Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
  • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/508—Rigid containers without fluid transport within
  • B01L3/5085—Rigid containers without fluid transport within for multiple samples, e.g. microtitration plates
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/02—Details
  • H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
  • H01J49/0409—Sample holders or containers
  • H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
• • H—ELECTRICITY
  • H01—ELECTRIC ELEMENTS
  • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
  • H01J49/00—Particle spectrometers or separator tubes
  • H01J49/26—Mass spectrometers or separator tubes
  • H01J49/34—Dynamic spectrometers
  • H01J49/40—Time-of-flight spectrometers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/06—Fluid handling related problems
  • B01L2200/0678—Facilitating or initiating evaporation
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
  • G01N2001/4027—Concentrating samples by thermal techniques; Phase changes evaporation leaving a concentrated sample
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
  • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
  • G01N2035/1027—General features of the devices
  • G01N2035/1034—Transferring microquantities of liquid

Definitions

• the present invention relates to the fields of biology and diagnosis.
• the present invention relates to a method of concentrating analytes.
• the majority of the analytical techniques used for the detection of analytes in solution consists in depositing a small volume drop of a solution containing analytes on a suitable substrate for the desired analysis technique.
• the evaporation of the drop results in a distribution of the analytes over the entire contact surface between the drop and the substrate, leading to what is commonly known as the "coffee stain effect". Indeed, during the evaporation of the drop, flows occur spontaneously due to non-uniform evaporation along the interface.
• the coffee stain effect is a physical phenomenon that occurs when a colloidal suspension evaporates on a solid substrate (Deegan et al., Nature, 1997, 389, 827-829): the suspended material is deposited preferably at the level of the line of contact between the drop and the substrate thus forming a deposition ring.
• the detections and their identifications are made difficult by this distribution of analytes at the contact line between the drop and the substrate and are then limited by the sensitivity of the analytical tools.
• the deposits must be homogeneous and reproducible from one drop to another.
• Another method for creating Marangoni flows is to create a surfactant concentration gradient that generates a surface tension gradient. It is known that the mixture of a colloidal suspension with a surfactant makes it possible to induce a solutocapillary effect which makes it possible to concentrate the analytes in the center of the deposition zone (Deegan et al., Physical Review E, 2000, 61, 475- 485). However, this method leads to pollution of the analyte solution by the surfactants and therefore does not facilitate their detection. Kutter et al.
• droplet deposition comprising an analyte on a MALDI plate for mass spectral analysis with a matrix assisted laser ionization source (Analytical Chemistry, 2013, 85, 1285-1289).
• the drops comprising the analyte are dispersed in an oily phase before being deposited.
• Such a method does not allow the concentration of analyte in the center of the deposition zone of the drop.
• Nguyen Truskett et al. describes the influence of surfactants on the evaporation of a drop, more precisely on the particle deposition pattern contained in the drop during evaporation. (Langmuir, 2003, 19, 8271-8279).
• the object of the present invention is therefore to provide an analyte concentration process allowing a homogeneous, reproducible, pollution-free deposit of said analytes and on all types of substrate in order to allow the detection of analyte concentrations lower than that permitted by classical methods.
• the invention relates to a method for concentrating at least one analyte comprising the following steps:
• the at least one surfactant is immiscible with the first phase.
• the at least one surfactant is non-volatile.
• the at least one non-volatile surfactant is fluorinated, selected from the following surfactants, but not limited to: 1H, 1H, 2H, 2H-perfluoro-1-octanol; 1H, 1H-perfluoro-1-tetradecanol; perfluorodecanoic acid; 2- (perfluorooctyl) ethyl alcohol; 2,2,3,3,4,4,4,4-heptafluoro-1-butanol; or a compound of the same family.
• the first phase does not include a surfactant.
• the first phase is an aqueous solution.
• the second phase is a volatile oil.
• the volatile oil is a fluorinated oil, preferably of the type or a mixture.
• the invention also relates to a method for detecting at least one analyte comprising:
• the concentration steps of at least one analyte as defined in the present invention may be repeated several times with the same at least one analyte prior to detecting said at least one analyte.
• the substrate comprises a plurality of wells each of dimensions configured to receive a plurality of drops of the first phase and the second phase.
• the invention also relates to a system implementing the method for detecting at least one analyte according to the present invention comprising:
• a microfluidic device configured to deposit at least one drop of the first phase and the second phase on a substrate
• a substrate said substrate being a plate comprising a plurality of wells each of dimensions configured to receive a plurality of drops of the first phase and the second phase; means for moving the substrate configured to move said substrate relative to the microfluidic device; and
• a device for physicochemical or biological detection of the at least one concentrated analyte a device for physicochemical or biological detection of the at least one concentrated analyte.
• Volatile relates to a compound which has a saturation vapor pressure value of from 10 mbar to 500 mbar at 20 ° C, preferably from 100 mbar to 500 mbar at 20 ° C; and evaporates at room temperature and atmospheric pressure.
• Non-volatile refers to a compound that does not evaporate at ambient temperature and atmospheric pressure.
• “Surfactant” relates to a molecule making it possible to lower the surface tension of a liquid phase.
• Analyte concerns a compound of interest present in a sample to be analyzed. It can be of any nature and any size among, without being limited to: crystals; biomolecules; peptides; proteins; nanoparticles or microparticles.
• the present invention relates to a method for concentrating at least one analyte.
• the method of concentrating at least one analyte comprises the steps of: preparing a first liquid phase comprising at least one analyte; depositing a drop of said first phase on a substrate;
• FIG. 1 The method of concentrating at least one analyte is illustrated in FIG.
• Evaporation of the drop of the second phase results in the concentration of at least one surfactant at the line of contact of the drop of the first phase on the substrate.
• This concentration of surfactant molecules creates a surface tension gradient by solutocapillary effect. This leads to the occurrence of recirculations generated by Marangoni flows from regions of lower surface tension to regions of greater surface tension, that is from the periphery of the drop to the apex of the drop.
• the analytes are then concentrated in the center of the surface initially occupied by the drop of the first phase, favoring their detection.
• the method of the present invention makes it possible to limit and / or prevent the pollution of the analyte present in the drop of the first phase because the surfactant is not present in the first phase.
• the steps of depositing a drop of said first phase and of depositing on said first-phase drop of a drop of a second phase are successive.
• the steps of depositing a drop of said first phase and of depositing on said first-phase drop of a drop of a second phase are independent. According to one embodiment, the steps of depositing a drop of said first phase and of depositing on said first-phase drop of a drop of a second phase are not successive.
• the steps of depositing a drop of said first phase and of depositing on said first-phase drop of a drop of a second phase are simultaneous. According to one embodiment, the steps of depositing a drop of said first phase and of depositing on said first-phase drop of a drop of a second phase are not independent.
• the examples of analytes include, but are not limited to: peptide, peptide mixture, high molecular weight polypeptide chain eg protein, or mixture thereof.
• the first phase is prepared by mixing a solution of at least one analyte with the liquid of the first phase.
• the first phase is prepared by mixing a solution of at least one analyte, as well as any other compound, except for a surfactant, such as a matrix for MALDI-TOF with the liquid of the first phase.
• a surfactant such as a matrix for MALDI-TOF
• the matrix for MALDI-TOF is selected from the following matrices, but without being limited thereto: 1,5-diaminonaphthalene; 2-mercaptobenzothiazole; 4-aminoquinaldine; 2- (2-aminoethylamino) -5-nitropyridine; 2 ', 4', 6'-trihydroxyacetophenone; aminoacridine; ⁇ -cyano-4-hydroxycinnamic acid or a compound of the same family.
• the deposition of the drops of the first phase and the second phase is carried out without being limited by a pipette or a microfluidic system.
• said drop is not encapsulated, contained, surrounded, wrapped or dispersed in the second phase.
• said drop is not encapsulated, contained, surrounded, wrapped or dispersed in the first phase.
• the drops of the first phase have a diameter ranging from 10 ⁇ m to 1 mm, preferably from 50 ⁇ m to 800 ⁇ m, even more preferentially from 100 ⁇ m to 400 ⁇ m.
• the drops of the second phase have a diameter of 10 microns. at 1 mm, preferably from 50 ⁇ m to 800 ⁇ m, still more preferably at 100 ⁇ m. at 400 ⁇ m ..
• the drops of the second phase have a diameter different from the diameter of the drops of the first phase.
• the drops of the second phase have a diameter greater than the diameter of the drops of the first phase. This embodiment has the advantage of allowing the second-phase drop to encompass the first-phase drop.
• the drops of the first phase have a volume ranging from 1 ⁇ l to 1, preferably from 1 ⁇ l to 10 ⁇ l.
• the drops of the first phase have a volume greater than 5 nL.
• the drops of the second phase have a volume ranging from 1 ⁇ l to 1, preferably from 1 ⁇ l to 10 ⁇ l. According to one embodiment, the drops of the second phase have a volume greater than 5 nL.
• the drops of the second phase have a volume different from the volume of the drops of the first phase.
• the drops of the second phase have a greater volume volume of drops of the first phase.
• the evaporation steps of the drops of the first phase and of the second phase take place at ambient temperature and atmospheric pressure.
• the evaporation of the drops of the first phase and / or the second phase can be accelerated by heating and / or by the application of a gas flow near the drops such as a flow of N2. , Ar, O2, air, or any gas known to those skilled in the art or a mixture thereof.
• the first phase does not include a surfactant. This embodiment makes it possible to prevent contamination of the first phase by external compounds such as surfactants.
• the first phase does not include the liquid of the second phase.
• the second phase does not include the liquid of the first phase.
• the first phase is an aqueous solution.
• the second immiscible phase with the first phase is an oil.
• the examples of aqueous solution include but are not limited to: a mixture of water and at least one water-miscible solvent, for example a water-acetonitrile mixture.
• a water-acetonitrile mixture is particularly advantageous because acetonitrile is miscible with water and allows easy dispersion of low hydrophilic compounds in the aqueous solution by its more hydrophobic nature of water.
• the second phase examples include but are not limited to: volatile oils such as fluorinated oils; water immiscible ethers such as, for example, ethyl ether; or light alkanes such as, for example, pentane or hexane.
• the surfactant is miscible in the second phase.
• the second phase is a volatile oil. This embodiment allows rapid and homogeneous evaporation of the second phase.
• the drop of volatile oil evaporates in less than one minute.
• the evaporation time of the volatile oil drop varies between 1 and 30 seconds, preferably between 1 and 10 seconds.
• the volatile oil is a fluorinated oil. It has advantages for this application, such as its saturation vapor pressure value, its miscibility with the surfactants used, the absence of contamination after evaporation, or its chemical inertness vis-à-vis the analyte solution.
• the fluorinated oil is preferably of the type
• the at least one surfactant is not miscible with the first phase.
• the at least one surfactant is miscible with the second phase.
• the contamination of the first phase by surfactant molecules is prevented, the noise on the analysis signal generated by such pollution is eliminated and the detection of the analytes is facilitated.
• the at least one surfactant is non-volatile.
• the nonvolatile surfactant following evaporation of the second phase, the nonvolatile surfactant is trapped mainly near the contact line of the first phase drop containing the analytes on the substrate without, however, entering it because no miscible in the latter.
• This concentration of surfactant molecules creates a surface tension gradient, and leads to the appearance of recirculations generated by Marangoni flows from the periphery of the drop to the apex of the drop.
• the at least one non-volatile surfactant is fluorinated, selected from the following surfactants, but without being limited thereto: 1H, 1H, 2H, 2H-perfluoro-1-octanol; 1H, 1H-perfluoro-1-tetradecanol; perfluorodecanoic acid; 2- (perfluorooctyl) ethyl alcohol; 2,2,3,3,4,4,4,4-heptafluoro-1-butanol; or a compound of the same family.
• the substrate is selected from, but without being limited to: metal; glass; silicon; polymer such as polydimethylsiloxane; teflon; stainless steel.
• the first phase and the substrate have opposite hydrophobicity characteristics.
• the substrate is hydrophobic, and conversely, if the first phase is an oily phase, so the substrate is hydrophilic. This embodiment prevents spreading of the drop on the substrate.
• the surface of the substrate undergoes a chemical and / or physical treatment before the droplets are deposited in order to ensure opposing hydrophobicity characteristics between said substrate and the first phase.
• Examples of chemical and / or physical treatment are well known to those skilled in the art and include, but are not limited to, chemical grafting or physical treatments (UV ozone, plasma etc ).
• the substrate may be a smooth surface plate or a plate comprising at least one well.
• the substrate is not a plate comprising hydrophilic zones in a hydrophobic coating.
• the substrate is not a steel plate covered with a hydrophobic teflon layer, said layer being structured by laser ablation to form hydrophilic zones on the plate.
• the at least one analyte is concentrated in the center of the drop zone of the first phase.
• the invention also relates to a method for detecting at least one analyte.
• the method for detecting at least one analyte comprises the following steps:
• the invention also relates to a method of concentrating and detecting at least one analyte.
• the method of concentrating and detecting at least one analyte comprises the following steps:
• the concentration steps of at least one analyte as defined in the first aspect of the present invention may be repeated several times with the same at least one analyte prior to detecting said at least one analyte.
• the first phase is prepared by mixing a solution of at least one analyte with the liquid of the first phase.
• the first phase is prepared by mixing a solution of at least one analyte, as well as any other compound, except for a surfactant, such as a matrix for MALDI-TOF with the liquid of the first phase.
• a surfactant such as a matrix for MALDI-TOF
• the matrix for MALDI-TOF is selected from the following matrices, but without being limited thereto: 1,5-diaminonaphthalene; 2-mercaptobenzothiazole; 4-aminoquinaldine; 2- (2-aminoethylamino) -5-nitropyridine; 2 ', 4', 6'-trihydroxyacetophenone;aminoacridine; ⁇ -cyano-4-hydroxycinnamic acid or a compound of the same family.
• the deposition of the drops of the first and second phases is carried out, without being limited thereto, by a pipette or a microfluidic system.
• said drop is not encapsulated, contained, surrounded, wrapped or dispersed in the second phase.
• said drop is not encapsulated, contained, surrounded, wrapped or dispersed in the first phase.
• the drops of the first phase have a diameter of 10 microns. at 1 mm, preferably 50 ⁇ m. at 800 ⁇ m. more preferably of 100 microns. at 400 ⁇ m. .
• the drops of the second phase have a diameter of 10 microns. at 1 mm, preferably 50 ⁇ m. at 800 ⁇ m, even more preferably 100 ⁇ m. at 400 ⁇ m ..
• the drops of the second phase have a diameter different from the diameter of the drops of the first phase.
• the drops of the second phase have a diameter greater than the diameter of the drops of the first phase. This embodiment has the advantage of allowing the second-phase drop to encompass the first-phase drop.
• the drops of the first phase have a volume ranging from 1 ⁇ l to 1, preferably from 1 ⁇ l to 10 ⁇ l.
• the drops of the first phase have a volume greater than 5 nL.
• the drops of the second phase have a volume ranging from 1 ⁇ l to 1, preferably from 1 ⁇ l to 10 ⁇ l.
• the drops of the second phase have a volume greater than 5 nL. According to one embodiment, the drops of the second phase have a volume different from the volume of the drops of the first phase.
• the drops of the second phase have a greater volume volume of drops of the first phase. According to one embodiment, the evaporation steps of the drops of the first and second phases take place at ambient temperature and atmospheric pressure.
• the stages of evaporation of the drops of the first and second phases can be accelerated by heating and / or by the application of a gas flow near the drops such as a flow of N2, Ar, O2, air, or any gas known to those skilled in the art or a mixture thereof.
• a gas flow near the drops such as a flow of N2, Ar, O2, air, or any gas known to those skilled in the art or a mixture thereof.
• the first phase does not include a surfactant.
• the first phase does not include the liquid of the second phase.
• the second phase does not include the liquid of the first phase.
• the first phase is an aqueous solution.
• the second immiscible phase with the first phase is an oil.
• the examples of aqueous solution include but are not limited to: a mixture of water and at least one water-miscible solvent, for example a water-acetonitrile mixture.
• a water-acetonitrile mixture is particularly advantageous because the acetonitrile is miscible with water and allows easy dispersion of low hydrophilic compounds in the aqueous solution by its more hydrophobic nature of water.
• the second phase examples include but are not limited to: volatile oils such as fluorinated oils; water immiscible ethers such as, for example, ethyl ether; or light alkanes such as, for example, pentane or hexane.
• the surfactant is miscible in the second phase.
• the second phase is a volatile oil.
• the drop of volatile oil evaporates in less than one minute.
• the evaporation time of the volatile oil drop varies between 1 and 30 seconds, preferably between 1 and 10 seconds.
• the volatile oil is a fluorinated oil.
• the at least one surfactant is not miscible with the first phase. In this embodiment, the at least one surfactant is miscible with the second phase.
• the at least one surfactant is non-volatile.
• the at least one non-volatile surfactant is fluorinated, selected from the following surfactants, but without being limited thereto: 1H, 1H, 2H, 2H-perfluoro-1-octanol; 1H, 1H-perfluoro-1-tetradecanol; perfluorodecanoic acid; 2- (perfluorooctyl) ethyl alcohol; 2,2,3,3,4,4,4,4-heptafluoro-1-butanol; or a compound of the same family.
• the substrate is selected from, but without being limited to: metal; glass; silicon; polymer such as polydimethylsiloxane; teflon; stainless steel.
• the first phase and the substrate have opposite hydrophobicity characteristics.
• the substrate is hydrophobic, and conversely, if the first phase is an oily phase, then the substrate is hydrophilic. This embodiment prevents spreading of the drop on the substrate.
• the surface of the substrate undergoes a chemical and / or physical treatment before the droplets are deposited in order to ensure opposing hydrophobicity characteristics between said substrate and the first phase.
• Examples of chemical and / or physical treatment are well known to those skilled in the art and include, but are not limited to, chemical grafting or physical treatments (UV ozone, plasma etc ).
• the substrate may be a smooth surface plate or a plate comprising at least one well.
• the at least one analyte is concentrated in the center of the drop zone of the first phase.
• the at least one analyte is concentrated in the center of the well in which the first drop has been deposited.
• the physico-chemical or biological analysis method for the detection of the at least one analyte can be chosen from, without being limited to: mass spectrometry, MALDI-TOF mass spectrometry, microscopy by fluorescence, microarrays, immunochemical assays or any other analytical analytical or biology analytical technique.
• the method of concentrating at least one analyte as defined in the present invention is particularly advantageous for the detection of analytes by the above methods of analysis.
• the homogeneity and reproducibility of analyte solution deposits are two important parameters for achieving an effective analysis.
• matrix-assisted laser desorption / ionization mass spectrometry coupled with a time-of-flight analyzer (MALDI-TOF-SM) an analysis technique commonly used in proteomics and in particular in all phases of development of biomarkers, has many advantages such as sensitivity, accuracy and speed of analysis.
• the detection sensitivity of the MALDI-TOF-MS technique is also strongly correlated with the mass of the biomolecule to be analyzed.
• this technique has some limitations regarding the reproducibility of the signal obtained mainly related to heterogeneity during the drying process of the samples.
• the concentration process according to the present invention makes it possible to respond advantageously to this disadvantage.
• the invention also relates to an analysis plate, in particular a MALDI plate.
• the analysis plate is smooth.
• the analysis plate comprises a plurality of wells.
• This analysis plate makes it possible to make homogeneous and reproducible deposits. It also has the advantage of allowing multiple deposits by repeating the deposit of drops of solution in the same well, leading to an increase in the concentration of analytes in said well.
• each well is configured to receive at least one drop of a solution.
• each well has a diameter ranging from 1 ⁇ to 10 mm, from 500 ⁇ to 1 mm or approximately 700 ⁇ .
• each well has a height of 1 ⁇ m. at 500 ⁇ m, 50 ⁇ m. at 250 ⁇ or about 100 ⁇ .
• each well has a volume ranging from 1.5 ⁇ L to 5 ⁇ M, preferably from 50 ⁇ L to 1, preferably from 1 ⁇ L to 500 ⁇ L. According to one embodiment, each well has a minimum volume of 100 nL.
• each well has a dimension of the order of 700 ⁇ m.
• each well has a height of 100 ⁇ m and a diameter of 700 ⁇ m.
• each well has a height of at least 100 ⁇ m. According to one embodiment, each well has a minimum diameter of 300 ⁇ m. According to one embodiment, each well has a diameter strictly greater than 300 ⁇ m.
• all the wells have the same height and / or the same diameter.
• the wells have different heights and / or diameters.
• the height and / or the diameter of the wells varies according to their position on the plate.
• the material of the analysis plate is selected from, but without being limited to: metal, glass, silicon, polymer, teflon, stainless steel.
• the analysis plate comprising a plurality of wells is manufactured by photolithography, preferably by photolithography of a silicon wafer surmounted by a photosensitive resin.
• the analysis plate is not a plate comprising hydrophilic zones in a hydrophobic coating.
• the analysis plate is not a steel plate covered with a hydrophobic teflon layer, said layer being structured by laser ablation to form hydrophilic zones on the plate.
• the analysis plate comprising a plurality of wells is manufactured by machining, preferably by machining a stainless steel plate.
• the plate according to the present invention is advantageously used as a substrate according to the first or second aspect of the present invention.
• each well is configured to receive a plurality of drops of the first phase and the second phase.
• This embodiment has the advantage of allowing multiple deposits by repeating the drop deposits in the same well, each of the deposits being separated by an evaporation step of the drop of the second phase and a step of evaporation of the drop. of the first phase.
• This embodiment leads to an increase in the concentration of analytes in said well.
• the invention also relates to a system implementing the method for detecting at least one analyte according to the present invention.
• the system implementing the method for detecting at least one analyte according to the present invention comprises:
• a microfluidic device configured to deposit at least one drop of the first phase and of the second phase comprising at least one surfactant on a substrate;
• said substrate being a plate comprising a plurality of wells each well being of dimensions configured to receive a plurality of drops, preferably at least two drops including a drop of the first phase and a drop of the second phase;
• a physio-chemical or biological detection device of the at least one concentrated analyte of the at least one concentrated analyte.
• the invention also relates to a system for concentration and detection of at least one analyte according to the present invention.
• the system for concentration and detection of at least one analyte according to the present invention comprises:
• a microfluidic device configured to deposit at least one drop of the first phase and of the second phase comprising at least one surfactant on a substrate;
• a substrate said substrate being a smooth plate or comprising a plurality of wells each well being configured to receive at least two drops including a drop of the first phase and a drop of the second phase;
• the system further comprises a first reservoir configured to receive the first phase comprising at least one analyte and a second reservoir configured to receive the second phase comprising at least one surfactant, each reservoir being in fluid communication with the device microfluidics.
• the first reservoir and the second reservoir are independent.
• the first reservoir and the second reservoir are tubes, channels or any container known to those skilled in the art.
• the microfluidic device comprises two tubes or channels (5, 6), the first tube (or channel) being configured to transport the first phase 1 independently of the second phase 4 and the second tube (or channel) 6 being configured to carry the second phase 4 independently of the first phase 1.
• the two tubes or channels (5, 6) meet before deposition at a junction 7 at which a drop of the first phase 1 is generated.
• the microfluidic device comprises a third tube or channel 8 in which the drop of the first phase 1 generated at the junction 7 of the first two tubes or channels (5, 6) is transported in the second phase 4.
• the microfluidic device is a digital microfluidic device.
• the microfluidic device is configured to deposit successively and independently at least one drop of the first phase and the second phase comprising at least one surfactant on a substrate. According to one embodiment, the microfluidic device is configured to deposit successively and independently at least one drop of the first phase and one drop of the second phase comprising at least one surfactant on a substrate, the drop of the second phase being deposited on the drop of the first phase.
• the substrate is the analysis plate according to the third aspect of the present invention.
• the substrate may be a smooth surface plate or a plate comprising at least one well.
• the substrate is an analysis plate as described above.
• the substrate comprises a plurality of wells.
• the analysis substrate makes homogeneous and reproducible deposits possible. It also has the advantage of allowing multiple deposits by repeating the deposit of drops of solution in the same well, leading to an increase in the concentration of analytes in said well.
• each well is configured to receive at least one drop of a solution.
• each well has a volume ranging from 1.5 ⁇ L to 5 ⁇ M, preferably from 50 ⁇ L to 1, preferably from 1 ⁇ L to 500 ⁇ L.
• each well has a minimum volume of 100 nL.
• each well has a dimension of the order of 700 ⁇ m. According to one embodiment, each well has a height of 100 ⁇ and a diameter of 700 ⁇ m.
• each well has a height of at least 100 ⁇ .
• each well has a minimum diameter of 300 ⁇ m.
• each well has a diameter strictly greater than 300 ⁇ m. According to one embodiment, all the wells have the same height and / or the same diameter.
• the wells have different heights and / or diameters.
• the height and / or the diameter of the wells vary according to their position on the plate. According to one embodiment, after evaporation, the at least one analyte is concentrated in the center of the drop zone of the first phase.
• the at least one analyte is concentrated in the center of the well in which the first drop has been deposited.
• the material of the substrate is selected from, but not limited to: metal, glass, silicon, polymer, teflon, stainless steel.
• the substrate is not a plate comprising hydrophilic zones in a hydrophobic coating.
• the substrate is not a steel plate covered with a hydrophobic teflon layer, said layer being structured by laser ablation to form hydrophilic zones on the plate.
• said drop is not encapsulated, contained, surrounded, wrapped or dispersed in the second phase.
• said drop is not encapsulated, contained, surrounded, wrapped or dispersed in the first phase.
• said drop is encapsulated, contained, surrounded, wrapped or dispersed in the second phase.
• said drop is encapsulated, contained, surrounded, wrapped or dispersed in the first phase.
• the drop of the first phase is formed independently of the drop of the second phase.
• the drop of the second phase is formed independently of the drop of the first phase. According to one embodiment, the drop of the first phase is deposited independently of the drop of the second phase.
• the drop of the second phase is deposited independently of the drop of the first phase.
• the drop of the first phase and the drop of the second phase are formed simultaneously.
• the drop of the first phase and the drop of the second phase are deposited simultaneously.
• the drop of the first phase is formed in the second phase.
• the drop of the second phase is formed in the first phase.
• the drop of the first phase is deposited in the second phase.
• the drop of the second phase is deposited in the first phase.
• the drop of the first phase is transported in the second phase.
• the means for moving the substrate relative to the microfiuidic device is a motorized mechanical means.
• the physio-chemical or biological detection device for the detection of the at least one analyte can be chosen from, without being limited to: mass spectrometry; MALDI-TOF mass spectrometry; fluorescence microscopy; microarray; immunochemical tests or any other analytical analytical chemistry or biology technique.
• FIG. 1 comprises a schematic representation of the drop deposit according to the prior art, of a deposit of two immiscible drops and the deposit according to the present invention, and the snaps corresponding to said deposits after the evaporation of the second phase and of the first phase.
• Figure 2 illustrates the influence of the method of concentration of at least one analyte on the detection of said analyte by MALDI-TOF mass spectrometry.
• Figure 3 illustrates the influence of the method of concentration of at least one analyte on the linearity and reproducibility of the signal obtained by MALDI-TOF mass spectrometry.
• Figure 4 illustrates the influence of the multi-deposition technique on the assay plate according to the present invention on the detection of said analyte by MALDI-TOF mass spectrometry.
• Figure 5 shows MALDI-TOF analysis spectra of a concentrated peptide mixture according to the method of the present invention.
• Figure 6 illustrates the influence of the method of concentration of at least one analyte of the present invention on the concentration of an analyte in the center of the deposition zone.
• FIG. 7 represents a microfluidic device comprising 2 tubes or channels (5, 6), a junction 7 and a third tube or channel 8.
• Figure 8 comprises a schematic representation of the deposit according to the present invention, and the plate corresponding to said deposit after the evaporation of the second phase and the first phase.
• MALDI (2-cyano-4-hydroxycinnamic acid 2 mg / ml) matrix solution comprising a mixture of acetonitrile and acidified distilled water in a 50/50 ratio;
• the matrix solution is prepared by diluting the matrix to a concentration of 2 mg / ml in a mixture of acetonitrile and acidified distilled water in a 50/50 ratio. The mixture requires 5 min of vortex, then 10 min in an ultrasonic bath so that the solubilization is complete.
• the peptide stock solution is prepared by diluting angiotensin II to a concentration of 1 pmol / L in a solution of 0.1% trifluoroacetic acid and stored at -20 ° C. Aliquots of peptide at 100 .mu.M are then prepared from a 1 pmol / .mu.l aliquot. by diluting it in a mixture of distilled water / acetonitrile (75/25). For each assay run, a fresh aliquot is diluted to the desired concentration with an acidified distilled water / acetonitrile (75/25) mixture containing 2 mg / ml matrix.
• a first deposit is carried out in a conventional manner, namely a drop of solution containing the peptides is deposited on a metal substrate using a pipette. Evaporation occurs, which will allow non-volatile analytes to become trapped on the surface of the substrate.
• a second deposit is made by successively depositing a drop of solution containing the peptides and a drop of volatile oil without surfactant on this first drop on a metal substrate using a pipette.
• a third deposit is performed according to the method of concentration of at least one analyte as defined in the present invention.
• the first deposit and the third deposit are then analyzed by MALDI-TOF-SM. Results
• the first two deposits described above result in a distribution of the analytes over the entire contact area between the drop and the substrate, often leading to the coffee staining phenomena described above.
• the method of concentration of at least one analyte as defined in the present invention allows a concentration of said analyte in the center of the area initially occupied by the drop.
• FIG. 1 illustrates the three deposits made:
• the deposition according to the prior art comprising a drop of the first phase (1) containing at least one analyte (11) deposited on a substrate (2);
• the second deposit comprising a drop of the first phase (1) containing at least one analyte (11) surmounted by a drop of the second phase (3), without surfactant, immiscible with the first phase (1) deposited on a substrate (2); the third deposit on a substrate (2) according to the method of concentration of at least one analyte (11) as defined in the present invention comprising a drop of the first phase (1) containing at least one analyte (11) surmounted by a drop of the second phase (4) containing at least one surfactant (41), said second phase (4) being immiscible with the first phase (1).
• the signal strength increases from 2085 to 60,000 for conventional deposition and deposition according to the method of the present invention, respectively, an increase in the signal by a factor of 30.
• Example 2 Deposition on Mal PI plate using a digital microfluidic system
• MALDI (2-cyano-4-hydroxycinnamic acid 2 mg / ml) matrix solution comprising a mixture of acetonitrile and acidified distilled water in a 50/50 ratio;
• a photosensitive resin SU-8 type 3035 On a silicon wafer, previously cleaned, is deposited a photosensitive resin SU-8 type 3035. The wafer is then placed in a spin-coater to obtain a uniform layer of resin on the silicon wafer. The wafer is then deposited on a hot plate for 30 minutes at 95 ° C. It is then placed in an ultra-violet machine and covered with a mask on which is printed the shape of the wells. The wafer then undergoes illumination for 17 seconds at a power of 20 mW / cm 2 . The wafer is then deposited in a bath containing the developing liquid. After 10 minutes, the resin that has not been irradiated is dissolved and the wells appear. The wafer is cleaned with isopropanol and dried.
• the plate thus obtained comprises a plurality of wells 700 ⁇ m in diameter and 100 ⁇ m in height. These wells make it possible to collect the drops generated by the digital microfluidic system. Several deposits according to the concentration method of the present invention are carried out in the same well.
• Figure 4 compares a "simple" deposition by the microfluidic system and a multi-deposition by the microfluidic system on the well plate, and both deposition were performed according to the concentration method of the present invention. The signal-to-noise ratio is improved and the intensity is increased by an order of magnitude.
• Deposition is performed by forming drops, generated by a digital microfluidic system, of solution containing the peptide mixture and then deposited according to the concentration method of the present invention on a stainless steel MALDI commercial plate for analysis. Evaporation occurs, which will allow non-volatile analytes to become trapped on the surface of the substrate.
• MALDI-TOF analysis of such deposits allows the detection of 10 peptides from a deposited amount of 250 attomoles of mixture.
• the gain of the present invention for the peptide mixture study is thus proved since the analysis involving a conventional deposition (drop of solution containing the peptides and drop of matrix solution are deposited on a metal substrate using a pipette) of a larger amount of the same mixture (2500 attomoles) allows the detection of only 4 peptides.
• Figure 5 shows the MALDI-TOF analysis spectra.
• the red markers correspond to the masses of the peptides detected.
• Example 5 Deposition of a MALDI Plate Protein Material and Methods Material
• MALDI matrix ⁇ -cyano-4-hydroxycinnamic acid at 2 mg / ml
• MALDI matrix ⁇ -cyano-4-hydroxycinnamic acid at 2 mg / ml
• analyte such as a polypeptide chain (MMP12 protein labeled with a fluorophore: fluorescein);
• the method of concentration of at least one analyte as defined in the present invention allows a concentration of a protein in the center of the surface initially occupied by the drop, which is not observed in the case of a deposit by a method classic ( Figure 6). Indeed, the deposition of the same protein solution by a conventional method (ie a drop of solution containing the protein is deposited on a metal substrate using a pipette) leads to a distribution of the protein over the entire contact surface between the drop and the substrate, often leading to coffee staining phenomena described above.

Landscapes

• Chemical & Material Sciences (AREA)
• Analytical Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Physics & Mathematics (AREA)
• Dispersion Chemistry (AREA)
• Hematology (AREA)
• Clinical Laboratory Science (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Optics & Photonics (AREA)
• Sampling And Sample Adjustment (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=57396729

Family Applications (1)

Country Status (4)

Families Citing this family (1)

Citations (1)

Family Cites Families (5)

• 2016
  • 2016-10-07 FR FR1659705A patent/FR3057354A1/fr not_active Withdrawn
• 2017
  • 2017-10-06 US US16/339,887 patent/US11402307B2/en active Active
  • 2017-10-06 EP EP17792118.6A patent/EP3523623A2/de not_active Withdrawn
  • 2017-10-06 WO PCT/FR2017/052748 patent/WO2018065741A2/fr not_active Ceased

Patent Citations (1)

Also Published As

Similar Documents

Legal Events