EP1343586B1 - Verfahren zum konzentrieren von analyten in proben - Google Patents

Verfahren zum konzentrieren von analyten in proben Download PDF

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Publication number
EP1343586B1
EP1343586B1 EP01998402A EP01998402A EP1343586B1 EP 1343586 B1 EP1343586 B1 EP 1343586B1 EP 01998402 A EP01998402 A EP 01998402A EP 01998402 A EP01998402 A EP 01998402A EP 1343586 B1 EP1343586 B1 EP 1343586B1
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EP
European Patent Office
Prior art keywords
analyte
container
bottleneck
volume
magnetic particles
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English (en)
French (fr)
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EP1343586A1 (de
Inventor
Pierre Puget
Patrick Pouteau
Frédéric Ginot
Patrice Caillat
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Biomerieux SA
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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Biomerieux SA
Commissariat a lEnergie Atomique CEA
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    • 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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/005—Pretreatment specially adapted for magnetic separation
    • B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/025—High gradient magnetic separators
    • B03C1/031—Component parts; Auxiliary operations
    • B03C1/033—Component parts; Auxiliary operations characterised by the magnetic circuit
    • B03C1/0332—Component parts; Auxiliary operations characterised by the magnetic circuit using permanent magnets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • 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
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/043—Moving fluids with specific forces or mechanical means specific forces magnetic forces
    • 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
    • B01L3/502723—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 venting arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications

Definitions

  • the present invention relates to a concentration process of an analyte present in a sample according to the claims 1 and 2.
  • many diagnostic tests in in vitro consist in chemically reacting an analyte searched with a suitable reagent. The one or products of the reaction is then detected directly or indirectly.
  • immunoassays in which the chemical reaction is a antibody / antigen recognition, or more general a protein / ligand reaction, and tests by probes of nucleic acid (s) in which we detect hybridization between nucleic acids.
  • a diagnostic test is all the better presents both sensitivity and specificity high. It is all the more sensitive as it allows detect a small amount of desired analyte. he is all the more specific as it is only positive for the desired analyte and not for analytes similar ..
  • Analyte means all or part of corpuscle or molecule that one wishes to isolate, to change medium and / or concentrate to be used and / or such as a microorganism, a bacterium, a fungus, a virus, a eukaryotic cell; a chemical compound ; a molecule such as a peptide, a protein, an enzyme, a polysaccharide, a lipid, a lipoprotein, a lipopolysaccharide, an acid nucleic acid, a hormone, an antigen, an antibody, a growth factor, a hapten; a cell such than a tumor cell etc.
  • Biologists have quite the means classical concentration of an analyte using including centrifugation techniques, filtration and / or magnetic sedimentation. These techniques require transfer of solutions and manipulations of the analyte that lead to a inevitable reduction in the amount of analyte analyzable.
  • the stages of centrifugation or magnetic sedimentation as such may need to be repeated several times time, the limit of the number of repetitions being imposed by the minimum volume of solution that can be easily and reliably handled with a pipette classic.
  • This minimum volume is of the order of ten micro-liters.
  • WO-A-9426414 discloses a method of transporting an analyte attached to magnetic particles from one container to a second.
  • the present invention meets this need, and not only offers the advantage of disadvantages mentioned above, but also many others advantages that the person skilled in the art will not fail to raise.
  • the analytes are defined above.
  • the preparation of solution A from the sample includes a step in which the analyte is preferably fixed reversibly on magnetic particles. The usefulness of this reversibility is explained below.
  • Magnetic particles have an adequate size particularly with the analyte to be isolated, and with the volume of solution A. They can be of sub-micrometric size for example when the analyte is a molecule.
  • the amount of particles used is function particular of the nature and amount of analyte to fix, it is preferably sufficient in number to fix all of the analyte.
  • the particles magnetic elements which can be used in the process of this invention may be for example products such than those of the trademark Dynabeads of the company Dynal (Norway) or MACS from Miltenyi Biotec (Germany), or products of the Immunicon Corp. company (USA).
  • magnetic particles usable are classically used in biology molecular and cellular. They must be in particular superparamagnetic in order to rebroadcast spontaneously after cancellation of the magnetic field.
  • thermosensitive magnetic particles having each a magnetic core covered with a layer intermediate.
  • the middle layer is itself covered by an outer layer based on a polymer likely to interact with at least one molecule biological, the outer polymer is thermosensitive and has a lower critical temperature of solubility (LCST) of between 10 and 100 ° C and preferably between 20 and 60 ° C.
  • LCST critical temperature of solubility
  • This layer external is synthesized from monomers cationic, which generate a polymer with the capacity to bind the nucleic acids.
  • This layer intermediate isolates the magnetic charges from the nucleus, to avoid the problems of inhibition of techniques amplification of these nucleic acids.
  • the magnetic particles released from the analyte can be moved out of the second container by means of a magnetic system. This can be useful for example to avoid any detrimental interaction of particles with released analytes and / or with chemical reagents and / or means used to highlight it.
  • the release of the analyte sought, or elution of the analyte can be performed for example in a buffer solution for example by heating or another suitable method.
  • the processes of release usable are all processes classic of the state of the art.
  • the techniques chromatographies offer a whole range of protein release techniques or other ligand usable in the process of the present invention, such as a pH change or a change in strength ionic, or a change of solvent, or the buffering containing EDTA or any other chelating substance of metal cations if the analyte is attached to the particle by a technique of metal-chelate.
  • the analyte is an oligonucleotide, can for example heat to a temperature of 50 to 60 ° C for an oligonucleotide of length 15 to 25 bases to dissociate all analytes from particles magnetic.
  • the magnetic system is a system for creating a fixed magnetic field or variable generating the application of a force on the magnetic beads, capable of immobilizing them or move them. It can consist of a set magnets or coils.
  • Coils of this type are for example Collectively manufactured using technologies mentioned above for making read / write heads hard drives.
  • magnetic particles after being transported, can be brought back into suspension, for example in the second container, by cancellation of the. magnetic field created by the system magnetic.
  • the analyte can be released in the second container, and the analyte can be moved either under transport of the liquid containing the analytes, with transport by displacement of a liquid second container to a third container.
  • the particles can be displaced magnetic released from the analyte of the second container to the first container via the bottleneck, or from the bottleneck to the first container, to the means of a magnetic system.
  • the analyte can be released from the magnetic particles by changing physical or chemical conditions for example by heating or by reaction with at least one substance in the other solution.
  • an immobilization agent of the analyte can be fixed on all or part of at least a wall of the second container or any support solid present in said second container.
  • media may consist of silica beads, hollow, solid glass beads or porous, quartz particles, grains of sand, vermiculite grains, zeolite and / or feldspar, glass wool and / or rock, clay balls, cork particles, marbles polystyrene, polyethylene, polypropylene, Small sintered polyethylene, porosity variable thickness, latex balls, balls coated with gelatin, and resin grains.
  • the bottleneck can be in the form of a capillary.
  • This form can to be interesting for example to limit the diffusion from the analyte from the second container to the first containing when said analyte was released from magnetic particles.
  • hybridization pads fixed on a surface of the second container so that they are accessible to the analyte when it is in the second container. This can be done by example in the form of an integrated microarray. So, according to the present invention, when the analyte to put in evidence is a nucleic acid it can be put in evidence by a nucleic acid chip technology.
  • the second container can therefore be a reservoir of a microcomponent, by example of a biochip, for example a DNA chip.
  • biochip we mean any solid support on which are fixed ligands, and in particular by a chip DNA means any solid support on which are fixed nucleic acids.
  • the method of fixing ligands can be achieved in different ways and especially by adsorption or covalence, for example in situ synthesis by the techniques of photolithography or by a piezoelectric system, by capillary deposition of preformed ligands.
  • examples of these biochips applied DNA chips are given in G.'s publications Ramsay, Nature Biotechnology, 16, p. 40-44, 1998; F. Ginot, Human Mutation, 10, p.
  • the second container can be also an entrance chamber to another containing a another process.
  • the second container can be connected directly or indirectly to another container used for other chemical reactions or stages of process targeting the analyte or a derivative thereof that a purification, an amplification, a marking, etc.
  • the other container or room may to be a PCR chamber to amplify a gene, possibly with then an analysis in a micro-total analysis system: MicroTAS).
  • reagents can be used, such as freeze-dried reagents, by example to make a homogeneous detection test of the analyte, for example by fluorescence transfer.
  • the analyte is defined above.
  • the volumes of the first and second containers are preferably adapted to volumes of solutions to handle. These volumes may be less than or equal to 10 ml.
  • These containers are used for example as reaction chambers.
  • the aforementioned techniques also make it possible to achieve capillaries of section from a few microns-squares to a few hundred thousand micron-squares for the transfer of solutions, or an analyte fixed on microparticles, according to the present invention, first container to a second container, for example from a reaction chamber to another chamber of reaction.
  • the ratio of the volumes ⁇ / ⁇ can be for example from 10 to 1000.
  • the first container can by example have a volume of about 0.1 to 100 ⁇ l.
  • the second container can have a volume of about 0.01 to 1 ⁇ l.
  • the invention therefore allows a reduction in volume which is 100 to 1000 times greater than what could to be achieved by laboratory or automated "macroscopic" systems of art prior art handling liquids with pipettes and bottles of a few tens of microliters. She allows to concentrate a sample by the same factor 100 to 1000.
  • photolithography techniques of solid substrates for example silicon, silica, or glass, or high-precision molding of plastics, allow the realization of containers of submillimetric dimensions, or even the order of a few microns in at least one direction, which may therefore have reduced volumes to micro-liter fractions.
  • the first container and / or the second container may / may have a shape that converges towards said bottleneck.
  • the bottleneck throttling can be for example in the form of a capillary as described in the previous paragraph.
  • the bottleneck may for example have a cross section of between 1 ⁇ m 2 to 1 mm 2 , preferably 100 ⁇ m 2 to 0.1 mm 2 .
  • the second container and / or the bottleneck can / may be provided with fluid inlet / outlet channels. These channels have heard a section adapted according to the volumes of solution they are meant to contain. So, by example for a highlighting of the analyte, when it comes to a nucleic acid, in the second containing by hybridization on capture probes supported by a solid support, these channels can be used to make the necessary washes before the reading step.
  • the aforementioned devices may include a capillary vent present at the level of the second container and connecting directly this one outside.
  • this vent serves to evacuate the fluid initially present in the container whether of air or liquid.
  • the presence of air in the second container is only a contingency. There may be events in other places, for example at bottleneck, first container etc.
  • These vent vents can be ordered by example by ball valves.
  • the invention therefore makes it possible through the use of micro-technology techniques to integrate into the devices called today labopuce, or "lab-on-a-chip” or even “micro-Total-Analysis-System” (MicroTAS) in the Anglo-Saxon terminology.
  • the device usable in the present invention can be combined with other functions to form a more complete system and more accurate biological analysis.
  • reaction chamber and transfer channels can be confused since these labs allow the realization of processes in for which reactions are capillaries, for example in certain techniques capillary electrophoresis and PCR.
  • the invention can for example be useful when the analyte sought is initially present in a sample of large volume but in limited quantity.
  • the proposed invention allows, for example, the implementation of the aforementioned micro-technologies, to concentrate a solution of molecules that we want detect, or move an analyte from a first solution to a second solution in a volume below the microliter, completely inaccessible by classical laboratory processes.
  • the present invention can be implemented by example in an in vitro diagnostic system automated, or a system for detecting contaminants in areas such as agribusiness and / or microbiological control industrial.
  • the invention can be applied for example to ultrasensitive detection without amplification of pathogens in a biological sample.
  • Acids nucleic acid pathogens potentially present in a sample can be extracted by techniques usual. They can then be purified and concentrated, always by standard techniques, up to a buffer volume of a few dozen microliters.
  • the use of the device or micro-component allows in this case to concentrate the biological material in the volume of the reaction chamber that corresponds to the second component. There, later stages of hybridization on plan and detection support can detect the presence or absence of nucleic acids of given sequence, characteristic of the infection of the sample.
  • the use of the invention thus allows to greatly increase the sensitivity of a test, to equal performance of the detection system.
  • the present invention may for example be applied to improve immunoassays. Indeed, for immunoassays in which a problem of sensitivity, the use of the invention, as previously described, concentrating the material in a very low volume, allows to greatly increase their sensitivity.
  • the use of the invention makes it possible to concentrate the sampling, and therefore to decrease the duration of the immunological reaction.
  • the biological sample is processed by means classical molecular biology to get a solution containing the target RNA molecules to detect; this solution has a volume of 200 microliters and the buffer solution is as follows: 10mM Tris, 1mM EDTA, 1M NaCl, 0.05% X-100 Triton, DNA salmon 0.14 mg / ml.
  • this capture oligonucleotide solution consists of: 10 mM Tris, 1 mM EDTA, pH 8, 10 11 ⁇ l capture oligonucleotide; the capture oligonucleotide is a 5 'biotynilated oligonucleotide of a sequence of, for example, 32 bases, complementary to a sub-sequence of the target DNA.
  • Example 2 Device according to a first mode of production
  • the device or component described in this example is a micro-component that can reduce a factor 100 to 1000 the volume of buffer in which is find a desired analyte while retaining the amount of analyte present in the original sample.
  • a vent 9 allows the evacuation of air fluids or liquid when filling or transferring liquids in the rooms.
  • the sample and the different reagents or tampons can be introduced into the devices of different ways. We give two here as examples.
  • This first embodiment is produced by practicing an orifice in the cover 11 of the device and equipping this orifice of a conical bowl 15.
  • a cylindrical part 13 serves to keep the conical bowl in position and to ensure sealing between the conical bowl and the device.
  • a pipette, a mouthpiece dilutor or syringe By applying for example a pipette, a mouthpiece dilutor or syringe, on the conical bowl, one can "push" the buffer or reagent inside the device by exerting pressure on the liquid. Air or other liquid or gaseous fluid initially present in the device will be removed from the 9. This vent leads here into the reaction chamber, but it can be placed according to the cases to other places of the device. We may even have several vents.
  • FIG. 2 A second embodiment for the introduction of liquid in the device is shown in the Figure 2.
  • the liquids are introduced by a capillary 17, itself connected to the outside of the device via an interface, not shown in the figure.
  • the lid 19 does not does not include an orifice.
  • the method described in this example allows reduce the buffer volume by a factor of 100 to 1000 in which there is a sought analyte, while retaining the amount of analyte present in the initial sample. It implements the device represented in the previous example.
  • the component is filled with buffer without analyte and without magnetic particle.
  • This buffer can be introduced by pouring the amount necessary in the conical bowl 15 shown on FIG. 1, and applying a pneumatic pressure in this conical bowl. Once the component filled, the excess buffer present in the bowl taper 15 is removed, for example using a pipette.
  • the sample composed of a certain amount of buffer, for example of the order of 30 .mu.l, in which the analytes sought were previously fixed on magnetic particles is deposited in the bowl conical 15.
  • the magnetic particles are then attracted towards the bottom of the introduction chamber 3 (FIG. 1) with the help of a magnet, for example the magnet 30 of the form shown in Figure 4 positioned under the device, in line with the conical bowl.
  • the magnetic particles are then gathered into one small pellet.
  • the magnet 40 of the form shown in Figure 5 arranged such so that the device is in its air gap 42, the pellet is attracted and transported from its position in the first introductory chamber 3, to through the capillary 5, in the reaction chamber 7.
  • the analyte is then released from the particles magnetic elements by heating (elution) inside the reaction chamber 7. During this operation, the magnetic particles are eventually put back into suspension in the reaction chamber 7 by removing the magnet.
  • Magnetic particles are again gathered in a pellet in the reaction chamber 7 by using a magnet again, for example from the form shown in Figure 4. They are then transported again through capillary 5 but in the opposite direction to that of the Chamber of reaction 7 to the introduction chamber 3, in using a magnet, for example of the form presented in Figure 5.
  • the component is previously filled with buffer without magnetic particle.
  • the sample composed of a certain amount of buffer, for example of the order of 30 .mu.l, in which the analytes sought were previously fixed on magnetic particles is deposited in the bowl 15.
  • Magnetic particles are attracted towards the bottom of the introduction chamber 3 (FIG. 1) using a magnet, for example in the form indicated in Figure 4. The magnetic particles are then gathered in a small pellet.
  • the analyte is released from magnetic particles by heating (elution) inside the capillary 5. During this operation, the magnetic particles are eventually resuspended in the capillary by removing the magnet.
  • Magnetic particles are again gathered in a pellet in the capillary using again a magnet, for example of the form presented in Figure 4.
  • the analytes are free in solution inside the capillary.
  • the analytes in solution are thus driven by the displacement of the liquid in the reaction chamber 7.
  • Magnetic particles they remain in position in the capillary, held in position as a base by the magnet fixed.
  • the particles magnetic are in the form of dry entities already present in the introductory chamber. entities are well described in patents US-A-5,750,338 and 4,672,040.
  • the introduction of the sample, in said chamber 3, solubilizes the magnetic particles that then bind to the analyte present from the beginning in said sample.
  • Example 5 Use of the invention to implement evidence the analyte in a homogeneous detection test
  • the analyte is highlighted in using the "Molecular Beacons” technique, such that it is described in Tyagi, S. and Kramer, F.R., Nat. Biotechnol. 14: 30-308, 1996.
  • probe sequence complementary to the target, is extended on both sides by two arms of a few nucleotides long, complementary to each other.
  • a fluorophore for example the EDANS group
  • a fluorescence inhibitor for example the group DABCYL is attached to the other arm.
  • both arms of the probe hybridize one on the other and the fluorescence of EDANS is extinguished by DABCYL.
  • DABCYL fluorescence inhibitor
  • both groups are distant from each other, and the fluorescence of EDANS is released. So the presence and even the concentration of the analyte, is revealed by the signal of fluorescence and the intensity of this signal.
  • the advantage of this procedure compared to the state of art is to concentrate in an alpha / beta ratio the analyte, for example 100 times, and therefore to decrease relatively the residual fluorescence of the probes "Molecular Beacons", and thereby increase the signal-to-noise ratio intrinsic to this technique highlighting an analyte.
  • the sensitivity of test is increased accordingly.
  • Example 6 Use of the invention to implement evidence the analyte using a DNA chip
  • the analyte is highlighted by hybridization on a DNA chip; the DNA chip has the advantage, compared to the marking technique example 5, to be able to do a lot hybridizations in parallel, thus offering the biologist a much greater analytical power.
  • the bottom of the second container 7 is a DNA chip, consisting for example of a twenty or so hybridization pads.
  • the chip is made by deposition of DNA according to the standard means of the state of the art of DNA chips.
  • the concentration of the analyte before its hybridization on the DNA chip allows a faster reaction of the analyte on the DNA chip.
  • This acceleration of kinetics by compared to the state of the art allows either to decrease the hybridisation time, ie to increase the sensitivity of detection of the system, since this sensitivity is in general limited by the kinetics of hybridization of the analyte on the DNA chip.
  • Example 7 Use of the invention as a point input of a ⁇ TAS
  • the invention serves as a point input to a more complex ⁇ TAS than a device composed of only two containers separated by a bottleneck.
  • ⁇ TAS the one presented by A. Northrup's team, who consists of an amplification chamber, followed by a Capillary electrophoresis of amplified products and detection (see Anal Chem, 1996, 68, 4081-4086).
  • the second container is in does a PCR amplification chamber, for example manufactured by means of microtechnologies.
  • This means that the second container has a means heating, cooling means, and a temperature sensor, which allows to apply to the liquid sample contained in the second containing 7 thermal cycles.
  • the input-output 21, 22 of fluid cutting this second container is the channel Electrophoresis injection of the sample amplified in the separation capillary.
  • the capillary separation is not shown on our figures (see Figure 1 of the above-mentioned article), nor the microreservoirs allowing the application of the fields necessary for the injection and then the electrophoresis separation.
  • the second container further contains pellets dry goods containing all the products necessary for PCR amplification; these products are "glassified” by well-known techniques, and glass-filled, ball-shaped caps containing the various products necessary for amplification, are deposited in the second container before setting up of the lid.
  • the manufacture of these caps is good described in the state of the art, for example US-A-4,678,812 and US-A-5,275,016.
  • the separation capillary further contains a separation gel, for example hydroxyethyl cellulose, which itself contains a marker fluorescent DNA, for example thiazole orange.
  • a separation gel for example hydroxyethyl cellulose
  • a marker fluorescent DNA for example thiazole orange.
  • the speed with which the whole chain of the order of a few minutes for magnetic transport, from 10 to 15 minutes for amplification and from 1 to 2 minutes for capillary electrophoresis, makes it possible to obtain results of excellent quality, without being necessary to isolate the compartments by valves.

Claims (9)

  1. Verfahren zum Konzentrieren eines Analyten, der in einer Probe enthalten ist, bei dem man
    ausgehend von der Probe, in einem ersten Behälter mit einem Volumen α, der durch einen engen Durchgang mit einem zweiten Behälter mit einem Volumen β verbunden ist, wobei das Volumen β kleiner ist als das Volumen α, eine Lösung A herstellt, in welcher der Analyt an magnetischen Teilchen fixiert ist,
    den an den magnetischen Teilchen des ersten Behälters fixierten Analyten mittels eines magnetischen Systems in den engen Durchgang überführt,
    den an den magnetischen Teilchen fixierten Analyten im Bereich des engen Durchgangs aus den Teilchen freisetzt und
    den Analyten durch Flüssigkeitsverdrängung aus dem engen Durchgang in den zweiten Behälter transportiert, wobei die magnetischen Teilchen im Bereich des engen Durchgangs zurückgehalten werden.
  2. Verfahren zum Konzentrieren eines Analyten, der in einer Probe enthalten ist, bei dem man:
    ausgehend von der Probe, eine Lösung A herstellt, in welcher der Analyt an magnetischen Teilchen fixiert ist,
    die Lösung A in einen ersten Behälter mit einem Volumen α einführt, der über einen engen Durchgang mit einem zweiten Behälter mit einem Volumen β verbunden ist, wobei das Volumen β kleiner ist als das Volumen α,
    den an den magnetischen Teilchen fixierten Analyten mittels eines magnetischen Systems aus dem ersten Behälter in den engen Durchgang überführt,
    den an den magnetischen Teilchen fixierten Analyten im Bereich des engen Durchgangs aus den Teilchen freisetzt und
    durch Flüssigkeitsverdrängung den Analyten aus dem engen Durchgang in den zweiten Behälter transportiert, wobei die magnetischen Teilchen im Bereich des engen Durchgangs zurückgehalten werden.
  3. Verfahren nach Anspruch 1 oder 2, bei dem man die von dem Analyten befreiten magnetischen Teilchen mittels eines magnetischen Systems aus dem engen Durchgang in den ersten Behälter transportiert.
  4. Verfahren nach Anspruch 1 oder 2, bei dem der Analyt aus den magnetischen Teilchen freigesetzt wird durch Modifizierung der physikalischen oder chemischen Bedingungen.
  5. Verfahren nach Anspruch 1 oder 2, bei dem man ein Agens zur Immobilisierung des Analyten an der Gesamtheit oder einem Teil mindestens einer Wand des zweiten Behälters oder irgendeinem festen Träger, der in dem zweiten Behälter vorhanden ist, fixiert.
  6. Verfahren nach Anspruch 1 oder 2, bei dem der enge Durchgang die Form einer Kapillare hat.
  7. Verfahren zum Nachweis eines Analyten in einer Probe, bei dem man
    den Analyten durch Anwendung eines Verfahrens nach einem der vorhergehenden Ansprüche konzentriert und
    den Analyten in dem zweiten Behälter oder in irgendeinem anderen Behälter, der direkt oder indirekt mit dem zweiten Behälter verbunden ist, nachweist.
  8. Verfahren nach Anspruch 7, bei dem der Analyt ausgewählt wird aus Mikroorganismen, wie z.B. einem Bakterium, einem Pilz, einem Virus, einer chemischen Verbindung, einem Molekül, wie z.B. einem Peptid, einem Protein, einem Enzym, einem Polysaccharid, einem Lipid, einem Lipoprotein, einem Lipopolysaccharid, einer Nucleinsäure, einem Hormon, einem Antigen, einem Antikörper, einem Wachstumsfaktor und einer Tumorzelle.
  9. Verfahren nach Anspruch 7, bei dem der Analyt, der eine Nucleinsäure ist, nachgewiesen wird durch eine Nucleinsäure-, vorzugsweise DNA-Chip-Technologie.
EP01998402A 2000-11-29 2001-11-27 Verfahren zum konzentrieren von analyten in proben Expired - Lifetime EP1343586B1 (de)

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FR0015417 2000-11-29
FR0015417A FR2817343B1 (fr) 2000-11-29 2000-11-29 Procede et dispositifs de transport et de concentration d'un analyte present dans un echantillon
PCT/FR2001/003743 WO2002043865A1 (fr) 2000-11-29 2001-11-27 Procedes et dispositifs de transport et de concentration d'un analyte present dans un echantillon

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AU2002222057A1 (en) 2002-06-11
ATE293494T1 (de) 2005-05-15
WO2002043865A1 (fr) 2002-06-06
US7569398B2 (en) 2009-08-04
DE60110256T2 (de) 2006-03-09
DE60110256D1 (de) 2005-05-25
FR2817343B1 (fr) 2003-05-09
US20040023273A1 (en) 2004-02-05
FR2817343A1 (fr) 2002-05-31

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