FR2795426A1 - Support for genetic analysis comprising reservoir(s) for a medium to be analyzed connected by passage(s) having temperature control device(s) to a test strip with analysis sites having biological probes - Google Patents

Abstract

A biological analysis support comprising reservoir(s) for a medium to be analyzed connected by passage(s) to a test strip with analysis sites fitted with biological probes and temperature control device(s) arranged along the passage(s), is new. An Independent claim is also included for manufacturing the new support comprising forming resistive heaters in a region on one face of a main substrate, forming the analysis sites on the same face but outside this region and applying a cap defining the reservoir to the first face.

Description

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AMPLIFICATION BIOLOGICAL ANALYSIS SUPPORT Technical field
The present invention relates to a biological analysis support, also called a biochip, and intended in particular for genetic analysis.

 Biochips generally include test ranges equipped with a plurality of analysis sites.

On these sites are grafted or fixed all or part of nucleic acids such as, for example, pieces of DNA or DNA strands. Each site can be equipped with strands, also called probes, whose code or genetic sequence is known.

 For analysis, the chip is brought into contact with a medium to be analyzed which also contains nucleic acids.

 The nucleic acids of the medium to be analyzed, called targets, then have the ability to selectively hybridize with probes from the biochip.

Hybridization takes place exclusively between targets and probes with a complementary genetic code.

 Thus, it is possible, by detecting the analysis sites on which a hybridization has taken place, to determine the biological material initially present in the medium to be analyzed.

 Biochips find applications in biological and genetic analysis, especially in the medical fields.

State of the art
As briefly mentioned above, biological analysis with biochips involves three steps

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 main which are the preparation of the analysis sites to fix the probes there, the putting in contact of the biochip with the medium to be analyzed, then the reading of the biochip to determine which sites were the object of a hybridization between probes and targets.

 Various techniques are known for attaching nucleic probes to biochip analysis sites. Among these techniques, mention may be made of, for example, those which use an intermediate compound such as silanes, poly-L-lysine or else a conductive polymer, copolymerized on each site with the probes. In the latter case, the fixing of the probes is initiated and controlled by the selective application on the sites of a bias voltage. The sites are then formed by a stud made of an electrically conductive material.

 These techniques, known per se, are illustrated for example by documents (1), (2) and (3), the references of which are given at the end of the description.

 Reading the chips, that is to say examining the various analysis sites to detect whether or not hybridizations have taken place, can be carried out by microscopy. Advantageously, however, hybridization can also be demonstrated by the detection of fluorescence light emitted by the targets fixed on the probes, in response to excitation light. This second possibility, however, requires preparation of the medium to be analyzed in order to fix fluorescent markers on the targets. On this subject, reference can also be made to the document (1) already mentioned.

 The medium to be analyzed, obtained by extraction of DNA or CDNA from a test sample, can undergo

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 other treatments prior to bringing it into contact with the biochip. Among these treatments, there may be mentioned a target multiplication treatment.

 In fact, the number of target DNAs or DNAs present in a test sample is generally low. It can be increased by a multiplication process based on the enzymatic duplication of the DNA or DNA strands. This process can be an "amplification" of the PCR (Polymerase Chain Reactionduplication polymerase) type.

 The amplification of the DNA or of the CDNA is carried out by adding appropriate enzymes and precursors to the medium and subjecting the medium to thermal cycles at determined temperatures. The amplification cycles can be carried out on miniaturized supports, such as chips, and can be associated with sorting by electrophoresis.

 An illustration of these techniques is given, for example, by documents (4), (5) and (6), the references of which are also given at the end of the description.

 All the operations linked to biological analyzes, and briefly described above, contribute to increasing the difficulty of implementing these analyzes, and therefore their cost.

 In particular, the DNA strand amplification operation proves to be delicate and requires a large number of manipulations.

Statement of the invention
The object of the invention is to propose an analysis support which integrates various means capable

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 to be implemented to carry out the various stages of preparation, analysis and reading.

 One goal is in particular to rationalize and simplify the preparation operations, in order to reduce the cost of the analysis and guarantee high reliability.

 Another object of the invention is to propose methods of manufacturing the analysis support.

 More specifically, the invention relates to a biological analysis support comprising at least one test range which is equipped with a plurality of analysis sites capable of being filled with biological probes, at least one reservoir intended to contain a medium to be analyzed, and at least one fluid passage from said reservoir to said test range, in which temperature control means are arranged along the fluid passage.

 The temperature control means are provided for fixing setpoint temperatures in different successive zones along the passage, so as to be able to initiate a multiplication, for example of the PCR type, of DNA or DNA strands capable of be present in the medium to be analyzed.

 When a plurality of temperature-controlled zones are provided, one or preferably several successive multiplication thermal cycles can be carried out.

 The medium to be analyzed introduced into the reservoir can thus be a medium which has not undergone a prior multiplication treatment, that is to say a medium relatively poor in targets to be detected.

 By flowing through the passage (s) between the reservoir and the test range, the DNA strands

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 or medium cDNA are multiplied so that they can be more easily detected at the analysis sites.

 The DNA strands are multiplied by means of enzymes added to the medium. These enzymes can be mixed directly with the medium introduced into the support tank, but can also be stored in additional support tanks.

 In this case, pipes inside the analysis support can be provided to mix the medium to be analyzed and the enzymes.

 According to a particular aspect of the invention, the fluid passage can comprise a plurality of juxtaposed channels, extending substantially parallel between the reservoir and the test range. The channels can also be folded back to describe meanders between the tank and the test range.

 The use of a plurality of channels makes it possible to treat a larger quantity of fluid while guaranteeing for each channel a low thermal inertia. A low thermal inertia of the medium to be analyzed makes it possible to quickly change its temperature from a given setpoint to a next setpoint of a thermal cycle. This characteristic favors the multiplication operation.

 According to another particular aspect of the invention, the temperature control means can comprise a plurality of heating elements, such as electrical resistors, for example. These define successive heating zones between the tank and the test range.

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 The heating elements can be arranged directly in the fluid passage or be separated from it by a wall.

 It should be noted that other means of controlling the temperature, by heating or by cooling, can also be provided. Among these, there may be mentioned, for example, channels traversed by thermostated heat transfer fluids or Peltier elements. The use of electrical resistors, however, allows improved electrical addressing and control. In addition, the size of the electrical resistances on the support is particularly reduced.

 The analysis sites of the chip can be lined with the molecules forming the probes, according to different techniques chosen, for example, from those mentioned in the description of the prior art.

 When the electrochemical deposition of probes is selected, the sites include conductive electrodes. Selective addressing of the sites by electrical means then makes it possible to apply successively or simultaneously to each site a potential capable of initiating the fixation of the probes.

 The electrodes, or some of them, can be associated with counter-electrodes formed in their vicinity. Thus, it is possible to fix the probes electrolytically by circulating an appropriate current respectively between each electrode and the counter-electrode which is associated with it.

 One or more counter electrodes can also be separated from the structure of the analysis support.

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 The electrical addressing of the electrodes can be direct addressing by associating with each electrode, or counter-electrode, an addressing terminal. The addressing can also be of the multiplexed type from a reduced number of addressing terminals.

 According to yet another particular embodiment of the analysis support, the analysis sites can be associated individually or collectively with electrical heating means. The heating means make it possible to bring each site, or a set of sites, to an optimal temperature for the hybridization of targets with the particular probes which equip these sites.

 The electrical addressing of the heating means, that is to say their supply control, can also be multiplexed or not.

 The invention also relates to a method of manufacturing an analysis support as described above.

 The method comprises the following steps: - the formation of heating resistors on a first face of a main substrate in a so-called multiplication region, and the formation of analysis sites on the first face, outside the multiplication region , - the formation of at least one inlet port on one side of the multiplication region distant from the analysis sites, and the formation of at least one outlet port on one side of the multiplication region facing the analysis sites, the orifices extending partially into the main substrate from the first face,

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 - The formation of at least one groove in the main substrate from a second face, opposite the first face, the groove extending in the multiplication region and connecting at least one inlet port to at least one outlet port , - after the formation of the groove, the transfer of the main substrate to a support substrate by the second face, so as to cover each groove, and thus form at least one channel, - the installation of an openwork cover s pressing on the first face of the main substrate, the cover delimiting at least one reservoir which surrounds at least one inlet orifice, and delimiting a test range which includes the analysis sites and at least one outlet orifice.

 The formation of the analysis sites may include the production of pads, pads or attachment electrodes, intended to receive probes.

 It should also be noted that the steps listed above do not necessarily have to be performed in the order in which they are presented.

 According to another possibility of implementing the invention, the analysis substrate can also be manufactured according to a method, in which: - at least one groove is etched in a support substrate, - the material is transferred onto the support, a main substrate so as to close the groove, - electrical resistances are formed on the main substrate in a so-called multiplication region coinciding with said groove,

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 - Analysis sites are formed in a region outside the multiplication region, in the vicinity of one end of the groove, - at least one entry orifice is made in the main substrate, passing through said main substrate and communicating with one end of at least one groove, facing away from the analysis sites, - at least one outlet orifice is made in the main substrate and communicating with the end of at least one groove, facing towards the sites analysis, - an openwork cover is put in place resting on the main substrate, the cover delimiting at least one reservoir which surrounds at least one inlet orifice, and delimiting at least one test range which includes sites for analysis and at least one outlet.

 As with the method described above, the order of the steps is not imperative.

 Furthermore, a step of thinning the main support may be provided after it has been transferred to the support substrate. This step makes it possible to keep only a smaller thickness of material, sufficient to withstand the electrical resistances, but which has low thermal inertia.

 Finally, according to a third possibility of implementing the method for manufacturing the analysis support, it may include the following steps: - the formation of electrical resistances on one face of a substrate in a so-called multiplication region,

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 the formation of analysis sites outside the multiplication region, the placement of at least one perforated cover on the substrate, the cover delimiting at least one reservoir outside the multiplication region, and delimiting a test range comprising the analysis sites, the cover being crossed in the multiplication region of at least one passage connecting the reservoir to the test range.

 As for the methods indicated above, the order of the stages indicated corresponds to a preferential order but not imperative.

 The passage in the wall portion which separates the test range from the tank can be formed by making a plurality of grooves in a part of the cover intended to cover the substrate between the tank and the test range and by transferring the cover to the substrate. so as to cover the grooves and thus form channels.

 Other characteristics and advantages of the invention will emerge more clearly from the description which follows, with reference to the figures of the appended drawings.

This description is given purely by way of non-limiting illustration.

Brief description of the figures - Figure 1 is a simplified schematic section of an analysis support according to the invention, - Figure 2 is a schematic representation corresponding to a top view of an analysis support according to the 'invention,

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 - Figures 3A to 3D are schematic sections of an analysis substrate during different stages of its manufacture, according to a first method according to the invention, - Figure 4 is a schematic section of an analysis substrate according to the invention, constituting a variant with respect to the support of Figure 3D, - Figures 5A to 5F are schematic sections of an analysis substrate during different stages of its manufacture, according to a second process in accordance with l 'invention.

Detailed description of modes of implementing the invention
The analysis support of FIG. 1 comprises a support substrate 10 on which a cover 12 is glued. The support substrate and the cover are for example made of silicon.

 The cover defines above the support substrate an inlet reservoir 14 intended to contain a medium to be analyzed and an outlet reservoir 16 which surrounds a test area 18 of the substrate 10.

 The opening of the inlet reservoir 14 is adjusted to receive, for example, a supply head of a syringe pump type system.

 The test area 18 is equipped with a plurality of analysis sites, only one of which is represented with the reference 20. In the example illustrated, the analysis site 20 comprises a metal electrode 22, for example made of gold, packed nucleic acid probes 24.

 The nucleic acid probes are, for example, strands of DNA fixed to the electrode by means of a conductive polymer 26.

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 It should be noted that, for reasons of clarity of the figures, the different elements represented are not shown on a uniform scale.

 The inlet tank 14 and the outlet tank 16 communicate by a network of channels 30. These allow the circulation of the medium to be analyzed from the inlet tank 14 where it is introduced, to the test range 18 equipped with the analysis sites.

 In the example illustrated, the channels 30 are etched, for example, in a polyimide layer 28, with a thickness of 5 to 30 μm, located at the base of the cover, in contact with the substrate. The cross-sectional dimensions of the channels are, for example, between 5 and 100 μm, for a channel length of the order of 5 mm.

The partition walls between the channels have a thickness of the order of 5 to 20 µm.

 The channels 30 correspond to an area of the analysis support dedicated to amplification, that is to say the multiplication of the nucleic targets initially present in the medium to be analyzed. To this end, the channels are equipped with a plurality of temperature control elements 31a, 31b, 31c, arranged so as to define a plurality of temperature-controlled zones 32a, 32b, 32c. These zones follow one another between the inlet of the channels, corresponding to the bottom of the inlet tank 14, up to the test range 18.

 The zones 32a, 32b, 32c of the channels are brought to different temperatures, generally included in a range from 40 ° C. to 95 ° C., so as to subject the liquid which crosses the channels to one or more thermal cycles adapted to a multiplication of PCR type.

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 In the example in the figure, only three zones 32a, 32b, 32c are indicated. These allow a single thermal cycle to be carried out. However, a greater number of zones, for example 90 zones, can be provided for carrying out on the order of 30 thermal cycles.

 The number of cycles and the temperature of the zones can be set by the temperature control elements 31a, 31b, 31c. The temperatures are determined as a function of the type of enzymes added to the medium for the amplification and / or as a function of the nucleic acids to be multiplied.

 According to a variant, it is also possible to provide a certain number of heating zones, for example three, and to give the channels a shape folded in meanders or in coils so as to traverse the heating zones several times.

 In the example described, the temperature control elements are equipped with electric heating resistors, for example in polycrystalline silicon, connected by conductive tracks to electrical terminals 40 provided on an edge of the substrate.

 The connections between electrical resistances and the electrical terminals 40 may be direct connections by conductive tracks 48, and / or connections made by means of a multiplexer circuit, shown schematically with the reference 42.

 Connection means, not shown, can also be provided for connecting the electrodes 22 of the analysis sites to addressing terminals provided on the edge of the substrate.

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 In the inlet tank there is an electrical resistance 33 intended to preheat the medium to be analyzed before it passes through the channels 30.

 In the same way, electrical resistors 35 are provided under the test range 18 in the vicinity of the analysis sites 20. These electrical resistors 35 make it possible to control the temperature in the vicinity of its sites in order to maintain the latter at a value favoring the hybridization between targets and probes.

 The electrical resistances in the inlet tank and in the outlet tank are, like those of the channels, selectively connected to electrical terminals 40 on the edge of the substrate 10, via the circuit 42.

 Finally, an electrical insulating layer 44, for example made of nitride, covers the resistors. It also surrounds the electrical terminals 40 and the electrodes 22 of the analysis sites.

 Figure 2 is a top view of an analysis substrate according to the invention.

 It is observed that the inlet reservoir 14 communicates with a plurality of inlet orifices 34 of a plurality of channels 30, adjacent and parallel to each other. The channels, shown in broken lines, extend rectilinearly under the cover 12 to terminate respectively in outlet orifices 36.

The outlet orifices are provided for discharging the medium to be analyzed into the outlet tank 16.

 The outlet orifices 36 are formed on the test area 18 in the vicinity of analysis sites.

 The analysis sites, which in this figure are devoid of lining, are each formed by a

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 electrode 22, in the form of a conductive metal pad, and by a metal counter electrode 23 which respectively surrounds the electrode 22 with which it is associated.

 The use of analysis sites comprising an electrode and a counter electrode respectively makes it possible to form on the sites a lining of target molecules by electrolytic means. Selective electrical addressing of the electrodes also makes it possible to choose the electrodes to be filled and thus more easily fix different probes at different sites.

 For addressing the electrodes, the support comprises a plurality of electrical contact terminals 40a formed on the edge of the substrate. These are connected by conductive tracks 48a, respectively to the plurality of electrodes 22. In the same way, terminals 40b are connected by tracks 48b to the counter-electrodes 23.

 Finally, a set of addressing terminals 40c are provided for applying an electric current to the temperature control elements, in this case the electrical resistances described with reference to the first figure.

 For reasons of clarity, these electrical resistances and the connection lines provided for their supply from the terminals 40c are not shown.

 The analysis support comprises, for example, 48 electrical terminals 40a, 40b, 40c in the form of metal studs with a length of 4 mm, a width of 200 μm and spaced from 100 μm.

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 These terminals are designed to connect the analysis support to external voltage and current generators.

 When the number of analysis sites is very large or when a large number of temperature control elements have to be supplied, electrical addressing by multiplexer can be envisaged.

 The filling of the sites can also take place by electro-polymerization.

 In this case, a mixture comprising for example a first pyrrole and a second pyrrole carrying probe molecules is deposited by pipetting on sites defined by one or more electrodes.

 A bias voltage applied between the electrode (s) and one or more counter electrodes makes it possible to cause the copolymerization of the pyrroles so as to fix the probe molecules on the sites.

 The test range can include, for example, a first electrically conductive plane forming an electrode, this plane being covered with a layer of electrical insulating material which surrounds and defines the location of the analysis sites. A second conductive plane formed above the layer of electrical insulating material can then constitute the counter electrode. The insulating material is, for example polyimide.

 Alternatively, the counter electrode can also be separated from the analysis support.

 The following description relates to a process for manufacturing an analysis support, the

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 main steps are illustrated in Figures 3A to 3D.

 The substrate 10 visible in FIG. 3A is designated by the main substrate. It is a SOI (silicon on insulator) type substrate comprising a thin layer of silicon 2 on the surface, separated from a thick layer 4, also made of silicon, by an insulating layer 6. The layer of intermediate insulator, made of silicon oxide, is also called "buried layer". It is used in particular as an etching stop layer in the etching operations which follow. The thick layer 4 is also designated by "rear layer".

 In a first step, the electrodes 22 of the analysis sites, the temperature control elements 31 and the electrical contact terminals 40 are formed on a main face of the substrate corresponding to the thin layer of silicon 2. The elements of temperature control are formed in a so-called multiplication region.

 The electrodes 22 of the analysis sites, in gold, for example, can be replaced, depending on the applications, by simple studs for attaching probes without conductive properties.

 During the first step of the method, the conductive tracks which connect the electrodes 22 and the temperature control elements 31 to the electrical contact terminals 40 are also formed. These tracks are not shown for reasons of clarity.

 A second step, illustrated in FIG. 3B, comprises the etching of orifices in the thin surface layer 2 of the substrate. This is particularly

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 inlet 34 and outlet 36 of the channels not yet made. The etching is for example a plasma etching with stopping on the buried oxide layer 6.

 The second step also comprises a thinning of the substrate consisting in reducing the thickness of the back layer 4. The total thickness of the substrate 10 is brought to approximately 250 μm.

 After thinning, the rear face of the substrate, that is to say the face opposite to the electrodes, is subjected to a local wet chemical etching in order to form channels 30 in the rear layer which extend respectively from an orifice of between 34, to an outlet 36.

 A second etching is provided to remove the buried oxide layer remaining at the bottom of the channels and to make the channels 30 communicate with the orifices 34, 36. The main substrate as obtained at the end of this etching is illustrated in FIG. 3C.

 It can be observed that the height of the channels is adjustable during the thinning of the rear layer 4. The method is suitable in particular for the production of deep channels, of the order of 20 to 500 μm.

 Furthermore, it is noted that the temperature control elements 31 are separated from the channels by a thin wall, constituted by the thin surface layer 2 of the substrate 10.

 FIG. 3D shows the transfer of the main substrate 10 onto a support substrate 1 of silicon.

The transfer is carried out by bonding the rear layer 4 of the main substrate to the support substrate, so that the latter constitutes a common bottom wall for the channels 30.

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 FIG. 3D also shows the transfer by gluing, of the front face of the main substrate 10 on a plate forming the cover 12.

 The cover 12 is cut so as to provide the inlet 14 and outlet 16 tanks there. A central portion 12a of the cover is supported on the upper face of the substrate to cover the temperature control elements 31 therein. This portion also separates the inlet tank 14 from the outlet tank 16. The inlet tank 14 coincides with the inlet ports 34 and the outlet tank coincides with the test area 18 which includes the outlet ports 36 and the sites analysis.

 According to a variant, the cover can be formed by depositing on the main substrate a thick layer of polyimide, then by etching the latter to form the inlet and outlet reservoirs there. The deposition of this layer and its etching then preferably take place before the etching of the inlet and outlet orifices of the channels.

 In the example of FIG. 3D, the support substrate 1 essentially has a function of maintaining the rigidity of the analysis support. According to a variant, however, represented in FIG. 4, the support substrate 1, also of the SOI type, may include temperature control elements 31a combined with those present on the main substrate.

 In the example illustrated, the support substrate has electrical resistances on its face facing the main substrate, in a region coinciding with the channels 30, that is to say with the multiplication region. Electric resistances

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 which form the temperature control elements 31a are electrically connected to terminals 41, formed on an edge of the support substrate. The connection tracks between the resistors and the terminals are not shown for reasons of clarity.

 However, it is observed that a wire connection 50 respectively connects terminals 41 of the support substrate to corresponding terminals 40 of the main substrate in order to combine the temperature control elements, located in the same zone of the channels 30.

 We now describe with reference to FIGS. 5A to 5F another possible manufacturing method for the analysis support, better suited to the production of shallow channels, that is to say with a depth of between 5 and 50 μm.

 FIG. 5A shows a first step of the method which consists in etching in a first substrate 1, or support substrate, one or more grooves.

The grooves, indicated with the reference 30 correspond to the channels of the analysis support.

 The channels are completed as shown in Figure 5B by sealing on the support substrate 1 a main substrate 10 which covers the grooves.

When the support and main substrates are both made of silicon, the seal can be a direct seal (Silicon Direct Bonding) without addition of material.

 An etching or polishing operation, in particular a chemical mechanical polishing makes it possible to reduce the thickness of the main substrate 10 to a value of the order of 1 to 100 m.

 This step, illustrated in FIG. 5C, makes it possible to reduce the thermal inertia of the main substrate,

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 in particular in the so-called multiplication zone, where the channels 30 are located.

 FIG. 5D shows the construction of the addressing terminals 40, the temperature control elements 31 and the electrodes 22 of the analysis sites. The temperature control elements 31 are resistors formed on the thinned main substrate, above the channels 30. Electrical connections between the terminals 40 and the temperature control elements 31 or the electrodes 22 are not shown, but are also carried out during the process step corresponding to FIG. 5D.

 We can refer to this subject in the previous description.

 Optionally, an electrical addressing of the analysis sites can be omitted. In this case, the electrodes 18 can be replaced by silicon oxide pads Si02 which allow good coupling with the poly-lysine or the silanes.

 The probes distributed by high precision system are then fixed on the pads previously silanized or covered with a lysine type polymer.

 FIG. 5E shows the formation of orifices 34, 36 for entering and leaving the channels. The orifices are formed on either side of the multiplication zone, and substantially coincide with the ends of the channels.

 A last FIG. 5F shows the production of a cover 12.

 As indicated above, this can be formed by depositing a thick layer of polyimide and then by etching this layer to form the

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 inlet and outlet tanks 14,16 and to release the test range 18.

CITED DOCUMENTS (1) "A DNA Microarray System for Analyzing Complex DNA
Samples Using Two-color Fluorescent Probe
Hybridization "
Analytical Methods & Instrumentation, Special Issue uTAS'96
Dari Shalon et al.

(2) "Covalent Attachment of Hybridizable
Oligonucleotides to Glass Supports "
Analytical Biochemistry 247,96-101 (1997), Article n AB972017
Beda Joos et al.

(3) "Electroconducting polymers for the construction of
DNA or peptide arrays on silicon chips "
Biosensors & Bioelectronics 13 (1998) 629-634
Thierry Livache et al., (4) "High-Density PCF and Beyond"uTAS'96 Analytical Methods & Instrumentation
Thimothy M. Woudenberg et al.,

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(5) "Continuous flow PCR on a chip" pages 7-10
Martin U. Kopp et al.,
Imperial College of Science, London.

(6) "Integrated Miniature DNA-Based Analytical
UTAS'96 instrumentation, pages 153-157
19-22 November 96 Basel
M. Allen Northrup, et al.

Claims (23)

1. Biological analysis support comprising at least one test area (18) equipped with a plurality of analysis sites (20) capable of being furnished with biological probes, at least one reservoir (14) intended to contain a medium to be analyzed and at least one fluid passage (30) from said reservoir to said test range, in which the fluid passage comprises temperature control means (31), arranged along the fluid passage.  2. Analysis support according to claim 1, in which the fluid passage comprises a plurality of juxtaposed channels (30) extending between the reservoir (14) and the test area (18).  3. Analysis support according to claim 1, in which the temperature control means (31) comprise a plurality of heating elements defining successive heating zones between the tank (14) and the test range ( 18).  4. Analysis support according to claim 3, in which the heating elements are electrical resistors.  5. Analysis support according to claim 3, in which the heating elements are arranged in the fluid passage.  6. Analysis support according to claim 3, in which the heating elements are separated from the passage of the fluid by a wall. <Desc / Clms Page number 25>  7. Analysis support according to claim 1, comprising a plurality of electrodes (22) forming said analysis sites.  8. Analysis support according to claim 7 comprising a plurality of counter-electrodes (23) formed respectively in the vicinity of said electrodes (22).  9. Analysis support according to claim 7, comprising an electrical addressing network multiplexed electrodes.  10. Analysis support equipped with at least a first conducting plane, forming an electrode, on which at least one analysis site is defined, and at least a second conducting plane forming a counter-electrode, a bias voltage being capable of 'be applied between the first and second conductive planes to cause a fixation of molecular probes deposited on said analysis site.  11. Analysis support according to claim 10, in which the first and second conductive planes are separated by a layer of electrical insulating material.  12. analysis support according to claim 10, in which the second plane is separated from a structure comprising the first conductive plane. <Desc / Clms Page number 26>  13. Analysis support according to claim 4, comprising an electrical addressing network (42) multiplexed electrical heating resistors.  14. Analysis support according to claim 1, comprising electrical means (35) for heating the analysis sites.  15. Analysis support according to claim 1, comprising electrical means (33) for heating associated with the reservoir.  16. A method of manufacturing an analysis support according to claim 1, comprising the following steps: - the formation of heating resistors on a first face of a main substrate in a so-called multiplication region, and the formation of sites analysis on the first face, outside the multiplication region, - the formation of at least one entry orifice on one side of the multiplication region distant from the analysis sites, and the formation of at least an outlet orifice on one side of the multiplication region facing the analysis sites, the orifices extending partially in the main substrate from the first face, - the formation of at least one groove in the main substrate from a second face, opposite the first face, the groove extending in the multiplication region and connecting at least one inlet orifice to at least one outlet orifice, <Desc / Clms Page number 27>  - after the formation of the groove, the transfer of the main substrate to a support substrate by the second face, so as to cover each groove, - the installation of an openwork cover resting on the first face of the main substrate , the cover delimiting at least one reservoir which surrounds at least one inlet orifice, and delimiting a test range which includes the analysis sites and at least one outlet orifice.  17. The method of claim 16, wherein a portion of the cover covers the heating resistors.  18. The method of claim 16, wherein the heating resistors (31a) are further formed on a face of the support substrate facing the main substrate during transfer, in a region coinciding with the multiplication region of the main substrate.  19. A method of manufacturing an analysis substrate according to claim 1, in which: - at least one groove is etched in a support substrate, - a main substrate is transferred onto the support substrate so as to close the groove, - electrical resistances are formed on the main substrate in a so-called multiplication region coinciding with said groove, - analysis sites are formed in a region outside the multiplication region, in the vicinity of one end of the groove, <Desc / Clms Page number 28>  - at least one entry orifice is made in the main substrate, passing through said main substrate and communicating with one end of at least one groove, turned away from the analysis sites, - at least one orifice is made outlet in the main substrate and communicating with the end of at least one groove, facing the analysis sites, - an openwork cover is put in place resting on the main substrate, the cover delimiting at least one reservoir which surrounds at least one inlet, and defining at least one test range which includes analysis sites and at least one outlet.  20. The method of claim 19, further comprising, after the transfer of the main substrate, a thinning thereof.  21. A method of manufacturing an analysis support according to claim 1, comprising the following steps: - the formation of electrical resistances on one face of a substrate in a so-called multiplication region, - the formation of analysis sites outside the multiplication region, - the installation of at least one perforated cover on the substrate, the cover delimiting at least one reservoir outside the multiplication region, and delimiting a test range comprising the sites of analysis, the hood being crossed in the region of <Desc / Clms Page number 29>  multiplication of at least one passage connecting the reservoir to the test range.  22. The method of claim 21, wherein, to form the passage, a plurality of grooves are practiced in a part of the cover intended to cover the substrate from the tank to the test range and the cover is transferred to the substrate so as to cover the grooves.  23. The method of claim 21, wherein the grooves are practiced in a layer (28) of polyimide of the cover.