WO2004005924A1

WO2004005924A1 - 3d microarray

Info

Publication number: WO2004005924A1
Application number: PCT/IB2002/003248
Authority: WO
Inventors: Mario Caria; Valérie MAZZA; Cinzia Melis; Alessio Sesselego
Original assignee: Spinelix
Priority date: 2002-07-03
Filing date: 2002-07-03
Publication date: 2004-01-15
Also published as: AU2002321720A1; EP1530723A1

Abstract

The invention relates to a new device and method for analyzing the interaction between reagent(s) and solutes or components, wherein said solutes or components diffuse across a matrix immobilizing said reagent(s), said matrix being located in a through hole within said device.

Description

3D MICRO ARRAY

Interactions between target organic or biological molecules and capture agents (that can be organic , biological molecules, such as nucleic acid, proteins, peptides, lipids, carbohydrates, cell extracts, viruses, bacteria, cells... called probes in this application) is a key point to build sensors for medical, pharmaceutical, biological, cosmetics or environment applications, such as human or animal diagnostics, allergological tests, risk screening, water or food quality control.. A tool in this type of analysis is parallel testing on the basis of microarrays. These arrays usually consist of a flat surface with capture probes at specific positions directed towards the targets that may be present in the sample.

In the particular case of protein microarrays several limitations are to be considered:

- According to the chemical properties of the probes, a spacer layer has to be deposited on the slide in order to efficiently capture these probes. US 6,365,418 and

US 6,329,209 (Arrays of protein-capture agents and methods of use thereof, Peter Wagner et al, Zyomyx Corporation) disclose methods of making and using arrays of protein-capture agents. A disadvantage of these spacer layers is their ability to non specifically capture targets, which contributes to a background signal that lowers the system selectivity. The use of specific buffers can reduce the non-specific signal still at the expense of simplicity.

- The planar geometry limits the achievable binding density. Hydrogel™ coated slides from Perkin Elmer are three dimensional substrates for protein microarrays and provide high probe loading capacity (Parallel immunoassays on Hydrogel biochips using microspot arrays, M. Sommer et al, to be published in Proceedings of SPIE Vol. 4626 (2002)). Biocept has also developed a similar technology (Method of making biochips and the biochips resulting therefrom," US 6,174,683, R. Fagnani et al). An alternative was to use porous matrices such as described in patent application EP 1 050 588 (A device for performing an assay, use of a membrane in the manufacture of such device, kit comprising said device and method for detection of an analyte using such device", Kreuwel et al, Akzo Nobel N.V), or WO 00/53625 (Microarrays of peptide affinity probes for analysing gene products and methods for analysing gene products, Donald Montgomery et al, Combimatrix corporation). In these systems though, probe molecules are just deposited on to these layers and the capture efficiency is not controlled and relies on electrostatic, thus weak interactions. - Another point that has to be addressed is the diffusion of the sample molecules towards the attached probes. It can be slow in the planar format (one night for DNA hybridization) and in porous matrices as well.

Metrigenix has disclosed a flow through device for which the diffusion time is reduced, and where the probes are bound inside the channels (WO 01/45843, Flow thru chip cartridge, chip holder, system and method thereof, Matthew Torres et al for Gene Logic Inc / Metrigenix). In the case of porous matrices, PamGene B.V. (US 6,383,748, WO 01/19517 and US20020025533, Analytical test device with substrate having oriented through going channels and improved methods and apparatus for using same, Wilhemus M Carpay et al,) describes a device based on capillary and pressure effects that force the sample fluid to pass through the porous wells. Nonetheless this technique is strongly sensitive to the rheological (surface tension, contact angle, density) properties of the sample fluid.

Moreover direct UV absorption is of great interest for probing molecular interactions, as shown in WO 01/66796 (Method and system for the simultaneous and multiple detection and quantification of the hybridization of molecular compounds such as nucleic acids, DNA, RNA, PNA and proteins, by Mario Caria). However, this technique relies on the measurement of absorption difference between probes alone and probe/target complexes. Sensitivity is a crucial issue, for example when protein-protein interactions are analyzed, as proteins exhibit low molar extinction coefficient compared to DNA: in conventional hydrogel based 3D protein microarray slides, the transmittance difference is about 10^"4. This shows that for direct UV detection, negligible amount of stray signal is acceptable. This implies the use of high performance rinsing technique, in particular when the fluid under analysis comprises a great number of compounds that may screen the signal.

A need thus exists to develop a system that would allow analysis of binding of target molecules to probe compounds, compatible with direct UV absoφtion without labeling, wherein high density of probe compounds can be achieved, with said probe compounds being chemically and/or biologically active, and wherein the rheological properties of the sample fluid would only slightly influence the efficiency of the interaction between the target molecules and the probe compounds. In the context of the invention, a target molecule is the molecule present in the sample fluid that is to be captured, via a specific interaction. A probe compound is a compound that captures a target molecule by physical or chemical interactions. A slab in the context of the invention presents empty through holes, and a probe chip is a slab presenting one or several holes filled with the active matrix. The invention therefore relates to a device for detecting the interaction of probe compound(s) with target molecule(s) present in a sample fluid, comprising a slab, preferably flat and non porous, presenting at least one through hole extending from one surface to the opposite one, wherein said probe compound is immobilized in a porous matrix within said through hole. In particular, the through hole is substantially peφendicular to the planar surface, which means that the angle between the axis of the hole and the surface of the slab is about 90° ± 10°. Such a probe chip is depicted in figure 1.

In the context of the present invention, a porous matrix is a two-phase material, with a solid phase and a pore phase. Typically, the minimum size of these pores is lnm. If Vs is the volume of the solid phase, V_p the volume of the pore phase, V the total volume, the volume fraction of the pore phase is commonly called the porosity, and is denoted Φ = V_p / V. The solid volume fraction is then 1 - Φ.

Thus the mean density of the porous material is p_s(l - Φ), where p_s is the density of the bulk solid phase. The preferred density is 0.8-1.2 g/cm³. In a specific embodiment, the device also comprises a reaction chamber wherein binding between target molecule(s) and probe compound(s) occurs and a rinsing chamber. The device of the invention allows to obtain a high density of probe molecules within each interaction channel (defined as a through hole filled with porous matrix immobilizing the probes), that leads to the possibility of detection of interaction and/or binding by UV absorption. In a specific embodiment, the device of the invention comprises a slab that comprises a plurality of holes, extending from one of the opposing surfaces to the other of the opposing surfaces.

In the preferred embodiment, the slab is a substantially flat surface in which an array of holes is made. In a specific embodiment the array of holes is organized in two sub arrays, one being used as a reference or for control, the other one being dedicated to targets/probes interactions, the distance between these two sub arrays being of the order of 1cm. Each of these holes may be considered as an interaction channel, as it is filled with the active matrix. The sample diffuses through these holes (called interaction channels) and interacts with the specific probe compound that is immobilized in the active matrix. The presence of multiple interaction channels allows to test, in the same experiment a unique sample fluid containing multiple targets against multiple probe compounds (each located in a different hole).

In a specific embodiment, the shape of the hole is a cylinder (Figure 2). In another embodiment, the shape of the hole is a parallelepiped. In another embodiment, the shape of the hole is a cone (Figure 3). The conical shape is interesting to match light focusing geometry when additional lenses are inserted in the light path to collect more photons. It also enables to cope with possible shrinking of the matrix during preparation process. The diameter dimension of the hole is preferably between lOμm and 10mm.

The depth dimension is preferably between lOμm and 10mm.

The matrix of the invention is preferably chosen such as to maintain the functionality of the probe compound. Two immobilization principles can be considered: physical entrapment in a porous matrix or chemical immobilization by covalent reaction.

In a preferred embodiment, said matrix is a sol-gel based matrix.

In a preferred embodiment, said matrix is a silicate sol-gel based matrix. Sol gel is a very good candidate for entrapment of a wide range of molecules or organisms. First its chemical synthesis can be made at low temperature (<60°C), which is compatible with the use of biological compounds. Secondly, the matrix pore size can be controlled by the preparation conditions and thus can be adjusted to the molecule; for example, it has been shown that entrapped antibodies keep most of their activity to interact with antigens or with organic molecules (J. Livage, CR. Acad. Sci., Paris, lib, 322 (1966) 323-334). Besides, in the first preparation steps (before polymerization), it is possible to include a fraction of a polymeric material (for example polydimethylsiloxane) in the solution in order to change the mechanical properties of the matrix.

Some examples of protein immobilization protocols are given in "Immobilized Biomolecules in Analysis : a practical approach" ed. T. CASS & F.S. LIGLER, Oxford University Press, chapter#7.

The method for obtaining doped sol gel matrix (with proteins in an active form) are known in the art and are described in patents applications "Doped sol-gel glasses for obtaining chemical interactions, US 5,824,526, YISSUM RES DEV CO (IL) Avnir et al", "Doped sol-gel glasses for obtaining chemical interactions US 5,292,801, YISSUM RES DEV CO (IL) Avnir et al,", "Doped sol-gel glasses for obtaining chemical interactions US 5,300,564 YISSUM RES DEV CO (IL) Avnir et al,", "Chromatography processes using doped sol gel glasses as chromatographic media US 5,308,495 YISSUM RES DEV CO (IL) Avnir et al,", "Qualitative and quantitative processes for reactive chemicals in liquids using doped sol-gel glasses US 5,300,564, YISSUM RES DEV CO (IL) Avnir et al,", "Doped sol-gel glasses for obtaining chemical interactions, US 5,650,311 , YISSUM RES DEV CO (IL) Avnir et al,", "Doped sol-gel glasses for obtaining chemical interactions, EP 439 318, , YISSUM RES DEV CO (IL) Avnir et al". The content of all these applications is incoφorated herein by reference, in particular the technical content and the examples describing the generation of a doped sol gel matrix.

In another embodiment, said matrix is a polymer (i.e. linear polyacrylamide).

In another embodiment, said matrix is a sieving matrix used for electrophoresis. In a specific embodiment, the probe compound immobilized in the matrix is a protein, in particular a native protein, an engineered protein, or a protein fragment.

In a specific embodiment, the probe compound immobilized in the matrix is a lipoprotein. In a specific embodiment, the probe compound immobilized in the matrix is an antibody.

In a specific embodiment, the probe compound immobilized in the matrix is an antigen.

In a specific embodiment, the probe compound immobilized in the matrix is an enzyme.

In a specific embodiment, the probe compound immobilized in the matrix is a protein from the CpnlO family as described in WO 00/69886 (Oligomeric chaperone proteins, Fergal Conan Hill et al) and WO 00/69907 (Protein scaffold and its use to multimerise monomeric polypeptides, Fergal Conan Hill et al). In a specific embodiment, the probe compound immobilized in the matrix is

DNA.

In a specific embodiment, the probe compound immobilized in the matrix is RNA.

In a specific embodiment, the probe compound immobilized in the matrix is PNA.

In a specific embodiment, the probe compound immobilized in the matrix is a bacteria.

In a specific embodiment, the probe compound immobilized in the matrix is a virus. In a specific embodiment, the probe compound immobilized in the matrix a cell or a collection of cells.

In a preferred embodiment, the molecule immobilized in the matrix in the device of the invention is a protein from the CpnlO family as mentioned above.

Slab material is chosen according to the detection method. For example, when UV absoφtion is used the slab must be opaque to UV light. When fluorescence is used, slab background fluorescence should be as small as possible. Useful slab materials include, e.g., glass, quartz and silicon, aluminium nitride, as well as polymeric slabs, e.g., plastics. The polymeric materials may have linear or branched backbones, and may be crosslinked or non-crosslinked. Examples of particularly preferred polymeric materials include, e.g., polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC) polystyrene, polysulfone, polycarbonate and such like.

Sol gel offers a smart solution because its physical and chemical properties can be easily tuned according to the fabrication conditions (temperature, pH, reagents). It's also possible to dope the matrix to get optimal optical properties (Klein LC (ed) 1988 Sol Gel Optics, Processing and Applications; Boilot JP et al,

1996, C. R. Acad. Sci., Paris lib, 322).

In a specific embodiment, one or two sides of the slab are coated with a sol- gel mixture in order to keep the matrix inside the holes (Figure 4) while enabling target molecule diffusion. The device of the invention allows to obtain high density of probe compounds within each interaction channel, a smart medium for target diffusion and large interaction area.

In a specific embodiment, the diffusion of the target through the probe chip is performed using an electric field. This method increases the speed of the reaction. The diffusion can also be performed by gravity or by forcing a pressure at the entry extremity of the hole.

In the preferred embodiment, the device of the invention also comprises a reaction chamber, wherein said reaction chamber comprises

• a bottom wall, • four side walls defining an inner volume,

• electrodes generating an electric field for diffusion of target molecules,

• a holder for holding said probe chip within said inner volume, wherein insertion of the chip on the chip holder defines two parts in the reaction chamber,

• preferably a joint to prevent the mixture of the fluids present in each part of the chamber, after inserting the chip on the chip holder. The dimensions of the chamber depend on the amount of liquid to be analyzed. In a specific embodiment, the internal dimension is about 2cm x 1cm x 4cm.

In a specific embodiment, two side walls of the reaction chamber are made of a conductive material (for example Platinum) and are facing each other, the probe chip is kept peφendicular to the electric field produced between the two electrodes, the other two sides walls are made of a non conductive material (Figure

5).

In a specific embodiment, the four side walls of the reaction chamber are made of a conductive material (for example Platinum), the probe chip is kept peφendicular to two facing walls (Figure 6).

The sample holder enables to keep the slab peφendicular to the electric field produced between the two conductive walls (electrodes).

The device of the invention may also comprise a similar chamber for rinsing operations (rinsing chamber).

In a specific embodiment, the sample containing the target compounds is chosen in the group consisting of a physiological fluid (which may in particular be .chosen between blood, serum, plasma, urine, tears, saliva, sweat), an excretion (such as expectorate, or phlegm) and a cell extract. In another embodiment, said sample is an alimentary fluid (such as meat juice for detection of contamination (toxins) in the food field).

In another embodiment, said sample is water (in particular for applications in the environmental field).

In a specific embodiment said target molecule is a protein. In a specific embodiment said target molecule is a labeled protein.

In a specific embodiment said target molecule is a labeled molecular entity.

In a specific embodiment said target molecule is a protein marker for cardiac damage.

In a specific embodiment said target molecule is human cardiac troponin T. In a specific embodiment said target molecule is human cardiac troponin I.

In a specific embodiment said target molecule is human myoglobin.

In a specific embodiment said target molecule is human cardiac fatty acid binding protein (cFabp). In a specific embodiment said target molecule is C-reactive protein (CrP).

In a specific embodiment said target molecule is human brain S-100 protein.

In a specific embodiment said target molecule is human serum albumin.

In a specific embodiment said target molecule is human transaminase GOT. In a specific embodiment said target molecule is human lactic dehydrogenase.

In a specific embodiment said target molecule is a molecular tumor marker present in blood or tissue.

In a specific embodiment said target molecule is human tumor Antigen. In a specific embodiment said target molecule is human Alpha- Fetoprotein.

In a specific embodiment said target molecule is human CA 125.

In a specific embodiment said target molecule is human CA19-9.

In a specific embodiment said target molecule is human Prostate-Specific Antigen. In a specific embodiment said target molecule is human immunoglobulin.

In a specific embodiment, said target compound is human cardiac troponin T, and said immobilized probe is its specific partner in the CpnlO engineered protein family.

The device of the invention has several advantages, as it is possible to capture multiple different target molecules present in the same sample fluid, the device is compatible with direct UV detection, the quantity of sample necessary is low, the method of using the device may shorten the reaction time, a minimal technical expertise is required for using the device, the device may be disposable.

The invention also relates to a method for detecting the presence of a target molecule in a sample fluid, wherein said target molecule interacts with a probe compound, comprising the steps of:

• diffusing said sample fluid through the interaction channels of a device according to the invention, and

• detecting the presence of capture of said target molecule by said probe compound.

Some additional steps can be envisioned:

An optional step of rinsing may be performed by diffusion of a rinsing buffer through the interaction channels, before the detection step. In particular, the rinsing step is performed using electric field to move unwanted species out of the matrix.

When the detection method is based on the use of labeled entities it may be necessary to diffuse a tracer solution through the interaction channels in order to label target/probe complexes.

This additional diffusion step may be followed by another rinsing step.

In a preferred embodiment the device of the invention used for performing the method according to the invention comprises a reaction chamber, said chamber comprising a bottom wall, four side walls defining an inner volume, wherein two walls are made in a conductive material and are facing each other, and the other two are made in a non conductive material, a holder for holding said probe chip within said inner volume, wherein said probe chip is parallel to the two walls made of a conductive material, and defines two parts in the reaction chamber, and preferably a joint to prevent the mixture of the fluids present in each part of the chamber, after insertion of the probe chip on the holder, and an electric field is applied between said two walls made of a conductive material for diffusing said sample.

In a preferred embodiment, said device also comprises a rinsing chamber, whose construction is similar to the reaction chamber.

First the probe chip is introduced inside the reaction chamber (figure 7. a). Two parts are thus defined in the reaction chamber separated by the probe chip. Water tightness is guaranteed by a joint so that the only way for a molecule to move from one part to the other is through the holes of the slab. Then one part is filled with the sample fluid and the other one is filled with a buffer, or both parts are filled with the sample fluid (figure 7.b). For diffusion of the samples through the matrix, using an electric field, the pH in solution is adjusted so that target molecules exhibit a non zero net charge. An electric field is applied between the two electrodes (walls made of a conductive material). During this phase all charged molecules and ions move under the effect of the electric field (figure 7.c). The electric field strength is about 50-250V/cm, preferably about 200V/cm.

The probe chip is then removed from the reaction chamber (figure 7.d).

When it is considered interesting to rinse, two steps can be envisioned for this process. First the probe chip is washed using a proper buffer in order to remove compounds that are not captured. When proteins are under investigation, sodium phosphate buffer, Tris buffer, Hepes buffer can be used. The choice of other buffers is within the skills of the person skilled in the art. Secondly it is put in a similar reaction chamber, which is filled with a washing buffer, similar to the buffers described above, and once again an electric field is applied (figure 7.e). This permits to reject the non specific ions and molecules out of the matrix and to keep only specifically bound target molecules, thus increasing the selectivity of the detection system. In particular, the invention relates to a method for detecting serum cardiac

Troponin T from human serum, comprising the step of performing the method of the invention with a device containing a protein from the CpnlO engineered protein family within the matrix, before detecting the interaction between Troponin T and CpnlO.

In a specific embodiment, binding of the target molecule to the probe in the matrix is detected by direct light (especially UV) absoφtion.

A method for performing this detection is the following. The light source is a discharge lamp (for example Deuterium lamp). A bandpass optical filter is used to select the spectral range corresponding to the maximum absoφtion efficiency of the target/probe complex. A diffractive element (e.g. a grating or a set of gratings) can also be used in particular when a very narrow spectral range is necessary (< lOnm).

A lens or a system of lenses enables to give the proper shape to light beam.

A beamsplitter (50/50) is used to dispatch two identical beams toward the holes used for reference or control and the ones used for measurement.

According to the number of holes on the probe chip, light transmitted through the holes is detected by using either a set of UV enhanced Si photodiodes, or arrays or matrix of UV enhanced Si photodiodes.

The optical design is such that all the light going through a hole should be collected by the detection element. This can be achieved either by working with a detection element that is significantly larger than the hole or by refocusing light with a lens or a microlens. Light intensity can be modulated (frequency of a few kHz) by using an optical chopper. The electrical signal delivered by the detectors is then analysed at the modulation frequency, which improves the signal to noise ratio.

Another method is described in detail in patent application WO 01/66796, the content of which is incoφorated herein by reference.

The method described in this patent application is intended for detecting a position of several binding sites on a support containing probes (biomolecules) possibly having bound targets, comprising the steps of :

- emitting a radiation from a source towards the support, - receiving a radiation coming from the support on a microelectrode detector sensitive to the radiation; and

- quantifying different sites of the support at the same time concerning possible hybridized targets.

The method described in this patent application, that is preferably performed in the framework of the present invention is such that the reception step comprises the step of receiving the radiation after it passed through the holes of the slab.

The detection device comprises a source for emitting a radiation towards the support, a microelectrode detector arranged to receive a radiation coming from the support and sensitive thereto and means for quantifying different sites of the support at the same time concerning possible bound targets.

It may also present one or more of the following characteristics:

- the source is a gas discharge lamp, the source is a laser source, preferably a semiconductor one or a gas one, it comprises a lens or a system of lenses arranged in the path of the radiation, before or after the support, it comprises a micro-lenses system to allow the passage of the maximum intensity of the incident radiation, arranged in the path of the radiation before or after the support, it comprises a monochromator or filter system for the selection of the passing energy of the incident radiation before or after the support, it comprises means for quantifying binding, especially the means for quantifying comprises an electronic reading circuit connected to the detector, preferably welded or glued directly to the detector or grown directly from the detector, the electronic reading circuit is of the VLSI ("Very Large Scale Integrated") design type, the microelectrode detector is formed by junctions on a semiconductor material, the semiconductor material is chosen from the group consisting of : high resistivity, Silicon, synthetic Diamond, a Gallium-based compound, or a compound containing Gallium and Aluminum, the semiconductor has contacts implanted to form junctions in diode type configurations, the distance between the microelectrodes is substantially the same, center to center, as the distance between the binding sites, the distance between the sites and/or between the microelectrodes ranges from 1 micrometer to 1 centimeter, the means for quantifying is arranged to transform the charge into electric current, the means for quantifying comprises an amplifying system, the slab comprising the through holes is made of glass with thin films of another material, the slab comprising the through holes is made a plastic polymer.

Detection techniques based on labels can also be used (radiolabels, enzyme labels, fluorescent and phosphorescent labels, chemoluminescent and bioluminescent labels (see" Immunoassays, essential data", edited by R. Edwards, Wiley 1996). The optical transmission can also be measured at specific wavelengths by using a spectrophotometer device.

Other features and advantages of the invention will appear in the following description of preferred embodiments thereof with reference to the drawings.

FIGURES

Figure 1 : Illustration of a probe chip according to the invention, with the slab, the holes and the porous matrix where probe compounds are encapsulated. It is 16mm x 20mm. Figure 2: Illustration of a slab with cylinder holes (side view).

Figure 3: Illustration of a slab with conical holes (side view).

Figure 4: Illustration of a probe chip according to the invention with a coating to keep the matrix inside the holes.

Figure 5: Description of a reaction chamber according to an embodiment, with the chip holder, the joint and two walls made of conductive material.

Figure 6: Description of a reaction chamber according to an embodiment, with the chip holder, the joint and four walls made of conductive material. Figure 7: Illustration of the different steps of the method according to an embodiment: introduction of the probe chip in the reaction chamber (a), filling of the reaction chamber with the sample fluid and with the buffer (b), diffusion of the target molecules by using electric field (c), removal of the probe chip (d) and rinsing of the probe chip by using electric field (e), in a rinsing chamber. Figure 8: Description of the probe chip used in example 1.

EXAMPLES

Example 1: Human cardiac troponin T capture for UV detection Principle

The device is designed to capture human cardiac TroponinT, that is thus the target molecule. The sample fluid is serum and may be blood. The probe compound is a specific engineered CpnlO protein, chosen by screening a phage display CpnlO library. In this example, the device is designed in order to optimise subsequent direct UV absoφtion detection.

Probe chip description Slab description

The slab is made of UV opaque material (transmittance smaller than 10-6 at wavelength between 250nm and 290nm). It is depicted in figure 8. Its dimensions are the folio wings:

Thickness: 500μm

Width x heigth: 5cm x 1.5cm It comprises two sets of 8 holes each (one set for control, one for human cardiac Troponin T capture). Holes characteristics

Diameter : 50μm

Depth : 500μm

Matrix characteristics and immobilization protocol

The probe is immobilized in a silicate sol-gel based matrix that fills the holes according to the following protocol: Step I 1) Dilute 15.27 g of TMOS in a double molar quantity of deionised water in a beaker.

2) Add 0.22 ml of 0.04M HCl and sonicate in an ice bath for 20 minutes to homogenize. 3) Cover with parafilm and store the mixture overnight in a refrigerator.

Step II

1) Put the beaker in an ice bath.

2) Dilute 16 mL of previously prepared TMOS sol in 24 mL of deionised water.

3) Add the solution of engineered CpnlO to the sol mixture to obtain doped sol.

4) Slowly Add 160 μL of 1 M NH₄OH and moderately shake the beaker.

5) Pour some drops of the doped sol-gel onto the holes. Pressure or capillary effects are used for injection inside the holes. 6) Store in a humid box in the refrigerator for 30 minutes.

7) Sol gel for dip coating o Put a little plastic cuvette (a few millilitres) in an ice bath. o Mix 50 parts of fresh prepared sol with 40 parts of methanol and 10 parts of a buffered solution (0.1 M acetate buffer and 0.1M HCl (ratio 3:1 v/v)). o Pour the mixture into the cuvette. o Dip the probe-chip slide in the sol gel and withdraw at a slow rate

(~10cm/min).

8) Let drying and aging for three days in a humid box.

This coating process enables to keep the active matrix inside the holes.

Reaction chamber description It comprises: • a bottom wall

• four side walls defining an inner volume

• two side walls are made of a Platinum and are facing each other • a holder for keeping the probe chip peφendicular to the electric field.

• a joint to prevent the mixture of the fluids present in each part of the chamber, after inserting the chip on the chip holder. The dimensions of the chamber are 2cmx 1 cmx4cm.

Target capture protocol

The probe chip ready to use is inserted inside the reaction chamber, which defines two parts (Figure 7. a). The sample fluid is poured into one of these parts, while a buffer is put into the other one (Figure 7.b).

This buffer is chosen among the followings : sodium phosphate buffer, Tris buffer, Hepes buffer.

Then a voltage of about 100-500V is applied between the two electrodes for a few minutes (Figure 7.c)

Washing step

In this step, the probe chip is removed from the reaction chamber (Figure 7.d) and washed with the buffer.

Rinsing step

The probe chip is introduced inside the rinsing chamber. Buffer is poured into the two chamber parts (Figure 7.e).

Then a voltage of about 10-500V is applied between the two electrodes for a few minutes.

Detection

After drying, UV light transmission of Troponin T/CpnlO complexes is then measured.

Half of the holes are used to detect and quantify the amount of target molecules. Each of them is initially filled with the same amount of probe compounds. In the first detection step corresponding to calibration, light transmitted by each of these holes is detected. This measurement can be made using either pixels from a matrix or discrete photodetectors.

SI and S2 stands for the corresponding signals. Then one of theses holes is put in contact with the sample fluid and rinsed according to the method described above.

In the final detection step, light transmitted by each of these holes is detected again.

SI ' and S2' stands for the corresponding signals. If the offset contributions from the detectors are Soffland Soff2, the relative transmission change between probe/target complexes and probes α is equal to:

_ S2 -Soff2 Sl -Soffl

(X — , Λ -

SI -Soffλ S2-Soff2 For better accuracy on α value, an average is made between the different holes.

Example 2: Human cardiac Troponin I capture for fluorescence detection

The manufacture of the probe chip is performed as in example 1. The target capture protocol, washing step and rinsing steps are as described in example 1.

The rinsing step is followed by different steps for preparing the probe chip for detection as described below. Labeling step

In this step, the probe chip is introduced inside another reaction chamber. A tracer solution (labeled specific protein + buffer) is poured into one of these parts, while a buffer is put into the other one. This buffer is chosen among the followings: sodium phosphate buffer, Tris buffer, Hepes buffer. The labeled protein may be a specific anti-Troponin I antibody that is labeled with a fluorescent moiety.

Then a voltage of about 100-500V is applied between the two electrodes for a few minutes.

Washing step

In this step, the probe chip is removed from the reaction chamber and washed with the buffer. Rinsing step

The probe chip is introduced inside the rinsing chamber. Buffer is poured into the two chamber parts. Then a voltage of about 10-500V is applied between the two electrodes for a few minutes.

After drying, the probe chip is ready for fluorescence detection.

Claims

1. A device for detecting the interaction between a target molecule present in a sample fluid and his specific probe compound, comprising a probe chip consisting of a slab presenting at least one through hole, extending from one surface to the opposite one of said slab, said through hole being filled with a porous matrix, wherein said probe compound is immobilized within said porous matrix.

2. The device of claim 1, wherein said slab comprises a plurality of holes, extending from one surface to the opposite one.

3. The device of claim 1 or 2, wherein said matrix is chosen in the group consisting of sol-gel based and silicate sol-gel based matrixes.

4. The device of claims 1 or 2, wherein said matrix is a polymer ( i.e. linear polyacrylamide)

5. The device of claim 1 to 4, wherein said slab is opaque to UV light

6. The device of any of claim 1 to 5, wherein said device comprises a reaction chamber, wherein said reaction chamber comprises

• a bottom wall,

• four side walls defining an inner volume, wherein two walls are made in a conductive material and are facing each other,

• a holder for holding said probe chip within said inner volume, wherein said probe chip is parallel to the two walls made of a conductive material.

7. The device of any claims 1 to 6, wherein all four side walls are made in a conductive material

8. The device of any claims 1 to 7, wherein said through hole is a cylinder.

9. The device of any claims 1 to 7, wherein said through hole is a cone .

10. The device of any claims 1 to 7, wherein said through hole is a parallelepiped.

11. The device of claim 8, wherein the diameter of said through hole is comprised between lOμm and 10mm.

12. The device of claim 9, wherein the larger diameter of said through hole is comprised between lOμm and 10mm.

13. The device of claim 10, wherein the side dimension of said through hole is comprised between lOμm and 10mm.

14. The device of any claims 1 to 13, wherein said probe compound is immobilized within said matrix by physical entrapment.

15. The device of any claims 1 to 13, wherein said probe compound is immobilized within said matrix by chemical cross-linking.

16. The device of any claims 1 to 15, wherein said probe compound is chosen in the group consisting of a protein, a protein fragment, a nucleic acid, a bacteria, a virus, a cell, and a collection of cells.

17. The device of claim 16, wherein said probe compound is an antigen.

18. The device of claim 16, wherein said probe compound is a protein from the CpnlO chaperonin family.

19. The device of any claims 1 to 18, wherein said sample fluid is chosen in the group consisting of a physiological fluid, an excretion and a cell extract.

20. The device of any claims 1 to 19, wherein said target molecule is chosen in the group consisting of human cardiac Troponin T , human cardiac Troponin I, human myoglobin, human cardiac fatty acid binding protein (cFABP), human protein C-reactive (CrP), human brain S-100 protein, human serum albumin, human Transaminase GOT, human Lactic dehydrogenase, a molecular tumor marker present in blood or tissue, human Tumor Antigen, human alfa- Fetoprotein, human CA 125, human CA 19-9, human Prostate Specific Antigen.

21. The device of any of claims 1 to 20 wherein said probe chip is coated with a porous sol gel layer.

22. A method for detecting the presence of a target molecule in a fluid sample, wherein said target molecule interacts with a probe compound, comprising the steps of:

• providing the device of any of claims 1 to 21,

• diffusing said sample through the through hole of said device, • optionally diffusing a rinsing buffer through the through hole of said device, and

• detecting the capture of said target molecule by said probe compound.

23. The method of claim 22, wherein said device comprises a reaction chamber, said chamber comprising a bottom wall, four side walls defining an inner volume, wherein two walls are made in a conductive material and are facing each other, a holder for holding said probe chip within said inner volume, wherein said probe chip is parallel to the two walls made of a conductive material, and defines two parts in the reaction chamber, and an electric field is applied between said two walls made of a conductive material for diffusing said target molecule present in said fluid sample.

24. The method of claim 23, wherein all side walls are made in a conductive material.

25. The method of any claims 22 to 24, wherein the presence of said target/probe complex is detected by direct UV absoφtion

26. The method of any claims 22 to 24, wherein the presence of said target/probe complex is detected by label dependent method.

27. The method of claim 26, wherein said label is a fluorophore.

28. The method of claim 26, wherein said label is an enzyme.

29. The method of claim 26, wherein said label is a radioisotope.

30. The method of claim 26, wherein said label is a luminescent compound.