WO2013023671A1

WO2013023671A1 - Microfluidic device and method for detecting analytes in a flow using electrochemical probes

Info

Publication number: WO2013023671A1
Application number: PCT/EP2011/063573
Authority: WO
Inventors: Nicolas DA MOTA
Original assignee: Da Mota Nicolas
Priority date: 2011-08-05
Filing date: 2011-08-05
Publication date: 2013-02-21

Abstract

According to the present invention, an embodiment uses a method to detect or quantify, or both, at least one analyte in a sample for analysis and comprises at least the following steps: Injection of at least one probe molecule in said sample, said probe molecule being able to interact with said analyte chemically (e.g. substitution, addition, elimination, recombination, rearrangement, acid-base, redox, radical, complexation, polymerization, chain, precipitation, adsorption, absorption, decomposition, or combustion reactions), physically (e.g. magnetic field, electrical polarity or agglomeration) or biologically (e.g. enzymatic reaction, antigen - antibody binding, addition reaction). Predetermined period during said interaction occurs between said probe molecule and analyte. Measurement of a relative amount of said probe molecule remaining after said period, into a microchannel, a microfluidic cell, inside or at the column end, or both. And, determination of a relative amount or the nature, or both, of said analyte from the measured relative amount of said probe molecule. Another embodiment according to the present invention is a device to detect or quantify, or both, at least one analyte in a sample for analysis. Said device comprises at least one microfluidic cell made from a substrate including at least one inlet and one outlet communicating by at least one microchannel. Said microchannel includes at least one injection area for the fluids, such as said sample and reagents, and at least one analysis area. Said reagent is at least one probe molecule that is able to react with said analyte. Said analysis area consists to measure a relative amount of said probe molecule remaining after at least one reaction with said analyte by at least one mean.

Description

MICROFLUIDIC DEVICE AND METHOD FOR DETECTING ANALYTES IN A FLOW USING

ELECTROCHEMICAL PROBES

5 FIELD OF THE ART

The present invention is about a method for analysing in a sample, by detection or quantification, or both, at least one specific compound, called analyte, and, also, about a device to implement this method, commonly called microfluidic electrochemical sensor 10 and aiming the detection of biological or chemical compounds.

BACKGROUND OF THE ART

Since the last decades, the microfluidic devices are known for the usefulness in some

15 domains, such as micro-technology, physics, chemistry, biology or pharmacology, with practical applications to the design of systems in which such small volumes of fluids will be used. The material fluid may be solid (e.g. particles), liquid, gas, material of some intermediate characteristics such as gel or sputum, tissue, organisms, or a combination of these. The devices structures present some advantages in domains such: the micro-

20 synthesis increasing the reaction efficiency to produce different compounds (molecules, particles, emulsions, etc.) from injected reagents inside a reaction chamber integrated in the microfluidic device, or the micro-analysis aiming the compounds detection in small amounts from various samples, in particular the biological fluids, where the microfluidic device focusing the sensor's detection area.

25 Usually, a complex system of micro-channels, with sections including at least one characteristic dimension from tens of nanometers to hundreds of micrometers, lengths from few millimeters to meters, series of inputs and outputs to be able to generate a flow, and sets of chemical or biological reaction chambers or any integrated systems corresponding to specific applications composed a microfluidic device.

30 Some complex microfluidic devices are often named as "lab-on-chip" or " ΤΑ8

(micro total analysis system)" to do series of various operations. For example, it's possible to perform different chemical or biological reactions in adding reagents, to filtrate the sample by an integrated porous membrane, to mix all the compounds by chaotic flows, to concentrate the analytes with a polymerase chain reaction (PCR), or to detect particles by flow cytometry or electrophoresis.

The miniaturisation of the reaction inside of these microsystems presents several advantages. Indeed, with their small volumes, small amounts of sample or reagents are necessary; it is low energy consumption and it reduces the cost of the implementation of the reaction.

Furthermore, some of these operations can be integrated in series on the same device, in associating the PCR and detection means for example.

Also, several analyses can be performed in parallel increasing the laboratories capacity (number of analysis done by day and by equipment).

At last, the miniaturisation of these systems allows for making portable equipments.

However, due to the small dimensions of the microfluidic devices, specific fluid dynamics properties are often observed. Under certain conditions, the flow is laminar and controlled by the solution viscosity. Indeed, the flows generated by pressure (e.g. gravity, overpressure by neutral gas, peristaltic, vacuum or syringe pumps) for incompressible fluids, such as aqueous solutions, are often referenced as Stokes flows, i.e. the fluid flow is everywhere parallel to the channel's walls (lubrication approximation), the wall friction involves the fluid velocity is zero only in macroscopic scales (no-slip condition) and the pressure does not vary in the thickness of the flow (lubrication approximation). These approximations imply that the incompressible Newtonian Stokes flow is organized according to a parabolic velocity field. In particular, flow inside a regular pipe or between two parallel plates, such flow is known as Poiseuille flow (see figure 1). So, for a channel with a regular section, only the component of the fluid velocity in the direction of the channel's axis is not negligible. In this case, the mass transfer in the perpendicular direction of the flow is only provided by the diffusion.

However, high molecular compounds, macromolecules or any species with large molecular mass in domains such as the biology (e.g. bio-polymers, peptides, proteins, DNA, RNA, cells, antibodies, enzymes) have a low diffusion coefficient. Besides the adsorption problem by affinity on the micro-channel's walls, most of the macromolecules are driving in the middle with a parabolic flow. Said molecules are invisibles for detection limited at the surface of the channel like electrodes integrated on its walls.

Even if the risks of adsorption can be reduced or controlled in appropriate solution (e.g. pH, ionic force or temperature controlled, or both) or in adding surfactant at the solution, such as TWEEN20©, or amphiphilic molecules to decrease the polarity of the walls and to influence their hydrophobicity (e.g. sodium dodecyl sulfate, n-dodecy-β-Ο- maltoside), the measurement of this kind of sensor can be imprecise if the molecules are not close to.

SUMMARY OF THE INVENTION

One purpose of the invention is to overcome these drawbacks by providing a method for detecting or quantifying, or both, at least one analyte in a sample and, also, a device for this purpose, with a simple design, inexpensive and permitting a high sensitivity measurement regardless of the analyte.

For this purpose, and according to the present invention, a method to detect or quantify, or both, at least one analyte in a sample is characterised by at least the following steps of:

- Injection of at least one probe molecule in the sample for analysis containing at least one analyte, said probe molecule is able to interact with said analyte by at least one chemical reaction (e.g. substitution, addition, elimination, recombination, rearrangement, acid-base, redox (oxidation-reduction), decomposition, combustion, complexation, polymerisation or radical reactions), or physical interaction (e.g. adsorption, absorption, electrostatic or magnetic interactions), or biologically (e.g. enzymatic reaction or recognition of an antigen by an antibody),

Reaction between said probe molecule and said analyte is during a determined period of incubation,

Said reaction with said analyte occurs inside of at least one micro-channel, microfiuidic device, a tube, a column or at their ends, or both,

The determination of relative amounts or properties, or both, of said analyte according to the measure of the relative amount of said probe molecule.

Said probe molecule injected with the sample for analysis containing at least one analyte in a dedicated space called a reaction chamber before the analysis section.

For a first embodiment of the method according to the present invention, said method includes at least one artificial activation step of at least one probe molecule to initiate the reaction with at least one analyte, said probe molecule is initially inert. Said artificial activation step is a chemical activation generated by addition of at least one chemical reagent inducing a spontaneous reaction (e.g. substitution, addition, elimination, recombination, rearrangement, acid-base, redox, decomposition, complexation, or radical reactions) with said probe molecule for example. Alternatively, said artificial activation step is a reaction issued from physical chemistry's domain, through third chemical reagents or not, such as electrochemistry (e.g. said activation is a redox reaction forced by at least one electrode), photochemistry (e.g. reaction by adsorption of an electromagnetic radiation for example), radiochemistry (e.g. activation of stable isotopes to create radioisotopes), thermochemistry (e.g. endothermic reaction) or biophysical chemistry, or both (e.g. electrochemiluminescence).

Advantageously, said probe molecule is activated in the main channel of the microfluidic device or the column after its injection in the sample for analysis.

According to another embodiment of the present invention, said probe molecule is artificially activated in at least one secondary channel in the microfluidic device or the column prior to its injection in the sample for analysis.

In addition, said probe molecule is activated inside the main or the secondary channel by at least one electrode, called generator and integrated in the aforesaid channel.

Preferably, the signal intensity issued from of at least one electrode, called collector and placed in the main channel of the microfluidic device or the column, measures the relative amount of the probe molecule.

Alternatively, the signal intensity issued from of at least one electrode, called collector and placed in at least one secondary channel below the main channel of the microfluidic device or the column, measures the relative amount of the probe molecule.

In addition, said probe molecule can be chosen from the following list. Ferric complexes and derivates, ferricinium, dimethylferrocene (DMF), ferrocene monocarboxylic acid (FCOOH), ferrocyanide, ferricyanide, ferrocenemethanol, osmium complexes and derivates, tris(2,2'-bipyridyl)osmium, osmium tetroxide, bis(4,4'-diamino- 2,2'-bipyridine)-(2'-3 '-dipyridophenazine)osmium, ruthenium complexes and derivates, tris(2,2'-bipyridyl)ruthenium, ruthenium tetroxide, ruthenocene, organic conductive salts, viologen, quinone and derivates, hydroquinone, benzoquinone and derivates, anthraquinone and derivates, 7,7,8, 8 -tetracyanoqui nodi methane (TCNQ), pyrroloquinoline quinine (PQQ), tetrathiafulvalene (TTF), N-methyl acidinium (NMA+), tetrathiatetracene (TTT), N-methylphenazinium (NMP+), 3-dimethylaminobenzoic acid (MBTH-DMAB), 3- methyl-2-benzothio-zolinone hydrazone, 2-methoxy-4-allylphenol, 4-aniinoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 3,3', 5,5'- tetramethylbenzidine (TMB), 2,2-azino-di-[3-ethyl-benz-thiazoline sulfonate], o- dianisidine, o-toluidine, 2,4-dichlorophenol, 4-amino phenazone, benzidine, metalloporphyrins, hydrogen peroxyde.

Another embodiment of the present invention is a device for the detection or the quantification, or both, of at least one analyte in a sample for analysis, said device comprising at least one microfluidic cell including a substrate with at least one input connecting with at least one output through at least one channel, said microchannel including at least one injection area and at least one detection area ; said device is characterized in that it comprises means for measuring a relative amount of at least one probe molecule which has not reacted with at least one analyte, said probe molecule being able to react with said analyte in a sample for analysis injected in the aforesaid microchannel.

Preferably, the device in the present invention includes at least one reaction chamber in which the probe molecule is injected in the sample for analysis, and is positioned upstream to the analysis area.

Furthermore, said device comprises means for artificially activating said probe molecule in order to initiate the reaction between said analyte and said probe molecule which is initially inert.

Said artificial activation means are preferably located in the microchannel of the microfluidic device.

Alternatively, said artificial activation means are located in at least one secondary channel of the microfluidic device, said secondary channel being positioned upstream to the main microchannel.

Said artificial activation means of said probe molecule are an electrochemical activation.

Furthermore, said electrochemical activation means consist in at least one electrode, called generator.

According to another embodiment of the present invention, said artificial activation means of the probe molecule consists in chemical activation means. Moreover, said analysis area includes at least one measuring device of a relative amount of the probe molecule.

Preferably, said analysis area is located in the microchannel of the microfluidic device.

Alternatively, said analysis area is located in at least one secondary channel of the microfluidic device, said secondary channel being located downstream of the main microchannel.

Furthermore, said measuring device consists in at least one electrode, called collector, and placed in the aforesaid main microchannel and/or the aforesaid secondary channel, the signal intensity measured through the aforesaid electrode allowing to determine a relative amount and the nature of the aforesaid probe molecules interacting with said electrode.

Advantageously, the device according to the invention includes several collectors distributed on the aforesaid analysis area inside the microchannel.

In addition, the probe molecule can be chosen from the following list: Ferric complexes and derivates, ferricinium, dimethylferrocene (DMF), ferrocene monocarboxylic acid (FCOOH), ferrocyanide, ferricyanide, ferrocenemethanol, osmium complexes and derivates, tris(2,2'-bipyridyl)osmium, osmium tetroxide, bis(4,4'-diamino- 2,2'-bipyridine)-(2'-3'-dipyridophenazine)osmium, ruthenium complexes and derivates, tris(2,2'-bipyridyl)ruthenium, ruthenium tetroxide, ruthenocene, organic conductive salts, viologen, quinone and derivates, hydroquinone, benzoquinone and derivates, anthraquinone and derivates, 7,7,8,8-tetracyanoquinodimethane (TCNQ), pyrroloquinoline quinine (PQQ), tetrathiafulvalene (TTF), N-methyl acidinium (NMA+), tetrathiatetracene (TTT), N-methylphenazinium (NMP+), 3-dimethylaminobenzoic acid (MBTH-DMAB), 3- methyl -2-benzothio-zolinone hydrazone, 2-methoxy-4-allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 3,3', 5,5'- tetramethylbenzidine (TMB), 2,2-azino-di-[3-ethyl-benz-thiazoline sulfonate], o- dianisidine, o-toluidine, 2,4-dichlorophenol, 4-amino phenazone, benzidine, metalloporphyrins, hydrogen peroxyde. BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages and features will be more apparent in the following description with several embodiments, given as non-limiting examples, of the method for detecting or quantifying, or both, at least one analyte in a sample for analysis and, also, a device for this purpose according to the present invention, referring to the drawings in annexes, in which:

- Figure 1 illustrates the rates profile of a Poiseuille flow in a microfluidic devices' s channel;

Figures 2.1-2.3 are schematics of devices implementing methods described in the present i n venti on ;

Figures 3.1-3.2 are schematics of device's embodiment with an electrochemical activation for the present invention;

- Figure 4 is a graphic showing the intensities variation vs. time for generator and collector electrodes;

- Figures 5 and 6 are schematics of several device's embodiments of the present invention;

- Figure 7 is a graphic showing the rationalization of the measured currents for several collector electrodes arrays;

Figure 8 is a schematic of a measurement set including the device for the present invention.

References Legend

(4) ® : Hi jno L'-.d analyte

a : Mat studied noteeuie

(3) — # * : Praise mofetaite (inert acivated farm)

< probe^* raweplej

(8) < A : "Anaty!e

% : "2nd anahrte / probe" complex

(6) : Generator electrode

(2) — : Codecior eteotesde

(5) — Ο / · : Ac*i*ator

o : Solid phase pd m*r)

(7) < : Membrane DETAILED DESCRIPTION OF THE INVENTION

Referring to figure 2.1, the device to detect or quantify, or both, at least one analyte in a sample for analysis according to the present invention include a microfluidic cell commonly formed from a non-adsorbent substrate having at least one input and at least one output communicating with a microchannel (1 ). Figure 2.1 shows only the microchannel (1). Said device includes means to measure (2) small amounts of at least one probe molecule (3) that did not react with the analyte (4), said probe molecule (3) being able to react with at least one analyte (4) in a sample for analysis injected inside the microchannel (1).

The matter of the aforesaid substrate is from the following non-exhaustive examples list: Silicon wafer, Silicon dioxide, Silicon nitride, glass or amorphous silicon, gallium arsenide, indium phosphoride, Aluminium, ceramics, polyimide, quartz, plastics, surfactants, silicones, resins, and polymers including Polydimethylsiloxane (PDMS), Poly(methyl methacrylate) (PMMA), acrylics, acrylates, polyethylene, polyethylene terephtalate, polycarbonates, polystyrenes and others copolymers of styrene, polypropylene, polyurethane, polyetrafluoroethy ene, liquid crystal polymers, polyolefins, alloys, superalloys, zircaloy, steel, stainless steel, Gold, Silver, Copper, Titanium, Tungsten, Molybdenum, Tantalum, Kovar®, Kevlar®, Kapton®, Mylar®, brass, etc..

Furthermore, said microchannel (1) is in a curvilinear geometric shape, its cross section is in various forms (e.g. square, rectangle or others polygons, circle, ellipse, parabola, hyperbola, or another non-regular surface). For example, the aforesaid microchannel (1) presents a characteristic dimension (e.g. width or height, or both) between 10 nm and 500 μιτι, and the length of said channel is between 500 μηι and several meters.

If necessary, the treatment of one part or all the surface of microchannel (1) walls by reagents (e.g. surfactants, spore-gel) modified the polarity to decrease possible undesired adsorptions or increase the analyte's affinity.

In addition and in the scope of the invention, the device can include at least one reaction chamber located upstream from the analysis area in which the probe molecule (3) injected in the sample for analysis. Said means of measurement (2) are at least one electrode, called collector, and placed in the microchannel (1). The intensity of the current, measured by said collector, is proportional to the probe molecules (3) number reaching said electrode (2) and that did not react with the aforesaid analyte (4) (see figure 2.3).

in thi s particular example of application according to the present invention, the device includes electrochemical activation means of the probe molecules (3) to initiate the reaction with the analyte (4), said probe molecule (3) being initially inert. Said activation means for the probe molecule (3) based on reactions with an activator (5) or electrochemical methods located in the microchannel (1) of the microfluidic device. Said electrochemical activation mean uses at least one electrode, called generator (6), and located upstream from said collector (2) on the same channel 's wall (figure 3.2).

So, the device according to the present invention gets an indirect detection method for molecule analysis (i.e analyte with a probe molecule). Said probe molecule (3), also called marker or indicator, is an intermediate molecular entities presenting an oxidation state. Said probe molecule can be a chemical species (e.g. molecules, ions, complexes as ferro- or ferricyanides, quinone and derivates) or biological compound (e.g. modified antibody with a specific antigen-binding site and an electrochemical graft from the species listed above). In this last example, said markers family allows a specific analysis of the studied analyte. As all the characteristics of the probe molecules are previously known, the study of the measured signal from the analytical device results to study indirectly the analyte both qualitatively (measure of the time-flight, specific interactions, kinetics) and quantitatively (measure of concentrations).

Said detection method measure a relative small amount of the probe molecule that did not react with the analyte in the sample for analysis, because the markers are in excess or the reaction kinetic between the compounds is too slow to be detected when the molecule passed near the collector with the flow.

if necessary, at least one technique (7) for the separation of mixtures (e.g. chromatography as HPLC, filtration, immobilization by affinity, and electrophoresis) can be used upstream from the microfluidic cell or integrated inside the microchannel (figure 2.2). These processes allow the separation and the refinement of the different compounds in the sample for analysis. According to another embodiment of the present invention, in reference at the figure 3.2, said electrochemical activation means (6) are located in the microchannel ( 1) in the opposite side from the collector (2).

So, two different operating modes for carrying out the present invention may be used. In the first operating mode, the probe molecule reacts directly with the analyte during the external preparation of the sample or inside the microchannel (1). In this last case, before the detection, a secondary channel injects the probe molecule in the main channel or in the reaction chamber.

In the second operating mode, the probe molecule is initially inert with the analyte and requires an electrochemical activation to generate the reaction between the aforesaid compounds. Said activation mean located inside of the device in a dedicated area symbolized as a unit or pre-unit.

This mode principle based on the electrochemical method of "generator-collector" (figure 3. 1). The application of a potential at one electrode (6), known as "generator" initiates a redox reaction with a molecule to change its oxidation state. The number of activated molecules can be determined in measuring the current of the generator by the electrons exchange between the aforesaid molecule and the electrode. In applying a potential on another electrode, known as "collector", said electrode (2) initiates the inverse redox reaction and come back to the initial oxidation state of said molecule. According to the present invention, the measurement of collector current allows to follow the variation of the probe molecule and so to follow indirectly the different reactions implying the aforesaid probe molecule.

The interaction between the probe molecule and the analyte can be a reaction in the chemical or physical chemistry's domain (e.g. substitution, recombination, rearrangement, acid-base, redox, radical, complexation (8), adsorption, absorption, decomposition, or combustion reactions) or in the physical domain (e.g. magnetic field, electrical polarity or agglomeration).

The main advantage of the method according to the present invention is to allow an indirect detection of low diffusive molecules that not necessary allow a redox reaction and so an extremely difficult electrochemical detection. Marked the studied target molecules, the analytes, depends of the interaction with the probe. In according of the reaction's kinetic of the couple analyte / probe molecule, a minimal length or a reaction chamber is necessary and may be optimised for said studied couple (specific device's development), or for all the possible couples (universal detection device).

The information concerning the analyte can be indirectly determined in measuring the variation of the collector's current. Independently of the specific information system (e.g. flow rate, dimensions, solvent viscosity, etc.), said variation is proportional to the number of probe molecules that reacted with analytes, the reaction kinetic and the initial concentrations of each compounds, the reagents and the analytes.

So, it is possible to determine only the analyte' s concentration.

Said collector, with the reference to the figure 4, probes a molecules layer in function of different parameters (e.g. electrode's width, flow rate and diffusion coefficient of said detected molecule). When several collectors are used to observe the same compound, even if the parameters of each electrode are considered, the respective gaps lengths between each one influence the analyzed solution' s depth. Indeed, every collector located downstream observes a deeper layer (figure 2.3). The signal measured at each electrode reflects the reaction that occurs in each observed respective layer. By this mean and according to the present invention, said detector allows a spatial analysis of samples. For example, it can indirectly analyze in discretizing, layer by layer, a solution where the molecules of different sizes organized according to the local flow rates. In addition, said mean allows a temporal analysis of samples For example, the detection, with several collectors in using the marking / tagging method, measures the reaction efficiency for a probe molecule / analyte couple at different electrodes positions inside the microchannel. Said measurements are a kinetic signature of said couple. Since the probe molecule is known, said signature gives additional qualitative information about the analyte and improves the quantification process.

The figures 5 and 6 are schematic representations of some embodiments of the present invention in which said device includes one or several generators (6), one or several secondary channels in contact with the main microchannel ( 1), and one or several collectors (2).

For example, said probe molecules are in the following list: Ferric complexes and derivates, ferricinium, dimethylferrocene (DMF), ferrocene monocarboxylic acid (FCOOH), ferrocyanide, ferricyanide, ferrocenemethanol, osmium complexes and derivates, tris(2,2'-bipyridyl)osmium, osmium tetroxide, bis(4,4'-diamino-2,2'-bipyridine)- (2'-3'-dipyridophenazine)osmium, ruthenium complexes and derivates, tris(2,2'- bipyridyl)ruthenium, ruthenium tetroxide, ruthenocene, organic conductive salts, viologen, quinone and derivates, hydroquinone, benzoquinone and derivates, anthraquinone and derivates, 7,7,8,8-tetracyanoquinodimethane (TCNQ), pyrroloquinoline quinine (PQQ), tetrathiafulvalene (TTF), N-methyl acidinium NMA+), tetrathiatetracene (TTT), N- methyl phenazinium (NMP+), 3-dimethylaminobenzoic acid (MBTH-DMAB), 3-methyl-2- benzothio-zolinone hydrazone, 2-methoxy-4-allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 3,3 ',5,5'-tetramethylbenzidine (TMB), 2,2-azino-di-[3-ethyl-benz-thiazoline sulfonate], o-dianisidine, o-toluidine, 2,4- dichlorophenol, 4-amino phenazone, benzidine, metalloporphyrins, hydrogen peroxyde.

Moreover, said probe molecule may be more specific for said analyte detection and present at least two distinct parts. The first part is specialized to react specifically to said analyte with a chemical function (e.g. amine, acid, carboxyl, carbonyl or peroxide groups, N-hydroxysuccinimide or any other interesting groups reacting specifically with the analyte) or with a biological compounds (e.g. amphiphilic molecules, enzymes, antigens, antibodies, peptides, nucleotides, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), cells, pathogens, virus and derivates). The second part of said molecule is the electrochemical probe allowing a redox reaction (e.g. a group derived from substances mentioned above).

Furthermore, several means can be used to automate the device for the manipulation and the preparation of the sample. Said means are external (e.g. solenoid valves) or integrated inside the device (e.g. microfluidic valves). In electrolytes excess, the mass transfer in solution is managed by the diffusion and the convection. For the convection, different means can move the sample or generate fluxes (e.g. gravity, syringe or peristaltic pumps, electro-osmosis or electrophoresis systems, over- or under-pressure between inputs and outputs microchannel by gas).

Related to the Reynolds and Schmidt's numbers, the Peclet number is a dimensionless number relevant in the study of transport phenomena in fluid flows. The Peclet number gives the predominant behaviour of the mass transfer between the diffusion and the convection of the studied compound: Pe = ^

D

where the parameters are:

D: Mass diffusion coefficient of the studied species; - L: Characteristic length {e.g. the height h of microchannel);

//: velocity (e.g. average linear velocity or flow rate for reference).

In addition, the device includes some different electrodes couples integrated inside at least one microchannel and respectively playing a specific function: one or several reference or pseudo-reference electrodes, called RE; one or several auxiliary (or counter) electrodes, called CE; and one or several working electrodes, called WE. RE is an electrode which has a stable and well-known electrode potential. The reaction of interest is occurring at the surface of WE. The CE, along with WE, provides circuit over which current is either applied or measured. If necessary, RE or CE, or both, are external but each RE/WE/CE couple always stays in contact together and with the solution by a salt bridge for example. When several WE in series used for the same prospect, they are commonly considered as an "array". In the embodiment of the present invention, said working electrode is integrated inside the microchannels device.

Said electrodes can be coated (e.g self-assembled monolayer modified electrodes) or made of materials in the following list: electrical conductive elements, metals (solid, liquid or porous), semiconducting materials, conductive ink or paste, conductive polymers or alloys (e.g. Ag, Al, Au, Cd, Co, Cr, Cu, Fe, Hg, Ir, Nb, Ni, Mo, Os, Pb, Pt, Pd, Ru, Si, Ti, Va, Zn, Zr, Carbon in graphite or diamond, glassy carbon, Indium Tin oxide, A1₂0₃, SiC, Si₃N₄, Zr0₂, MgO, etc.). In addition, the RE's materials can be made of those currently used in electrochemistry (e.g. Standard Hydrogen Electrode (SHE), Normal Hydrogen Electrode (NHE), Reversible Hydrogen Electrode (RHE), Saturated Calomel Electrode (SCE), Copper / Copper(II) Sulfate Electrode, Silver / Silver Chloride Electrode, pH- electrode (in case of pH buffered solutions), Palladium-Hydrogen Electrode, Dynamic Hydrogen El ectrode (DHE)) .

The potential applied at the surface of the WE depends of the probe molecule. For an array, the potential applied at each electrode can be different for the analysis (generator- collector method) or for the technical purpose (e.g. IR-drop), or both.

The electrodes are connected individually or in group, according to their function, with an electrical contact (same electrodes material or not) to a measurement equipment called commonly potentiostat (for one WE), bipotentiostat (for two WE), multipotentiostat (for several WE), and polypotentiostat (for several potentiostats used in series or parallel, or both, circuits). The WE behaviour in a microchannel is in a non-dimension representation. Nondimensionalization allows to considerate the majority of microfluidic devices designs and determines in a systematic manner the characteristic units of the system. For this example, the characteristic length is the smallest dimension of the channel, its height h.

In this dimensionless system, the Cartesian coordinates are also normalized by this characteri and Y=y/h) and the dimensionless time is defined by:

The flow profile becomes:

U_x(Y) = 6PeY{\ - Y)

The electrode's current is proportional to the concentration's gradient at its surface. The c e is defined by:

In said system, the dimensionless current becomes:

/^'(f) w f dC(T) ^

dX

nFDic⁰ H ay

where the parameters are:

n: number of electrons in the redox reaction;

F: Faraday constant;

- D: Mass diffusion coefficient of the studied species;

h: microchannel' s height;

1: microchannel's width;

w: electrode length;

- W = w / h;

c°: Initial concentration of the species.

For a microchannel electrode, the steady state current is limited between two extreme and disctinct behaviors, the thin layer effect and the totally mass-transfer-limited condition, commonly called the Levich's behavior.

In the dimensionless system, the thin-layer current equation is: ψ_τι = Ρθ

And for the Levich's behaviour, the current equation of a microband is:

ψ, = 1.468W^{2/ 3} Pe' ³

In addition of said behaviors, the dimensionless representation permit to rationalize most of the steady-state current behaviours in only one curve, depending of the used electrodes' number, presented in the figure 7. However, in some specific cases (i.e. at low rates when the diffusion is not anymore negligible in the microchannel axis) the steady- state current can be amplified.

Furthermore, the measurement of the electric charge value 0 corresponding at the sum of currents transferred at the electrode surface can increase the signal if the detection sensitivity of the equipment is limiting.

The amount of molecules reacting with the electrode is related to the steady-state current by the following equation:

Pe ^h

where C is the homogenous concentration of the redox species, normalized by c°, downstream from the electrode.

The analyzed solution depth can be determined in measuring a blank solution of redox species with a same diffusion coefficient or with a numerical simulation. Said dimensionless depth H (figure 2.3) and current are related by the following equation:

w ϊ Γ ^H

Ψ = \~ dX = \ Pe_x (Y)dY where Ρβχ ϊ) is the flow profile equation.

For an array of n similar electrodes, the equation is:

∑ _i = "\ Pe_x (Y)dY

'=1 0

So, in resolving these equations, the dimensionless depth H can be determined. For example, the solution depth observed by a microbands array in a parabolic flow is:

By recurrence, the different observed depth can be determined for each electrode of the array with the respective current.

The figure 8 is a schematic representation of a measuring chain example: A computer linked to a multipotentiostat to record the signal, for the data post-treatment and, so, to give the desired information. Independently, the computer can also pilot and control the different processes to manipulate and prepare the samples. If necessary, usable software applications can manage all or several processes of said actions automatically or through a user interface.

Obviously, different embodiments exist according to the present invention with detection methods other than the amperometry, such as the voltammetry, the potentiometry, the coulometry, the polarography, or the impedance spectroscopy, etc.

For the present invention, the device presents different interests in several domains, such as medical applications (e.g. diagnosis of auto-immune deceases, survey of neoplasms behavior such as cancers, detection of endogenous substances or pathogens, drug tests), in food science (e g quantification of allergens: rheomorphic proteins such as the casein from cow milk, β-barrel proteins such as the peanut Ara hi, proteins with disulphide bonds such as β-lactoglobulin, or prolamins, etc.), or for the environment (e.g. measurement of oxygen or pesticides concentrations).

Example 1: Analytic device after a High-performance liquid chromatography (HPLC)

For the analytical device's fabrication, the microfluidic device made from a glass substrate and a structure in PDMS including two reservoirs (3 mm of diameter) connected by a linear microchannel (length L = 2 cm, width / = 540 pm, height h - 17 pm). By photolithography and the "lift-off' technique, electrodes are deposited at the substrate surface; for a good adhesion between the electrodes materials and the glass, a titanium sublayer of 5 nm thick was used. These electrodes are microbands perpendicular to the channel. From upstream to downstream, the electrodes are: a silver / silver chloride reference electrode (100 pm wide), six similar working electrodes array (E1-E6) in platinum (w, = 20 pm), and an auxiliary electrode in platinum (250 pm wide). The gap from the array to the Ref is 100 pm, and to the CE is 200 pm. According to the chosen working electrodes pair, the gap between them respectively is: ΙΟΟμπι (E2-E1), 400μιη (E3-E1), 700μιη (E4-E1), ΙΟΟΟμηι (E5-E1), 1300 μπι (E6-E1). To avoid any non-specific adsorptions on microchannel walls and electrodes, a conservative solution is injected in the microfluidic device outside any analysis, i.e. a buffer solution of 0.5 mol/L carbonic acid / bicarbonate (pH 7.3) and 0.05% TWEEN20©. For the electrochemical detection, a multipotentiostat is used.

For the analysis, plasma or serum samples from the patient blood is used. This sample is respectively dilute (i.e. 1 : 100, 1 :1000 andl : 10000) in a 0.5 mol/L potassium phosphate buffer solution (pH 7.0) and 0.05% TWEEN20©. Individually, 20 μΕ of each preparation is injected in a HPLC column to separate the individual components. The column, 9TSKgel QC-PAK GFC 399GL0, was 15 cm long and 8 mm in diameter and the separation was carried out at a flow rate of 1 mL/min. The mobile phase composed with 0.5 mol/L KC1 in 0.05 mol L potassium phosphate buffer at pH 7.0.

At regular intervals, 0.5 mL of the mobile phase is automatically collected from the column outlet. In each collected volumes, a concentrated solution is added to obtain a preparation of 1 mL. This concentrated solution contains a buffer solution and the inert probe molecule: hydroquinone (H2QN). Each preparation contains with the collected solution, 1 mmol/L H2QN, 0.5 mol L KC1, 0.05 mol/L potassium phosphate buffer at pH 7.0. With an external loop sampling valve and a peristaltic pump, 10 μΐ, of each preparation is successively injected inside the device's microchannel, at a flow rate of 10 μί/πηη, and fractioned between 100 μΕ of another mobile phase. Said mobile phase composed with 0.5 mol L KC1, 0.05 mol/L potassium phosphate buffer (pH 7.0) and 0.05% TWEEN20©. The electrode El is polarized at 0.55 V vs. Ref to initiate the redox reaction with H2QN; at this potential, two electrons are exchanged with the electrode to transform these species into parabenzoquinone, C6H4O2, often called p-quinone or simply quinone (pQN). The others electrodes (from E2 to E6) are polarized at 0.35 V vs. Ref. to force the inverse redox reaction; pQN transform back into H2QN still with two exchanged electrons. El, in contact with each fraction containing H2QN, forces the reaction to generate pQN. In using the chronoamperometry method, the El current measurement gives the amount of activated probe molecules. During the time where the front is in the gap (E2-E1), pQN reacts with the analytes, contained in each fraction, in function of their chemical properties. Then, with the flow, when the others electrodes are in contact with each fraction, they force the reaction with a relative amount of remaining pQN. The respective current measurements at each electrode give the different (pQN / H2QN) ratios for each (En - El) gap. In considering all the manipulations and dilutions, said measurements give, for each fraction collected from the liquid chromatography, the analytes concentration and a specific kinetic signature to these species. In addition, with these results and the retention time of each fraction at the column end, each species contained in the initial sample are identified with the help of a database gathering all the kinetic signatures, retention times and information about determined species.

Additionally, a diagnosis kit contains:

a microfluidic device with integrated electrodes;

- a buffer solution;

concentrated cleaning and conservation solutions;

a control serum with bio-chemical compounds for testing;

a solution for calibration ;

- a concentrated solution of probe molecules;

- an analytical equipment (i.e. multipotentiostat).

Furthermore, the means for the injection of solutions (e.g. conservative, cleaning, calibration solutions) or for the samples preparation, or both, can be partially or totally integrated into the microfluidic device. These processes can be managed by the equipment for the automation of the analysis.

Example 2: Diagnosis device for the Multiple Sclerosis disease (MS)

MS is an immune-mediated disorder mediated by a complex interaction of the individual's genetics and as yet unidentified environmental insults. The immune system attacks the nervous system, possibly as a result of exposure to a molecule with a similar structure to one of its own. Said embodiment for the present invention is a device for the detection or quantification, or both, of antibodies generated by the immune system specific to this autoimmune disease.

For the detection of these antibodies, the probe molecule is a functionalized peptide

(FP) in referring to the method used by F. Real-Fernandez et al. in « Ferrocenyl Glycopeptides as Electrochemical Probes to Detect Autoantibodies in Multiple Sclerosis Patients' Sera » (Peptide Science, 90, 4, p. 488-495, 2008). Said FP synthesis based on a CSF1 14(Glc) sequency, the peptidic part with the immunogen group recognized by the antibodies related to the MS disease, and an ending part specialized for the electrochemical detection based on ferrocenyl group.

The diagnosis system is composed of a 10 μ]_, external loop sampling valve, a peristaltic pump, a separation column with a nanoporous membrane, used as a filter, and a microfluidic device connected together with biocompatible silicone tubings. The nanoporous membrane have a pore size of 10 ~ 20 nm in diameter with molecular weight cut off (MWCO) of 10000 Dalton (Da). The MWCO is small enough to stop the antibody and the antibody-antigen complex, but too high for FP alone. The microfluidic device, made as in the first example, include a three-electrode cell: Ag/AgCl reference electrode "Ref (150 μιη wide), gold working electrode "El" (50 μιη wide) and gold auxiliary electrode "CE" (200 μηι wide). The applied potential at El (E = 0.5 V vs Ref) force the electrons exchange between the ferrocenyl group on the probe molecule and the electrode surface.

For the diagnosis, sera from patient blood are used. These sera are respectively dilute

(i.e. 1 : 100, 1 : 1000 and 1 : 10000) in a buffer solution FBS (FBS 10%, NaCl 9 g L, Tween20© 0.05%). For each diluted sera, two samples are prepared: a testing sample and a control sample. Each sample contains 8 μΐ, of the analyzed sera and respectively:

2 μί of FP in the buffer solution;

- 2 μΐ_^ of buffer solution.

The FP's amount is known to obtain a final concentration of 5 mmol/L in the sample. During an incubation of two hours at 23 °C, the antibody binds to the specific antigen forming a high molecular complex. Then, each sample is injected in the separation column at a flow rate of 10 μΕ/ι ίη to separate the complexes from the free probe molecules FP. The column end connecting to the microfluidic device, the remaining molecules are detected electrochemically by the three-electrode cell. After each injection, the nanoporous membrane is replaced to avoid any contamination. The difference of currents, measured by El, between the testing and control samples allows the quantification of free FP. Considering the different manipulations and dilutions, this set up allows to indirectly measure, qualitatively and quantitatively, the presence of antibodies from patient's serum with the MS disease.

Additionally, a diagnosis set up includes:

a column of separation; a microfluidic device with integrated three-electrode cell;

cleaning and conservative solutions;

control sera (positive and negative testing solutions);

functionalized peptides specific for the MS diagnosis;

an analytical equipment (i.e. potentiostat).

Furthermore, the means for the injection of solutions (e.g. mobile phase, conservative, cleaning solutions) or for the samples preparation, or both, can be integrated partially or totally into the device. Also, said processes can be managed by the equipment for the automation of the diagnosis.

Finally, said examples are only particular illustrations and do not limit the scope of the present invention.

Claims

1 - A method to detect or quantify, or both, at least one analyte in a sample for analysis comprising at least the following steps:

Injection of at least one probe molecule in the sample for analysis compri sing at least one analyte, said probe molecule being able to react with said analyte chemically (e.g. substitution, addition, elimination, recombination, rearrangement, acid-base, redox, radical, complexation, polymerization, chain, precipitation, adsorption, absorption, decomposition, or combustion reactions), physically (e.g. magnetic field, electrical polarity or agglomeration) or biologically (e.g. enzymatic reaction, antigen - antibody binding, addition reaction);

- Predetermined period during the reaction occurs between said probe molecule and analyte;

- Measure a relative amount of said probe molecule remaining in a microchannel, a microfluidic cell, inside or at the column end, or both;

- Determination of a relative amount or the nature, or both, of the analyte from the relative amount measurement of the probe molecule.

2 - The method of claim 1 , wherein said molecule probe is injected in the sample for analysis comprises at least one analyte into a said reaction chamber located upstream from the analysis area.

3 - The method of claim 1 or claim 2, wherein said probe molecule is artificially activated by at least one mean to initiate the reaction with said analyte, said probe molecule being initially inert.

4 - The method of claim 3, wherein said artificially activation mean is a spontaneous reaction, e g by reagents addition, such as chemical or biological species.

5 - The method of claim 3, wherein said artificially activation mean is a controlled reaction, e.g. by electrochemistry, photochemistry, radiochemistry, thermochemistry or biophysical chemistry.

6 - The method of any one of claims 1 to 5, wherein said probe molecule is artificially activated into the main channel of the microfluidic cell or the column after injection of said probe molecule in the sample. 7 - The method of any one of claims 1 to 5, wherein said probe molecule is artificially activated into at least one secondary channel of the microfluidic cell or the column before the injection of said probe molecule in the sample.

8 - The method of claim 6 or claim 7, wherein said probe molecule is activated by at least one electrode, called generator, into said main or secondary channel .

9 - The method of any one of claims 1 to 8, wherein said relative amount of the aforesaid probe molecule is obtained from the current intensity measurement of at least one electrode, called collector, into said main channel of the microfluidic cell or column.

10 - The method of any one of claims 1 to 8, wherein said relative amount of said probe molecule is obtained from the current intensity measurement of at least one electrode, called collector, into at least one secondary channel downstream from the main channel of the microfluidic cell or column.

1 1 - The method of any one of claims 1 to 10, wherein said probe molecule comprises the chemical species and derivates in the following list: Ferric complexes and derivates, ferricinium, dimethylferrocene (DMF), ferrocene monocarboxylic acid (FCOOH), ferrocyanide, ferricyanide, ferrocenemethanol, osmium complexes and derivates, tris(2,2'-bipyridyl)osmium, osmium tetroxide, bis(4,4'-diamino-2,2'- bipyridine)-(2'-3 '-dipyridophenazine)osmium, ruthenium complexes and derivates, tris(2,2'-bipyridyl)ruthenium, ruthenium tetroxide, ruthenocene, organic conductive salts, viologen, quinone and derivates, hydroquinone, benzoquinone and derivates, anthraquinone and derivates, 7,7,8, 8-tetracyanoquinodimethane (TCNQ), pyrroloquinoline quinine (PQQ), tetrathiafulvalene (TTF), N-methyl acidinium (NMA+), tetrathiatetracene (TTT), N-methylphenazinium (NMP+), 3- dimethylaminobenzoic acid (MBTH-DMAB), 3-methyl-2-benzothio-zolinone hydrazone, 2-methoxy-4-allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4- aminoantipyrene, 4-methoxynaphthol, 3,3 ',5,5'-tetramethylbenzidine (TMB), 2,2- azino-di-[3-ethyl4jenz-thiazoline sulfonate], o-dianisidine, o-toluidine, 2,4- dichlorophenol, 4-amino phenazone, benzidine, metalloporphyrins, hydrogen peroxide.

12 - A device to detect or quantify at least one analyte into a sample comprising: at least one probe molecule reacting with said analyte;

at least one microfluidic cell including a substrate; - at least one inlet and at least one outlet ports for the injection and extraction of fluids as the said sample or the said probe molecule, and formed in said substrate;

- at least one microchannel formed in said substrate and connecting the said ports; at least one analysis area comprising at least one mean to measure a relative amount of said probe molecule remaining after said reaction with said analyte.

13 - The device of claim 12, wherein said probe molecule and analyte is injected in at least one reaction chamber upstream from said detection area.

14 - The device of claim 12 or claim 13, wherein said probe molecule is artificially activated by at least one mean to initiate the reaction with said analyte, said probe molecule being initially inert.

15 - The device of claim 14, wherein said artificial activation mean occur into said microchannel upstream from said detection area.

16 - The device of claim 14, wherein said artificial activation mean occur into a secondary channel connected to said microchannel upstream from said analysis area. 17 - The device of any one of claims 14 to 16, wherein said artificial activation mean of said probe molecule is from a controlled reaction such as an electrochemical activation mean.

18 - The device of claim 17, wherein said electrochemical activation comprises at least one reaction between said probe molecule and at least one electrode, called generator.

19 - The device of any one of claims 14 to 16, wherein said artificial activation of said probe molecule is from a spontaneous reaction, e.g. with chemical or biological reagents.

20 - The device of any one of claims 12 to 19, wherein said analysis area comprises at least one mean to measure a relative amount of said probe molecule.

21 - The device of claim 20, wherein said analysis area is into said microchannel of said microfluidic cell.

22 - The device of claim 20, wherein said analysis area is into a secondary channel connected downstream to said microchannel.

23 - The device of any one of claims 12 to 22, wherein said mean to measure the relative amount of said probe molecule comprises at least one electrode, called collector, located into said main or secondary microchannels, or both. 24 - The device of any one of claims 12 to 23, wherein said probe molecule comprises the chemical species and derivates in the following list: Ferric complexes and derivates, ferncinium, dimethyl ferrocene (DMF), ferrocene monocarboxylic acid (FCOOH), ferrocyanide, ferricyanide, ferrocenemethanol, osmium complexes and derivates, tris(2,2'-bipyridyl)osmium, osmium tetroxide, bis(4,4'-diamino-2,2'- bipyridine)-(2'-3 '-dipyridophenazine)osmium, ruthenium complexes and derivates, tris(2,2'-bipyridyl)ruthenium, ruthenium tetroxide, ruthenocene, organic conductive salts, viologen, quinone and derivates, hydroquinone, benzoquinone and derivates, anthraquinone and derivates, 7,7,8, 8-tetracyanoquinodimethane (TCNQ), pyrroloquinoline quinine (PQQ), tetrathiafulvalene (TTF), N-methyl acidinium (NMA+), tetrathiatetracene (TTT), N-methylphenazinium (NMP+), 3- dimethylaminobenzoic acid (MBTH-DMAB), 3-methyl-2-benzothio-zolinone hydrazone, 2-methoxy-4-allylphenol, 4-aminoantipyrin (AAP), dimethylaniline, 4- aminoantipyrene, 4-methoxynaphthol, 3,3 ' ,5,5'-tetramethylbenzidine (TMB), 2,2- azino-di-[3-ethyl-benz-thiazoline sulfonate], o-dianisidine, o-toluidine, 2,4- dichlorophenol, 4-amino phenazone, benzidine, metalloporphyrins, hydrogen peroxide.