WO2002088735A2 - Procede de depot d'un spot d'un produit d'interet, et application pour l'isolement et/ou la determination d'un analyte - Google Patents
Procede de depot d'un spot d'un produit d'interet, et application pour l'isolement et/ou la determination d'un analyte Download PDFInfo
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- WO2002088735A2 WO2002088735A2 PCT/FR2002/001444 FR0201444W WO02088735A2 WO 2002088735 A2 WO2002088735 A2 WO 2002088735A2 FR 0201444 W FR0201444 W FR 0201444W WO 02088735 A2 WO02088735 A2 WO 02088735A2
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
- G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
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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
- B01L3/02—Burettes; Pipettes
- B01L3/0241—Drop counters; Drop formers
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00351—Means for dispensing and evacuation of reagents
- B01J2219/00387—Applications using probes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00457—Dispensing or evacuation of the solid phase support
- B01J2219/00459—Beads
- B01J2219/00466—Beads in a slurry
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00277—Apparatus
- B01J2219/00497—Features relating to the solid phase supports
- B01J2219/005—Beads
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00596—Solid-phase processes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00646—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports
- B01J2219/00648—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports by the use of solid beads
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00655—Making arrays on substantially continuous surfaces the compounds being bound to magnets embedded in or on the solid supports
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00659—Two-dimensional arrays
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00583—Features relative to the processes being carried out
- B01J2219/00603—Making arrays on substantially continuous surfaces
- B01J2219/00677—Ex-situ synthesis followed by deposition on the substrate
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
- B01J2219/00718—Type of compounds synthesised
- B01J2219/0072—Organic compounds
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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
- B01L2400/00—Moving or stopping fluids
- B01L2400/02—Drop detachment mechanisms of single droplets from nozzles or pins
- B01L2400/021—Drop detachment mechanisms of single droplets from nozzles or pins non contact spotting by inertia, i.e. abrupt deceleration of the nozzle or pin
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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
- 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
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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
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
- B01L3/50857—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using arrays or bundles of open capillaries for holding samples
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- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
- C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
Definitions
- the present invention relates generally to the deposition, for example in the dry state, on a substrate, of at least a predetermined quantity, or dose, of at least one product of interest, distributed over the surface of the substrate and circumscribed according to an elementary area, of small value, in particular at most equal to 35 mm 2 , and preferably between 0.001 and 3 mm 2 , called spot or task.
- the product of interest is a pigment or a dye
- the juxtaposition of spots of color and / or of different intensities reproduces any image or characters of an alphabet
- analyte in particular of a bio-molecule, in particular bio-macro-molecule such as nucleic acid or protein
- reaction wells for example on a microplate
- the product of interest is a reagent for capturing the analyte, for example a ligand which specifically binds to the analyte; in the latter case, the spot or spots obtained are arranged for example on the bottom of one or more microtiter wells,
- a liquid (or fluid) medium is prepared or available comprising a vehicle, a solvent for example, and the product of interest distributed (dispersed and / or dissolved) homogeneously in said vehicle, b) from the liquid medium, according to any suitable technique, a drop is formed whose volume is determined in correspondence with the predetermined quantity of the product of interest to deposit in the spot; and the drop is placed or deposited, with or without acceleration (for example by gravity), on the surface of the substrate, then in contact over a very limited area with the liquid medium of the drop, c) at least partially, or even completely, the vehicle is eliminated from the drop, by all appropriate means, for example by drying, while it remains on the surface of the substrate, so as to leave the desired spot on the latter. , consisting of or comprising the product of interest, covering the surface of the substrate.
- the surface tension between the liquid medium and the outside varies according to the interface of the drop, which generates a internal micro-circulation of the liquid medium, having the effect of concentrating the product of interest on the periphery of the drop.
- this phenomenon has the effect of generating a deposit of the ligand, having the shape of a crown, in which the distribution and the concentration of ligand do not can be mastered, nor reproducible, which goes against the reliability sought in any method of analysis, in particular in the field of molecular biology.
- the Applicant has carried out various tests to suppress or limit the MARANGONI effect, in the process of forming a spot of a ligand on the bottom of a well of an analysis microplate:
- the MARANGONI effect is certainly limited, but the drop does not dry or requires a drying time that is not compatible with an automated or industrial process, 2) on the other hand, by heating the substrate, so that the product of interest does not have time to migrate towards the edge of the drop, we nevertheless observe the formation of a halo-shaped spot,
- the present invention therefore aims to remedy the MARANGONI effect, by counteracting the latter.
- a magnetic support is distributed or is distributed in the form of particles, homogeneously, in the vehicle,
- the product of interest is linked to the magnetic support, whereby the liquid medium, from which the drop is formed, comprises transport particles, distributed in the vehicle, each comprising both the magnetic support and the product of interest ,
- a magnetic field is generated crossing the surface of the substrate in a section comprising the contact area between the drop and the substrate, whereby the spot obtained comprises the transport particles , and therefore the product of interest, for example in the dry state, according to a uniform surface distribution.
- Such an advantage is important for the determination of an analyte of the bio-molecule type, knowing that the spot is often washed, sometimes several times, before the ligand-bound analyte is determined by any suitable method, for example with a labeled reagent.
- isolated or “isolation” is meant generically any technique making it possible to separate an analyte, but also to enrich or concentrate in said analyte in any fraction or liquid part containing it. However, it is also understood, possibly in conjunction with the preceding definition, any technique making it possible to determine the analyte, in the sense of detection and / or quantification thereof, from the liquid medium containing it.
- analyte means any entity, in particular biological entity, to be isolated.
- analytes considered below by the present invention mention will be made of cells, organelles, viruses and bacteria, antibodies, antibody fragments, antigens, haptens, lectins, sugars, ribo- acids, deoxyribonucleics, proteins, in particular A or G, hormones, hormone receptors, and in general all molecules or macromolecules, natural or synthetic, or the like, to be determined, ie to detect and / or quantify.
- ligand is understood to mean an element capable of forming, through a chemical or physical bond, a complex with an analyte.
- a ligand mention may be made of antibodies, antibody fragments, antigens, haptens, lectins, sugars, ribo- and deoxyribonucleic acids, proteins in particular A or G, hormones, hormone receptors, biotin, avidin or streptavidin and, in general, natural or synthetic ligands, and analogs of modified ligands, which can compete with ligands.
- support means any type of support, polymer, inorganic or metallic.
- polymer supports mention may be made of plastic supports based on polystyrene, poly (meth) acrylates, polybutadiene, polypropylene, or others, alone or in the form of copolymers.
- inorganic supports mention may be made of silica, silicon, mica, glass, quartz, etc.
- metallic supports mention may be made of gold, l silver, titanium oxide, vanadium oxide,
- the immobilization of the ligands on the support can be carried out either by simple adsorption on the support, native or modified, or by means of a chemical reaction (functionalization), or physical, allowing the surface of the support to be modified, and thus allow the attachment of the ligand by covalent bonds, or other traditional means well known to those skilled in the art.
- particle means any particle of a polymeric, inorganic or metallic support, onto which it is possible to graft a ligand.
- the particles being able to be separated by magnetic means. From the above definition, small particles, in particular superparamagnetic particles, whose sedimentation rate under the effect of gravity is less than thermal agitation, but which can constitute aggregates by any process allowing them to be joined together, or to assemble them on larger particles, separable by magnetic means.
- polymer particles By way of example of polymer particles, mention may be made of particles obtained by emulsion polymerization such as latexes, or particles of larger size, but magnetic.
- metallic particles that may be mentioned include ferro-, ferri-, para- or superparamagnetic particles, whether or not covered with natural or synthetic polymers, the composition of which comprises iron or other metals such as cobalt, nickel or others, alone or in the form of alloys, but magnetic.
- inorganic particles By way of example of inorganic particles, mention may be made of particles based on silica or silicon, magnetic or not.
- the magnetic particles used by the present invention can be classified into two categories, namely particles of relatively large diameter, for example of the order of one or a few microns, and those of relatively small diameter, for example of order of a few tens of nanometers, and in the colloidal state.
- Magnetic particles of relatively large diameter when placed in a magnetic field move towards the place where the field is the highest and at a speed sufficient to be separated from their environment by this means.
- the particles described in document EP-A-0 125 995 are obtained by precipitation of ferrous and ferric salts in basic medium, followed by a silanization reaction in methanol. Their final diameter is between 0.1 and 1.5 ⁇ m and their density of 2.5 g / cm 3 .
- the particles described in documents EP-A-0 106 873, EP-A-0 585 868 and US-C-5,356,713, are obtained by different polymerization processes, or also those described in document US-C- 4,297,337 use a porous glass matrix in which magnetic pigments are dispersed.
- Magnetic particles of relatively small diameter are practically not attracted to a simple permanent magnet within a reasonable time. These particles are in particular widely used for the magnetic separation of cells.
- those described in document US-C-4,230,685 are obtained by emulsion of a mixture of albumin, protein A and Fe 3 O 4 particles with a diameter of 15-20 nm, and can immobilize antibodies by the intermediate of protein A.
- the document US-C-4,452,773 describes another type of particles, obtained by precipitation of ferrous and ferric salts in basic medium, and in the presence of a polysaccharide. These particles can immobilize antibodies, oligonucleotides, lectins or other biomolecules, by coupling to the polysaccharide, using known grafting methods.
- HGMS High Gradient Magnetic Separation
- HGMS High Gradient Magnetic Separation
- the product of interest is a ligand, which can be an oligonucleotide for capturing a target nucleic acid.
- the product of interest is linked to the magnetic support, in the form of particles, by functionalization of the support, then bond, for example covalent bond, between the functionalized support and the product of interest.
- the substrate is a wall of a micro-titration or microanalysis well.
- the method according to the invention is part of an isolation method, for example of determining an analyte, and in such a case the product of interest is a specific ligand of the analyte, and the substrate is for example the wall of a micro-titration well, as obtained at the end of a process according to the invention.
- an isolation device for example for determining an analyte, comprising or incorporating at least one well, at least part of the wall of which, for example its bottom, constitutes a substrate for which is deposited at least one spot obtained by a method according to the invention.
- FIG. 1 and 2 show two stages of a deposition process according to the prior art, the index a being reserved for a top view, and the index b being reserved for a sectional view;
- FIG. 2c represents on an enlarged scale a detail A of FIG. 2a,
- FIG. 3c represents a detail B, on an enlarged scale of FIG. 3b
- FIGS. 6a to 6c represent the method of forming and placing a drop, as required by a deposition process according to the invention.
- a liquid medium (4) is prepared or available, comprising a vehicle, for example a solvent, and the product of interest (2) distributed (dissolved and / or suspended) homogeneously in the vehicle,
- a drop (5) whose volume is determined in correspondence with the predetermined amount of the product is formed by any suitable means, for example with those described below with reference to FIG. 6 interest to deposit, and this drop is placed on the surface (1a) of the substrate, in contact with the liquid medium (see Figure 1) c) at least partially, or even entirely, the vehicle is eliminated from the drop (5), while it remains on the surface (1 a) of the substrate (1), for example by drying, so as to leave the spot (3), for example in the dry state; as shown in Figure 2, in practice this spot has the shape of a halo or crown, consisting of a heap of the product of interest (2).
- the procedure is for example as follows:
- the liquid medium (4) is loaded into a reservoir (10), provided at its lower end with an orifice (12), of dimensions adapted so that the medium (4) does not flow by gravity, when the reservoir (10) is at rest,
- the tank (10) is caused to move from its starting position (see Figure 6a) to an arrival position (see Figure 6b), determined by the contact between the stop (11) (from side of the tank (10)) and the substrate (1)
- the formation and the deposition of the drop can be carried out according to any process different from that previously described, for example by deposition from a capillary, by simple contact between the latter containing the liquid medium and the substrate.
- a magnetic support (6) is prepared or available, distributed in the form of particles, in a homogeneous manner, in the vehicle (cf. FIG. 3), 2 - the product of interest (2) is linked, by any appropriate means, to the magnetic support (6), whereby the liquid medium (4), from which the drop (5) is formed, comprises particles (7 ) of transport, distributed in the vehicle, each comprising and the magnetic support (6) and the product of interest (2) (cf. Figures 3a to 3c).
- a magnetic field (8) is generated, with a magnet, chosen and positioned so that the magnetic field (8) passes through, practically perpendicularly, the surface (1a) of the substrate (1), immobile, according to a section (1c) comprising, and therefore wider than the contact area (1d) between the drop (5) and the substrate (1) (cf. Figure 4a and 4b); after drying, if necessary total, the spot (3) obtained comprises the transport particles (7), in the dry state, these transport particles being uniformly distributed inside the spot (3), as shown in the Figures 5a and 5b.
- - magnetic latexes are one example, among others, of a magnetic support divided into particle forms
- the magnet used generates the magnetic field for the implementation of the invention
- each micro-titration well constitutes the substrate
- the analyte is a nucleic target, for example an amplicon post RT-PCR,
- the product of interest is a ligand, namely capture oligonucleotide, immobilizable on any support by a reactive pair, namely streptavidin, biotin; this oligonucleotide is specific, that is to say complementary to the nucleic target, or non-specific for the previously exemplified analyte, the transport particles correspond to the conjugation product, or conjugate, between the magnetic particles and the abovementioned capture oligonucleotide,
- the analyte linked to the capture oligonucleotide is detected by any appropriate means, for example with a labeled reagent binding to the immobilized analyte
- Example 1 Magnetic latex deposits. Influence of the presence of a magnet under the deposition surface on the morphology of the spot.
- the deposits are analyzed under an optical microscope at 5X magnification, and in a bright background.
- An image of each spot is acquired using a CCD camera connected to the microscope tube.
- Each pixel of this image is associated with a gray level, from 0 (black) to 256 (white), the lower the density of material.
- a cross section of the spots thus makes it possible to determine the distribution of the particles according to the mode of deposition.
- the latexes are preferably distributed on the periphery of the spot, in the form of a halo (in the manner of FIG. 2c), during drying.
- a magnet under the surface of the deposit, during drying, and inducing a magnetic attraction force perpendicular to the surface, avoids the movement of convection of the particles towards the edge of the spot. A uniform distribution of the particles is observed after drying.
- a streptavidin solution at 1 mg / ml is prepared in 0.2M borate buffer pH9.2. This is deposited in the form of spots in several wells of microtitration plate using the deposition device described above, and equipped with a head comprising four capillaries making it possible to simultaneously deposit four spots whose diameter is around 1 mm. As said above, such spots can be obtained by depositing drops, from a simple contact between capillary tubes and the bottom of the well. The spots are left to dry for 15 minutes, then are rinsed with a 0.05% PBS-tween solution.
- Immobilization of biotinyl capture oligonucleotides 30 ⁇ l of a 5.10 solution 15 copies / ml in PBS buffer of specific (A) or non-specific (B) oligonucleotides of a nucleic target, and both 5 'biotinyls are introduced into the functionalized wells. The immobilization reaction takes place for 30 minutes at 37 ° C. with stirring. The wells are then rinsed in PBS-tween buffer.
- the oligonucleotide A has the sequence: 5'BIOTINE-TTG GAT TGG CCA TCC AGT
- the oligonucleotide B has the sequence: 5'BIOTINE-CAT GTG CTA CTT CAC CAA CGG
- biotinyl oligonucleotides 5.10 14 copies of a solution of specific (A) or non-specific (B) biotinyl oligonucleotides are added to 100 ⁇ l of magnetic particles-streptavidin. The immobilization reaction takes place for 30 minutes at 37 ° C. with stirring. The latexes are then washed by magnetization and taken up in borate buffer, before being deposited in the form of spots in the wells of a microplate with the capillary deposition device described above. Magnets are placed under some of the wells when depositing and drying the spots.
- This fluorescent oligonucleotide is complementary or not, respectively oligonucleotides A or B.
- 30 ⁇ l of this solution are deposited in each of the activated wells. The hybridization reaction takes place for 30 minutes at 37 ° C. with stirring. The wells are then rinsed in PBS-tween buffer, then in PBS buffer. They are analyzed under a fluorescence microscope in the presence of 50 ⁇ l of PBS in each well.
- the fluorescent signal is distributed uniformly inside the spot, thanks to the homogeneous distribution of the capture sites.
- the signal generated by the spot is analyzed using simple image processing, which consists in calculating the average gray level inside a window defined by the user.
- Each gray level n- is associated with a number of pixels pj.
- the average gray level of a window is defined by:
- Example 3 Detection of post-RTPCR amplicons on magnetic latex spots.
- RNA of the Sabin3 poliovirus (7 kB), described by its sequence in GENBANK No. X00596, is carried out by RT-PCR (Access kit from Promega), in the presence of biotinylated primers allowing the obtaining of amplicons DNA labeled at their 5 'end with biotin.
- the amplicons (200 bases), described according to the sequence below, are checked (quantity and size) on agarose gel:
- the amplicons are denatured for 5 minutes at 94 ° C., then added to the wells comprising the latex spots.
- the hybridization reaction takes place for 30 minutes at 37 ° C. with stirring.
- the wells are then rinsed in PBS-tween buffer.
- a 0.1 mg / ml streptavidin-fluorescein solution in 0.2% PBS / tween / BSA is prepared. 30 ⁇ l of this solution are added to each well. Non-specific controls are obtained on wells which have not undergone the hybridization of the PCR products. Incubation takes place for 30 minutes at 37 ° C with shaking. The wells are then rinsed in PBS-tween buffer, then in ammonium carbonate buffer before being dried by air jet and analyzed under a fluorescence microscope.
- the first image shows a spot of homogeneous and relatively strong intensity, which proves the presence of i) a specific capture of the amplicons on the magnetic support particles and ii) an effective detection of these biotinyl amplicons by streptavidin. It is here confirmed that the use of magnetic magnetic latexes makes it possible to obtain a uniform distribution of the particles and therefore of the detection signal inside the spot, without corona effect, which considerably simplifies the necessary image analysis. for quantification of the fluorescence emitted.
- each particle of combined transport in the form of a complex - a magnetic particle comprising the magnetic support, a first ligand (oligo-nucleotide specific for a nucleic target), and a fluorophore marker
- non-magnetic particle comprising a non-magnetic support, and another fluorophore marker, together constituting the product of interest, linked to the nucleic target by a second ligand (another specific oligo-nucleotide, in another region, of the nucleic target )
- the method according to the invention can be implemented for printing or depositing a paint on any surface positioned in a magnetic field.
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/475,873 US20040170757A1 (en) | 2001-04-26 | 2002-04-25 | Method for depositing a spot product of a product of interest, and use for isolating and/or determining an analyte |
EP02726283A EP1381862A2 (fr) | 2001-04-26 | 2002-04-25 | Procede de depot d'une molecule sur une surface |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR01/05639 | 2001-04-26 | ||
FR0105639A FR2824001B1 (fr) | 2001-04-26 | 2001-04-26 | Procede de depot d'un spot d'un produit d'interet, et application pour l'isolement et/ou la determination d'un analyte |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2002088735A2 true WO2002088735A2 (fr) | 2002-11-07 |
WO2002088735A3 WO2002088735A3 (fr) | 2003-09-25 |
Family
ID=8862731
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2002/001444 WO2002088735A2 (fr) | 2001-04-26 | 2002-04-25 | Procede de depot d'un spot d'un produit d'interet, et application pour l'isolement et/ou la determination d'un analyte |
Country Status (4)
Country | Link |
---|---|
US (1) | US20040170757A1 (fr) |
EP (1) | EP1381862A2 (fr) |
FR (1) | FR2824001B1 (fr) |
WO (1) | WO2002088735A2 (fr) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100723425B1 (ko) * | 2006-04-13 | 2007-05-30 | 삼성전자주식회사 | 기판상에 생체분자 액적을 프린팅하는 장치 및 방법 |
JP2011135009A (ja) * | 2009-12-25 | 2011-07-07 | Tokyo Electron Ltd | 基板乾燥装置及び基板乾燥方法 |
WO2021011944A2 (fr) * | 2019-07-18 | 2021-01-21 | Essenlix Corporation | Dosage homogène faisant appel à l'imagerie |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000049382A2 (fr) * | 1999-02-16 | 2000-08-24 | The Perkin-Elmer Corporation | Systeme de distribution de billes |
WO2001015154A1 (fr) * | 1999-08-23 | 2001-03-01 | Burstein Technologies, Inc. | Procedes et appareil permettant de former physiquement des structures non operationnelles sur un disque optique |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6586193B2 (en) * | 1996-04-25 | 2003-07-01 | Genicon Sciences Corporation | Analyte assay using particulate labels |
HU225069B1 (en) * | 1997-09-09 | 2006-06-28 | Lyotropic Therapeutics | Coated particles, methods of making and using |
-
2001
- 2001-04-26 FR FR0105639A patent/FR2824001B1/fr not_active Expired - Fee Related
-
2002
- 2002-04-25 WO PCT/FR2002/001444 patent/WO2002088735A2/fr not_active Application Discontinuation
- 2002-04-25 EP EP02726283A patent/EP1381862A2/fr not_active Withdrawn
- 2002-04-25 US US10/475,873 patent/US20040170757A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000049382A2 (fr) * | 1999-02-16 | 2000-08-24 | The Perkin-Elmer Corporation | Systeme de distribution de billes |
WO2001015154A1 (fr) * | 1999-08-23 | 2001-03-01 | Burstein Technologies, Inc. | Procedes et appareil permettant de former physiquement des structures non operationnelles sur un disque optique |
Also Published As
Publication number | Publication date |
---|---|
FR2824001B1 (fr) | 2003-10-10 |
WO2002088735A3 (fr) | 2003-09-25 |
EP1381862A2 (fr) | 2004-01-21 |
US20040170757A1 (en) | 2004-09-02 |
FR2824001A1 (fr) | 2002-10-31 |
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