EP1381670A1

EP1381670A1 - Miniature device for separating and isolating biological objects and uses thereof

Info

Publication number: EP1381670A1
Application number: EP02727702A
Authority: EP
Inventors: Alexandra Fuchs; Patrice Caillat; Daniel Dupret; Fabrice Lefevre
Original assignee: Commissariat a lEnergie Atomique CEA
Current assignee: Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Priority date: 2001-04-27
Filing date: 2002-04-26
Publication date: 2004-01-21
Also published as: FR2823999A1; US20040168916A1; FR2823999B1; WO2002088299A1; JP2004535176A

Abstract

The invention concerns a miniature device for separating and isolating biological objects, the use of said device for isolating, separating, culturing and/or analysing biological objects and a method for separating and isolating biological objects using said device.

Description

MINIATURE DEVICE FOR SEPARATING AND ISOLATING OBJECTS

BIOLOGICAL AND USES

The present invention relates to a miniature device for separating and isolating biological objects, to a method for separating and isolating biological objects using this device as well as to its applications.

Biology, and in particular genomics, is currently experiencing a revolution in the way of generating and processing data from its analyzes. While more and more genetic sequence data are available thanks to major sequencing projects by organisms, players in biology, the medical world and the pharmaceutical industry are seeking to integrate all of this data in analyzes with large scale and multi-parameter.

The world of microtechnologies, in particular that of microsystems, has the means to meet this demand thanks to its achievements in miniaturization, surface functionalization, microfluidics and large-scale and low-cost manufacturing techniques.

Currently, the marriage of these two worlds has given rise to multi-parameter analysis tools known as DNA chips. These tools are dedicated to the analysis of biological macromolecules (proteins and mainly DNA). ^

There is a significant number of researches around the world in very varied fields which integrate, via microtechnologies, biological protocols with increasingly reduced dimensions.

For example, microtiter plates have gone from a standard format of 96 wells to a format of 384 and then 1536 wells as robotics progresses. The use of these increasingly miniaturized microtechnologies makes it possible to reduce the volumes of reagents used and thus reduce the analysis costs.

In the particular case of DNA chips, the principle of analysis consists in ordering XY matrixing of nucleic probes at increasingly smaller steps, of the order of 20 μm. Similarly, the step of processing biological samples is undergoing the race for size reduction with the increasingly common integration of polymerase chain reactions (PCR) in DNA chips or cell lysis function. This is how it has already been proposed, notably in the patent

US 6,071,394 to separate and fix cells on electronic chips by dielectrophoresis, the cells suspended in an appropriate buffer being separated according to their dielectric properties. However, this technique does not make it possible to separate and isolate single biological objects for individualized analyzes. The current trend which involves a reduction in the volumes of reagents used also results in the use of analyte tests on biological reagents fixed on solid surfaces, so as to gradually abandon the use of tube tests with homogeneous solutions. .

Thus, various solutions have already been proposed for the attachment of biological molecules of interest to different materials such as glass, plastic or metal. For example, for the attachment of nucleic probes to a substrate, three main approaches are currently known:

- ^ Use of a glass substrate coated with poly-L-lysine which is a polymer having an affinity with nucleic probes (Schena M. et al., science, 1995, 270 (5235), 4θ * 70),

- the use of glass covered with a functionalized silane, the nucleic probe then carrying a complementary function so as to form a covalent bond with the silane (O'Donnell MJ. et al, Anal. Chem., 1997, 69, 2438 -2443)

- The use of metal electrodes, in gold for example, allowing the copolymerization of a simple monomer and a monomer carrying the nucleic probe.

Similarly, in the field of proteomics, it has in particular been reported the attachment of peptides via conductive polymers carrying pyrrole functions (polypyrroles), (Livache T. et al, Biosensors & Bioelecrronics, 1998, 13, 629 -634). It is also possible to fix living biological objects such as bacteria or eukaryotic cells on solid substrates by different techniques:

- grafting of specific antibodies from the cell to be fixed on a solid substrate; there are indeed antibodies against microorganisms commonly used in molecular biology (bacteria, yeasts), such as anti-E antibodies. coli 1434 sold by Fitzgerald Industries International, Inc., as well as antibodies against surface receptors (CD receptors) of higher eukaryotic lymphoid cells, - grafting of peptides specific for certain cell types on the support (Holland J. et al. , Biomaterials, 1996, 17, 2147-2156),

- non-specific functionalization of the support by polymers allowing cell adhesion (Aframian D.J. et al, Tissue Εng., 2000, 6 (3), 209-216). These techniques are interesting insofar as they generate the formation of specific bonds between the biological object to be fixed and the support, however they require numerous stages of preparation and isolation before these can finally be fixed individually on the support.

The inventors therefore set themselves the goal of providing a new miniature device for separation and isolation of biological objects making it possible to maintain a matrix approach with a large number of points in which each point contains one and only one type or category of biological object, but in which the preliminary operations of preparation, separation or isolation can be avoided or reduced, thus considerably reducing the number of manipulations and pipetting of the biological object to be treated.

The present invention therefore relates to a miniature device for separation and / or isolation of biological objects, comprising at least one first electrode integrated into the device, constituted by a structure provided with a matrix of reaction microcuvettes, each microcuvette comprising a bottom constituting a reception area, characterized in that said bottom has no hole and that the maximum surface of said bottom of each microcuvette is defined so as to isolate a single biological object, said structure being linked to a supply circuit for creating a potential difference between said first electrode and at least one second electrode integrated or external to the device.

Thanks to this device, it is now possible to fix only one biological object per microcuvette, given that the hanging surface of the biological object to be fixed completely covers the reception area, each microcuvette therefore containing only one only type of biological object selected and fixed which can then be treated collectively.

Consequently, according to the invention, the attachment zone of the biological object to be fixed has either a surface substantially identical to the surface of the reception zone, or a surface greater than the surface of the reception zone.

According to the invention, the maximum surface of the bottom of each microcuvette is preferably less than or equal to twice the smallest surface of the biological object to be isolated. In a preferred embodiment of the invention, the surface of said base is less than or equal to the smallest surface of the biological object to be isolated.

More particularly, this surface is generally between 1 μm ² and 400 μm ² , in particular between 1 and 50 μm ² .

According to the invention, the maximum surface of the bottom of each microcuvette is preferably less than the smallest surface of the biological object to be isolated.

According to the invention, a biological object is characterized by its container and its content. The container corresponds to any element making it possible to compartmentalize the content. The container can for example be the wall of a bacterial cell, the envelope of a virus, the membrane of a cell, a lipid double layer, micelles, a phospho lipid bilayer crossed by intrinsic proteins, etc. the content corresponds to the biological material isolated in a compartment that is the container. The content can for example correspond to nucleic acids, proteins, ribosomes, membrane vesicles or to a complex mixture of these. As an example of a biological object, mention may be made of any cell, healthy or not, whether it is prokaryotic or eukaryotic, viruses, liposomes, etc. By way of example of a cell, mention may be made of bacteria, yeasts, fungi, microalgae and also cells of plant, animal and human origin.

Examples of viruses include the HIV virus, bacteriophages, etc.

The device according to the invention can be advantageously used in the field of cell analysis by fixing a single biological object of interest to the reception zone which in fact constitutes a trap zone or by subsequent fixing of one or more several elements derived from the biological object of interest previously fixed, these derivatives including the products resulting from the possible lysis of the biological objects, from their treatment by localized PCR or any other biological, chemical or electrical treatment. When these derivatives correspond to nucleic acids, we speak of DNA chips and when these derivatives correspond to proteins, we speak of protein chips. The matrix of reaction microcuvettes can be surmounted at least in part by one or more layers of insulating materials and / or by an attached biocompatible plastic grid, so as to form a matrix of microreservoirs, each microreservoir containing at least one microcuvette. These microreservoirs can for example be produced by lithography of the layer of insulating material. The insulating materials can for example be chosen from insulating polymers such as polyimides and resins such as, for example, SU-8 resins.

The size of the microreservoirs is defined so as to process, in a minimal volume, the single isolated biological object. These microreservoirs generally have a width and / or a length of between 5 and 500 μm and preferably between 5 and 100 μm.

According to a particular embodiment of the invention, the miniature device can include alternating conductive layers (electrodes) and layers of insulating materials. According to one embodiment of the invention, one face of the first electrode integrated into the device can constitute the bottom of the microcuvettes. According to another embodiment of the invention, the bottom of the microcuvettes of the device consists of a layer of glass, plastic or silicon.

When the device according to the invention has a second integrated electrode, it is deposited on a first layer of insulating material and is located in a plane spaced from the bottom of the microcuvettes.

When the device according to the invention comprises a second external electrode, the latter may be integral with a cover or a cover, preferably consisting of one or more layers of insulating material. Consequently, one of the layers of insulating materials may not form an integral part of the device according to the invention, but be in the form of a removable insert (cover, cover, cover) which covers at least partially said device and containing optionally at least one electrode.

The device according to the invention may also include at least a third electrode integrated into the device, a second layer of insulating material being interposed between the second and the third electrode. In this case, and according to a variant of the invention, the device can comprise several second and / or third electrodes isolated from each other.

The device according to the invention can also be equipped with an integrated multiplexing circuit ^ 'at least some of said electrodes.

The multiplexing circuit integrated into this device can be used for different functions: fixing different reagents within the same device, heating isolated from the reception areas, local pH measurement, reading an electrical signal, etc. In the devices according to the invention, at least one edge of one of the second and / or third electrodes and / or of one of the first and / or second layers of insulating material can constitute at least part of an edge d '' a microreservoir.

According to the invention, the first, second and third electrodes, as well as the external electrode are constituted by at least one metallic layer, for example made of chromium, gold or platinum. These metal layers generally have a thickness of between 0.1 and 10 μm.

According to one embodiment of the invention, a reagent capable of fixing the biological object to be isolated is fixed on at least part of the zone for receiving the reaction microcuvettes.

The nature of the reagent used for fixing biological objects may vary depending on the nature of the objects to be fixed and the nature of the bottom of the microcuvettes.

In fact, when the bottom of the microcuvettes consists of an electrode as described above, the reagent used is preferably chosen from conductive copolymers, such as for example polypyrroles, to which proteins, peptides or all are attached molecules specific to the type of biological object to be fixed, such as for example antibodies, receptors, glycoproteins, lectins, cell adhesion molecules ("Cell Adhesion Molecules": CAM), laminin, fibronectin, integrins , sugars, etc ...

The conductive copolymers are for example described in international application WO 94/22889.

The poly pyrroles are particularly preferred according to the invention.

The specific molecules attached to the monomers of the conductive copolymer can in particular be chosen from protein A, protein G, fibronectin and more generally, from cell adhesion proteins and antibodies directed against surface receptors.

The fixing of these molecules on the monomers of the conductive copolymer and in particular on the pyrrole monomers can be carried out according to different techniques:

- either the specific molecules are attached directly to the monomers of a conductive polymer, said monomers carrying -NHS or aldehyde functions capable of reacting with the primary amine functions of the molecule used, - or the specific molecules are indirectly attached to the monomers of a conductive polymer carrying the biotin function, by means of a successive chemical stacking of streptavidin-biotin-specific molecule. For carry out this chemical stacking, the device according to the invention is then treated collectively so as to carry out the copolymerization of the monomers of the conductive polymer carrying the biotin function, then to treat said device with streptavidin and then with a specific molecule linked to the biotin, to obtain pyrrole-biotin-streptavidin-biotin-specific molecule copolymers.

In a first form of implementation, the reagent used to fix the biological object can be specific of this one to allow a direct interaction: reagent of the microcuvette-biological object.

In a second form of implementation, the reagent used is not specific to the biological object. The latter must therefore be functionalized.

For this purpose, the biological objects to be fixed may, for example, be previously functionalized by specific antibodies which can react with the reagents used. In this case, protein A or G fixed only on the trap zone via the conductive polymer, will recognize the Fc fragment of the antibodies fixed beforehand on the objects to be immobilized.

Among the peptides which can be attached to the monomers of the conductive copolymer, mention may in particular be made of the binding peptides specific for the membrane receptors on the surface of the biological object to be fixed, such as for example peptides containing the arginine-glycine-aspartate sequence ( RGD) and which have an affinity for integrins (cell adhesion proteins on the surface of eukaryotic cells).

When the bottom of the microcuvettes consists of a layer of glass, plastic or silicon, the reagent used is preferably:

- a polymer not specific to the type of object to be fixed, such as for example poly-L-lysine or fibronectin; said polymer being deposited locally on the reception areas ("lift-off" technique: deposition of a photo-imageable resin, localized exposure then deposition of the polymer on the resin, then unblocking of the resin),

- a protein or a peptide; in this case the proteins and the peptides are fixed to the said glass, plastic or silicon layer covered with a layer of silane modified by -NHS or aldehyde functions on which is fixed said reagent; the proteins and peptides used in this case being of the same nature as those described above.

According to a second embodiment of the invention, the zone for receiving the reaction microcuvettes does not comprise a reagent capable of fixing the biological object which it is desired to isolate.

In this case, the fixation of biological objects is directly carried out by means of an electric field.

This embodiment is particularly advantageous because it avoids the prior functionalization of the devices in accordance with the invention with a reagent capable of fixing the biological object to be isolated. This embodiment is particularly well suited to the isolation and fixation of bacteria.

As described above, the device according to the invention may include several first and / or second and / or third electrodes. These electrodes can be either independent, microreservoir by microreservoir, to allow the reading of an electrical signal in response to a reaction which took place in the microcuvette, or connected together to allow an identical treatment in all the microcuvettes, such as for example the application of an electric field for lysis of biological objects or an electric field of copolymerization.

The different levels of electrodes can allow the specific fixation of biological objects, then once the objects are fixed in the microcuvettes, the lysis of these objects then, when it is for example biological cells, the fixation by electrical copolymerization of nucleic acid probes from a nucleotide amplification carried out directly in each of the microreservoirs, so as to obtain microreservoirs carrying nucleic acid probes in large quantities. When the device according to the invention is equipped with an integrated multiplexing circuit, it is then possible to provide for the fixing of different reagents in the microcuvettes of the same device and the processing of all the signals emitted by the electrodes. of each microreservoir.

When equipped with an integrated multiplexing circuit, the device according to the invention can therefore comprise microcuvettes containing different reagents so as to allow the attachment of biological objects of different types to a single device. The presence of electrodes associated with an integrated multiplexing circuit also allows electrical detection at the level of a microcuvette or a microreservoir, this detection being for example associated with monitoring the electrical behavior of a biological object or the release of molecules. in response to chemical or physical aggression.

The miniature device according to the invention can be equipped with a closure means, such as for example a cover or a transparent film, making it possible to close all or all of the microreservoirs individually or collectively.

Other characteristics of the miniature device in accordance with the invention appear on the attached 1 to 9 in which:

- Figure 1 shows a miniature device according to the invention, equipped with a support 7 and an electrical supply circuit 103, in which the bottom of each microcuvette 5 is constituted by a first electrode 1 forming a reception area 9 to which a reagent is optionally fixed, the first electrode 1 being surmounted by a first layer of insulating material 2 on which a second electrode 3 rests surmounted by a second layer of insulating material 4 forming microreservoirs 6, FIG. 2 represents a miniature device according to the invention, equipped with a support 27 and an electrical supply circuit 103, in which the bottom of each microcuvette ^ 5 is constituted by a first electrode 21 forming a receiving zone 29 on which is optionally attached a reagent, the first electrode 21 being surmounted by a first layer of insulating material 22 on which a second layer rests of insulating material 24 forming microreservoirs 26, this device being equipped with an external electrode 28, - FIG. 3 represents a miniature device according to the invention, equipped with a support 37 and an electrical supply circuit 103, in which the bottom of each microcuvette 35 is constituted by a first electrode 31 forming a receiving zone 39 to which a reagent is optionally fixed, the first electrode 31 being surmounted by a first layer of insulating material 32 on which a second electrode 33 rests surmounted by a second layer of insulating material 34 forming microreservoirs 36, this device being equipped with an external electrode 38, - Figure 4 shows a miniature device according to the invention, equipped with a support 47 and an electrical supply circuit 103, containing a plurality of first electrodes 41 electrically isolated between them and in which the bottom of each microcuvette 45 consists of a first electrode 41 forming a receiving area 49 to which a reagent is optionally attached, the first electrodes 41 being partly surmounted by a first layer of insulating material 42 on which a second electrode 43 surmounted by a second layer of insulating material 44 forming microreservoirs 46, this device being equipped with an external electrode 48, - FIG. 5 represents a miniature device according to the invention, equipped with a support 50 and an electrical supply circuit 103, containing a plurality of first electrodes 51 electrically insulated therebetween and in which the bottom of each microcuvett e 55 consists of a first electrode 51 forming a receiving zone 59 to which a reagent is optionally fixed, the first electrodes 51 being partly surmounted by a first layer of insulating material 52 on which a second electrode 53 surmounted by a second layer of insulating material 54 forming microreservoirs 56, this device being equipped with an external electrode 58 and an integrated multiplexing circuit 104,

FIG. 6 represents a miniature device according to the invention, equipped with a support 67, in ^ equel the bottom of each microcuvette 65 is constituted by a first electrode 61 forming a reception zone 69 on which a reagent is optionally attached, the first electrode 61 being surmounted by a first layer of insulating material 62 on which rests a second electrode 63 surmounted by a second layer of insulating material 64 itself surmounted by a third electrode 101 on which rests a third layer of insulating material 102 forming microreservoirs 66,

- Figure 7 shows a miniature device according to the invention identical to that shown in Figure 1 except that it further comprises a removable closure means 100 for closing each of the microreservoirs 76, - Figure 8 shows a miniature device according the invention identical to that shown in FIG. 5 except that it further comprises a means of removable closure 100 making it possible to close each of the microreservoirs 86 in which an external electrode 88 is integrated,

- Figure 9 shows a miniature device according to the invention, equipped with a support 97 and an electrical supply circuit 103, in which the bottom of each microcuvette 95 is constituted by a layer of glass or silicon 93 forming a reception area 99 to which a reagent is fixed, said layer of glass or silicon 93 being surmounted by a first layer of insulating material 92 forming microreservoirs 96 on which a first electrode 91 rests itself surmounted by a second layer of insulating material 94, this device being equipped with an external electrode 98.

It is of course understood that the devices illustrated in these figures correspond to particular embodiments of the invention and in no way constitute a limitation thereof.

The methods of manufacturing such devices are known and described for example in patent application FR-A-2 781 886.

The invention also relates to the use of at least one miniature device in accordance with the invention for the isolation, separation, culture and / or analysis of biological objects.

By way of example, the miniature devices in accordance with the invention, and in particular the devices ^ of the type of those represented in FIG. 2, can be used to fix a single biological object by microcuvette such as for example a biological cell, these cells then being cultured directly on the device to amplify cells by successive cell divisions. A device is thus obtained comprising a homogeneous population of cells in each microreservoir. The daughter cells from cell divisions can then be recovered while the mother cells remain fixed on the bottom of the microcuvettes.

The devices in accordance with the invention therefore make it possible to recover only daughter cells corresponding therefore only to cell lines capable of dividing. This use is advantageous insofar as it makes it possible to eliminate dead cells from a bacterial culture transformed by plasmids and treated with an antibiotic. The devices in accordance with the invention can also be used as a means of analyzing the content of a heterogeneous panel of cells, by immobilizing different cells in an ordered matrix at the rate of one cell by microcuvette, then extraction of the macromolecules that the 'we want to analyze. With this in mind, we can

- either use devices provided with a plurality of first independent electrodes such as the devices of FIGS. 4 and 5, to fix, different reagents specific to each type of cell to be immobilized,

- Either use a device such as that of FIG. 9 comprising microcuvettes, the bottom of which consists of a glass layer carrying a chemical coupling function or a device such as those represented by the figures

1 and 3, and locally pipette specific reagents according to each type of cell to be immobilized.

The immobilization of the cells is then carried out for example by soaking the device in a heterogeneous cell culture or by successive soaking in different homogeneous cell cultures, the presence of reagents specific to each type of cell making it possible to order the cell matrix.

The second electrode present in all the devices used to immobilize these cells can either be collectively pre-functionalized (for example by specific antibodies (to extract from each cell type a type of protein), or more generally be used to electrochemically fix a product originating from the cell following, for example, a PCR reaction.

The devices illustrated in FIGS. 4 and 5 can also be used to carry out high throughput screening (HTS) of chemical or biological reagents on the cells. In this case, the plurality of first independent electrodes allows individualized electrical measurement in response to the action of chemical or biological reagents on the cells in the microreservoirs.

These HTS screens can be carried out on animal cells in culture and in this case, the surface of the bottom of the microcuvettes is equal to or less than the smallest section of the cells to be tested, ie approximately 100 μm ² for conventional animal cells. In addition, the devices according to the invention can be used to carry out fleeting electroporation of the cells.

The subject of the invention is also a method of separation and / or isolation of biological objects, characterized in that it consists: - in a first step, in bringing at least one miniature device as defined above, into contact with a solution of biological objects homogenized, in particular with a culture solution of biological cells, to allow the fixing of said objects at the bottom of the microcuvettes on the reception areas, at the rate of at most one biological object per microcuvette, - then in a second step, washing the unbound biological objects, so as to obtain a miniature device on which the objects to be isolated are immobilized.

The biological objects thus isolated and fixed on the device can then be studied according to the techniques described above, for example by measuring the variation in their electrical properties under the effect of an active principle.

When the miniature device used according to this method comprises a multiplexing circuit, it is then possible to carry out individualized electrical measurements in each microcuvette.

According to a first embodiment of the method according to the invention, the fixing of biological objects is carried out by means of an electric field. In this case, devices are preferably used in which the first electrode integrated into the device constitutes the bottom of the microcuvettes.

According to a second embodiment of the method according to the invention, the fixing of the biological objects is carried out by means of a reagent fixed on at least part of the bottom of the reaction microcuvettes. In this case, the bottom of the microcuvettes may equally well be constituted by a first electrode or by a layer of glass, plastic or silicon.

The process according to the invention may possibly include a third step during which the objects fixed to the bottom of the microcuvettes, in particular when it is a question of biological cells, are lysed so as to release the genetic material which they contain in the microreservoir corresponding to the microcuvette where they were fixed. The lysis of fixed objects can be carried out by electric shock, thermal shock or sonication.

The genetic material thus released can then, in a fourth step, be collectively amplified by PCR by introduction into the microcuvettes of the various reagents necessary for a PCR reaction, these reagents comprising in particular at least one primer functionalized with pyrrole groups. The amplified sequences thus obtained are then fixed, in a fifth step, on an electrode by electropolymerization, collectively.

According to a particular embodiment of this method, the third and fourth steps can be carried out simultaneously.

According to yet another particular embodiment of the invention, and when using devices such as those illustrated in Figures 1 and 3 or a device such as that shown in Figure 9, it is possible to use these devices as a screening tool for the search for protein or nucleotide ligands of a given target.

In the case of the search for a protein ligand, the starting cell culture is then an expression cell bank and the cells of the bank are immobilized as described above.

The devices used according to this variant are previously functionalized by the target at the level of an electrode. The target may be a molecule such as a peptide, a protein, a nucleotide sequence, a peptidoglycan, a sugar or any other chemical molecule. This target can also be functionalized by a pyrrole group and thus be fixed on an electrode by electropolymerization. In each microcuvette, a recombinant protein will be expressed by the immobilized cell and released from the cell, either by secretion or by lysis of the cell. During this expression within each cell, it is possible to incorporate labeled protein precursors (such as for example methionine

S35) so that the protein thus expressed is also labeled. Proteins showing affinity with the target will specifically bind to the electrode functionalized with the target. If the proteins have been labeled during their expression, they can then be detected. Otherwise, the detection of the protein / ligand interaction must be carried out according to an additional step consisting for example of reacting a labeled universal anti-epitope antibody, and in this case, an expression library expressing all the recombinant proteins must be used with this epitope universal Positive wells thus contain the potential protein ligands of the target. The level of affinity of this ligand can for example be estimated by means of successive increasingly stringent washes or by competition with other known ligands.

Once the reaction as described above has been carried out, it remains to recover the clones corresponding to the positive wells.

If the immobilized cell is still viable, this recovery can be done by simply culturing the device and pipetting the daughter cells into the positive wells as described above.

If the reaction as described above is harmful for cell division, care will have been taken beforehand to make a duplication of the initial device containing the individualized cells of the bank (chip A).

A duplication method consists, for example, in culturing the chip A and then transferring the daughter cells in a directed fashion to a new identical device (chip B), the microreservoirs of the chip A possibly being placed opposite each other with the microreservoirs of chip B, with stirring.

The chip A then undergoes the treatment as described above in order to determine the wells containing the protein ligand of interest while the chip B makes it possible to recover the clones corresponding to these positive wells, for example by pipetting. By way of example of this particular embodiment of the invention, the cell expression bank is an expression bank, secretor of antibodies or of antibody subdomains. The target fixed on the electrode is a protein, a peptide, a virus, an oligonucleotide, against which an antibody is sought. In addition to the foregoing provisions, the invention also comprises other provisions which will emerge from the description which follows, which refers to examples of immobilization of bacteria on miniature devices in accordance with the invention as well as an example describing the protocol for preparing a DNA chip on a device in accordance with the invention.

EXAMPLE 1 ISOLATION AND FIXATION OF BACTERIA ON A MINIATURE DEVICE VIA PROTEIN A A solution of protein A is prepared at 0.1 mg / ml in phosphate buffer (PB S).

Using a pipette, a drop of this protein A solution is then deposited on a miniature device in accordance with the invention and as described in attached FIG. 1, so that said drop covers all of the pits.

On this device, each microreservoir has a diameter of 230 μm and a depth of 40 μm; the bottom surface of each microcuvette being 40 μm ² .

Then an electric field is applied for 10 seconds between the two electrodes of the chip: potential of + 2.9 V on the electrode where it is desired to fix the protein A, the other electrode being grounded.

When the fixation is carried out, the device is then rinsed with a PBS solution.

Furthermore, a PBS solution containing a bacteria / antibody complex is prepared.

To do this, we first prepare a solution of E. Coli DH5α in PBS (10 ⁹ bacteria / ml), as well as a solution of the corresponding antibody Anti Ε. Coli (Dako) at 0.5 mg / ml.

These two solutions are then mixed (V / V) and left stirring at room temperature for 1 hour and thirty minutes in order to form the bacteria / antibody complex. The bacteria / antibody complex is then concentrated by centrifugation, the excess antibodies being removed by removing the supernatant. The operation is repeated three times, after resuspension of the bacteria / antibody complex in PBS. Using a pipette, a drop of the solution containing the bacteria / antibody complex is then deposited on the miniature device functionalized by protein A, so that said drop covers all of the microcuvettes. The miniature device is then left to incubate for 1 hour and 30 minutes at room temperature, in order to allow the immobilization of the bacteria / antibody complex at the bottom of the microcuvettes.

At the end of the incubation, the device is then rinsed thoroughly with PBS in order to remove the bacteria / antibody complexes which have not reacted with protein A.

A device is obtained on which E. Coli bacteria are immobilized, on the basis of a bacterium by microcuvette.

The miniature device according to the invention thus prepared can then be used in various biological applications.

EXAMPLE 2: ISOLATION AND FIXATION OF BACTERIA ON A MINIATURE DEVICE UNDER THE ACTION OF AN ELECTRIC FIELD

1) Fixation of bacteria by electric field

A suspension of E. Coli DH5α bacteria is prepared in permuted water at the rate of 10 ⁹ bacteria / ml.

A miniature device identical to that used above in Example 1 is then immersed in this suspension of bacteria.

Then an electric field is applied for 10 seconds between the two electrodes of the chip: potential of + 0.9 V on the electrode where one wishes to fix the bacteria, the potential of the other electrode being fixed at - 2V .

When the fixing is carried out, the device is then rinsed with water and dried with a nitrogen blower.

A device is obtained on which E. Coli bacteria are immobilized, on the basis of a bacterium by microcuvette. The miniature device according to the invention thus prepared can then be used in various biological applications.

EXAMPLE 3 PREPARATION OF A DNA CHIP BY PCR FROM A MINIATURE DEVICE CONFORMING TO THE INVENTION

This example describes the general protocol for preparing a DNA chip on a miniature device in accordance with the invention.

1) Depositing a sense primer on a miniature device conforming to the invention A 2.3 ^" M solution of a forward primer modified in position 5 'is prepared by a pyrrole group (0.77 μM) in 2.3 ^{" 2} M lithium perchlorate.

This solution is deposited on the miniature device in accordance with the invention and as prepared above in Example 2.

An electric field is then applied for 3 seconds between the two electrodes of the chip: potential of + 2.9 V applied to the electrode where it is desired to fix the primer, the other electrode being grounded.

The miniature device is then rinsed with water and dried with a nitrogen blower.

2) Lysis of bacteria

This step is carried out by heating the bacteria at a temperature of 94 ° C for 2 minutes.

3) Carrying out of the PCR The PCR is carried out using the following solution: Tris-HCL

1 mM, 5 mM KC1, 2 mM MgCl ₂ , 0.8 mM dNTP; antisense primer marked with biotin in the 5 ′ position: 0.1 μM, BSA 1 mg / ml, Taq DNA polymerase from ROCHE at 0.02 μm tees / μl and sense primer at 0.01 μM.

The chip is then immersed in the oil. The PCR is carried out under the following conditions: 3 minutes at 94 ° C then 30 cycles at 94 ° C for 30 seconds, 60 ° C for 30 seconds and 72 ° C for 1 minute and 30 seconds; then 72 ° C for 3 minutes and finally 25 ° C for 30 seconds. The cycles are performed in a Hybaid thermal cycler.

The miniature device is then rinsed with water after the end of the PCR cycles.

The amplified DNA is fluorescently labeled with Streptavidin phycoerythrin.

The fluorescence is then visualized using a fluorescence microscope.

Claims

1. Miniature device for separation and / or isolation of biological objects comprising at least one first electrode integrated into the device, constituted by a structure provided with a matrix of reaction microcuvettes, each microcuvette comprising a bottom constituting a reception zone, characterized in that said bottom has no hole and that the maximum surface of said bottom of each microcuvette is defined so as to isolate a single biological object, said structure being linked to a supply circuit to create a potential difference between said first electrode and at least one second electrode integrated or external to the device.

2. Device according to claim 1, characterized in that the maximum surface of the bottom of each microcuvette is preferably less than or equal to twice the smallest surface of the biological object to be isolated.

3. Device according to claim 2, characterized in that the surface of said bottom is less than or equal to the smallest surface of the biological object to be isolated.

4. Device according to any one of claims 1 to 3, characterized in that the maximum surface of the bottom of each microcuvette is between 1 μm ² and 400 μm ² .

5. Dispositîrselon to claim 4, characterized in that the maximum area of the bottom of each microcuvette is between 1 and 50 microns ^2.

6. Device according to any one of the preceding claims, characterized in that the matrix of reaction microcuvettes is surmounted at least in part by one or more layers of insulating materials and / or by an attached biocompatible plastic grid, so as to form a matrix of microreservoirs.

7. Device according to any one of the preceding claims, characterized in that one face of the first electrode integrated into the device constitutes the bottom of the microcuvettes.

8. Device according to any one of claims 1 to 6, characterized in that the bottom of the microcuvettes consists of a layer of glass, plastic or silicon.

9. Device according to any one of claims 6 to 8, characterized in that the insulating materials are chosen from polyimides and resins.

10. Device according to any one of claims 6 to 9, characterized in that the microreservoirs have a width and / or a length of between 5 and 500 μm.

11. Device according to any one of the preceding claims, characterized in that it comprises several said first electrodes electrically isolated from each other.

12. Device according to any one of the preceding claims, characterized in that the second electrode is integrated into the device and that it is deposited on a first layer of insulating material and located in a plane spaced from the bottom of said microcuvettes.

13. Device according to any one of the preceding claims, characterized in that it comprises at least a third electrode integrated into the device, a second layer of insulating material being interposed between the second and the third electrode.

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14. Device according to claim 13, characterized in that it comprises several said second and / or third electrodes isolated from each other.

15. Device according to any one of claims 1 to 11, characterized in that the second electrode is external is that it is integral with a cover or a cover.

16. Device according to any one of the preceding claims, characterized in that it is equipped with an integrated circuit for multiplexing at least some of said electrodes.

17. Device according to any one of the preceding claims, characterized in that a reagent capable of fixing the biological object to be isolated is fixed on at least part of the zone for receiving the reaction microcuvettes.

18. Device according to claim 17, in combination with claim 7, characterized in that said reagent is chosen from conductive copolymers to which proteins, peptides or any molecules specific to the type of cell to be fixed are attached.

19. Device according to claim 18, characterized in that the conductive copolymers are chosen from polypyrroles.

20. Device according to claim 18 or 19, characterized in that the reagent is a pyrrole-biotin-streptavidin-biotin-specific molecule copolymer.

21. Device according to claim 17, taken in combination with claim 8, characterized in that the said reagent is chosen from polymers not specific to the type of cell to be fixed.

22. Device according to claim 21, characterized in that the said polymers are poly-L-lysine.

23. Device according to claim 17, taken in combination with claim 8, characterized in that the reagent is a protein or a peptide and that said layer of glass, plastic or silicon is covered with a layer of modified silane by -NHS or aldehyde functions to which said reagent is attached.

24. Device according to claim 17, characterized in that it comprises microcuvettes containing different reagents.

25. Device according to any one of the preceding claims, characterized in that it is equipped with a closing means.

26. Use of at least one miniature device as defined in any one of the preceding claims, for the isolation, separation, culture and / or analysis of biological objects.

27. A method of separation and / or isolation of biological objects characterized in that it consists:

- in a first step, bringing into contact at least one miniature device as defined in any one of claims 1 to 25 with a solution of biological objects homogenized, in particular with a solution for culturing biological cells, to allow the fixing of said objects to the bottom of the microcuvettes on the reception zones, at the rate of at most one biological object per microcuvette, - Then in a second step to wash the unbound biological objects, so as to obtain a miniature device on which the objects to be isolated are immobilized.

28. The method of claim 27, characterized in that the fixing of biological objects is carried out by means of an electric field.

29. Method according to claim 27, characterized in that the fixing of the biological objects is carried out by means of a reagent fixed on at least part of the bottom of the reaction microcuvettes.

30. Method according to any one of claims 27 to 29, characterized in that one uses a device comprising microreservoirs and that it comprises a third step during which the objects fixed to the bottom of the microcuvettes are lysed so as to release the genetic material which they contain in the microreservoir corresponding to the microcuvette where they have been fixed.

31. Method according to claim 30, characterized in that it comprises a fourth step during which the released genetic material is amplified by PCR.

32. Method according to claim 31, characterized in that the third and fourth steps are carried out simultaneously.

33. Method according to claim 31 or 32, characterized in that the amplified sequences So t fixed, during a fifth step, on an electrode by electropolymerization.

34. Method according to any one of claims 27 to 33, characterized in that the device used comprises a multiplexing circuit and that individualized electrical measurements are carried out in each microcuvette.

35. Method according to any one of claims 27 to 34, characterized in that the device used comprises a multiplexing circuit and that the biological objects to be isolated come from a heterogeneous panel of cells.

36. Use of at least one device as defined in any one of claims 1 to 25, as a screening tool for the search for protein or nucleotide ligands of a given target.