WO2013023671A1 - Dispositif microfluidique et procédé de détection d'analytes dans un flux à l'aide de sondes électrochimiques - Google Patents

Dispositif microfluidique et procédé de détection d'analytes dans un flux à l'aide de sondes électrochimiques Download PDF

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WO2013023671A1
WO2013023671A1 PCT/EP2011/063573 EP2011063573W WO2013023671A1 WO 2013023671 A1 WO2013023671 A1 WO 2013023671A1 EP 2011063573 W EP2011063573 W EP 2011063573W WO 2013023671 A1 WO2013023671 A1 WO 2013023671A1
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probe molecule
analyte
reaction
derivates
sample
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PCT/EP2011/063573
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English (en)
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Nicolas DA MOTA
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Da Mota Nicolas
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • 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.
  • 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-
  • 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.
  • micro total analysis system micro total analysis system
  • PCR polymerase chain reaction
  • 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.
  • a method to detect or quantify, or both, at least one analyte in a sample is characterised by at least the following steps of:
  • 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),
  • chemical reaction e.g. substitution, addition, elimination, recombination, rearrangement, acid-base, redox (oxidation-reduction), decomposition, combustion, complexation, polymerisation or radical reactions
  • physical interaction e.g. adsorption, absorption, electrostatic or magnetic interactions
  • 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,
  • 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.
  • 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.
  • 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).
  • 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
  • biophysical chemistry e.g. electrochemiluminescence
  • said probe molecule is activated in the main channel of the microfluidic device or the column after its injection in the sample for analysis.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • said electrochemical activation means consist in at least one electrode, called generator.
  • said artificial activation means of the probe molecule consists in chemical activation means.
  • said analysis area includes at least one measuring device of a relative amount of the probe molecule.
  • said analysis area is located in the microchannel of the microfluidic device.
  • 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.
  • 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.
  • the device according to the invention includes several collectors distributed on the aforesaid analysis area inside the microchannel.
  • 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,
  • FIG. 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.
  • FIG. 4 is a graphic showing the intensities variation vs. time for generator and collector electrodes
  • FIG. 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.
  • the device to detect or quantify, or both, at least one analyte in a sample for analysis 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
  • 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).
  • the aforesaid microchannel (1) presents a characteristic dimension (e.g. width or height, or both) between 10 nm and 500 ⁇ , and the length of said channel is between 500 ⁇ and several meters.
  • reagents e.g. surfactants, spore-gel
  • the treatment of one part or all the surface of microchannel (1) walls by reagents modified the polarity to decrease possible undesired adsorptions or increase the analyte's affinity.
  • 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).
  • the device in thi s particular example of application according to the present invention, 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).
  • 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).
  • said markers family allows a specific analysis of the studied analyte.
  • 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.
  • At least one technique (7) for the separation of mixtures 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.
  • said electrochemical activation means (6) are located in the microchannel ( 1) in the opposite side from the collector (2).
  • the probe molecule reacts directly with the analyte during the external preparation of the sample or inside the microchannel (1).
  • a secondary channel injects the probe molecule in the main channel or in the reaction chamber.
  • the probe molecule 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.
  • 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 chemical or physical chemistry's domain e.g. substitution, recombination, rearrangement, acid-base, redox, radical, complexation (8), adsorption, absorption, decomposition, or combustion reactions
  • 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.
  • the specific information system e.g. flow rate, dimensions, solvent viscosity, etc.
  • Said collector probes a molecules layer in function of different parameters (e.g. electrode's width, flow rate and diffusion coefficient of said detected molecule).
  • parameters e.g. electrode's width, flow rate and diffusion coefficient of said detected molecule.
  • 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.
  • 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.
  • said mean allows a temporal analysis of samples
  • 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.
  • FIGS. 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).
  • 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-te
  • 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).
  • Said means can be external (e.g. solenoid valves) or integrated inside the device (e.g. microfluidic valves).
  • electrolytes excess the mass transfer in solution is managed by the diffusion and 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).
  • Pe ⁇
  • D Mass diffusion coefficient of the studied species
  • - L Characteristic length ⁇ e.g. the height h of microchannel
  • 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.
  • WE When several WE in series used for the same prospect, they are commonly considered as an "array".
  • 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, A1 2 0 3 , SiC, Si 3 N 4 , Zr0 2 , MgO, etc.).
  • electrical conductive elements e.g self-assembled monolayer modified electrodes
  • metals solid, liquid or porous
  • semiconducting materials e.g. Ag, Al, Au, Cd, Co, Cr, Cu, Fe, Hg, Ir, Nb, Ni, Mo, Os, Pb, Pt, P
  • 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)) .
  • SHE Standard Hydrogen Electrode
  • NHE Normal Hydrogen Electrode
  • RHE Reversible Hydrogen Electrode
  • SCE Saturated Calomel Electrode
  • Copper / Copper(II) Sulfate Electrode Silver / Silver Chloride Electrode
  • pH- electrode in case of pH buffered solutions
  • Palladium-Hydrogen Electrode in case of pH buffered solutions
  • DHE Dynamic Hydrogen El ectrode
  • the potential applied at the surface of the WE depends of the probe molecule.
  • 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.
  • the flow profile becomes:
  • the electrode's current is proportional to the concentration's gradient at its surface.
  • the c e is defined by:
  • n number of electrons in the redox reaction
  • 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.
  • 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.
  • the steady- state current can be amplified.
  • 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:
  • 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:
  • the dimensionless depth H can be determined.
  • the solution depth observed by a microbands array in a parabolic flow is:
  • 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.
  • 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, ⁇ -barrel proteins such as the peanut Ara hi, proteins with disulphide bonds such as ⁇ -lactoglobulin, or prolamins, etc.), or for the environment (e.g. measurement of oxygen or pesticides concentrations).
  • medical applications e.g. diagnosis of auto-immune deceases, survey of neoplasms behavior such as cancers, detection of endogenous substances or pathogens, drug tests
  • food science e.g quantification of allergens: rheomorphic proteins such as the casein from cow milk, ⁇ -barrel proteins such as the peanut Ara hi, proteins with disulphide bonds such as ⁇ -lacto
  • Example 1 Analytic device after a High-performance liquid chromatography (HPLC)
  • the gap from the array to the Ref is 100 pm, and to the CE is 200 pm.
  • the gap between them respectively is: ⁇ (E2-E1), 400 ⁇ (E3-E1), 700 ⁇ (E4-E1), ⁇ (E5-E1), 1300 ⁇ (E6-E1).
  • 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 ⁇ .
  • a multipotentiostat is used for the electrochemical detection.
  • 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 ⁇ 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.
  • 0.5 mL of the mobile phase is automatically collected from the column outlet.
  • 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).
  • H2QN hydroquinone
  • 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.
  • 10 ⁇ of each preparation is successively injected inside the device's microchannel, at a flow rate of 10 ⁇ / ⁇ , and fractioned between 100 ⁇ 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.
  • a diagnosis kit contains:
  • an analytical equipment i.e. multipotentiostat
  • the means for the injection of solutions e.g. conservative, cleaning, calibration solutions
  • solutions e.g. conservative, cleaning, calibration solutions
  • samples preparation e.g., samples preparation, or both
  • microfluidic device e.g.
  • 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.
  • 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 ⁇ ]_, 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).
  • MWCO molecular weight cut off
  • 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 ⁇ wide), gold working electrode “El” (50 ⁇ wide) and gold auxiliary electrode “CE” (200 ⁇ 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.
  • FBS FBS 10%, NaCl 9 g L, Tween20 ⁇ 0.05%).
  • a testing sample i.e. 1 : 100, 1 : 1000 and 1 : 10000
  • FBS buffer solution
  • the FP's amount is known to obtain a final concentration of 5 mmol/L in the sample.
  • the antibody binds to the specific antigen forming a high molecular complex.
  • each sample is injected in the separation column at a flow rate of 10 ⁇ / ⁇ ⁇ 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.
  • 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.
  • a diagnosis set up includes:
  • an analytical equipment i.e. potentiostat
  • the means for the injection of solutions e.g. mobile phase, conservative, cleaning solutions
  • solutions e.g. mobile phase, conservative, cleaning solutions
  • samples preparation e.g., a sample preparation, or both
  • said processes can be managed by the equipment for the automation of the diagnosis.

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Abstract

Selon la présente invention, un mode de réalisation utilise un procédé pour détecter ou quantifier, ou les deux, au moins un analyte dans un échantillon pour analyse et comprend au moins les étapes suivantes : injection d'au moins une molécule de sonde dans ledit échantillon, ladite molécule de sonde étant apte à interagir avec ledit analyte de manière chimique (par exemple, réactions de substitution, d'addition, d'élimination, de recombinaison, de réarrangement, acide-base, redox, radicale, de complexation, de polymérisation, en chaîne, de précipitation, d'adsorption, d'absorption, de décomposition ou de combustion), de manière physique (par exemple, champ magnétique, polarité électrique ou agglomération) ou de manière biologique (par exemple, réaction enzymatique, liaison antigène-anticorps, réaction d'addition). La période prédéterminée durant ladite interaction a lieu entre ladite molécule de sonde et l'analyte; mesure d'une quantité relative de ladite molécule de sonde demeurant après ladite période, dans un microcanal, une cellule microfluidique, à l'intérieur ou sur l'extrémité de colonne, ou les deux; et détermination d'une quantité relative ou de la nature, ou les deux, dudit analyte à partir de la quantité relative mesurée de ladite molécule de sonde. Un autre mode de réalisation selon la présente invention est un dispositif pour détecter ou quantifier, ou les deux, au moins un analyte dans un échantillon pour analyse. Ledit dispositif comprend au moins une cellule microfluidique faite d'un substrat comprenant au moins une entrée et une sortie en communication par au moins un microcanal. Ledit microcanal comprend au moins une zone d'injection pour les fluides, tels que ledit échantillon et des réactifs, et au moins une zone d'analyse. Ledit réactif est au moins une molécule de sonde qui est apte à réagir avec ledit analyte. Ladite zone d'analyse consiste à mesurer une quantité relative de ladite molécule de sonde demeurant après au moins une réaction avec ledit analyte par au moins une mesure.
PCT/EP2011/063573 2011-08-05 2011-08-05 Dispositif microfluidique et procédé de détection d'analytes dans un flux à l'aide de sondes électrochimiques WO2013023671A1 (fr)

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CN111537055A (zh) * 2020-05-18 2020-08-14 商丘师范学院 一种用于超高压冲击波测量探针布设实验装置及其实验方法
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CN111676131A (zh) * 2020-06-17 2020-09-18 清华大学 用于活体单细胞局部微区原位自由基刺激的微流控装置

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