WO1998029740A1

WO1998029740A1 - Micro system for biological analyses and method for making same

Info

Publication number: WO1998029740A1
Application number: PCT/FR1997/002440
Authority: WO
Inventors: Jean-Frédéric Clerc; Claude Massit
Original assignee: Commissariat A L'energie Atomique
Priority date: 1996-12-30
Filing date: 1997-12-29
Publication date: 1998-07-09
Also published as: EP0948744A1; FR2757949B1; FR2757949A1; JP2001510564A

Abstract

The invention concerns a micro system for biological analysis for detecting analytes, for example antigens, in a sample. The micro system comprises: an isolating substrate (1) coated with a first conductor (3) forming a first electrode, and a second conductor (7) bearing a plurality of conductive elements (7) extending above the first conductor so as to form a second coplanar electrode arranged at a distance d from the first conductor; and means (13, 15) for polarising the first and second conductors. The electrode(s) are covered with a specific ligand. After a sample has been introduced on the substrate, the presence of analyte in the sample can be detected by measuring the impedance between electrodes 3 and 7, which will indicate the formation or not of an analyte-ligand layer on the electrode(s).

Description

MICROSYSTEM FOR BIOLOGICAL ANALYSIS AND METHOD

MANUFACTURING

DESCRIPTION

Technical area

The present invention relates to a microsystem for biological analyzes, usable in particular in the health sector, the food industry and the environment.

It applies more particularly to the production of microsystems for biological analyzes intended for in vitro diagnosis, in particular in the analysis of infectious diseases (detection of HIV, ycobacteria, etc.).

In these areas where it is a question of saving health, we are currently looking for single-use microsystems of analysis, which are simple to use, use very small volumes of sample and are based on the principle of direct detection requiring neither detection reagent (marker or other), nor amplification of the detection signal.

State of the prior art.

Over the past four years, new test formats (multi-affinity, integration of functions such as gene amplification, separation by electrophoresis) have appeared and their industrial applications seem important, although that today they are mainly located in the sequencing of the human genome.

These new tests - owe their success above all to the introduction of microtechnologies in the biological field, thus making it possible, through integration and parallelization, to achieve high performance, speeds and sensitivities. In addition, microtechnologies lead to new solutions, both technical (miniaturization, integration) and economic (mass production), likely to boost the development of biosensors.

Thus, tests were carried out recently for direct immunochemical detection, consisting of thin layers of semiconductor and silica with antibodies covalently linked to these layers, which make it possible to detect the presence of an antigen capable of reacting with these antibodies by measuring the capacity of the whole (see reference

1: Battaillard et al, in Analytical Chemistry, 60, 1988, pages 2374-2379). Another microsystem of the same type has been described by Schyberg et al, in the reference

2: Sensors and Actuators, B26-27, 1995, pages 457-460.

The documents referenced 3: FR-A-2 598 227 and 4: EP-A-244 326 also describe a method for detecting and / or identifying a biological substance in a liquid sample using electrical measurements. . According to this method, the sample is brought into contact with a reagent-carrying plate comprising a ligand specific for the biological substance to be detected, this plate possibly being made of a semiconductor material such as silicon, and being coated with a silica insulating layer, then we measure the C and / or R components of the electrical impedance of the system to detect the presence of the biological substance in the sample.

All these systems call upon the recognition reaction between the biological substance to be detected and a specific ligand for this substance in order to carry out a direct detection of the latter without having to use other reagents or means of detection (markers, reactions of signal amplification etc). In fact, this recognition reaction results in the formation of an active layer of very thin thickness, for example from 100 Å to 1000 Å, which has electrical characteristics, for example a capacity and an impedance, different from those of the system in the absence of said layer. However, with currently known systems, the distance between the measurement electrodes placed on either side of the active layer remains large relative to the thickness of this active layer; and this affects detection sensitivity. The present invention specifically relates to a biological analysis microsystem, based on the same principle, that is to say on the recognition reaction of the biological substance or analyte to be detected with a specific ligand, which makes it possible to perform the measurement with a distance between the measurement electrodes much more. low and thereby improve the sensitivity of the device.

Statement of the invention

According to the invention, the device for detecting an analyte comprises: an insulating support coated with a first conductor forming a first electrode, and a second conductor supporting a plurality of conductive elements extending above the first conductor so as to form a second coplanar electrode arranged at a distance d from the first driver, and

- polarization means of the first and second conductors.

According to the invention at least one of the electrodes, preferably both, are covered with a specific ligand L of the analyte to be detected.

In this way, when a sample to be analyzed on contact with the two electrodes is deposited on the insulating support provided with the electrodes, the analyte A possibly present in this sample will react with the ligand L to form an LA complex, ie an active layer. on the electrodes. The formation of this layer can be detected by measuring the impedance between the electrodes. With the device of the invention, the particular coplanar structure of the electrodes makes it possible to arrange the two electrodes at a very short distance d, for example from 20 to 500 nm, which is of the order of 2 to 5 times the thickness of the active layer formed by reaction of the analyte with the specific ligand on the electrodes. So, . the impedance measurement will relate to the layer itself and not to a layer of air or of fluid not representative of the phenomenon of recognition between the analyte and the specific ligand.

This particular arrangement of the electrodes on the same support makes it possible to obtain a multiplicity of overlap zones between the first and second electrodes resulting in a large total surface of distributed covering. The distribution of the covering surface and the absence of a second support allows the analyte quick access to the electrodes compared to a device where the electrodes are each on a separate support and where the fluid must enter a space between two thick structures. The smaller this space, the more the fluid is hampered, hence problems of access time and homogeneity. Furthermore, this arrangement makes it possible to increase the developed surface of the electrodes and thus to obtain, when measuring the impedance of the active layer formed, a high signal / noise ratio.

Finally, as will be seen below, the conductive elements, produced for example in the form of mushrooms, have good mechanical strength, which makes it possible to preserve the distance between the electrodes.

According to a preferred embodiment of the device of the invention, the first conductor has the shape of a comb with wide teeth, the second conductor has the shape of a comb with narrower teeth, the teeth of the second conductor being interposed between the teeth of the first conductor and supporting conductive elements extending above the teeth of the first conductor.

The device of the invention can be used for the detection of analytes of various types. By way of example of such analytes, in particular in the medical field, mention may be made of antigens, haptens, antibodies, peptides, nucleic acid fragments (DNA or RNA), enzymes and enzyme substrates. . For this detection, in accordance with the invention, at least one of the electrodes of the device is covered with a ligand specific for the analyte to be detected, for example by direct or indirect grafting of this ligand on the electrode (s) . When the electrode (s) thus covered are in contact with a sample containing the corresponding analyte, an analyte-ligand complex, or active layer, is formed on the electrode (s), and the presence of this complex is directly detected by an electrical impedance measurement.

The specific ligands covering the electrode (s) are those which have at least one site for recognition of the analyte and which are capable of binding to the latter. The ligand-analyte pair can thus belong to the antigen-antibody, hapten-antibody, hormone receptor, DNA- _C DNA, RNA- _C RNA, enzyme-substrate, or any other association of biological molecules or not, capable of forming between they are complex.

The invention also relates to a method of manufacturing the device described above. This process comprises the following steps: a) forming on an insulating support a first conductor and a second conductor spaced apart from each other, b) coating the insulating support comprising the first and second conductors with an insulating layer, c) etch this insulating layer to expose areas of the second conductor which will serve as electrical contact pads, d) forming on these zones and above the insulating layer conductive elements by galvanic growth of a metal, and e) removing the insulating layer by dissolving in a solvent, dissolving neither the first and second conductors, nor the metal conductive elements.

According to an alternative embodiment of this method, it further comprises depositing a metal layer above the insulating layer, which will serve as funds for the galvanic growth of the conductive elements and will have dimensions corresponding to those of the elements. conductors to be trained. According to this variant, steps d) and e) of the process defined above are replaced by the following steps: d ') depositing on the assembly a metallic layer, then a photolithography resin layer, e') exposing the resin and develop it to expose areas of the metal layer having the dimensions of the conductive elements to be formed, f ') forming on these areas of the metal layer the conductive elements by galvanic growth of a metal, g') removing the resin and the metal layer except on the places which correspond to the conductive elements, and h ') remove the insulating layer by dissolving in a solvent, neither dissolving nor first and second conductors, nor the metal of the elements.

To implement the method of the invention, one can use either an insulating support in glass, or an insulating support in silicon.

In the case of an insulating glass support, the first and second conductors are preferably formed by depositing metal on the insulating support at the corresponding places. This can be done by metallization followed by etching to define the conductive areas corresponding to the first and second conductors.

In the case where one starts from a silicon insulating support, the first second conductors are preferably formed on this support by implantation of ions, for example boron or phosphorus ions at the desired locations.

In both cases, an insulating layer, for example of silica, is then deposited on the support comprising the first and second conductors, which is then etched to define the contact pads of the conducting elements which will form the second on the second conductor. electrode. These can be obtained by galvanic growth of a metal such as gold.

As will be seen below, the various stages of this manufacturing process can be carried out by the techniques conventionally used in microelectronics.

After these steps have been carried out, a complementary step of coating the first conductor and the conductive elements, ie the electrodes of the device, is generally carried out with a specific ligand for the analyte to be detected. The embodiment of this step depends, on the one hand, on the material constituting the electrodes and, on the other hand, on the specific ligand used.

Other characteristics and advantages of the invention will appear better on reading the description which follows, given of course by way of illustration and not limitation.

Brief description of the drawings.

Figure 1 is a perspective view of a detection device according to the invention.

FIG. 2 is a view in vertical section of the device of the invention along the line XX ′ in FIG. 1.

Figures 3 to 7 illustrate the main stages of manufacturing the device of Figure 1 in the case where the insulating support is a glass.

FIGS. 8 to 12 illustrate an alternative embodiment of the manufacturing process illustrated by FIGS. 3 to 7.

Figures 13 to 17 illustrate another embodiment of the device of Figure 1 suitable for the use of a silicon support.

Detailed description of the embodiments

In Figure 1, there is shown in perspective a microsystem for biological analysis according to one invention.

This microsystem comprises an insulating support 1 provided on its upper surface with a first conductor 3 and. of a second conductor 5. The first conductor 3 which constitutes one of the electrodes of the microsystem, has the shape of a comb whose teeth 3a are relatively large compared to the space between successive teeth. The second conductor 5 also has the shape of a comb comprising teeth 5a interposed between the teeth 3a of the first conductor 3, these teeth 5a are narrower than the teeth 3a and they support conductive elements 7 having the form of fungi which s 'extend above the first conductor 3 at a distance d therefrom. The conductive elements 7 form the second electrode of the microsystem. The electrodes 3 and 7 can be connected to an external electrical circuit respectively by the conductors 13 and 15 in order to polarize the electrodes and to perform impedance measurements between them.

In Figure 2 which is a vertical section of the device of Figure 1 along line XX ', we see that the conductive element 7 in the form of a mushroom is disposed at a distance d from the teeth 3a of the first conductor 3, being supported by the teeth 5a of the second conductor. As can be seen in this figure, the shape of the fungus makes it possible to have a large electrode surface for the recognition reaction of the analyte to be detected.

In Figures 3 to 7, there is shown schematically the embodiment of the microsystem of Figure 1 starting from an insulating glass support. The production is carried out by a "cold" process, the manufacturing steps being carried out at temperatures not exceeding 300K.

FIG. 3 illustrates the realization of the first step of the manufacturing process according to which the first and second conductors in the form of combs are deposited on the support 1. This can be done by metallization of the support 1, followed by etching which makes it possible to define the conductive areas which will constitute the first comb-shaped conductor 3 with its teeth 3a and the second comb-shaped conductor 5 with the teeth 5a. These combs can be made of gold, a gold-chrome alloy or a gold-nickel-chrome alloy. In fact, the use of gold or of a gold alloy makes it possible, on the one hand, to then obtain good galvanic growth of the fungi and, on the other hand, to graft under good conditions the electrodes of biological molecules which will constitute the specific ligand of the analyte to be detected.

In Figure 4, there is shown the implementation of steps b) and c) of the method of the invention.

In step b) an insulating layer 6, for example of silica, is deposited on the coated support obtained in the first step, which is then etched to expose the areas 7a corresponding to the contact pads between the second conductor and the mushroom shaped elements. This can be done by cold depositing layer 6, for example by PECVD (chemical vapor deposition activated by plasma) or by spinning with colloidal silica. The holes corresponding to the contact pads are then made in layer 6 by photolithography.

It is possible for this purpose to use an organic resin photosensitive to ultraviolet and to eliminate the silica layer at the desired locations corresponding to the contact pads by dissolution in hydrofluoric acid. We get this. the configuration represented in FIG. 4 where the holes 7a correspond to the contact pads of the conductive elements 7.

In Figure 5, there is illustrated step d) of the method of the invention. In this step, the conductive elements 7 are formed in the form of mushrooms by galvanic growth of metal, using the contact pads as deposition electrodes. When the metal is gold or a gold alloy, it can be carried out at room temperature with a bias voltage of the order of a volt. This galvanic growth directly develops the form of fungus.

In Figure 6, there is shown step e) of the method of the invention in which the silica layer 6 is removed by dissolution using hydrofluoric acid. The device shown in FIG. 1 is thus obtained.

With a view to its use for the detection of analyte, this device is subjected to a final step of coating the two electrodes with a specific ligand for the analyte to be detected. For this purpose, the surface of the electrodes is modified, for example with thioalkanes when the electrodes are made of gold. In Figures 8 to 12, there is shown an alternative embodiment of the method described above.

In this variant, the first steps of the process are carried out as illustrated in FIGS. 3 and 4, but after etching the layer of silica and obtaining the configuration shown in FIG. 4, a continuous layer of metal is deposited on the support. FIG. 8 illustrates this step which can be carried out by sputtering of a metal. In this figure, we see the metal layer 8.

After this deposition, the assembly is covered with a photolithography resin which will then be hardened by irradiation so as to become insoluble in certain areas of the substrate, the uncured resin being eliminated by an appropriate solvent.

Figures 9 and 10 show these steps. In Figure 9, we see the support covered with the metallic continuous layer 8 which is coated with the resin layer 9, for example the organic resin photosensitive to ultraviolet.

By exposure to the desired areas identified by arrows, this resin is hardened to make it insoluble.

In Figure 10, the assembly is shown after removal of the uncured resin, which exposes the metal layer 8 on areas which correspond to the conductive elements 7 to be formed.

FIG. 11 illustrates the production of the conductive elements 7. This can be carried out by galvanic growth, for example of gold, under the same conditions as those described above. The structure shown in FIG. 11 is obtained where the elements 7 are delimited in the hardened resin layer 9.

Of course, it is possible to use instead of the hardened resin, a resin capable of being degraded and made soluble by irradiation, the irradiated zones being in this case inverted.

In FIG. 12, the step of eliminating the hardened resin layer 9 and the metal layer 8 is shown on the zones which do not correspond not to the conducting elements 7. This can be done by applying an oxygen plasma.

The structure shown in FIG. 12 is obtained, which corresponds substantially to that of FIG. 5.

The step of eliminating the silica layer 6 and the step of grafting a ligand are then carried out as in the case of FIGS. 6 and 7.

In FIGS. 13 to 17, another embodiment of the device in FIG. 1 has been described, using an insulating silicon support. The process used is a process of silicon microelectronics type. The manufacturing steps are compatible with the CMOS circuit manufacturing lines. FIG. 13 illustrates the first step of the method according to which the first and the second conductors are formed on the support. In this case, the conductive zones corresponding to the combs 3 and 5 are produced by implanting ions in the silicon support to make it conductive on these zones represented by 3a and 5a in the figure. The ions used for implantation are, for example, boron or phosphorus ions, and implantation is carried out through a mask defining the zones to be implanted using sufficient energy to make the silicon conductive on these - zones, over a thickness from a few 1000 Â to 1 micron.

FIG. 14 illustrates step b) of the method, in which an insulating layer 6 is formed on the assembly. This insulating layer is silica which can be formed by thermal oxidation of the silicon on the entire support (implanted zones and not established). After this step, the thickness of the zones implanted is weaker. FIG. 9 also illustrates step c) of the method of the invention in which the insulating layer 6 is etched to expose certain areas 7a of the second conductor which will serve as contact pads. This engraving can be carried out by photolithography in. the same conditions as before.

In Figure 15, there is shown the intermediate step of metallization of the contact pads 10 which can be performed by a complete metallization followed by localized etching corresponding to the location of the contact pads.

FIG. 16 represents the step of forming the conductive elements above the layer 6. This can be carried out by galvanic growth as in the case of FIG. 5.

FIG. 17 represents step e) of elimination of the silica layer, for example by means of hydrofluoric acid. The last step of the process can be carried out as previously by grafting on the conductive elements 7 and on the electrode formed by the first conductor 3 a ligand specific for the analyte to be detected. When the conductor 3 and the elements 7 are made of gold or of a gold alloy, the surface of the electrodes is modified with thioalkanes, then the ligand is fixed on the gold surfaces thus modified by reaction between the ligand and the terminal chains. of thioalkanes.

When the first conductor 3 is made of silicon, this fixing can be carried out by conventional techniques, for example by adsorption on the electrode after having oxidized it very superficially, or by forming a covalent bond between the electrode and the ligand using a bifunctional coupling reagent capable of reacting both with the electrode and with the ligand.

The use of such reagents is well known. As an example of a reagent suitable for coupling ligands constituted by antibodies, proteins and peptides, on silicon, mention may be made of silane derivatives comprising an alkoxysilane group and an NH ₂ group separated from one 'other by a hydrocarbon chain. Techniques of this type are described in references 1, 3 and 4 cited above.

Thus, the silicon can be chemically modified by an alkoxy or a chlorosilane comprising a function capable of reacting with the specific ligand, for example a terminal group-CN, -NH2 or -SH. Indeed, these groups are capable of reacting, after activation by an appropriate functional agent, with the groups of molecules such as antibody fragments and oligonucleotides.

By way of example of an alkoxy silane which can be used, mention may be made of the compounds of formulas:

R1

C2H5— O— Si - (CH ₂ ) _n - ^L R ²

^R1 R1

where R ¹ represents CH ₃ , C ₂ H ₅ , OCH ₃ or OCH ₂ H ₅ , R ² represents -CN, -NH ₂ or -SH, and n is an integer from 1 to 17. Such compounds are fixed on the silicon by reaction with the free hydroxyls, according to the reaction scheme:

, 2

When R "represents NH, an oligonucleotide fragment can be attached to this group by conventional reactions.

2 When R represents SH, an antibody fragment can also be attached to this group by conventional coupling reactions.

It should be noted that the techniques that can be used to carry out the various stages of manufacturing the microsystem are those of microelectronics.

The use of microtechnologies allows in particular:

- improve the sensitivity of the device (precise control of geometries, optical or electrical parameters),

- improve the specificity of the detection by using several devices of the same type on a single silicon wafer (redundancy, multiple detection), - to improve the reliability of the detection by overcoming the problems of localized pollution or non-specific reactions, and

- to obtain a reduction in the manufacturing cost (miniaturization of sensitive elements, use of collective hybridization and packaging techniques developed for microsystems).

Thus, these devices manufactured by microtechnology processes will offer end users, in particular to decentralized analysis laboratories, the advantage of high practicability.

The device of the invention whose conductive elements 7 and possibly the first conductor 3 have been coated with an appropriate specific ligand, can be used for the detection of analyte in the following manner.

A drop of the sample to be analyzed is placed on the support plate 1. Given the dimensions of the wafer, the drop covers the elements 7 and the electrode 3. If the sample contains the analyte A, an active layer is formed on the surface of the electrodes 3 a and 7 by formation of the LA complex. which occupies practically the entire thickness d between the electrodes. The presence of this layer is detected by measuring the impedance between these electrodes.

This can be done by applying an appropriate voltage to electrodes 3 and 5 and by measuring the intensity of the current flowing between these electrodes. By comparing this measurement with a measurement carried out under the same conditions in the absence of analyte, one can verify whether the thickness of the sensitive layer has increased and deduce therefrom the presence or not of analyte. References cited:

(1) Battaillard et al, Analytical Chemistry, 60, 1988, pages 2374-2379.

(2) Schyber et al, Sensors and Actuators, B26-27, 1995, pages 457-460.

(3) FR-A-2 598 227

(4) EP-A-244 326

Claims

1. Device for detecting an analyte, comprising: - an insulating support (1) coated with a first conductor (3) forming a first electrode, and a second conductor (5) supporting a plurality of conductive elements ( 7) extending above the first conductor so as to form a second coplanar electrode disposed at a distance d from the first conductor, and

- polarization means (13, 15) of the first and second conductors.

2. Device according to claim 1, in which at least one of the electrodes is covered with a specific ligand L of the analyte A to be detected.

3. Device according to claim 1 or 2, wherein the first conductor has the shape of a comb with wide teeth (3a), the second conductor has the shape of a comb with narrower teeth (5a), the teeth of the second conductor being interposed between the teeth of the first conductor and supporting conductive elements (7) extending above the teeth of the first conductor.

4. Device according to any one of claims 1 or 2, in which the distance d is from 20 to 500 nm.

5. Device according to any one of claims 1 to 4, wherein the support is made of glass, the first and second conductors are made of metal and the conductive elements are of metal.

6. Device according to claim 5, in which the metal is gold or a gold alloy.

7. Device according to any one of claims l to 4, wherein the support is made of silicon, the first and second conductors are made of silicon made conductive by ion implantation, and the conductive elements forming the second electrode are made of metal .

8. Device according to claim 7, in which the metal is gold.

9. A method of manufacturing a device according to claim 1, comprising the following steps: a) forming on an insulating support (1) a first conductor (3) and a second conductor (5) spaced apart from one other, b) coating the insulating support comprising the first and second conductors with an insulating layer (6), c) etching this insulating layer to expose areas (7a) of the second conductor which will serve as electrical contact pads, d ) forming in these areas and above the insulating layer of the conductive elements (7) by galvanic growth of a metal, and e) removing the insulating layer (6) by dissolving in a solvent, ne ^'or dissolving the first and second conductors, nor the metal of the conductive elements.

10. A method of manufacturing a device according to claim 1, comprising the following steps: a) forming on an insulating support (1) a first conductor (3) and a second conductor (5) spaced from each other, b) coating the insulating support comprising the first and second conductors with an insulating layer ( 6), c) etching this insulating layer to expose areas (7a) of the second conductor which will serve as electrical contact pads, d) deposit on the assembly a metal layer (8), then a layer (9) of photolithography resin, e) exposing the resin and developing it to expose areas of the metal layer having the dimensions of the conductive elements (7) to be formed, f) forming on these areas of the metal layer the conductive elements (7) by galvanic growth of a metal, g) remove the resin (9) and the metal layer (8) except on the places which correspond to the conductive elements (7), and h) remove the insulating layer (6) by dissolving in a solvent, dissolving neither the first and second conductors, nor the meta l elements.

11. Method according to any one of claims 9 and 10, further comprising a step of coating the first conductor and the conductive elements with a ligand L specific for the analyte to be detected.

12. Method according to any one of claims 9 to 11, in which step a) consists depositing a metal on the insulating support at the locations corresponding to the first and second conductors.

13. Method according to any one of claims 9 to 11, wherein the support being made of silicon, the first and the second conductors are formed on this support by ion implantation.

14. Method according to any one of claims 9 to 13, in which in step b), a layer of silica is deposited on the support comprising the first and second conductors, this layer is etched in step c) by photolithography, and this layer is removed at the end of the operation by dissolution in hydrofluoric acid.

15. The method of claims 11 and 12, wherein the metal of the conductive elements being gold, it is modified by thioalkanes before coating the elements with the specific ligand of one analyte to be detected.