WO2003100423A2 - Better organised biochip - Google Patents

Abstract

The invention relates to a biochip (1) comprising a support (2). One useful surface (2a) of the aforementioned support comprises an operating surface (3) having a network of basic sites (Xn) disposed thereon and numerous ligands which are fixed to the respectively different basic sites. The inventive biochip is characterised in that the basic sites are distributed between a central biological analysis area (4) and a peripheral area (5) which defines said central area and which comprises corners that are free of any basic sites.

Description

 BIOPUCE WITH AN IMPROVED ORGANIZATION

The present invention relates to biochips, and more particularly the mode of organization of its binding sites, to serve as tools in molecular biological analysis.

By “biochip”, is meant a functional unit, for single use or not, generally implemented for the purposes of molecular biological determination, arranged for and / or intended to cooperate with at least one distinct and complementary device or instrument, and receiving a sample of interest, liquid or fluid, immobile or in motion, suspected of containing at least one target species, in suspension or in solution, for example a biomolecule, possibly labeled, and delivering at least one output signal in relation to the presence, and / or the nature, and / or the structure, and / or the quantity of said target species. This biochip includes at least:

- a support comprising a useful face comprising an operating surface in contact with the sample; the material or the material of the support is inert, and not substantially electrically conductive, at least according to said operating surface, in the sense that said material practically does not interact with the sample, and in particular the target species, by strong bond of the covalent chemical bond type, or by weak bond, for example hydrogen bond, other interactions of physical type, such as surface tension, not being otherwise excluded; the material constituting such a support is for example silicon, a glass, or a plastic material, for example a thermoset or thermoplastic synthetic resin, for example a polypropylene, a polystyrene or a polyacrylamide resin,

a network or matrix of elementary sites distributed in a methodical or orderly fashion on the operating surface, in the manner of pixels, along at least two reference axes, each elementary site being addressed, that is to say identified by coordinates which are unique to it in any benchmark formed by the axes of reference; these elementary sites are themselves treated or not, with a view to fixing or anchoring the ligands, which will be discussed below; this treatment may for example consist of a coating with a layer of an electronic conductive polymer, for example modified polypyrrole, according to the techniques described in the documents FR 2703359, WO9422889, EPO 691 978, FR 2 787 582, EP 1 141 391, and WO 0036145,

- Where appropriate, a set of connections, or circuit, for example electrical, with the various elementary sites respectively, said set of connections being arranged to connect each elementary site individually, independently from the other elementary sites, for example by addressing (see document FR-A-2741475), said set of connections is for example an electrical circuit multiplexed with electrodes and counter-electrodes disposed respectively in said elementary sites;

- a multiplicity of ligands, or probes, identical attached or anchored, by any appropriate means, in each elementary site;

- Optionally, means forming with the operating surface a circulation cavity or stay of the liquid sample, comprising at least one inlet and one outlet for the latter;

- optionally, means of observation, transmission, or emission of the signal or output signals, corresponding to the binding of the target species with the ligands, respectively at one or more binding sites; This biochip can also comprise, in an integrated manner or not with the operating surface and its ligands or probes, various means, brought to the scale of the biochip, conventionally used in the laboratory, for:

- obtain or prepare the sample of interest, from another so-called starting sample, by denaturation, separation, concentration, purification, etc. ;

- Treat the sample of interest, for example by amplification, and / or labeling with a marker, before bringing it into contact with the ligands or probes;

- Process the complex (s) formed between the target species and respectively the ligand (s), for example by labeling, in order to obtain, simultaneously or after the formation of the complexes, respectively the output signal (s).

When the biochip, or "biochip", or "micro-array", is associated or further comprises these laboratory means, in the technical sector concerned, we will then speak of "labopuce" (or "lab on a chip"). In such a case, the support comprises a set of channels and / or sinks, for example reactive, linked together and with the circulation cavity or stay, as well as various reagents or treatment agents, in the dry or liquid state, arranged on the support according to the circulation and the treatment or treatments retained for the sample starting point or sample of interest. The dimensions of these biochips, for example of the order of 1 mm ² to a few cm ² , normally require, for their production or production, the implementation of techniques known as “micro” or “nano-technologies”, for example by lithography or micro-machining.

However, the Applicant does not intend to be limited to particular dimensions, in particular of the order of μm or of nm, when it uses the term "biochip" in the present description and in the appended claims, considering that the same structure or the same arrangement as that defined below can be implemented with dimensions of the order of a few mm ² , as with much larger dimensions. Of course, a biochip as considered by the present invention can not function autonomously, except to embark with it its own source of energy. Consequently, this biochip is designed to cooperate, for example removably, on the one hand with external means making it possible to circulate or remain, in contact with the operating surface and the ligands, the liquid sample of interest, but also other fluids or liquids such as washing liquid, and on the other hand with means for detecting and processing the output signal (s), the whole being generally controlled and controlled, by external electronic means, analog or IT, for example according to any processing logic or flowchart.

By "biomolecule" is meant any entity, in particular biochemical, or biological, identical to or derived from any molecular species existing in nature. Among the biomolecules considered by the present invention, mention may be made of certain biopolymers, for example DNA, RNA, oligo and polynucleotides, functional or structural proteins, peptides, oligo and polypeptides, polysaccharides, etc.

By “marking” or “marked” is meant the characteristic according to which a marker, that is to say a substituent or residue allowing, is fixed on an entity, for example the target species, covalently or otherwise, with or without the help of an external means, such as illumination, with or without a subsequent step, such as contacting a substrate, to produce a signal, previously called the output signal.

The preferred markers according to the present invention are:

- haptens (example: biotin fixing a streptavidin-phycoerythrin conjugate)

- fluorophores, for example fluorescein, cyanine, phycoerythrin - luminophores: luminol, isoluminol, ABEI (_N-4-amino-butyl-N- ethyl-isoluminol)

- enzymes: for example oxidation of a chromogen; confer horse-radish peroxidase, alkaline phosphatase

By “determination” is meant the identification, qualitative and / or quantitative, the detection, the description (for example sequencing), the separation, or the enrichment of the target species, which can be called “analyte” in the case qualitative and / or quantitative identification. According to the present invention, the expression "determination" means any sequencing of a DNA or polypeptide type biomolecule.

The output signal or signals considered by the present invention may have any suitable nature, depending on the markers used, and the type of detection required. They may be light, visible or invisible, electrical, electro-optical, electrochemical, etc. signals. Furthermore, these signals may, if necessary, be detected separately, taking into account, on the one hand, the addressing elementary sites of the biochip, and on the other hand of the connection assembly respectively with the various elementary sites, possibly present on the biochip.

By "ligand" is meant any entity, cellular, biological, or biomolecule having an affinity, specific or not, for a target species. Affinity expressing that it forms, under the conditions (in particular temperature, pH, ionic strength, etc.) of bringing the target species into contact with the ligand, a stable pairing or complex between said target species and said ligand. By way of example of a ligand, mention will be made of any oligonucleotide capable of binding by weak bonds, in this case it is said to hybridize, with a strand of DNA (target species), comprising a sequence complementary to that of the ligand. Each ligand is attached or anchored at each elementary site of the biochip, optionally after functionalization of the operating surface of the support, by any suitable means, for example chemical, by covalent bond, for example by means of a spacer arm, or by adsorption, absorption, etc.

In the case of elementary sites coated as indicated above with a polymer of modified polypyrrole or polythiophene type, and electrically addressed, the binding of the ligands can be carried out according to the electrochemical techniques described in documents FR 2789401, EP 1 152 821 and WO 0047317, FR2 742 451, EP 0 868 464 and WO 9722648.

By target species is meant any cell, biological or biochemical species capable of binding by weak bond to one or more ligands.

Being a biochip, in the current state of the technical sector considered, we can distinguish two ways of obtaining respectively different ligands, each fixed in multiple number respectively on the different elementary sites:

- an in situ way, which consists, by a series of successive operations, incremented, in synthesizing on the operating surface itself, the different ligands, together, elementary motif by elementary motif, for example sea by sea, starting from 'a first motif fixed at different elementary sites, and this in the order of the sequences respectively chosen for the different ligands; in this regard, reference is made to documents FR 2703359, EP 0 691 978, W09422889, US 5744305, US 6 015 880, WO 9525116, and EP 0 750 629,

- And an ex situ way, which consists in synthesizing or obtaining the respectively different ligands outside the operating surface, and in fixing each in multiple number the different ligands in their elementary sites respectively different, by printing methods (with or without contact) or by differentiated electrical activation, because addressed, of said elementary sites; confer documents FR 2703359, EP 0 691 978 and

WO9422889, WO 0151689, US 6,090,933.

Today, biochips are tools, simple or complex, well suited to all kinds of molecular biology analysis; confer "DNA chips: a new tool for genetic analysis and diagnostics". M.Cuzin. Clinical and Biological Transfusion 2001; 8: 291-6. "How to make a DNA chip" Mickael C Pirrung. Angew.chem.lnt.ed 2002, 41, 1276, 1289. In general, a biochip as described above is placed at the bottom of a well, for example a micro-titration plate, within which the liquid sample comprising the target species is introduced, stays, then is discharged. The object of the present invention is to organize the operating surface, the network of elementary sites, and the ligands or probes of the biochip, so as to be able not only to determine one or more target species, but also to control on the support of the biochip this determination and its operating conditions. In other words, the subject of the invention is a biochip embedding the means making it possible to control the biological determination carried out principally with said biochip.

Furthermore, the present invention relates to a biochip, for which the output signals, for example of the light type, are not disturbed by the geometrical environment of said biochip, whatever the elementary site considered.

A biochip according to the invention generally comprises a support, one useful face of which comprises an operating surface with a network of elementary sites disposed on the operating surface and a multiplicity of ligands each fixed in multiple number at respectively different elementary sites.

According to the invention, the elementary sites are distributed between, on the one hand, a central zone assigned to the biological determination of at least one target species, comprising active elementary sites, each comprising in multiple numbers so-called active ligands, respectively different , intervening directly in the biological determination, and a peripheral zone (5) circumscribing at least in part the central zone, comprising elementary so-called control sites, comprising where appropriate so-called control ligands. Preferably, the contour of the operating surface has a generally polygonal shape, for example rectangular, with rounded angles. And, for example, for this purpose, the elementary sites are distributed between a central zone whose contour has a generally polygonal shape, for example rectangular, and the peripheral zone circumscribing at least partially the central zone, whose contour usually has the shape of a line broken, for example rectangular, open or closed, said peripheral zone having angles free of all elementary sites.

The present invention is now described, with reference to the accompanying drawing, in which: FIG. 1 represents a biochip according to the invention, seen from above and on an enlarged scale, diagrammatically FIG. 2 and FIG. 3 represent the deposition plans of ligands or probes in biochips identical to the representation according to FIG. 1. In accordance with FIG. 1, a biochip according to the invention comprises a support (2) of which a useful face (2a), for example upper, comprises a operating surface (3) supporting a network of elementary sites (Xn), arranged on said surface, and a multiplicity of ligands (not shown, and therefore not numerically referenced), each fixed in multiple number at respectively different elementary sites.

As shown in FIG. 1, the elementary sites each identified by a letter (A to Z) and by a number (0 to 13) are distributed between, on the one hand a central zone 4, of rectangular shape, corresponding to the rows A to H and columns 1 to 12, assigned to a biological determination of at least one target species, comprising elementary so-called active sites, since it intervenes directly in the biological determination of the target species, each comprising in multiple number of said ligands active for the same reasons, respectively different, and on the other hand a peripheral zone (5), constituted by rows I and Z and columns 0 and 13, circumscribing at least partially the central zone (4), comprising sites so-called control elementals, possibly including so-called control ligands, in the sense that said sites and ligands are not directly involved in the biological determination of said target species, and so optional a number y of elementary sites called identification of the type of biochip thus produced. As shown in Figure 1, the contour of the operating surface

(3) therefore has a generally polygonal shape, for example rectangular, with rounded angles. And to this end, all the elementary sites (Xn), are distributed between the central zone (4) and the peripheral zone (5), previously defined, circumscribing at least in part the central zone (4), and whose outline has generally the shape of a broken line, for example rectangular, open or closed, this peripheral zone (5) having angles free of all elementary sites.

By way of examples, the following is described the obtaining, the construction and the use of biochips identical to that described with reference to FIG. 1 and to the preceding description. These biochips are produced and obtained from a silicon-based support, according to the technique generally described in documents FR2787582, EP1141391 and WO0036145 with the following main essential steps:

a) Structuring of a substrate so as to obtain microcuvettes on this substrate comprising in their bottom a layer of a material capable of initiating and promoting the adhesion thereon of a film of a pyrrole copolymer functionalized by electropolymerization, b) Collective electropolymerization, so as to form an electropolymerized film of a copolymer of functionalized pyrrole and pyrrole on the bottom of said microcuvettes, on the layer of said material, from a solution of functionalized pyrrole and pyrrole , in the presence of chemical reagents suitable for said electropolymerization, c) Direct or indirect fixing of a biological probe on the functionalized pyrrole, by injection of a solution of the biological probe, optionally into one or more microcuvette (s) in the presence of chemical reagents necessary for the direct, or indirect, fixation of this biological probe on the functionalized pyrrole.

EXAMPLE 1 Biological Model for Demonstrating the Differential Profiles of Messenger RNAs and their Detection Threshold

In order to highlight the changes that take place at the level of the profiles of messenger RNAs within a biological unit (for example between two different physiological situations), we have implemented a biological model containing genes whose expression levels involve three classes of messenger RNA 1. very abundant mRNA (10%); 2. moderately abundant mRNA (1%); 3. sparingly abundant mRNA (of the order of one copy or more). We have chosen the cadmium detoxification system in yeast (Saccharomyces cerevisiae) as a biological model. This well-documented system contains ten genes whose expression levels perfectly meet our criteria. These ten genes are divided into 5 classes according to their response to the presence of cadmium:

Constituent class: Act1 (1 X), Opi3 (1 X) Moderately induced class: Ynl208W, Ydr411C (= 5 X) Strongly induced class: Seo1, Gre1 (> 30 X) Moderately repressed class: Rps31, Krr1 (≡1 / 5 X) Highly repressed class: Yef3, YJI200C (≡1 / 16 X) Table 1: Number of copies of mRNA per cell corresponding to these genes

(According to the reference Holstege et al. (1998) Cell 95: 717-728). The coding sequences of these 10 genes were extracted from the SGD website dedicated to the complete yeast genome (http: // qenome- www.stanford.edu/ Saccharomyces /, Deval A. Lashkavi et al. (1997) PNAS 94: 13057-13062).

The choice of yeast lies on the one hand in that it is an ancestral eukaryotic organism, and on the other hand in its organizational proximity of the genome with that of higher eukaryotic cells. In addition, it is easy to manipulate and its genome is fully sequenced (6430 genes distributed on 16 chromosomes). It is also well documented and widely used as a biological model in pharmacology, toxicology and oncology studies.

Yeast culture, extraction of total RNAs and labeling of

cDNA

This study was carried out using the YPH499 strain (MAT α ura3-52 his3-Δ200 ade2-101 uaa trpi - Δ 63 Iys2-801 uag Ieu2- Δ 1). The cells were cultured in a YPD liquid medium containing 6.7 g / l of nitrogenous yeast base and 5 g / l of ammonium sulfate without amino acids (Qbiogen, France), 2% glucose, 30 mg / l of uracil, adenine, lysine, histidine, tryptophan and leucine. Growth takes place until the middle of the exponential phase (DO600 = 0.4) at 30 ° C, then the cells are exposed to 1 mM CD ²⁺ for 1 h (Fauchon et al., 2002), harvested, washed twice in 10 ml of sterile water and resuspended in the buffer S (Sorbitol, phosphate buffer, and 1 μl of Mercaptoethanol). The addition of Zymoliase (100U / mL) in buffer S for 30 min at 30 ° C allows destruction of the cell wall. The extraction of total RNAs is carried out using TRIAZOL (Abgene®, UK). The integrity of the RNAs is checked on agarose gel and by spectrophotometer at 260 and 280 nm.

The total RNAs (approximately 20 μg) of the test and reference samples are back-transcribed using the Superscript II Reverse-Transcription kit.

(Invitrogen Corporation; Carlsbad-CA) in the presence of dNTPs and a dNTP-biotin. The cDNAs thus labeled are purified on Microcon filters according to the manufacturer's recommendations (Milipore Corp., Canton, Massa.).

II. Choice of pyrrole probes

The probes were chosen using OLIGO 6 software which takes into account their affinity, their percentage of G-C and their Tm (melting temperature). Initially, these probes terminated by an amino group were qualified on a glass slide with PCR products and cDNA. The single probes thus retained are 50 seas on which was grafted a spacer of type dT 10 terminated by a pyrrole group in position 5 ′.

III Choice of calibrators The internal calibrators C1 (Porphyromonas gingivalis W83), C2

(Arabidopsis Thaliana) and C3 (Plectovirus) were chosen according to the same criteria set for the yeast pyrrole probes. These calibrators as well as the corresponding single targets have no significant homology with our sequences of interest, in this case those of the yeast genome. The targets of calibrators C1, C2 and C3 are added in this order at concentrations allowing to obtain increasing signals with factors of 102. (exp: Signal C3 = (Signal C2) 2 = (Signal C1) 4. This will allow to estimate the number of target copies present in the biological sample of interest. IV. Addressing of pyrrole probes

The addressing is carried out using pyrrole monomers whose nitrogen is substituted by 50-mer oligonucleotides synthesized and checked beforehand. All the probes will carry at their 5 'end a spacer of the dT10 type adjacent to the polypyrrole group and which ensures maximum accessibility. The 128 microelectrodes are successively addressed according to the deposition plan described in the present invention. The electrocopolymerization takes place in an aqueous solution containing an inert salt: 0.2 M LiCIO4 as electrolyte and a mixture of pyrrole 20 mM and 1 μM of ODN-pyrrol. The electropolymerization takes place under a potential of 1 V vs / DHW. Between each deposit, the chip is thoroughly rinsed with deionized water. At the end of this stage, the matrix is dried, stored under Argon at 4 ° C. until it is used.

V. Hybridization on MICAM chip

Hybridization solution (100 μl) is composed of: 1 μl of biotinylated target (10 μM); 99 μl of hybridization buffer (1.3% Denhart 5X; 66.7% Salmon sperm; 31.9% of buffer A)

The hybridization is carried out as follows: 20 μl of the hybridization solution are deposited on the chip. The chip is then covered with a coverslip for maximum sealing. Incubation is carried out for 45 minutes at 45 ° C. in a humid chamber.

The post-hybridization washing is carried out with 4 × 25 μl of the buffer (0.01 M PBS; 0.534 M NaCl; 2.7 mM KCI; 0.05% Tween 20). Streptavidin-Phycoerythrin (0.1 mg / ml) is used for 10 min in the dark for development.

Post-revelation washes are carried out in 4 X 25 μl of washing buffer.

Buffer A: PBS: 0.02 M; NaCl

Nucleotide sequences of pyrrole probes and calibrators (the probe number corresponds to its position on the coding sequence of the corresponding gene).

Le plan de dépôt des sondes est montré à la figure 2, dans la zone centrale (4) correspondant aux rangées A à H , et aux colonnes 1 à 12. Cette disposition reproduit exactement le format des 96 positions d'une plaque de microtitration. Il conserve l'indexation qui sert de référence à l'homme du métier.

The probe deposition plan is shown in Figure 2, in the central zone (4) corresponding to rows A to H, and to columns 1 to 12. This arrangement reproduces exactly the format of the 96 positions of a microtiter plate. It retains the indexing which serves as a reference for those skilled in the art.

Results on PCR Products:

First, we verified the hybridization steps with long PCR products of the order of 500 bp. The resulting signals are very specific between the probe and the PCR product which covers it (Fig. 4). FIG. 4 shows the specificity of the hybridization signals on a Micam® chip in the presence of long PCR fragments. (fig 4 a: Hybridization ACT 560 Bio (6 ng / μl) fig 4 b: Hybridization Gre 36 Bio (6 ng / μl) fig 4 c: Hybridization Rps 38 Bio (3 ng / μl) + T3 (Plectovirus) (0.5 ng / .mu.l)). The% standard responses are ranged between 80 and 100% as shown in the table below.

Stallion 1361

Example 2

Detection of point mutations by mini-sequencing of the p53 and K-ras genes, the most frequently described in colorectal human cancers

These point mutations are defined in the table below.

ACAS CTTAATGTCA CGCACGATTTCCC

ACAS CTTAATGTCA CGCACGATTTCCC

AGAATGCCA

P72S GAGGCTGCTCCCC Human

GCTGGTGCAG P72AS GGGCCACG Human

GCACATGACG

175S1 GAGGTTGTGAGG Human

CAGCACATGAC

175S2 GGAGGTTGTGAGGC Human

GCTCATGGTGG

175AS1 GGGCAGC Human

CGCTCATGGTGG

175AS2 GGGCAG Human

TGCATGGGCGGC

248S1 ATGAAC Human

GCATGGGCGGCA

248S2 TGAACC Human

GATGGTGAGGAT

248AS1 GGGCCTCC Human

GATGATGGTGAG

248AS2 GATGGGCCTC Human

CTGAATATAAACT

TGTGGTAGTT

12S1 GGAGCT Human

TGAATATAAACTT

GTGGTAGTTGGA

12S2 Human GCTG

GGCACTCTTG

12AS1 CCTACGCCAC Human

AGGCACTCTT

Humain

12AS2 GCCTACGCCA Human 13S1

Human

Hybridization is carried out at 45 ° C. for 1 h 30 min in the MICAM hybridization buffer from 15 μl of the aliquot fraction of a purified PCR product (alkaline phosphatase, exonuclease I) and fragmented (digestion with a restriction enzyme).

In the previous tables, the sense and antisense probes are described by their respective sequences, and constitute the active ligands within the meaning of the general definition of the invention explained above. The target species is a genomic fragment of a patient comprising the exons of interest of the p53 or Kras genes.

The addressing is carried out from pyrrole monomers whose nitrogen is replaced by oligonucleotide sequences whose size is between 15-30 seas previously synthesized. All the probes will carry at their 5 'end a linker which follows the polypyrrole grouping thus ensuring maximum accessibility. The 96 microelectrodes are successively addressed according to the deposition plan described in FIG. 3. The electrocopolymerization takes place in an aqueous solution containing an inert salt: 0.2M LiCIO ₄ as the electrolyte and a mixture of pyrrole 20mM and 1 μM of ODN-pyrrol. The electropolymerization takes place at a potential of 1 V vs / DHW. Between each deposit, the chip is rinsed with deionized water. At the end of this process, the matrix is dried, stored under Argon at 4 ° C until it is used.

The deposition plan for the sense and antisense probes is shown in Figure 3, in the central zone (4) corresponding to rows A to H, and to columns 1 to 12. This arrangement reproduces exactly the format of the 96 positions of a microtiter plate. It retains the indexing which serves as a reference for those skilled in the art.

With a genomic sample of a patient, obtained for example after amplification, the operating procedure for the implementation of the biochip previously described, with a view to detecting the mutations previously defined, comprises the following essential steps:

Hybridization is carried out at 45 ° C. for 1 h 30 min in the MICAM hybridization buffer from 15 μl of the aliquot fraction of a purified PCR product (alkaline phosphatase, exonuclease I) and fragmented (digestion with a restriction enzyme).

The post-hybridization washing is done with 4 X 250 μl of PBS.

In a final volume of 20 μl, the extension is carried out for 1 H 30 min at 45 ° C. in the presence of Thermosequenase (8U), of the buffer of thermosequenase, of ddNTP (10 μM final) including one biotinylated. The post-extension washes are done for 2 X 90 sec with H2O at

80 ° C. Streptavidin-Phycoerythrin (0.1 mg / ml) is used for 10 min in the dark for development.

The post-revelation washes are carried out in 4 X 250 μl of washing buffer.

For these two examples previously, or at the same time as the deposition of the probes, or after said deposition, various elementary sites, belonging to the peripheral zone (5), are generally assigned to the control of the biological determination carried out elsewhere in the central area (4).

To this end, in the peripheral zone (5), ten elementary sites (10), corresponding to row I according to FIG. 1 and at positions 2 to 11, are assigned together to the digital identification of the biochip. To this end, each elementary identification site (10) comprises a marker fixed or linked via the pyrrole, capable of generating an identification signal, for example a light signal. The combination of identification signals, obtained during the processing of the biochip, generates an identification code in binary format. As shown by way of example in FIG. 3, the sites 7; 8 and 11 have a marker attached or linked via the pyrrole (1), then that the other sites in row I do not have one (0). Hence in Figure 3 the code 0000011001, which is a binary coding whose number is 1 x 2 ⁴ + 1 x 2 ³ + 1 x 2 ° = 16 + 8 + 1 = 25. This number is therefore characteristic of plan for depositing and / or allocating the chip to a given analysis.

In the peripheral zone (5), two elementary sites (11 and 12) called positive control and negative control respectively, predetermined in positions (D0 and C0) respectively comprise a ligand capable of binding with the target species, and a ligand not capable of binding with said target species, under the conditions of contact with binding between the above-mentioned target species and the other ligands in the central zone (4). the ligands attached in zones 11 and 12 are each different from the ligands or probes present in the central zone (4).

This arrangement makes it possible to validate the stages of preparation of the biological sample to be studied and / or the stage of hybridization

In the peripheral zone (5) two elementary sites 13 and 14, for quality control, predetermined in position (B0 and G0).

It is about quality control of the different stages of the realization of the biochip. It can therefore be, as in Example 1, elementary quality control sites comprising an electrolyte which makes it possible to validate the process for manufacturing the biochip as to whether the biochip is indeed potentially addressable.

These can be elementary quality control sites of the detection system, for example comprising a pyrrole - oligonucleotide - biotin on which avidin is capable of clinging to the avidin coupled to a marker.

Finally, these can be basic quality control sites for sample preparation and hybridization. These ligands therefore undergo the same treatment as the sample to be analyzed.

Still in the peripheral zone (5), an elementary site (15), said to be untreated, predetermined in position (E0), remains free from any treatment, and or an elementary site (16), said to be treated, predetermined in position

(F0), remains free of any ligands, for example with a layer of an electronic conductive polymer. This arrangement makes it possible to appreciate the "background noise" likely to be generated by the support itself or its processing, in particular in the elementary sites previously defined.

In the peripheral zone (5), ten elementary calibration sites (17), predetermined in position (the whole row Z, positions

2 to 11), are assigned together to the quantitative measurement of the so-called output signals generated by one of the markers fixed or linked, via the pyrrole, on the support (2).

These elementary identification sites comprise respectively different quantities (from 10 ⁴ to 10 ⁹ molecules) of one or more species of markers. This arrangement makes it possible to establish a calibration of the reading.

In the peripheral zone (5), six elementary sites (18), called calibration sites, predetermined in position (column 13, positions B to G), are assigned together to the internal calibration of the biochip. These elementary calibration sites comprise respectively different quantities (from 10 ⁴ to 10 ⁹ molecules) of a “calibrator” target species, having no homology with the targets of interest. This arrangement makes it possible to appreciate the quantity and the efficiency of the hybridization. Finally, the support (2) comprises a member (19), said polarizing, deposited in the peripheral zone (5) on the support (2), which can consist of an additional elementary site, and which makes it possible to identify in relative position the biochip (1).

As the preceding description shows, in the central zone (4) the elementary sites are one hundred and twenty-eight in number and distributed along two orthogonal axes, namely in eight rows referenced A to H along an axis, and twelve columns referenced 2 to 11, along another perpendicular axis.

The peripheral zone (5) comprises two lines or rows referenced Z and I, and two columns referenced 0 and 13, surrounding the central zone (4) of rectangular shape.

Claims

1) Biochip (1) comprising a support (2) of which a useful face (2a) comprises an operating surface (3) with a network of elementary sites (Xn) disposed on said operating surface, and a multiplicity of ligands each fixed in number multiple in respectively different elementary sites, characterized in that the elementary sites (Xn) are distributed between, on the one hand a central zone (4) assigned to a biological determination of at least one target species, comprising so-called elementary sites active, each comprising a multiple number of so-called active ligands, respectively different, directly involved in the biological determination, and a peripheral zone (5) at least partially circumscribing the central zone, comprising elementary so-called control sites, comprising where appropriate so-called control ligands. 2) Biochip according to claim 1 characterized in that the contour of the operating surface (3) has a generally polygonal shape, for example rectangular, with rounded angles 3) Biochip according to claim 1, characterized in that, in the central zone (4), the elementary sites, the number of X multiplied by n, are distributed along two orthogonal axes, for example in four, eight or sixteen (X = 4, 8, 16) rows along one axis, and six, twelve or twenty-four columns respectively (n = 6, 12, 24) along another axis 4) Biochip according to claim 1, characterized in that, in the peripheral zone (5), there elementary identification sites (10), predetermined in position, are assigned together to the digital identification of said biochip, each elementary site of identification comprising or not a labeled ligand capable of generating an identification signal, the combination of the identification signals present or absent respectively in elementary identification sites generating an identification code in binary format of the biochip 5) Biochip according to claim 1, characterized in that the peripheral zone (5) comprises two rows and two columns, framing the central zone of quadrangular shape 6) A biochip according to claim 1, characterized in that, in the peripheral zone (5), two elementary sites, (11, 12) called positive control and negative control respectively, predetermined in position, respectively comprise two ligands capable and not capable respectively of binding with a target species, under the conditions of contact with bond between said target species and at least one other ligand in the central zone (4) 7) Biochip according to claim 1, characterized in that, in the peripheral zone (5), an elementary site (14) said to be untreated, predetermined in position, remains free from any treatment and / or any electrode, and / or an elementary site (15), said treated, predetermined in position, remains free of any ligand, for example coating with a layer of an electronic conductive polymer 8) A biochip according to claim 1, characterized in that, in the peripheral zone (5), z elementary sites (17) said to be of calibration, predetermined in position, are assigned together to the quantitative measurement of the signal or signals, called output, generated by one or more markers attached or linked, directly or indirectly, to the support (2), said elementary identification sites comprising respectively different quantities of the same ligand labeled with said marker 9) A biochip according to claim 1, characterized in that, in the peripheral zone (5), t elementary sites (18) called calibration sites, predetermined in position, are assigned together to the internal calibration of said biochip, said elementary sites calibration comprising respectively different quantities of the same target species, as a ligand 10) Biochip according to claim 1, characterized in that the support comprises at least one member (19), said polarizing device, arranged in the peripheral zone (5) on the support