EP1381862A2

EP1381862A2 - Method for depositing a spot of a product of interest, and use for isolating and/or determining an analyte

Info

Publication number: EP1381862A2
Application number: EP02726283A
Authority: EP
Inventors: Agnès PERRIN; Alain Theretz; Thierry Delair; Bernard Mandrand
Original assignee: Biomerieux SA
Current assignee: Biomerieux SA
Priority date: 2001-04-26
Filing date: 2002-04-25
Publication date: 2004-01-21
Also published as: WO2002088735A2; FR2824001B1; WO2002088735A3; US20040170757A1; FR2824001A1

Abstract

The invention concerns a method for depositing on a substrate (1) a product of interest (2), in a small elementary surface (3), called spot, which consists in: a) preparing or providing a liquid medium (4) comprising a carrier and the product of interest (2); (b) from the liquid medium, forming a drop (5), and setting said drop on the surface (1a) of the substrate, in contact with the liquid medium; (c) eliminating the carrier of the drop (5), for example by drying, so as to allow said spot to remain. The invention is characterised in that it consists, in combination, in: (1) preparing or providing a magnetic support (6), distributed in the carrier; (2) binding the product of interest (2) to the magnetic support (6), so that the liquid medium, wherefrom the drop (5) is formed, comprises transport particles (7); (3) at least while eliminating the carrier of the drop (5), generating a magnetic field (8) passing through the surface (1a) of the substrate (1), so that the resulting spot (3) comprises the transport particles, and hence the product of interest.

Description

METHOD FOR DEPOSITING A SPOT OF A PRODUCT OF INTEREST, AND APPLICATION FOR ISOLATING AND / OR DETERMINING AN ANALYTE

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.

Different techniques require such a deposit, in the dry state, among which we can cite:

- the printing of a paper-type substrate, by ink jet, in which case the product of interest is a pigment or a dye, and the juxtaposition of spots of color and / or of different intensities reproduces any image or characters of an alphabet,

- isolation and / or determination (qualitative and / or quantitative) of an analyte, in particular of a bio-molecule, in particular bio-macro-molecule such as nucleic acid or protein, by the use of reaction wells , for example on a microplate, in which case 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,

Conventionally, whatever the technique concerned, such a spot is obtained according to the following general method: a) 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.

Such a method involves a substantial difficulty, which in particular has been identified and analyzed in the field of inkjet printing; cf. US-A-5,695,820

Due to the evaporation and / or condensation of the vehicle, during elimination, or for other reasons, 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.

In the field of inkjet printing, this phenomenon, known as the MARANGONI effect, has the effect of giving each spot a halo form.

In the field of the (qualitative and / or quantitative) determination of a bio-molecule, 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.

Prior to the present invention, 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:

1) by cooling the substrate, 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,

3) by drying the drop in a humid atmosphere, we find the disadvantages according to 1),

4) by increasing the viscosity of the liquid medium, with an appropriate additive, the drop does not dry or badly.

The present invention therefore aims to remedy the MARANGONI effect, by counteracting the latter.

According to the present invention, it has been discovered that the effect

MARANGONI practically stopped, in any process of forming a spot from a drop, as soon as in combination the following operating conditions are implemented:

1) a magnetic support is distributed or is distributed in the form of particles, homogeneously, in the vehicle,

2) 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 ,

3) at least during the elimination of the vehicle from the drop, 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.

Thanks to the invention, and as shown by the experimental protocol described below in the field of determination of an analyte, of the biomolecule type, a homogeneous spot is obtained in which almost all of the product of interest is well distributed on the surface, via the transport particles, having exactly the same distribution on the surface of the spot.

Thanks to the action of the magnetic field, there is also a better retention of the spot on the surface of the substrate, no doubt due to the penetration or anchoring of the transport particles in the surface layer of the substrate.

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.

Throughout this description, the following vocabulary is explained.

By "isolate" 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.

By "analyte" means any entity, in particular biological entity, to be isolated. Among the 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.

The term “ligand” is understood to mean an element capable of forming, through a chemical or physical bond, a complex with an analyte. By way of example of 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.

The term "support" means any type of support, polymer, inorganic or metallic. By way of example of 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. By way of example of inorganic supports, mention may be made of silica, silicon, mica, glass, quartz, etc. As an example of 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.

The term "particle" means any particle of a polymeric, inorganic or metallic support, onto which it is possible to graft a ligand. In particular, are considered to fall within the scope of the present invention, 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.

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. Examples of 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.

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.

By way of example, mention may be made of the particles described in document EP-A-0 125 995. They 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 μm and their density of 2.5 g / cm ³ . Similarly, 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. Other patents also describe the use of particles of small sizes, but voluntarily aggregated in order to increase the magnetic mass as in the document US-C-5, 169,754. The article by PA Risφen et al, Protein Expression and Purification, 6 (1995), 272-277 also describes magnetic gels.

Placed in a magnetic field, all these relatively large particles generate a movement towards the place where the field is the most intense. A simple permanent magnet or equivalent arrangements as described for example in document EP-A-0 317 286 can be used. These particles are commonly used for the separation of cells or molecules, as well as in immunoassays as described in document EP-A-0 528 708.

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. For example, 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. Their use has often been resumed, as in documents US-C-5,543,289 or WO-A- 88/00060, or they are used in particular applications such as those described in documents FR-A-2 710 410 and FR-A -2,732,116. Document US-C-4,795,698 describes a modification of the Molday procedure, by replacing, for example, the polysaccharide by another polymer of protein nature. The proteins present on the surface of the particles can thus be used for the subsequent immobilization of antibodies by coupling methods known to those skilled in the art.

These particles of relatively small diameter require the use of special arrangements, making it possible to locally increase the magnetic field gradient. This technique is known to those skilled in the art under the name of HGMS (for High Gradient Magnetic Separation) and uses a device consisting, for example, of an open container, filled with a matrix of fibers or metallic grains. . Placed in an external magnetic field, this device makes it possible to obtain a very large field gradient on the very surface of the fibers. Even superparamagnetic particles of limited dimensions can be retained on such a device, provided that the network of fibers is sufficiently tightened and that the path of a particle brings it in the vicinity of the matrix. By way of example, such devices are described in documents US-C-4,375,407, US-C-5,543,289, WO-A-96/26782, WO-A-96/26011 or US-C-5, 186.827, and publications like that of BL Hirschbein et al. (CHEMTECH, March 1982, pp 172-179). Montages can also be found commercially, for example, under the name MACS column at Miltenyi Biotec (Bergisch Gladbach, Germany).

In accordance with the present invention, by way of example, the product of interest is a ligand, which can be an oligonucleotide for capturing a target nucleic acid.

Preferably, 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.

By way of example, and as described below in the experimental protocol, the substrate is a wall of a micro-titration or microanalysis well.

Preferably, and by way of example, 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.

In such a case, an isolation device is used, 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.

The present invention is now described with reference to the accompanying drawing, of schematic and explanatory nature, in which: - Figures 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,

- Figures 3, 4 and 5 show three stages of a deposition process according to the present invention; as before, the index a is reserved for a top view, and the index b for a section view; FIG. 3c represents a detail B, on an enlarged scale of FIG. 3b,

- By way of example, FIGS. 6a to 6c represent the method of forming and placing a drop, as required by a deposition process according to the invention.

In accordance with the prior art and in Figures 1 and 2, it involves depositing in the dry state on a substrate (1) at least a predetermined quantity of at least one product of interest, distributed over the surface ( 1 a) of the substrate and circumscribed according to an elementary area (3), of low value, for example having a diameter of the order of 0.3 mm.

In accordance with this prior process

a) 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,

b) from the liquid medium, 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).

As shown in Figures 6a to 6c, to form the drop (5) from the liquid medium (4), 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)

- when the movement of the reservoir (10) is stopped, the mechanical shock thus created on the latter makes it possible to extract by the orifice (12) a drop of the liquid medium (4), which will move in the air before depositing on the substrate (1) (see Figure 6c).

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.

According to the invention, in accordance with FIGS. 3 to 5, and by difference to the method according to the prior art:

1 - 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).

3 - at least during the at least partial elimination of the vehicle from the drop (5), 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.

The present invention and its advantages are demonstrated by the experimental protocol below, with the following correspondence between the vocabulary of the claims and that of said protocol:

- 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,

- the bottom of 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.

Preparation of magnetic latexes

A magnetic latex solution (diameter = 300 nm, solid content = 0.1%) is prepared in a 0.2M borate buffer, pH 9.2, is commercially available from the French company Ademtech, in Pessac (France), under the reference AD F112 / E212E / P212.

This solution is introduced into a device as described with reference to Figures 6a to 6c, adaptable on a micro-titration plate (Nunc Maxisorb, polystyrene support, 96 wells). This system makes it possible to eject drops towards the bottom of a well, through a nozzle of 50 μm in diameter, after a path in the air of approximately 500 μm.

Deposit under magnetic field

Under the bottom of the well of this plate, stationary in position, is deposited a cylindrical magnet, developing a magnetic field, crossing perpendicularly the bottom of the well, whose cylindrical head has a surface exceeding the section of the well (diameter = 12 mm). The latexes are deposited in the form of spots at the bottom of the surface of this well, and left to dry for approximately 10 minutes. Deposit without magnetic field

The same type of deposit is made in the absence of a magnet under the well.

Analysis

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.

Results

In the absence of a magnet, 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. There is a very large heterogeneity of particle distribution. On the other hand, the presence of 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.

Intensive washing shows that the spots are not affected and that the particles are very strongly adsorbed on the surface after drying. This makes it possible to resolve an important point observed during deposits of non-magnetized particles: the crown (halo) which they form when drying, can be torn off during the washes, to the point that this ring is only partial. Example 2: Detection of nucleic targets on magnetic particle spots. Interest of the deposit of magnetic particles compared to a direct deposit.

Direct deposit

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 μl 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

Deposit via magnetic particles

We start with a magnetic emulsion (Ademtech, Pessac, France reference AD F112 / E212E / P212). The particles of this emulsion are then functionalized on the surface, by covalent grafting of sptreptavidin using a heterobifunctional coupling agent, namely carbodiimide.

They are then washed by magnetization and taken up in buffer.

PBS at a solid level of 0.2%.

Fixation of biotinyl oligonucleotides: 5.10 ¹⁴ copies of a solution of specific (A) or non-specific (B) biotinyl oligonucleotides are added to 100 μl 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.

Capture and spot detection of fluorescent nuclei targets

A target oligonucleotide having the following sequence:

5'FITC-ACT GGA TGG ATC CAA

is therefore labeled at its 5 ′ end with fluorescein, with a concentration of 7.10 ¹⁴ copies / ml. This fluorescent oligonucleotide is complementary or not, respectively oligonucleotides A or B. 30 μl 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 μl of PBS in each well.

Results

Direct deposit No fluorescence signal could be detected in the case of direct deposition under the conditions used. It is likely that the amount of streptavidin adsorbed on the surface of the microplate is insufficient to then allow effective capture of the labeled targets.

• Case of deposition via magnetic particles

In both cases, an image of the fluorescent spots is obtained in the presence of the specific oligonucleotide. However, the morphology of the image is very different depending on whether the deposition took place in the presence or absence of the magnet under the microplate.

In the absence of a magnet, the same heterogeneous distribution of the signal is observed as that described in Example 1 in the absence of a magnet, that is to say a high concentration of magnetic particles and therefore of capture oligonucleotides. at the periphery of the drop.

In the presence of a magnet, 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:

The fluorescence levels generated on the spots of magnetic particles were thus analyzed.

The advantage of depositing magnetic particles under a magnetic field appears clearly. It is possible in this case to differentiate clearly the “specific” spots of the “non-specific” spots, whereas this is not possible in the case of spots of non-magnetized particles.

Example 3: Detection of post-RTPCR amplicons on magnetic latex spots.

Amplification of nuclear targets

The amplification of 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:

this tcσggcccct gaatgσggct aattctaacc atggagcagg cagctgcaaσ ccagcagcca gcctgtcgta acgcgcaagt ccgtggcgga accgactact ttgggtgtcc gtgtttcctt ttattcttga atggctgctt atggtgacaa tcatagattg ttatcataaa gcgagttgga ttggccatcc agt

Synthesis of magnetic particles conjugated to a capture oligonucleotide

5.10 ¹⁴ copies of a capture oligonucleotide solution, complementary to the PCR products to be detected, are added to 100 μl of magnetic particles-streptavidin obtained as described in example 2. The capture 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 micro-titration plate with the capillary deposition device previously described. Magnets are placed under the wells before deposition.

Capture of amplicons on magnetic latex spots

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.

Detection of biotinylated PCR products

A 0.1 mg / ml streptavidin-fluorescein solution in 0.2% PBS / tween / BSA is prepared. 30 μl 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.

Results

Two images show spots obtained respectively in the presence and in the absence of the amplicons. 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.

With the method according to the present invention, it is possible to deposit several spots, juxtaposed but separated from each other on the same flat support, as it is possible to superimpose several spots on each other.

As an indication, with regard to the molecular determination of a nucleic material, for example of bacterial type, in accordance with French patent FR 2797 690 (the content of which is incorporated into the present description as necessary), 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

- a 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.

Claims

1. Method for depositing on a substrate (1) at least a predetermined quantity of at least one product of interest (2), distributed on the surface (1a) of the substrate and circumscribed according to an elementary area (3) of low value, called spot, process according to which:

® a liquid medium (4) is prepared or available, comprising a vehicle and the product of interest (2) distributed homogeneously in said vehicle,

© from the liquid medium, a drop (5) is formed, the volume of which is determined in correspondence with the predetermined quantity of the product of interest to be deposited, and said drop is placed on the surface (1 a) of the substrate contact with the liquid medium

characterized in that, in combination:

© the product of interest (2) is linked to the magnetic support (6), whereby the liquid medium, from which the drop (5) is formed, comprises transport particles (7), distributed in the vehicle, each comprising and the magnetic support (6) and the product of interest (2),

© at least during the elimination of the vehicle from the drop (5), a magnetic field (8) is generated crossing the surface (1 a) of the substrate (1) according to a section (1c) comprising the contact area (1d ) between the drop (5) and the substrate (1), whereby the spot (3) obtained comprises the particles (7) of transport, and therefore the product of interest (2), according to a homogeneous distribution on the surface.

2. Method according to claim 1, characterized in that the product of interest (2) is a ligand, for example an oligonucleotide for capturing a target nucleic acid

3. Method according to claim 1, characterized in that the product of interest (2) is linked to the magnetic support (6), in the form of particles, by functionalization of said support, then bond, for example covalent bond, between the support functionalized and the product of interest

4. Method according to claim 1, characterized in that the substrate (1) is a wall of a micro-titration or micro-analysis well

5. Isolation method, for example of determining an analyte, characterized in that the product of interest (2) is a specific ligand for an analyte, and a substrate capable of being obtained by a method according to any of claims 1 to 4.

6. Isolation device, for example for determining an analyte, characterized in that it comprises or incorporates a well of which at least part of the wall, for example its bottom, constitutes a substrate on which is deposited at least a spot capable of being obtained by a process according to any one of claims 1 to 4