisant l'ionisation en nébulisation ou en désorption.The polypeptides are analyzed by mass spectrometry before or after enzymatic digestion (Cf. Roepstroff P. Mass spectrometry in protein studies from genome to function. Current Opinion in Biotechnology, 1997, 8, 6-13), by mass spectrometry techniques using ionization in nebulization or desorption.
Les modifications post-traductionnelles des protéines peuvent être étudiées en soumettant les analytes à des phosphatases ou des glycosylases (Cf. Qin J, Chait BT. Identifications and characterization of posttranslational modifications of proteins by MALDI Ion Trap mass spectrometry . Anal Chem, 1997, 69, 4002-4009.) Lorque l'analyse par spectrométrie de masse a lieu après digestion avec une endopeptidase donnée, on peut comparer les spectres des masses observées avec les banques des spectres des masses théoriques des résidus de digestion avec ladite endopeptidase.Post-translational modifications of proteins can be studied by subjecting the analytes to phosphatases or glycosylases (Cf. Qin J, Chait BT. Identifications and characterization of posttranslational modifications of proteins by MALDI Ion Trap mass spectrometry. Anal Chem, 1997, 69 , 4002-4009.) When the analysis by mass spectrometry takes place after digestion with a given endopeptidase, one can compare the spectra of the masses observed with the banks of the spectra of theoretical masses of the digestion residues with said endopeptidase.
En spectrométrie de masse MALDI, les échantillons peuvent être déposés sur des membranes de poly(vinylidene difluoride), ou de polyurethane (Cf. Me Comb ME, Oleschuk RD, Manley DM, Donald L, Chow A, O'neil JD, Ens W, Stabding KG, Perreault H. Use of non-porous polyurethane membrane as a sample support for matrix-assisted laser désorption ionisation time-of-flight mass spectrometry of peptides and proteins. Rapid Commun Mass Spectrom, 1997, 11 (15), 1716-22) . Tout comme les peptides et les protéines, on peut prévoir d'analyser les glycoproteines par spectrométrie de masse (Cf. Ninh J, Loyaux D, Redeker N, Rossier R.. Sequencing of branched peptides with CLD/PSD MALDI-TOF in the low picomoles range: application to the structural study of the posttranslational polyglycylation of tubulin. Anal. Chem, 1997, 69, 3979-3985; Harvey DJ. Identification of protein-bound carbohydrates by mass spectrometry. Proteomics 2001, 1, 311-328; Yamagaki T, Nakanishi H. Ion intensity analysis of post-source decay fragmentation in curved-field reflectron matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of carbohydrates: for structural characterization of glycosylation in proteome analysis. Proteomics 2001, 1, 329, 339).In MALDI mass spectrometry, the samples can be deposited on poly (vinylidene difluoride) or polyurethane membranes (Cf. Me Comb ME, Oleschuk RD, Manley DM, Donald L, Chow A, O'neil JD, Ens W , Stabding KG, Perreault H. Use of non-porous polyurethane membrane as a sample support for matrix-assisted laser désorption ionisation time-of-flight mass spectrometry of peptides and proteins. Rapid Commun Mass Spectrom, 1997, 11 (15), 1716 -22). Like peptides and proteins, we can plan to analyze glycoproteins by mass spectrometry (Cf. Ninh J, Loyaux D, Redeker N, Rossier R .. Sequencing of branched peptides with CLD / PSD MALDI-TOF in the low picomoles range: application to the structural study of the posttranslational polyglycylation of tubulin. Anal. Chem, 1997, 69, 3979-3985; Harvey DJ. Identification of protein-bound carbohydrates by mass spectrometry. Proteomics 2001, 1, 311-328; Yamagaki T, Nakanishi H. Ion intensity analysis of post-source decay fragmentation in curved-field reflectron matrix-assisted laser desorption / ionization time-of-flight mass spectrometry of carbohydrates: for structural characterization of glycosylation in proteome analysis. Proteomics 2001, 1, 329, 339).
Sur la figure 4, les références aux éléments semblables à la figure 3 ont été reprises. Un premier support 20, sous la forme d'une plaque plane, comprend un canal d'alimentation 4 prévu pour l'alimentation en phase mobile et comprenant une zone d'enrichissement 21 de la phase mobile en échantillon. Le premier support 20 comprend une micro-colonne de fractionnement 2 comprenant une portion de micro-canal 3 muni d'un orifice d'introduction 3a en communication fluidique avec un canal d'alimentation 4 en aval d'une zone d'enrichissement 18 d'une phase mobile en échantillon, et un orifice d'évacuation 3b. Le premier support 20 comprend un micro-canal de capture 8 intersectant la micro-colonne de fractionnement 2 au niveau d'un élément terminal 9, et un canal d'alimentation en éluant secondaire 15 en communication fluidique avec une entrée du micro-canal de capture 8 en amont de l'intersection avec la micro-colonne de fractionnement 2. Un second support 22, sous la forme d'une plaque plane, comprend un micro-conduit 23 débouchant à une extrémité dans une micro-colonne de fractionnement secondaire en communication fluidique du côté opposé avec une zone de détection 11 pourvue de micro-leviers 13.In FIG. 4, the references to elements similar to FIG. 3 have been repeated. A first support 20, in the form of a flat plate, comprises a supply channel 4 intended for supply in the mobile phase and comprising an enrichment zone 21 of the mobile phase in the sample. The first support 20 comprises a fractionation micro-column 2 comprising a micro-channel portion 3 provided with an introduction orifice 3a in fluid communication with a supply channel 4 downstream of an enrichment zone 18 d 'a mobile phase in sample, and an evacuation orifice 3b. The first support 20 includes a capture micro-channel 8 intersecting the fractionation micro-column 2 at a terminal element 9, and a secondary eluent supply channel 15 in fluid communication with an inlet of the micro-channel of capture 8 upstream of the intersection with the fractionation micro-column 2. A second support 22, in the form of a flat plate, comprises a micro-conduit 23 opening at one end into a secondary fractionation micro-column in fluid communication on the opposite side with a detection zone 11 provided with micro-levers 13.
Un troisième support 24, également sous la forme d'une plaque plane, comprend un canal d'évacuation 6.A third support 24, also in the form of a flat plate, comprises a discharge channel 6.
Les second et troisième supports 22, 24 sont disposés de part et d'autre du premier support 20, parallèlement au premier support, en étant accolés à ce dernier, de façon que le canal d'évacuation 6 du troisième support 24 est en communication fluidique avec l'orifice d'évacuation 3b de la micro-colonne de fractionnement 2, et le micro-conduit 23 du second support est en communication fluidique avec le micro-canal de capture 8, en aval de l'intersection avec la micro-colonne de fractionnement 2.The second and third supports 22, 24 are arranged on either side of the first support 20, parallel to the first support, being attached to the latter, so that the discharge channel 6 of the third support 24 is in fluid communication with the discharge orifice 3b of the fractionation micro-column 2, and the micro-conduit 23 of the second support is in fluid communication with the capture micro-channel 8, downstream of the intersection with the micro-column fractionation 2.
Sur la figure 5, où les références aux éléments semblables à ceux de la figure 4 ont été reprises, un premier support 20 comprend une pluralité de microcolonnes de fractionnement 2, et une pluralité de micro-canaux de capture 8 associés. Le second support 22 comprend une pluralité de micro-conduits en communication fluidique avec les micro-canaux de capture 8, et une pluralité de zone de détection 11. Le troisième support 24 comprend un canal d'évacuation 6 en communication fluidique avec l'ensemble des orifices d'évacuation 3b des micro-canaux 3. Les premiers, second et troisième support 20, 22, 24 sont disposés parallèlement.In FIG. 5, where the references to the elements similar to those of FIG. 4 have been repeated, a first support 20 comprises a plurality of fractionation microcolumns 2, and a plurality of associated capture micro-channels 8. The second support 22 comprises a plurality of micro-conduits in fluid communication with the capture micro-channels 8, and a plurality of detection zones 11. The third support 24 comprises a discharge channel 6 in fluid communication with all of the discharge orifices 3b of the micro-channels 3. The first, second and third supports 20, 22, 24 are arranged in parallel.
Dans une variante de disposition des supports illustrée par les figures 6 et 7, les second et troisième support 22, 24 sont agencés perpendiculairement au premier support 20. Sur la figure 6, le premier support 20 comprend une unique micro-colonne de fractionnement 2. Sur la figure 7, le premiers support 20 comprend une pluralité de micro-colonnes de fractionnement 2, les second et troisièmes supports 22, 24 étant adaptés en conséquence.In a variant arrangement of the supports illustrated by FIGS. 6 and 7, the second and third supports 22, 24 are arranged perpendicular to the first support 20. In FIG. 6, the first support 20 comprises a single fractionation micro-column 2. In FIG. 7, the first support 20 comprises a plurality of fractionation micro-columns 2, the second and third supports 22, 24 being adapted accordingly.
Sur la figure 8, un support comprend quatre lots de micro-colonnes de fractionnement 2 agencés sensiblement en étoile. Les micro-colonnes de fractionnement 2 du premier lot s'inscrivent dans une fourchette de longueur petites. Les microcolonnes de fractionnement 2 du deuxième lot s'inscrivent dans une fourchette de longueurs plus grandes, celles du troisième lot s'inscrivent dans une fourchette de longueurs encore plus grandes, celles du quatrième lot s'inscrivent dans une fourchette de longueurs encore supérieures. Le support comprend micro-colonne d'enrichissement centrale 25 de forme carrée, en communication fluidique d'où est issue chacune des micro-colonnes de fractionnement des lots, au centre de laquelle débouche un canal d'introduction de l'échantillon situé dans un plan vertical, et non représenté sur la figure 8. Afin d'améliorer encore la sensibilité du dispositif d'analyse, c'est-à-dire sa capacité à détecter des protéines, on peut prévoir des extractions préalables des constituants de l'échantillon, avant l'introduction de l'échantillon dans les microcolonnes de fractionnement.In FIG. 8, a support comprises four lots of fractionation micro-columns 2 arranged substantially in a star. The fractionation micro-columns 2 of the first batch fall within a range of small lengths. The fractionation microcolumns 2 of the second batch fall within a range of longer lengths, those of the third batch fall within a range of even longer lengths, those of the fourth batch fall within a range of even longer lengths. The support comprises a central enrichment micro-column 25 of square shape, in fluid communication from which each of the batch fractionation micro-columns comes, at the center of which opens a channel for introducing the sample located in a vertical plane, and not shown in FIG. 8. In order to further improve the sensitivity of the analysis device, that is to say its ability to detect proteins, it is possible to provide for preliminary extractions of the constituents of the sample , before introducing the sample into the fractionating microcolumns.
Sur la figure 9, un étage d'extraction préalable 30 comprend un support 31 représenté partiellement et pourvu d'une pluralité de mico-colonnes de fractionnement préalable 32 rectilignes parallèles et de même longueur, d'un canal d'alimentation 33 en communication fluidique avec les micro-colonnes de fractionnement 32, et un canal d'évacuation 34 en communication fluidique avec les micro-colonnes de fractionnement 32 du côté opposé au canal d'alimentation 33. Les micro-colonnes de fractionnement 32 sont formées par des portions de micro-canaux munies de moyens de séparation intermédiaires. Le support 31 comprend de moyens de capture fluidique 35 sous la forme de micro-canaux de capture 36, alimenté en éluant d'un côté à partir d'un conduit d'alimentation 37, et en communication fluidique du côté opposé avec un conduit de récupération 38 commun. Chaque micro-canal de capture traverse transversalement une micro-colonne de fractionnement 32 au niveau d'un élément terminal situé à proximité d'un orifice d'évacuation de la micro-colonne de fractionnement 32 du côté du canal d'évacuation 34. L'échantillon transporté par une phase mobile est amené par le canal d'alimentation 33, circule dans les micro-colonnes de fractionnement 32, où il subit une séparation en fonction d'une sélectivité des moyens de séparation des micro-colonnes de fractionnement 32. L'association d'une pluralité de micro-colonnes de fractionnement 32 permet une séparation importante dans des micro-colonnes de fractionnement 32 de faible diamètre, sans limiter un débit d'une phase mobile enrichie en échantillon. Les moyens de captures 35 permettent des captures successives des constituants de l'échantillon présents à un instant donné de capture au niveau de l'élément terminal d'intersection des micro-canaux de capture 36 avec les micro- colonnes de fractionnement 32.In FIG. 9, a preliminary extraction stage 30 comprises a support 31 shown partially and provided with a plurality of prior fractionation mico-columns 32 rectilinear parallel and of the same length, with a supply channel 33 in fluid communication with the fractionation micro-columns 32, and a discharge channel 34 in fluid communication with the fractionation micro-columns 32 on the side opposite to the feed channel 33. The fractionation micro-columns 32 are formed by portions of micro-channels provided with intermediate separation means. The support 31 comprises fluid capture means 35 in the form of capture micro-channels 36, supplied by eluting on one side from a supply line 37, and in fluid communication on the opposite side with a supply line recovery 38 common. Each capture micro-channel transversely passes through a fractionation micro-column 32 at the level of a terminal element situated near an evacuation orifice of the fractionation micro-column 32 on the side of the evacuation channel 34. The sample transported by a mobile phase is brought through the feed channel 33, circulates in the fractionation micro-columns 32, where it undergoes separation according to a selectivity of the separation means of the fractionation micro-columns 32 The association of a plurality of fractionation micro-columns 32 allows a significant separation in fractionation micro-columns 32 of small diameter, without limiting a flow rate of a mobile phase enriched in sample. The capture means 35 allow successive captures of the constituents of the sample present at a given capture time at the terminal element of intersection of the capture micro-channels 36 with the fractionation micro-columns 32.
Après une capture, on récupère dans le conduit de récupération 38 une portion des constituants de l'échantillon, qui est acheminée vers des micro-colonnes de fractionnement et des zones de détection tels que décrit précédemment. Les constituants séparés lors l'extraction préalable dans un lot de micro-colonnes de fractionnement préalable 32 possédant une sélectivité particulière pourront être mieux séparés par la suite en adaptant la sélectivité d'un ou plusieurs lots de micro-colonnes de fractionnement d'analyse vers lequel la portion capturée est acheminée.After a capture, a portion of the constituents of the sample is recovered in the recovery conduit 38, which is conveyed to fractionation micro-columns and detection zones as described above. The constituents separated during the prior extraction in a batch of preliminary fractionation micro-columns 32 having a particular selectivity can be better separated thereafter by adapting the selectivity of one or more batches of analysis fractionation micro-columns to which the captured portion is routed.
Dans un lot de micro-colonnes de fractionnement d'extraction préalable, les micro-colonnes sont de longueurs égales afin de récupérer en extrémité de chaque micro-colonne sensiblement les mêmes constituants. En outre, les produits capturés sur différentes portions sont évacués collectivement dans un conduit de séparation. Au contraire, dans un lot de micro-colonne de fractionnement d'analyse, les micro-colonnes de fractionnement peuvent être de longueur différentes pour permettre un fractionnement différentiel, et chaque micro-canal de capture communique avec une zone de détection associée.In a batch of prior extraction fractionation micro-columns, the micro-columns are of equal length in order to recover at the end of each micro-column substantially the same constituents. In addition, the products captured on different portions are collectively evacuated in a separation conduit. On the contrary, in a batch of analysis fractionation micro-column, the fractionation micro-columns can be of different length to allow differential fractionation, and each capture micro-channel communicates with an associated detection zone.
Dans un dispositif d'analyse, on peut prévoir une pluralité de lots de microcolonnes de fractionnement préalable possédant chacun une sélectivité de séparation différente, et communiquant chacun avec une pluralité de lots de micro-colonnes de fractionnement d'analyse, chaque lot de micro-colonnes de fractionnement d'analyse possédant une sélectivité particulière, de préférence adaptée en fonction de la sélectivité du lot d'extraction préalable.In an analysis device, it is possible to provide a plurality of batches of preliminary fractionation microcolumns each having a different separation selectivity, and each communicating with a plurality of batches of analysis fractionation micro-columns, each batch of micro- analysis fractionation columns having a particular selectivity, preferably adapted as a function of the selectivity of the batch of prior extraction.
Sur la figure 10, où les références aux éléments semblables à ceux de la figure 9 ont été reprises, un support 31 comprend un unique micro-canal de capture 36 traversant successivement les micro-colonnes de fractionnement préalable 32 d'un même lot, et débouchant finalement dans le conduit de récupération 38.In FIG. 10, where the references to elements similar to those of FIG. 9 have been repeated, a support 31 comprises a single capture micro-channel 36 successively passing through the prior fractionation micro-columns 32 of the same batch, and finally opening into the recovery duct 38.
Sur la figure 11, où les références aux éléments semblables à ceux de la figure 9 ont été reprises, chaque micro-canal de capture 36 comprend une portion amont 36a située entre le conduit d'alimentation 37 et l'intersection avec la micro-colonne de fractionnement préalable 32, et une portion aval 36b située entre la micro-colonne de fractionnement préalable 32 et le conduit de récupération 38. La portion amont 36a et la portion aval 36b débouchent sur la micro-colonne de fractionnement préalable 32 en des points décalés, aux extrémités d'un segment de capture 40. La portion amont 36a débouche à l'extrémité aval du segment de capture 40, la portion aval 36b débouchant à l'extrémité amont du segment de capture 40.In FIG. 11, where the references to the elements similar to those of FIG. 9 have been repeated, each capture micro-channel 36 comprises an upstream portion 36a situated between the supply duct 37 and the intersection with the preliminary fractionation micro-column 32, and a downstream portion 36b situated between the preliminary fractionation micro-column 32 and the recovery duct 38. The upstream portion 36a and the downstream portion 36b open onto the preliminary fractionation micro-column 32 at offset points, at the ends of a capture segment 40. The upstream portion 36a opens at the downstream end of the capture segment 40, the downstream portion 36b opening at the upstream end of the capture segment 40.
Lors d'une capture, les constituants séparés de l'échantillon situés au niveau du segment de capture 40 seront capturés. Ainsi, on capture un plus grand nombre de constituants lors d'une même capture.During a capture, the separate components of the sample located at the capture segment 40 will be captured. Thus, a larger number of constituents is captured during the same capture.
Ces moyens de capture avec micro-canal de capture à portion aval et amont décalées peut également être appliqués sur des micro-colonnes de fractionnement principal, comme cela est représenté sur la figure 14.These capture means with offset micro-channel with downstream and upstream portion offset can also be applied to main fractionation micro-columns, as shown in FIG. 14.
Sur la figure 14, où les références aux éléments semblables à ceux de la figure 3 ont été reprises, un support 1 comprend des micro-colonnes de fractionnement 2, des micro-canaux de captures 8 associés munis de portions amonts 8a et de portion avals 8b décalées, débouchant dans chaque micro-colonne de fractionnement 2 respectivement en aval et en amont d'un segment de capture 40.In FIG. 14, where the references to elements similar to those of FIG. 3 have been repeated, a support 1 comprises fractionation micro-columns 2, associated capture micro-channels 8 provided with upstream portions 8a and downstream portion 8b offset, opening into each fractionation micro-column 2 respectively downstream and upstream of a capture segment 40.
La portion aval 8b d'un micro-canal de capture 8 débouche en aval sur une zone de détection 11 munie de micro-leviers 13. Un canal de sortie 45 relie l'ensemble des micro-canaux de capture 8 en aval des zones de détection 11, pour une évacuation des phases mobiles et des constituants non retenus par les micro-leviers sélectifs. Le support 1 comprend un micro-conduit de lavage 46 en communication fluidique avec chacun des micro-canaux de capture 8, directement en amont des zones de détection 11. Lors d'une capture, un éluant de capture, différent de la phase mobile enrichie en échantillon circulant dans les micro-colonnes de fractionnement 2, circule à contre-courant dans le segment de capture 40. L' éluant possède avec les constituants situés dans le segment de capture 40 à l'instant de la capture une affinité différente de la phase mobile. Les moyens de séparation étant cependant les mêmes, i.e. ceux de la micro-colonne de fractionnement 2, la sélectivité obtenue lors du passage de l' éluant de capture dans le segment de capture 40 est différente de la sélectivité obtenue pendant le passage de la phase mobile. En conséquence, pendant la capture, on réalise une séparation secondaire des produits de fractionnement situés dans le segment de capture 40. Pour améliorer encore la détection et la séparation des constituants, on peut prévoir qu'une micro-colonne de fractionnement comprend une portion terminale pourvue d'une sélectivité différente de la sélectivité de la portion amont de la micro- colonne. Compte tenu de la sélectivité de la portion amont, on sait que des constituants possédant certaines caractéristiques migreront plus rapidement et atteindront en premier la portion terminale. La sélectivité de la portion terminale est alors adaptée pour une séparation supplémentaire des constituants parvenant sensiblement au même moment en bout de la micro-colonne de fractionnement.The downstream portion 8b of a capture micro-channel 8 leads downstream to a detection zone 11 provided with micro-levers 13. An outlet channel 45 connects all of the capture micro-channels 8 downstream of the zones of detection 11, for evacuation of the mobile phases and of the constituents not retained by the selective micro-levers. The support 1 comprises a washing micro-duct 46 in fluid communication with each of the capture micro-channels 8, directly upstream of the detection zones 11. During a capture, a capture eluent, different from the enriched mobile phase as a sample circulating in the fractionation micro-columns 2, flows against the current in the capture segment 40. The eluent has, with the constituents located in the capture segment 40 at the time of capture, an affinity different from the mobile phase. However, the separation means being the same, ie those of the fractionation micro-column 2, the selectivity obtained during the passage of the capture eluent in the capture segment 40 is different from the selectivity obtained during the passage of the phase mobile. Consequently, during the capture, a secondary separation of the fractionation products located in the capture segment 40 is carried out. To further improve the detection and the separation of the constituents, provision may be made for a fractionation micro-column to comprise a terminal portion. provided with a selectivity different from the selectivity of the upstream portion of the micro- column. Given the selectivity of the upstream portion, it is known that constituents having certain characteristics will migrate more quickly and reach the terminal portion first. The selectivity of the terminal portion is then adapted for additional separation of the constituents arriving at substantially the same time at the end of the fractionation micro-column.
Cette différence de sélectivité d'une portion d'une micro-colonne de fractionnement peut être appliquée à une micro-colonne de fractionnement, une microcolonne de fractionnement secondaire, une micro-colonne de fractionnement préalable. Par portion terminale, on entend une portion située juste en amont d'une sortie ou de moyens de capture.This difference in selectivity of a portion of a fractionation micro-column can be applied to a fractionation micro-column, a secondary fractionation microcolumn, a prior fractionation micro-column. By terminal portion is meant a portion located just upstream of an outlet or of capture means.
Dans le cas d'une application aux micro-colonnes de fractionnement ou aux micro-colonnes de fractionnement préalable, on pourra de façon avantageuse prévoir une portion terminale à sélectivité différente juste en amont d'un élément terminal de capture d'une micro-colonne de fractionnement préalable. Ainsi, on réalise un fractionnement ou une extraction préalable précis, permettant d'isoler des constituants d'un échantillon à partir d'un nombre de constituants initial très important, pour une détection plus précise par la suite.In the case of an application to fractionation micro-columns or to preliminary fractionation micro-columns, it is advantageous to be able to provide a terminal portion with different selectivity just upstream of a terminal element for capturing a micro-column prior splitting. Thus, a precise fractionation or prior extraction is carried out, making it possible to isolate constituents of a sample from a very large initial number of constituents, for more precise detection thereafter.
Sur la figure 13, où les références aux éléments semblables à ceux de la figure 1 ont été reprises, un support d'analyse 1 comprend des moyens d'alimentation en échantillon et en phase mobile séparés.In FIG. 13, where the references to elements similar to those of FIG. 1 have been repeated, an analysis support 1 comprises means for supplying the sample and the mobile phase separately.
Un support 1 représenté partiellement comprend un canal d'alimentation en échantillon 4 comprenant un orifice d'introduction 4a et un orifice d'évacuation 4b. Les orifices d'introduction 3a des micro-colonnes de fractionnement 2 débouchent dans le canal d'ahmentation en échantillon 4. Le support 1 comprend également un canal d'alimentation en phase mobileA support 1 partially shown comprises a sample supply channel 4 comprising an introduction orifice 4a and a discharge orifice 4b. The introduction orifices 3a of the fractionation micro-columns 2 open into the sample increase channel 4. The support 1 also includes a mobile phase supply channel
41, et des micro-conduits d'alimentation en phase mobile 42 comprenant un orifice d'entrée 43 débouchant dans le canal d'alimentation en phase mobile 41, et un orifice de sortie 44 débouchant dans le canal d'alimentation en échantillon 4. Chaque orifice d'évacuation d'un micro-conduit d'alimentation en phase mobile 42 débouche dans le canal d'alimentation en échantillon 4 en regard d'un orifice d'introduction 3a d'une micro-colonne de fractionnement 2.41, and mobile phase supply micro-conduits 42 comprising an inlet orifice 43 opening into the mobile phase supply channel 41, and an outlet orifice 44 opening into the sample supply channel 4. Each discharge orifice of a mobile phase supply micro-duct 42 opens into the sample supply channel 4 opposite an introduction orifice 3a of a fractionation micro-column 2.
Chaque micro-conduit d'alimentation en phase mobile 42 prolongé par une micro-colonne de fractionnement 2 forme un micro-canal intersectant le canal d' ahmentation en échantillon 4. En d'autres termes, un micro-conduit d'alimentation en phase mobile 42 peut être envisagé comme une portion d'un micro-canal dénuée de moyens de séparation, et située en amont d'une portion de micro-canal munie de moyens de séparation et formant ainsi une micro-colonne de fractionnement 2, le micro-canal d'alimentation en échantillon 4 intersectant l'ensemble des micro-canaux au niveaux de l'orifice d'introduction 3a des micro-colonnes de fractionnement 2.Each mobile phase supply micro-duct 42 extended by a fractionation micro-column 2 forms a micro-channel intersecting the sample-raising channel 4. In other words, a phase supply micro-duct mobile 42 can be envisaged as a portion of a micro-channel devoid of separation means, and located upstream of a portion of micro-channel provided with means of separation and thus forming a fractionation micro-column 2, the sample supply micro-channel 4 intersecting all of the micro-channels at the level of the introduction orifice 3a of the fractionation micro-columns 2.
En fonctionnement, un échantillon circule dans le canal d'alimentation en échantillon 4, de l'orifice d'introduction 4a vers l'orifice d'évacuation 4b. Pour provoquer le passage d'une phase mobile enrichie en échantillon dans les microcolonnes de fractionnement 2, en vue d'une séparation et d'une détection, on provoque une circulation de phase mobile dans les micro-conduits d'alimentation en phase mobile 42. La phase mobile traverse transversalement le canal d'alimentation en échantillon 4 en s' enrichissant en échantillon, puis est récupérée en aval par les micro-colonnes de fractionnement 2.In operation, a sample circulates in the sample supply channel 4, from the introduction orifice 4a to the discharge orifice 4b. To cause the passage of a mobile phase enriched in the sample in the fractionation microcolumns 2, with a view to separation and detection, a circulation of mobile phase is caused in the mobile phase supply micro-conduits 42 The mobile phase crosses the sample supply channel 4 transversely, enriching itself in the sample, then is recovered downstream by the fractionation micro-columns 2.
Les micro-conduits d'alimentation en phase mobile 42 permettent à un instant donné une injection simultanément dans toutes les micro-colonnes de fractionnement 2 d'une même quantité de phase mobile enrichie en échantillon. Une différence de débit de phase mobile enrichie d'une micro-colonne de fractionnement 3 à une autre pourrait entraîner des variations de détection en amont des micro-colonnes de fractionnement 3, notamment si on a prévu une capture simultanée de produits de fractionnement au niveau d'éléments terminaux des micro-colonnes de fractionnement. Dans le cas d'un dispositif d'analyse comprenant un étage d'extraction préalable, on peut bien entendu prévoir en amont de l'étage d'extraction préalable une zone de capture d'échantillon tel que décrit ci-dessus.The mobile phase supply micro-conduits 42 allow at a given instant an injection simultaneously into all of the fractionation micro-columns 2 of the same quantity of mobile phase enriched in sample. A difference in mobile phase flow rate enriched from one fractionation micro-column 3 to another could lead to detection variations upstream of the fractionation micro-columns 3, in particular if provision is made for a simultaneous capture of fractionation products at the level of terminal elements of the fractionation micro-columns. In the case of an analysis device comprising a preliminary extraction stage, it is of course possible to provide, upstream of the preliminary extraction stage, a sample capture zone as described above.
On a décrit indifféremment une détection de protéines ou de peptides. Une cellule biologique comporte un grand nombre de protéines, pouvant générer après digestion un nombre plus grand encore de peptides. Dans le cas où l'on souhaite analyser les peptides, on prévoit une digestion enzymatique préalable de protéines, par exemple par la trypsine.A detection of proteins or peptides has been described indifferently. A biological cell contains a large number of proteins, which can generate an even greater number of peptides after digestion. In the case where it is desired to analyze the peptides, provision is made for prior enzymatic digestion of proteins, for example with trypsin.
Afin d'améliorer la détection des peptides en grand nombre, on peut prévoir en amont de micro-colonnes une ou plusieurs micro-colonnes de tri, et notamment une micro-colonne de tri par chromatographie par exclusion de taille, déjà évoquée auparavant.In order to improve the detection of large numbers of peptides, one or more sorting micro-columns can be provided upstream of micro-columns, and in particular a micro-column for sorting by size exclusion chromatography, already mentioned previously.
Dans la suite de la description, des exemples d'analyses possibles à l'aide d'un dispositif d'analyse selon un aspect de l'invention sont fournis. Exemple 1In the following description, examples of possible analyzes using an analysis device according to one aspect of the invention are provided. Example 1
On peut analyser deux échantillons biologiques, chacun à l'aide de 8 supports du type représenté sur la figure 8. Sur les supports, les échantillons sont séparés par électrochromatographie supplée par pression complémentaire.Two biological samples can be analyzed, each using 8 supports of the type shown in FIG. 8. On the supports, the samples are separated by electrochromatography supplemented by additional pressure.
Par exemple, chacun des supports contients 4 lots de 1000 microcolonnes de fractionnement présentant un gradient de longueur, avec une différence de longueur minimale de 20 microns entre deux micro-colonnes, si bien qu'entre la plus première et la dernière des 1000 micro-colonnes, il existe une différence de longueur de 20 mm. Les 1000 microcolonnes de fractionnement du premier lot présentent des longueurs comprises entre 12 et 14 cm. Les 1000 microcolonnes du deuxième lot présentent des longueurs comprises entre 14 et 16 cm. Les 1000 microcolonnes du troisième lot présentent des longueurs comprises entre 16 et 18 cm. Les 1000 microcolonnes du quatrième lot présentent des longueurs comprises entre 18 et 20 cm. On peut nommer un tel format de support par l'abréviation (FC, 4, 1000, 20, 12-14, 14- 16, 16-18, 18-20) ou de façon plus compacte par (FC, 4, 1000, 20, 12-20).For example, each of the supports contains 4 lots of 1000 fractionation microcolumns having a length gradient, with a minimum length difference of 20 microns between two micro-columns, so that between the earliest and the last of the 1000 micro-columns columns, there is a length difference of 20 mm. The 1000 microcolumns in the first batch have lengths between 12 and 14 cm. The 1000 microcolumns in the second batch have lengths between 14 and 16 cm. The 1000 microcolumns in the third batch have lengths between 16 and 18 cm. The 1000 microcolumns in the fourth batch have lengths between 18 and 20 cm. We can name such a support format by the abbreviation (FC, 4, 1000, 20, 12-14, 14- 16, 16-18, 18-20) or more compactly by (FC, 4, 1000, 20, 12-20).
Les supports employés comprennent des micro-canaux de capture. Les produits de fractionnement adsorbés à l'instant t sur une micro-colonne de fractionnement à l'endroit d'intersection avec ledit micro-canal de capture correspondant sont capturés simultanément, et subissent des micro ou nano-élutions secondaires, orthogonales, parallèles, terminales, simultanées.The media used include micro-capture channels. The fractionation products adsorbed at time t on a fractionation micro-column at the point of intersection with said corresponding capture micro-channel are captured simultaneously, and undergo secondary, orthogonal, parallel micro or nano-elutions, terminal, simultaneous.
Les micro-canaux de capture débouchent sur des zones de détection à microleviers avec détection optique.The capture micro-channels lead to detection zones with microlevers with optical detection.
Les phases stationnaires des micro-colonnes de fractionnement du premier support (FC, 4, 1000, 20, 12-20) sont greffées avec des molécules chaînes alkyl C30. Un tel support peut être dénomé (FC, 4, 1000, 20, 12-20)-C30.The stationary phases of the fractionation micro-columns of the first support (FC, 4, 1000, 20, 12-20) are grafted with C30 alkyl chain molecules. Such a support can be called (FC, 4, 1000, 20, 12-20) -C30.
Les phases stationnaires des micro-colonnes de fractionnement du second support (FC, 4, 1000, 20, 12-20) sont greffées avec des molécules butyl. (FC, 4, 1000, 20, 12-20)-butyl. Les phases stationnaires des micro-colonnes de fractionnement du troisième support (FC, 4, 1000, 20, 12-20) sont greffées avec des molécules cyclo-hexyl. Un tel support peut être dénomé (FC, 4, 1000, 20, 12-20)-cyclohexyl.The stationary phases of the fractionation micro-columns of the second support (FC, 4, 1000, 20, 12-20) are grafted with butyl molecules. (FC, 4, 1000, 20, 12-20) -butyl. The stationary phases of the fractionation micro-columns of the third support (FC, 4, 1000, 20, 12-20) are grafted with cyclo-hexyl molecules. Such a support can be called (FC, 4, 1000, 20, 12-20) -cyclohexyl.
Les phases stationnaires des micro-colonnes de fractionnement du quatrième support (FC, 4, 1000, 20, 12-20) sont greffées avec des molécules phenyl. Un tel support peut être dénomé (FC, 4, 1000, 20, 12-20)- phenyl. Les phases stationnaires des micro-colonnes de fractionnement du cinquième support (FC, 4, 1000, 20, 12-20) sont greffées avec des molécules ethyl. Un tel support peut être dénomé (FC, 4, 1000, 20, 12-20)-ethyl.The stationary phases of the fractionation micro-columns of the fourth support (FC, 4, 1000, 20, 12-20) are grafted with phenyl molecules. Such a support can be called (FC, 4, 1000, 20, 12-20) - phenyl. The stationary phases of the fractionation micro-columns of the fifth support (FC, 4, 1000, 20, 12-20) are grafted with ethyl molecules. Such a support can be called (FC, 4, 1000, 20, 12-20) -ethyl.
Les phases stationnaires des micro-colonnes de fractionnement du sixième support (FC, 4, 1000, 20, 12-20) sont greffées avec des molécules amino-propyl. Un tel support peut être dénomé (FC, 4, 1000, 20, 12-20)-amino-propyl.The stationary phases of the fractionation micro-columns of the sixth support (FC, 4, 1000, 20, 12-20) are grafted with amino-propyl molecules. Such a support can be called (FC, 4, 1000, 20, 12-20) -amino-propyl.
Les phases stationnaires des micro-colonnes de fractionnement du septième support (FC, 4, 1000, 20, 12-20) sont greffées avec des molécules dihydroxypropyl. Un tel support peut être dénomé (FC, 4, 1000, 20, 12-20)-dihydroxypropyl Les phases stationnaires des micro-colonnes de fractionnement du huitième support (FC, 4, 1000, 20, 12-20) sont greffées avec des molécules cyanopropyl. Un tel support peut être dénomé (FC, 4, 1000, 20, 12-20)- cyanopropyl.The stationary phases of the fractionation micro-columns of the seventh support (FC, 4, 1000, 20, 12-20) are grafted with dihydroxypropyl molecules. Such a support can be called (FC, 4, 1000, 20, 12-20) -dihydroxypropyl The stationary phases of the fractionation micro-columns of the eighth support (FC, 4, 1000, 20, 12-20) are grafted with cyanopropyl molecules. Such a support can be called (FC, 4, 1000, 20, 12-20) - cyanopropyl.
Chaque zone de détection associé à un micro-canal de capture comprend huit microleviers chacun muni d'un revêtement spécifique. Par exemple, le premier peut avoir un revêtement à base de chaînes alkyl C30, le deuxième un revêtement à base de chaînes octadecyl, le troisième un revêtement à base de chaînes octyl, le quatrième à bases de chaînes butyl, le quatrième à base de chaînes phenyl, le cinquième à base de chaînes cyclo-hexyl, le cinquième à base de chaînes ethyl, le sixième à base de chaînes amino-propyles, le septième à base de chaînes dihyroxypropyl, le huitième à base de chaînes cyanopropyl.Each detection zone associated with a capture micro-channel comprises eight micro-levers each provided with a specific coating. For example, the first may have a coating based on C30 alkyl chains, the second a coating based on octadecyl chains, the third a coating based on octyl chains, the fourth based on butyl chains, the fourth based on chains phenyl, the fifth based on cyclo-hexyl chains, the fifth based on ethyl chains, the sixth based on amino-propyl chains, the seventh based on dihyroxypropyl chains, the eighth based on cyanopropyl chains.
Les solvants de base pour les élutions primaires des chromatographies de fractionnement effectuées sur les supports de format (FC 4, 1000, 20, 12-20) peuvent être des mélanges ternaires eau, acide trifluoro-acétique (TFA), acétonitrile. On peut prévoir six pas d'élution primaire, l'éluant utilisé lors de chaque pas présentant successivement les variantes de composition suivantes : (eau, 10% acétonitrile, TFA 0,1%). (eau, 15% acétonitrile, TFA 0,1%), (eau, 20% acétonitrile, TFA 0,1%), (eau, 25% acétonitrile, TFA 0,1%), (eau, 30% acétonitrile, TFA 0,1%), (eau, 35% acétonitrile, TFA 0,1%).The basic solvents for the primary elutions of the fractionation chromatographies carried out on format supports (FC 4, 1000, 20, 12-20) can be ternary mixtures of water, trifluoroacetic acid (TFA), acetonitrile. Six primary elution steps can be provided, the eluent used in each step having successively the following composition variants: (water, 10% acetonitrile, 0.1% TFA). (water, 15% acetonitrile, 0.1% TFA), (water, 20% acetonitrile, 0.1% TFA), (water, 25% acetonitrile, 0.1% TFA), (water, 30% acetonitrile, TFA 0.1%), (water, 35% acetonitrile, 0.1% TFA).
On prévoit cinq paliers d'élution secondaire pour chaque palier d'élution primaire avec des éluants présentant successivement les variantes de composition suivantes : (eau, 15% acétonitrile, TFA 0,1%), (eau, 16% acétonitrile, TFA 0,1%), (eau,Five secondary elution stages are provided for each primary elution stage with eluents having successively the following composition variants: (water, 15% acetonitrile, TFA 0.1%), (water, 16% acetonitrile, TFA 0, 1%), (water,
17% acétonitrile, TFA 0,1%), (eau, 18% acétonitrile, TFA 0,1%), (eau, 19% acétonitrile, TFA 0,1%).17% acetonitrile, TFA 0.1%), (water, 18% acetonitrile, TFA 0.1%), (water, 19% acetonitrile, TFA 0.1%).
On réalise pour chaque échantillon les séries d'empreintes successives évoquées ci-dessus. La série d'empreintes successives du premier échantillon est comparée ensuite à la série d'empreintes successives du deuxième échantillon, les séries d'empreintes de détection étant ensuite archivées dans une base de données informatiques.The successive series of imprints mentioned above are produced for each sample. The series of successive fingerprints of the first sample is then compared to the series of successive fingerprints of the second sample, the series detection fingerprints then being archived in a computer database.
Là où sont détectées des différences, les fractionnements sont prélevés et analysés par l'une des nombreuses méthodes d'analyse connues de l'Homme de l'Art. Le procédé est applicable à toute recherche d'expression différentielle de protéines pour un tissu donné notamment pour la comparaison d'un individu sain et d'un individu souffrant d'une pathologie. Il est applicable également à la comparaison de l'expression des protéines dans deux situations physiologiques différentes. Il aussi applicable à la comparaison de l'expression de protéines chez deux souches de micro- organismes (virus, bactéries, levures), ou applicables à la détection d'expressions différentielles de protéines sur micro-organismes (virus, bactéries, levures) soumis à des stimuli précis.Where differences are detected, the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art. The method is applicable to any search for differential expression of proteins for a given tissue, in particular for the comparison of a healthy individual and an individual suffering from a pathology. It is also applicable to the comparison of protein expression in two different physiological situations. It is also applicable to the comparison of protein expression in two strains of microorganisms (viruses, bacteria, yeasts), or applicable to the detection of differential expression of proteins on microorganisms (viruses, bacteria, yeasts) subjected to specific stimuli.
Exemple 2Example 2
On peut utiliser le dispositif pour comparer des empreintes de protéines basiques entre deux échantillons. Chaque échantillon est analysé sur un support du type représenté sur la figure 8, et de format (FC, 4, 1000, 20, 12-20), conformément à la dénomination utilisée dans l'exemple 1.The device can be used to compare fingerprints of basic proteins between two samples. Each sample is analyzed on a support of the type shown in FIG. 8, and of format (FC, 4, 1000, 20, 12-20), in accordance with the name used in Example 1.
Ces supports sont du type en plastique et des micro-colonnes de fractionnement monolithes macroporeuses synthétisées in situ. Les micro-colonnes de fractionnement sont à caractère fortement zwitterionique et contiennent des copolymeres à base de sulfoalkylbetaine (N,N-dimethyl-N-methacryloyloxyethyl-N-(3- sulfopropyl) ammonium betaine (Cf. Niklund C, Sjorgen A, Irgum K, Νes I. Anal. Chem. 2001. Feb 1, 73,(3) , 444-52).These supports are of the plastic type and macroporous monolithic fractionation micro-columns synthesized in situ. The fractionation micro-columns are strongly zwitterionic in nature and contain copolymers based on sulfoalkylbetaine (N, N-dimethyl-N-methacryloyloxyethyl-N- (3-sulfopropyl) ammonium betaine (Cf. Niklund C, Sjorgen A, Irgum K , Νes I. Anal. Chem. 2001. Feb 1, 73, (3), 444-52).
Les protéines basiques sont séparées dans les micro-colonnes de fractionnement selon différentes méthodes avec des éluant primaires (éluant A :eau; éluant B: eau, 10 mM phosphate de sodium). Les produits de fractionnement sont séparés dans des micro-colonnes de fractionnement secondaire située en aval de moyens de capture. Les élutions secondaires sont modulées par des ions thiocyanate (éluant primaire + 10 mM thiocyanate) ou par des ions perchlorate (éluant primaire + 10 mMperchlorate).The basic proteins are separated in the fractionation micro-columns according to different methods with primary eluent (eluent A: water; eluent B: water, 10 mM sodium phosphate). The fractionation products are separated in secondary fractionation micro-columns located downstream of capture means. Secondary elutions are modulated by thiocyanate ions (primary eluent + 10 mM thiocyanate) or by perchlorate ions (primary eluent + 10 mMperchlorate).
La série d'empreintes successives du premier échantillon est comparée à la série d'empreintes successives du deuxième échantillon, les séries d'empreintes étant ensuite archivées dans une base de données informatiques. Là où sont détectées des différences, les fractionnements sont prélevés et analysés par l'une des nombreuses méthodes d'analyse connues de l'Homme de l'Art.The series of successive fingerprints of the first sample is compared with the series of successive fingerprints of the second sample, the series of fingerprints then being archived in a computer database. Where differences are detected, the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art.
Exemple 3Example 3
On peut comparer spécifiquement les empreintes de peptides et de protéines membranaires de deux échantillons.The fingerprints of peptides and membrane proteins from two samples can be compared specifically.
Par exemple, on prévoit que chaque échantillon est analysé sur deux support du type représenté sur la figure 8, possédant un format du type (FC, 4, 1000, 20, 12-20). Les supports intégrés possède des micro-colonnes de fractionnement greffées avec des chaînes alkyles C4.For example, it is expected that each sample is analyzed on two supports of the type shown in FIG. 8, having a format of the type (FC, 4, 1000, 20, 12-20). The integrated supports have fractionation micro-columns grafted with C4 alkyl chains.
Dans chaque premier support d'analyse d'un échantillon, les peptides membranaires sont dissouts dans le dichlorométhane (CH2C12)-hexafluoro-2-propanol (HFIP) (4:1) contenant des traces de pyridine, puis séparés dans micro-colonnes de fractionnement en utilisant différentes élutions primaires lors de différentes empreintes successives.In each initial support for analyzing a sample, the membrane peptides are dissolved in dichloromethane (CH2C12) -hexafluoro-2-propanol (HFIP) (4: 1) containing traces of pyridine, then separated in micro-columns of fractionation using different primary elutions in different successive fingerprints.
Les élutions primaires sont à base de mélanges de l' éluant A (acide formique-eau (2:3)) d'une part et de l'éluant B (acide formique -2-propanol (4:1)) d'autre part. Les élutions primaires présentent successivement les variantes de composition suivantes : (A 100%, B 0%); (A 80%, B 20%); (A 60%, B 40%); (A 40%,The primary elutions are based on mixtures of eluent A (formic acid-water (2: 3)) on the one hand and eluent B (formic acid -2-propanol (4: 1)) on the other go. The primary elutions successively present the following composition variants: (A 100%, B 0%); (A 80%, B 20%); (A 60%, B 40%); (At 40%,
B 60%); (A 20%, B 80%); (A 0%, B 100%);B 60%); (A 20%, B 80%); (A 0%, B 100%);
A chaque fois qu'une séparation primaire est effectuée, on procède à trois paliers d'élution secondaire dans les micro-colonnes de fractionnement secondaire, avec des élutions secondaires présentant successivement les variantes de composition suivantes : (A 95%, B 5%); (A 90%, B 10%); (A 85%, B 15%).Each time a primary separation is carried out, three stages of secondary elution are carried out in the secondary fractionation micro-columns, with secondary elutions successively having the following composition variants: (A 95%, B 5%) ; (A 90%, B 10%); (A 85%, B 15%).
Dans le deuxième support d'analyse d'un échantillon, les protéines membranaires, après extraction au Triton X-114, précipitation à l'éthanol à 90% et redissolution dans l'acide formique à 65%, sont séparées dans les micro-colonnes de fractionnement en utilisant différentes élutions primaires lors de différentes empreintes successives.In the second analysis support for a sample, the membrane proteins, after extraction with Triton X-114, precipitation with 90% ethanol and redissolution in 65% formic acid, are separated in the micro-columns fractionation using different primary elutions in different successive fingerprints.
Les élutions primaires sont à base de mélanges de l'éluant A (acide formique-eau (65:35)) et de l'éluant B (acétonitrile-eau (65:35)). Les élutions primaires présentent successivement les variantes de composition suivantes : (A 100%, B 0%); (A 80%, B 20%); (A 60%, B 40%); (A 40%, B 60%); (A 20%, B 80%); (A 0%, B 100%).The primary elutions are based on mixtures of eluent A (formic acid-water (65:35)) and eluent B (acetonitrile-water (65:35)). The primary elutions successively present the following composition variants: (A 100%, B 0%); (A 80%, B 20%); (A 60%, B 40%); (A 40%, B 60%); (A 20%, B 80%); (A 0%, B 100%).
A chaque fois qu'une séparation primaire est effectuée, on procède à trois paliers d'élution secondaire dans les micro-colonnes de fractionnement secondaire, avec des élutions secondaires présentant successivement les variantes de composition suivantes : (A 95%, B 5%); (A 90%, B 10%); (A 85%, B 15%)..Each time a primary separation is carried out, three stages of secondary elution are carried out in the secondary fractionation micro-columns, with secondary elutions successively presenting the following variants of composition: (A 95%, B 5%); (A 90%, B 10%); (A 85%, B 15%) ..
La série d'empreintes successives du premier échantillon est comparée ensuite à la série d'empreintes successives du deuxième échantillon, les séries d'empreintes de détection étant ensuite archivées dans une base de données informatiques. Là où sont détectées des différences, les fractionnements sont prélevés et analysés par l'une des nombreuses méthodes d'analyse connues de l'Homme de l'Art.The series of successive fingerprints of the first sample is then compared to the series of successive fingerprints of the second sample, the series of detection fingerprints then being archived in a computer database. Where differences are detected, the splits are removed and analyzed by one of the many methods of analysis known to those skilled in the art.
Exemple 4Example 4
On peut utiliser les données acquises lors d'analyses effectuées à l'aide de dispositif selon les exemples 1 à 3 décrits ci-dessus, pour configurer un test rapide sur consommable miniaturisé. Pour un tel test, on peut utiliser un dispositif muni de supports pourvus seulement des microcolonnes de fractionnement où de nombreuses comparaisons ont montré des différences d'empreintes reproductibles pour une pathologie donnée.The data acquired during analyzes carried out using a device according to examples 1 to 3 described above can be used to configure a rapid test on miniaturized consumable. For such a test, it is possible to use a device provided with supports provided only with fractionating microcolumns where numerous comparisons have shown differences in reproducible fingerprints for a given pathology.
Un individu sain montre une empreinte A, l'individu atteint de la pathologie montrant une empreinte B. Un premier support (sA) est dédié à la reconnaissance de l'empreinte A, un deuxième support (sB) étant dédié à la reconnaissance de l'empreinte B.A healthy individual shows an A imprint, the individual suffering from pathology showing a B imprint. A first support (sA) is dedicated to the recognition of the A imprint, a second support (sB) being dedicated to the recognition of the 'imprint B.
Tant pour la fabrication dudit support (sA) que pour la fabrication dudit support (sB), les seules micro-colonnes où il y a toujours une même différence sont conservées. Les supports restent pourvus chacune de micro-canaux de capture débouchant sur des zones de détection munies de micro-leviers.Both for the manufacture of said support (sA) and for the manufacture of said support (sB), the only micro-columns where there is always the same difference are kept. The supports each remain provided with capture micro-channels leading to detection zones provided with micro-levers.
Dans ce test ciblé sur ladite pathologie, le nombre de microcolonnes, de microcanaux et de microleviers est très réduit. Les micro-colonnes de fractionnement choisies, plus précisément les sélectivité des méthodes de séparation en relation avec les composition des phases solides et liquides utilisées, sont adaptés pour mettre en évidence la présence dans un échantillon de protéines dont la présence ou l'absence est caractéristique de la pathologie ciblée.In this test targeted at said pathology, the number of microcolumns, microchannels and microlevers is very small. The fractionation micro-columns chosen, more specifically the selectivity of the separation methods in relation to the composition of the solid and liquid phases used, are adapted to demonstrate the presence in a sample of proteins of which the presence or absence is characteristic. of the targeted pathology.
Un dispositif d'analyse est adapté pour l'analyse comparative chimique ou biochimique de deux échantillons de nature chimique, ou bien deux échantillons de nature biochimique comme des extraits cellulaires crus ou issus d'une extraction préalable ou ayant subi une digestion enzymatique.An analysis device is suitable for the comparative chemical or biochemical analysis of two samples of a chemical nature, or else two samples of a biochemical nature such as cell extracts which are raw or derived from a prior extraction or which have undergone enzymatic digestion.
Un échantillon biologique est caractérisé en particulier par sa composition en chacun(e) des protéines, glycoproteines, phosphoprotéines, lipoprotéines, lipides, polysaccharides, hormones, vitamines synthétisés de manière permanente ou occasionnelle selon le tissu ou l'état physiologique ou pathologique envisagée. Un dispositif d'analyse, selon un aspect de l'invention, permet la détection de ces constituants par séparation et par l'obtention d'empreintes. Un dispositif d'analyse peut s'appuyer sur un gradient de longueur d'un grand nombre de micro-canaux ou de micro-colonnes de séparation par exemple par micro-électrophorèse, micro-chromatographie ou micro-électrochromatographie.A biological sample is characterized in particular by its composition in each of the proteins, glycoproteins, phosphoproteins, lipoproteins, lipids, polysaccharides, hormones, vitamins synthesized permanently or occasionally depending on the tissue or the physiological or pathological condition envisaged. An analysis device, according to one aspect of the invention, allows the detection of these constituents by separation and by obtaining fingerprints. An analysis device can rely on a length gradient of a large number of micro-channels or micro-separation columns, for example by micro-electrophoresis, micro-chromatography or micro-electrochromatography.
Un ensemble de micro-colonnes de fractionnement peut être associé à un deuxième, voire un troisième ensemble de micro-canaux ou micro-colonnes, chacun ou chacune des micro-canaux ou microcolonnes de séparation du premier ensemble étant individuellement couplé à chacun ou chacune des micro-canaux ou micro-colonnes de séparation dudit deuxième ensemble de micro-canaux ou micro-colonnes.A set of fractionation micro-columns can be associated with a second or even a third set of micro-channels or micro-columns, each or each of the micro-channels or microcolumns of separation of the first set being individually coupled to each or each of the micro-channels or micro-columns for separating said second set of micro-channels or micro-columns.
On a décrit une détection par micro-leviers sélectifs qui permet d'adapter la sélectivité des micro-leviers à la sélectivité de micro-colonnes de fractionnement auxquelles ils sont associé. Un détection supplémentaire peut être effectuée par spectrométrie de masse. Le dispositif d'analyse peut également utiliser des autres moyens de détection connus de l'Homme de l'Art tels que fluorescence, résonance plasmonique de surface (SPR), résonance magnétique nucléaire (NMR), électrochimie, spectrophotométrie, cette liste n'étant pas limitative. Une détection supplémentaire peut être effectuée par spectrométrie de masse. Le dispositif d'analyse peut également utiliser des autres moyens de détection connus de l'Homme de l'Art tels que fluorescence, résonance plasmonique de surfaceA detection by selective micro-levers has been described which makes it possible to adapt the selectivity of the micro-levers to the selectivity of the fractionation micro-columns with which they are associated. Additional detection can be performed by mass spectrometry. The analysis device can also use other detection means known to those skilled in the art such as fluorescence, surface plasmon resonance (SPR), nuclear magnetic resonance (NMR), electrochemistry, spectrophotometry, this list not being not limiting. Additional detection can be performed by mass spectrometry. The analysis device can also use other means of detection known to those skilled in the art such as fluorescence, surface plasmon resonance.
(SPR), résonance magnétique nucléaire (NMR), électrochimie, spectrophotométrie, cette liste n'étant pas hmitative. Grâce à l'invention, on obtient un dispositif d'analyse permettant des séparations des constituants d'un échantillon, selon des sélectivités différentes, et une détection des constituants.(SPR), nuclear magnetic resonance (NMR), electrochemistry, spectrophotometry, this list is not hmitative. Thanks to the invention, an analysis device is obtained allowing separation of the constituents of a sample, according to different selectivities, and detection of the constituents.
On peut également réaliser des séparations successives avec des sélectivités différentes pour mieux séparer des constituants d'un échantillon. Un dispositif d'analyse permet une analyse exhaustive et rapide d'un échantillon et sa comparaison avec un autre échantillon.It is also possible to carry out successive separations with different selectivities in order to better separate the constituents of a sample. An analysis device allows an exhaustive and rapid analysis of a sample and its comparison with another sample.
La détection par micro-leviers reliés à des moyens d'analyse permet un stockage et une comparaison de données enregistrées. Un dispositif selon l'invention est adapté pour une miniaturisation. Bien entendu, l'invention n'est pas limitée aux modes de réalisation et aux variantes décrits ci-dessus. Des modifications peuvent être apportées sans sortir du cadre de l'invention. Detection by micro-levers linked to analysis means allows storage and comparison of recorded data. A device according to the invention is suitable for miniaturization. Of course, the invention is not limited to the embodiments and to the variants described above. Modifications can be made without departing from the scope of the invention.

Claims

Revendications claims
1 . Dispositif d'analyse chimique ou biochimique d'échantillons biologique ou chimique, notamment pour une analyse comparative d'au moins deux échantillons, comprenant une pluralité de micro-colonnes de fractionnement1. Device for chemical or biochemical analysis of biological or chemical samples, in particular for a comparative analysis of at least two samples, comprising a plurality of fractionation micro-columns
(2) de constituants d'un échantillon, chaque micro-colonne de fractionnement (2) comprenant au moins une portion de micro-canal munie de moyens de séparation intermédiaires, la portion de micro-canal comprenant un orifice d'introduction d'une phase mobile enrichie en échantillon et un orifice d'évacuation situé à une extrémité terminale, caractérisé par le fait qu'il comprend des moyens fluidiques de capture (7) de produits de fractionnement au niveau d'un élément terminal de chaque micro-colonne de fractionnement (2) situé en amont de son orifice d'évacuation, des micro-canaux de capture destinés à récupérer les produits de fractionnement capturés, et des ensembles de micro-leviers sélectifs (13) associés aux micro-colonnes de fractionnement (2) et situés en aval des micro-canaux de capture, un micro-levier (13) comportant des moyens de détection reliés à des moyens d'analyse.(2) of constituents of a sample, each fractionation micro-column (2) comprising at least one portion of micro-channel provided with intermediate separation means, the portion of micro-channel comprising an orifice for introducing a mobile phase enriched in sample and a discharge orifice located at a terminal end, characterized in that it comprises fluidic means for capturing (7) fractionation products at the level of a terminal element of each micro-column of fractionation (2) located upstream of its discharge orifice, capture micro-channels intended to recover the captured fractionation products, and sets of selective micro-levers (13) associated with the fractionation micro-columns (2) and located downstream of the capture micro-channels, a micro-lever (13) comprising detection means connected to analysis means.
2. Dispositif selon la revendication 1, caractérisé par le fait qu'une micro-colonne de fractionnement (2) ou un groupe (3) de micro-colonnes de fractionnement de même longueur diffère par sa longueur des autres micro- colonnes de fractionnement (2) ou groupes (3) de micro-colonnes de fractionnement, les éléments terminaux étant situés sur chaque micro-colonne de fractionnement (2) à une distance donnée de l'extrémité terminale de la microcolonne de fractionnement (2).2. Device according to claim 1, characterized in that a fractionation micro-column (2) or a group (3) of fractionation micro-columns of the same length differs in its length from the other fractionation micro-columns ( 2) or groups (3) of fractionation micro-columns, the terminal elements being located on each fractionation micro-column (2) at a given distance from the terminal end of the fractionation microcolumn (2).
3 . Dispositif selon l'une quelconque des revendications 1 ou 2, caractérisé par le fait que chaque micro-colonne de fractionnnement (2) diffère d'une micro-colonne de fractionnement (2) immédiatement plus longue par un élément de longueur donné.3. Device according to either of Claims 1 and 2, characterized in that each fractionation micro-column (2) differs from an immediately longer fractionation micro-column (2) by an element of given length.
4. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait qu'il comprend des micro-colonnes de fractionnement secondaire (20) situées en aval des moyens fluidiques de capture (7) et en amont de l'ensemble de micro-leviers (13) associé à une micro-colonne de fractionnement (2), et destinées au fractionnement secondaire des produits de fractionnement capturés.4. Device according to any one of the preceding claims, characterized in that it comprises secondary fractionation micro-columns (20) located downstream of the fluidic capture means (7) and upstream of the set of micro-levers (13) associated with a fractionation micro-column (2), and intended for the secondary fractionation of the captured fractionation products.
5. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait qu'il comprend plusieurs lots de micro-colonnes de fractionnement (2), chaque lot de micro-colonnes de fractionnement (2) possédant une sélectivité déterminée par les moyens de séparation de ses micro-colonnes de fractionnement (2) comprenant une phase stationnaire revêtue ou non et/ou des moyens électriques de séparation.5. Device according to any one of the preceding claims, characterized in that it comprises several batches of fractionation micro-columns (2), each batch of fractionation micro-columns (2) having a selectivity determined by the means for separating its fractionation micro-columns (2) comprising a stationary phase coated or not and / or electrical separation means.
6. Dispositif selon la revendication 5, caractérisé par le fait qu'il comprend un support (1) portant plusieurs lots de micro-colonnes de fractionnement (2), des moyens de captures (7) et des ensembles de micro-leviers (13) associés, et un canal d'alimentation de l'ensemble des lots de micro-colonnes de fractionnement.6. Device according to claim 5, characterized in that it comprises a support (1) carrying several lots of fractionation micro-columns (2), capture means (7) and sets of micro-levers (13 ) associated, and a supply channel for all the lots of fractionation micro-columns.
7. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait que les micro-leviers sélectifs (13) comprennent des moyens de détection en fonction de leur état de surface ou de l'état de surface d'un revêtement, de leur nature chimique ou de la nature chimique d'un revêtement.7. Device according to any one of the preceding claims, characterized in that the selective micro-levers (13) comprise detection means according to their surface state or the surface state of a coating, of their chemical nature or the chemical nature of a coating.
8. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait que les micro-colonnes (2) présentent un diamètre compris entre 1 microns (μm) et 100 microns (μm).8. Device according to any one of the preceding claims, characterized in that the micro-columns (2) have a diameter between 1 microns (μm) and 100 microns (μm).
9. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait qu'il comprend un support de fractionnement (1) portant les micro-colonnes de fractionnement (2) et un support de détection (8) portant les micro-leviers (13), les supports étant sensiblement plans, et les supports étant sensiblement parallèles ou perpendiculaires entre eux.9. Device according to any one of the preceding claims, characterized in that it comprises a fractionation support (1) carrying the fractionation micro-columns (2) and a detection support (8) carrying the micro-levers (13), the supports being substantially planar, and the supports being substantially parallel or perpendicular to each other.
10. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait qu'il comprend en amont des micro-colonnes de fractionnement (2) au moins un étage de fractionnement préalable comprenant au moins une micro-colonne de fractionnement préalable (32), des moyens de capture fluidiques (36, 37, 38) au niveau d'un élément terminal de la microcolonne de fractionnement préalable (2), et un canal de récupération des extraits préalables prévu pour amener les extraits préalables vers les micro-colonnes de fractionnement.10. Device according to any one of the preceding claims, characterized in that it comprises, upstream of the fractionation micro-columns (2) at least one prior fractionation stage comprising at least less a prior fractionation micro-column (32), fluidic capture means (36, 37, 38) at a terminal element of the prior fractionation microcolumn (2), and a channel for recovering the preliminary extracts provided to bring the previous extracts to the fractionation micro-columns.
11. Dispositif selon la revendication 10, caractérisé par le fait qu'un étage de fractionnement préalable comprend une pluralité de micro-colonnes de fractionnement préalable (32), chacune intersectée par un micro-canal de capture (36), les micro-canaux de capture (36) étant reliés à un canal de récupération (38).11. Device according to claim 10, characterized in that a prior fractionation stage comprises a plurality of micro-columns of prior fractionation (32), each intersected by a capture micro-channel (36), the micro-channels capture (36) being connected to a recovery channel (38).
12. Dispositif selon la revendication 10, caractérisé par le fait qu'un étage de fractionnement préalable comprend une pluralité de micro-colonnes de fractionnement préalable (32), et un micro-canal de capture (36) intersectant successivement les micro-colonnes de fractionnement préalable, et débouchant dans un canal de récupération (38).12. Device according to claim 10, characterized in that a preliminary fractionation stage comprises a plurality of micro-columns of prior fractionation (32), and a capture micro-channel (36) successively intersecting the micro-columns of prior fractionation, and opening into a recovery channel (38).
13. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait qu'une micro-colonne de fractionnement comprend une portion terminale munie de moyens de séparation différents des moyens de séparation intermédiaires.13. Device according to any one of the preceding claims, characterized in that a fractionation micro-column comprises an end portion provided with separation means different from the intermediate separation means.
14. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait que des moyens de capture fluidique associés à une micro- colonne de fractionnement (3, 32) comprennent un micro-canal de capture (8, 36) comprenant une portion amont (8a, 36a) débouchant à une extrémité aval d'un segment de capture (40) de la micro-colonne (3, 32), et une portion aval (8b, 36b) débouchant à une extrémité amont du segment de capture (40).14. Device according to any one of the preceding claims, characterized in that fluid capture means associated with a fractionation micro-column (3, 32) comprise a capture micro-channel (8, 36) comprising a portion upstream (8a, 36a) opening at a downstream end of a capture segment (40) of the micro-column (3, 32), and a downstream portion (8b, 36b) opening at an upstream end of the capture segment ( 40).
15. Dispositif selon l'une quelconque des revendications précédentes, caractérisé par le fait qu'il comprend un micro-conduit (70) de lavage de microleviers sélectifs débouchant dans des micro-canaux de capture (8) directement en amont de micro-leviers sélectifs. 15. Device according to any one of the preceding claims, characterized in that it comprises a micro-duct (70) for washing selective microlevers opening into micro-capture channels (8) directly upstream of micro-levers selective.
16. Ensemble d'analyse comparative chimique ou biochimique d'au moins deux échantillons biologiques ou chimiques caractérisé par le fait qu'il comprend au moins deux dispositifs comprenant une pluralité de micro-colonnes de fractionnement (2) de constituants d'un échantillon, chaque micro-colonne de fractionnement (2) comprenant au moins une portion de micro-canal munie de moyens de séparation intermédiaires, la portion de micro-canal comprenant un orifice d'introduction d'une phase mobile enrichie en échantillon et un orifice d'évacuation situé à une extrémité terminale, des moyens fluidiques de capture (7) de produits de fractionnement au niveau d'un élément terminal de chaque micro- colonne de fractionnement (2) situé en amont de son orifice d'évacuation, des micro-canaux de capture destinés à récupérer les produits de fractionnement capturés, et des ensembles de micro-leviers sélectifs (13) associés aux microcolonnes de fractionnement (2) et situés en aval des micro-canaux de capture, un micro-levier (13) comportant des moyens de détection reliés à des moyens d'analyse.16. A set of chemical or biochemical comparative analysis of at least two biological or chemical samples, characterized in that it comprises at least two devices comprising a plurality of fractionation micro-columns (2) of constituents of a sample, each fractionation micro-column (2) comprising at least one micro-channel portion provided with intermediate separation means, the micro-channel portion comprising an orifice for introducing a mobile phase enriched in sample and an orifice for evacuation situated at a terminal end, fluidic means of capture (7) of fractionation products at the level of a terminal element of each micro-fractionation column (2) located upstream from its evacuation orifice, of micro-channels of capture intended to recover the fractionation products captured, and sets of selective micro-levers (13) associated with the fractionation microcolumns (2) and located downstream micro-capture channels, a micro-lever (13) comprising detection means connected to analysis means.
1 7 Procédé d'analyse chimique ou biochimique d'échantillons biologique ou chimique, caractérisé par le fait qu'on réalise des fractionnements différentiels d'une phase mobile enrichie en échantillon, on capture simultanément différents produits de fractionnement obtenus, et on analyse chacun des produits de fractionnement à l'aide d'un ensemble de micro-leviers sélectifs.1 7 Process for chemical or biochemical analysis of biological or chemical samples, characterized in that differential fractionations of a mobile phase enriched in sample are carried out, different fractionation products obtained are simultaneously captured, and each of the fractionation products using a set of selective micro-levers.
18. Procédé selon la revendication 17, caractérisé par le fait que l'on fractionne un produit de fractionnement capturé avant de l'analyser.18. The method of claim 17, characterized in that a captured fractionation product is fractionated before analyzing it.
19. Procédé selon l'une quelconque des revendications 17 ou 18, caractérisé par le fait que l'on détecte des constituants d'un produit de fractionnement à l'aide des micro-leviers (13), selon des caractéristiques de polarité, solvophobicité ou porosité du matériau qui les constitue ou d'un revêtement des micro-leviers, ou selon des caractéristiques de polarité, solvophobicité, échange d'ion ou affinité avec des groupement fonctionnels greffés sur les micro-leviers. 19. Method according to any one of claims 17 or 18, characterized in that components of a fractionation product are detected using the micro-levers (13), according to characteristics of polarity, solvophobicity or porosity of the material of which they are made or of a coating of the micro-levers, or according to characteristics of polarity, solvophobicity, ion exchange or affinity with functional groups grafted onto the micro-levers.
20. Procédé selon l'une quelconque des revendications 17 à 19, caractérisé par le fait que l'on fractionne l'échantillon par chromatographie, par micro-électrophorèse, ou par interaction avec des nano-électrodes.20. Method according to any one of claims 17 to 19, characterized in that the sample is fractionated by chromatography, by micro-electrophoresis, or by interaction with nano-electrodes.
21 . Procédé selon l'une quelconque des revendications 17 à 20, caractérisé par le fait que l'on analyse la déviation ou la fréquence de vibration des micro-leviers (13).21. Method according to any one of Claims 17 to 20, characterized in that the deviation or the frequency of vibration of the micro-levers (13) is analyzed.
22. Procédé selon l'une quelconque des revendications 17 à 21, caractérisé par le fait que l'on analyse les éléments de fractionnement par spectrométrie de masse, avant ou après l'analyse à l'aide des micro-leviers (13).22. Method according to any one of claims 17 to 21, characterized in that the fractionation elements are analyzed by mass spectrometry, before or after the analysis using micro-levers (13).
23. Procédé selon l'une quelconque des revendications 17 à 22, caractérisé par le fait que l'on analyse un premier échantillon, on analyse un second échantillon, et on compare les résultats d'analyse des deux échantillons.23. Method according to any one of claims 17 to 22, characterized in that a first sample is analyzed, a second sample is analyzed, and the analysis results of the two samples are compared.
24. Procédé selon la revendication 23, caractérisé par le fait qu'on analyse les premier et second échantillons en vue de la comparaison d'empreintes de protéines des échantillons, à l'aide de micro-leviers sélectifs aptes à montrer une empreinte de protéines différentielle.24. The method of claim 23, characterized in that the first and second samples are analyzed for the comparison of protein fingerprints of the samples, using selective micro-levers capable of showing a protein imprint differential.
25 . Procédé selon l'une quelconque des revendications 17 à 24, caractérisé par le fait que l'on effectue une extraction préalable sur un échantillon avant un fractionnement différentiel de l'échantillon. 25. Process according to any one of Claims 17 to 24, characterized in that a preliminary extraction is carried out on a sample before a differential fractionation of the sample.
EP02745510A 2001-06-08 2002-06-10 Device for analysing a chemical or biological sample, comparative analysis assembly, and related analysis method Withdrawn EP1393059A1 (en)

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FR0107537 2001-06-08
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Publication number Priority date Publication date Assignee Title
US6337479B1 (en) * 1994-07-28 2002-01-08 Victor B. Kley Object inspection and/or modification system and method
US20080315092A1 (en) * 1994-07-28 2008-12-25 General Nanotechnology Llc Scanning probe microscopy inspection and modification system
US5751683A (en) * 1995-07-24 1998-05-12 General Nanotechnology, L.L.C. Nanometer scale data storage device and associated positioning system
US6339217B1 (en) * 1995-07-28 2002-01-15 General Nanotechnology Llc Scanning probe microscope assembly and method for making spectrophotometric, near-field, and scanning probe measurements
US7196328B1 (en) 2001-03-08 2007-03-27 General Nanotechnology Llc Nanomachining method and apparatus
US6802646B1 (en) * 2001-04-30 2004-10-12 General Nanotechnology Llc Low-friction moving interfaces in micromachines and nanomachines
EP1196939A4 (en) * 1999-07-01 2002-09-18 Gen Nanotechnology Llc Object inspection and/or modification system and method
US7329388B2 (en) * 1999-11-08 2008-02-12 Princeton Biochemicals, Inc. Electrophoresis apparatus having staggered passage configuration
US6406604B1 (en) * 1999-11-08 2002-06-18 Norberto A. Guzman Multi-dimensional electrophoresis apparatus
US6813937B2 (en) * 2001-11-28 2004-11-09 General Nanotechnology Llc Method and apparatus for micromachines, microstructures, nanomachines and nanostructures
US7282329B2 (en) * 2002-08-22 2007-10-16 Massachusetts Institute Of Technology Suspended microchannel detectors
JP2005538855A (en) * 2002-09-09 2005-12-22 ジェネラル ナノテクノロジー エルエルシー Fluid delivery of a scanning probe microscope
US7094345B2 (en) * 2002-09-09 2006-08-22 Cytonome, Inc. Implementation of microfluidic components, including molecular fractionation devices, in a microfluidic system
US6989134B2 (en) * 2002-11-27 2006-01-24 Velocys Inc. Microchannel apparatus, methods of making microchannel apparatus, and processes of conducting unit operations
US20050014134A1 (en) * 2003-03-06 2005-01-20 West Jason Andrew Appleton Viral identification by generation and detection of protein signatures
EP1616189A4 (en) * 2003-03-31 2007-10-17 Cytonome Inc Implementation of microfluidic components, including molecular fractionation devices, in a microfluidic system
US20040203126A1 (en) * 2003-04-08 2004-10-14 Yokogawa Electric Corporation Method and apparatus for separating and purifying biopolymers
US20050051489A1 (en) * 2003-08-20 2005-03-10 California Institute Of Technology IC-processed polymer nano-liquid chromatography system on-a-chip and method of making it
US7422910B2 (en) * 2003-10-27 2008-09-09 Velocys Manifold designs, and flow control in multichannel microchannel devices
US20050095602A1 (en) * 2003-11-04 2005-05-05 West Jason A. Microfluidic integrated microarrays for biological detection
US8007724B2 (en) * 2003-11-07 2011-08-30 Princeton Biochemicals, Inc. Electrophoresis apparatus having at least one auxiliary buffer passage
WO2005047882A2 (en) * 2003-11-07 2005-05-26 Princeton Biochemicals, Inc. Multi-dimensional electrophoresis apparatus
TWI253352B (en) * 2003-12-26 2006-04-21 Ind Tech Res Inst Solid-phase micro extraction device
US8592219B2 (en) * 2005-01-17 2013-11-26 Gyros Patent Ab Protecting agent
JP4403853B2 (en) * 2004-03-26 2010-01-27 いすゞ自動車株式会社 Analytical pretreatment method and apparatus for analysis of polycyclic aromatic hydrocarbons
US7305850B2 (en) * 2004-07-23 2007-12-11 Velocys, Inc. Distillation process using microchannel technology
US20060051265A1 (en) * 2004-09-08 2006-03-09 Health Research, Inc. Apparatus and method for sorting microstructures in a fluid medium
US7524672B2 (en) * 2004-09-22 2009-04-28 Sandia Corporation Microfluidic microarray systems and methods thereof
US7592139B2 (en) 2004-09-24 2009-09-22 Sandia National Laboratories High temperature flow-through device for rapid solubilization and analysis
US7943046B2 (en) 2004-10-01 2011-05-17 Agilent Technologies, Inc Methods and systems for on-column protein delipidation
US7449116B2 (en) * 2004-10-01 2008-11-11 Agilent Technologies, Inc. Methods and systems for protein separation
JP2008534914A (en) * 2005-01-17 2008-08-28 ユィロス・パテント・アクチボラグ Multipurpose channel
US20060183166A1 (en) * 2005-02-11 2006-08-17 Michael Mayer Arrays of supported biomembranes and uses thereof
US7641865B2 (en) * 2005-04-08 2010-01-05 Velocys Flow control through plural, parallel connecting channels to/from a manifold
US8574432B2 (en) 2005-04-14 2013-11-05 California Institute Of Technology Integrated chromatography devices and systems for monitoring analytes in real time and methods for manufacturing the same
WO2007002237A1 (en) * 2005-06-22 2007-01-04 Adhesives Research, Inc. Molecularly imprinted polymer and use thereof in diagnostic devices
US20070028668A1 (en) * 2005-07-20 2007-02-08 National Institute Of Advanced Industrial Science And Technology Molecule detection sensor, detection sensor, and gas transferring pump
JP2007248323A (en) * 2006-03-17 2007-09-27 National Institute Of Advanced Industrial & Technology Molecule detection sensor
US20070048795A1 (en) * 2005-08-26 2007-03-01 Xiangming Fang Immunoaffinity separation and analysis compositions and methods
US9007213B2 (en) * 2006-04-19 2015-04-14 The United States Of America As Represented By The Secretary Of The Navy Methods and systems for object identification and for authentication
US20070246106A1 (en) 2006-04-25 2007-10-25 Velocys Inc. Flow Distribution Channels To Control Flow in Process Channels
US7709264B2 (en) * 2006-09-21 2010-05-04 Philip Morris Usa Inc. Handheld microcantilever-based sensor for detecting tobacco-specific nitrosamines
JP4984849B2 (en) * 2006-11-27 2012-07-25 パナソニック株式会社 Component separation device and chemical analysis device using the component separation device
CN101622532B (en) * 2007-04-27 2013-08-14 爱科来株式会社 Analysis chip and analysis device
US20100032297A1 (en) * 2007-04-27 2010-02-11 Arkray, Inc. Electrophoresis Chip and Electrophoresis Apparatus
US8778059B2 (en) * 2007-12-03 2014-07-15 Schlumberger Technology Corporation Differential acceleration chromatography
US8047829B1 (en) * 2009-01-26 2011-11-01 Sandia Corporation Method for forming polymerized microfluidic devices
US8974651B2 (en) 2010-04-17 2015-03-10 C.C. Imex Illuminator for visualization of fluorophores
US9176012B2 (en) * 2012-04-16 2015-11-03 David Samuel Lieberman Methods and systems for improved membrane based calorimeters
ES2921604T3 (en) * 2013-03-15 2022-08-30 Haemonetics Corp Hemostasis Test Cartridge
KR20150052898A (en) * 2013-11-06 2015-05-15 연세대학교 산학협력단 Sample plate using MALDI-TOF mass spectrometer and manufacturing method of the sample plate
US9835587B2 (en) 2014-04-01 2017-12-05 C.C. Imex Electrophoresis running tank assembly
US10768176B2 (en) * 2014-06-17 2020-09-08 Anteo Technologies Pty Ltd Hetero functional binding systems
ES2970505T3 (en) * 2016-06-17 2024-05-29 Koninklijke Philips Nv Compact gas analysis device and method
US10746696B2 (en) 2016-12-19 2020-08-18 Analog Devices, Inc. Self-calibrated heavy metal detector
CN108732271B (en) * 2018-05-22 2023-03-31 福州大学 On-line solid phase extraction detection method for adenosine triphosphate and phosphorylation metabolite thereof
GB2576077B (en) 2018-05-31 2021-12-01 Micromass Ltd Mass spectrometer
CN114152655B (en) * 2021-12-31 2023-11-14 常州大学 Polystyrene and L-Phe compound modified electrode for chiral recognition of glutamic acid enantiomer and preparation method thereof
WO2023178222A1 (en) * 2022-03-16 2023-09-21 Inso Biosciences Inc. Devices, methods, and systems for enriching cellular samples by physical properties

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2191110B (en) * 1986-06-06 1989-12-06 Plessey Co Plc Chromatographic separation device
DE3724966C3 (en) * 1986-07-29 1996-03-21 Sharp Kk sensor
US4908112A (en) * 1988-06-16 1990-03-13 E. I. Du Pont De Nemours & Co. Silicon semiconductor wafer for analyzing micronic biological samples
US4935040A (en) * 1989-03-29 1990-06-19 The Perkin-Elmer Corporation Miniature devices useful for gas chromatography
US6054277A (en) * 1996-05-08 2000-04-25 Regents Of The University Of Minnesota Integrated microchip genetic testing system
US6120666A (en) * 1996-09-26 2000-09-19 Ut-Battelle, Llc Microfabricated device and method for multiplexed electrokinetic focusing of fluid streams and a transport cytometry method using same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO02101382A1 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11355331B2 (en) 2018-05-31 2022-06-07 Micromass Uk Limited Mass spectrometer
US11373849B2 (en) 2018-05-31 2022-06-28 Micromass Uk Limited Mass spectrometer having fragmentation region
US11437226B2 (en) 2018-05-31 2022-09-06 Micromass Uk Limited Bench-top time of flight mass spectrometer
US11476103B2 (en) 2018-05-31 2022-10-18 Micromass Uk Limited Bench-top time of flight mass spectrometer
US11538676B2 (en) 2018-05-31 2022-12-27 Micromass Uk Limited Mass spectrometer
US11621154B2 (en) 2018-05-31 2023-04-04 Micromass Uk Limited Bench-top time of flight mass spectrometer
US11879470B2 (en) 2018-05-31 2024-01-23 Micromass Uk Limited Bench-top time of flight mass spectrometer
US12009193B2 (en) 2018-05-31 2024-06-11 Micromass Uk Limited Bench-top Time of Flight mass spectrometer
US12027359B2 (en) 2018-05-31 2024-07-02 Micromass Uk Limited Bench-top Time of Flight mass spectrometer

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