Patent Application
20070240986
Miniaturisation of analytical devices has become a trend in analytical chemistry for two main reasons: reducing the time required for single analyses and reducing the size of the sample/waste. Many developments have been shown over the last years in the fabrication of microfluidic device and their use for developing assays.
One bottleneck of miniaturisation of analytical systems is to ensure a low limit of detection of the low number of molecules present in the small volume of the microfluidic device. Different detection means including optical, mass spectrometry or electrochemical detection have been implemented with success to detect rather large concentrations of analyte. For example, many microsystems exist for the detection of glucose in microfluidic devices, for example the system developed by Therasense and which allows one to perform a coulometric detection in only 0.3 μL of capillary blood. Detecting low concentrations while ensuring large dynamic ranges necessitates the optimisation of the geometry of the microfluidic device as well as the method of detection. This invention aims at a specific method and related device that enable the detection of lower concentration of redox active molecules, particularly applied to enzyme and immunological assays (immunoassays),
The present invention relates to an electrochemical microfluidic device and method to optimise electrochemical detection in a microstructure (and, preferably, a microchannel or a network of microchannels). The essential feature of the device is to minimise the ohmic resistance of the microstructure(s). Minimisation of the ohmic resistance (or even of the impedance) of a microstructure allows one to improve the electrochemical detection and notably amperometric measurements since the over-potential to apply to compensate for the ohmic resistance can also be minimised, which allows an improved quality of the electrical signal.
One aim of the invention is thus to optimise electrochemical detection in a microfluidic device. Electrochemical detection in microfluidic devices has already been shown as an attractive solution for the detection of redox-active molecules in small volumes. This technique can for instance be used as a detection means after separation or for enzyme or immunoassay analyses. One of the constraints of microfluidic systems is that the typical dimensions of the microstructures are quite unfavourable against the conduction of current. Indeed, when for instance a microstructure consists of a tubular capillary having typical dimensions of one or few centimeters in length (L) and few tens of micrometer in diameter (d), the ohmic resistance (R) is large even if the resistivity of the solution (ρ) is rather low, as expressed by the ohmic law of equation 1:
R=ρL/A Equation 1
where A is the cross-section area of the tube capillary with A=πd2/4.
As an example, when the capillary radius is 20 μm and the length of the capillary to transport the solution is 1 cm, the factor L/A is equal to 8×106 cm−1. With a solution of 100 mM phosphate, the resistance along such a microchannel would already be 106Ω, so that only small current densities could be transported along this channel.
The large resistance present in microfluidic systems is an important drawback for electrochemical applications. Indeed, this resistance may distort the responses, necessitate feed-back voltage to compensate for the drop due to the ohmic resistance or even prevent large signals to be measured accurately. For such electrochemical applications, and in particular, for electrochemical biosensors, it would thus be of great advantage to have microfluidic systems with reduced resistance.
In our invention, the microstructure dimensions are in the same order of magnitude as those given in the above example (channel length in the centimeter range and channel diameter of a few tens of μm). However, an electrically conductive means is positioned in a portion of the microstructure or along the entire microchannel such as to conduct the current from one point of the channel to another one. In this case, the current is no more transported only by the ionic current through the channel, but it can also be transported through the electrically conductive means.
Experiments made with and without conductive means in microstructures having a microchannel of dimensions similar to those mentioned above show that the current intensity that can be passed without ohmic resistance (or “iR drop”) is larger in the case where the microstructure comprises an electrically conductive means. In some cases, the electrically conductive means can be connected as a counter-electrode such as to enable counter reaction to take place inside the channel and hence regeneration of the product of the reaction taking place at the working electrode.
As will be shown in further detail below, it has also been put into evidence that excellent electrochemical responses can be obtained even when the electrically conductive means is not connected and hence is not part of the ensemble of electrodes serving for the detection (hereinafter also referred to as “electrode system”). For the sake of clarity, a 2-electrode system comprises only working and pseudo-reference electrodes and a 3-electrode system comprises working, counter and reference electrodes. In the invention, the microfluidic device comprises an electrically conductive means which may be present in addition to the 2-electrode or 3-electrode ensemble, and this electrically conductive means is then not connected to any of these electrodes. In such a case, when the microfluidic device is filled with a solution, a contact will be created between the ensemble of electrodes and the electrically conducting means, which becomes thus part of the global electrical circuit. In the invention, the electrically conductive means may itself constitute a counter-electrode or a pseudo-reference electrode and then be part of the electrode ensemble. In both configurations, the electrically conductive means shall be adapted to provide an extremely low-resistance path for the current, so that the global resistance of the microstructure is minimised, even for a microstructure of very small cross-section. As will be further described below, it appears that the resistance of the microstructure is thus reduced even when the electrically conducting means is not directly connected.
This phenomenon is very interesting to prevent perturbation of the electrochemical detection signal in a microsystem, and it is likely to be explained by the fact that the presence of an electrically conductive means along the microstructure creates a system that can be schematically represented by two resistances in parallel (one large resistance, Rm, resulting from the small dimension of the microstructure and from the relatively high resistance of the solution, and one very low resistance, Rc, due to the extremely low resistivity of the conducting material serving as electrically conductive means). These resistances work in parallel, and this even if the electrically conductive means is not connected to the electrochemical detection circuit. Thus, the resulting global resistance, Rg, is approximately equal to Rc, so that the electrically conductive means acts as a kind of by-pass of resistance, which provides a more favourable route for the current. The applied potential can thus be maintained approximately constant along the entire microstructure (even in the presence of large currents) because of the very low global resistance of the system. The integration of an electrically conductive means thus prevents perturbation of the electrochemical signal due to iR drop, and the present invention thus provides a powerful means to improve the quality of the signals that can be obtained for electrochemical detection in a microsensor system.
It should be noted here that electrophoresis would not be possible in the device of this invention, since the electrically conductive means would maintain an approximately constant potential along the microstructure, so that almost no gradient of electric field could be generated, thereby hindering electroosmosis as well as electrophoretic separation.
This invention provides a microfluidic device comprising at least one microstructure which comprises one or a series of working electrode(s), as well as an electrically conductive means integrated inside the microstructure(s) in such a manner as to reduce the ohmic resistance within this microstructure. Reduction of the ohmic resistance is particularly needed in sensors of small dimensions (e.g. in microchannels) or when large current densities are used, because the ohmic resistance disturbs the signal that can be measured electrochemically. The microfluidic device of this invention is directed to electrochemical sensors having reduced ohmic resistance, thereby enabling improved electrochemical responses.
In one embodiment of the invention, the electrically conductive means is connected as the counter-electrode, which can advantageously be used during reduction or oxidation (or “redox”) reactions to regenerate the analyte to detect. In an alternative embodiment, this conductive means is not connected to an external electric meter (e.g. a potentiostat, a power supply, etc.). Hence, in such a configuration, the electrically conductive means is not an electrode (since it is not connected), but only a tool added to the microfluidic sensor device in order to conduct the current around a path of high electrical resistance (e.g. a solution in a microchannel), thereby allowing a decrease in the overall resistance of the system. Such an electrically conductive means can reduce the ohmic resistance in the microstructure and hence optimise the signal that can be obtained for a redox reaction. For instance, with a microchannel comprising an electrically conductive means along its entire length and a counter electrode or a pseudo-reference electrode placed at the inlet or outlet of the -microchannel while the working electrode is integrated within a wall portion of the microchannel, the electrically conductive means (even when it is not connected) enables transport of the current along the microchannel and hence over the distance separating the working electrode from the counter or pseudo-reference electrode placed at the inlet or outlet, which enables a minimised resistance and hence an optimised electrochemical signal that can be obtained with such a device.
It should be stressed here that the device of the invention does not necessarily contain the reference or pseudo-reference electrode. Indeed, the reference or pseudo-reference electrode can be provided by another piece of instrumentation, and hence is not an integral part of the microfluidic device. For example, the reference or pseudo-reference electrode can be a silver/silver chloride wire that is placed in a reservoir at the inlet or outlet of the microstructure or in a means serving for dispensing solution into the microfluidic device (such as e.g. a syringe), in such a manner that this reference or pseudo-reference electrode is in contact with the analyte solution during electrochemical detection. This can advantageously achieved when the microfluidic device of the invention is intended to be disposable and hence thrown away after each assay or after a well-defined series of experiments, while the reference or pseudo-reference is intended to remain even when the microfluidic device is replaced by a new one.
A further aspect of this invention provides a method of fabricating a microfluidic device, including integrating electrically conductive means to be in contact with a solution to be present in the microstructure so as to minimise the ohmic resistance within the microstructure. In one embodiment, the electrically conductive means is formed with at least one through-hole serving as a mask to manufacture the microstructure in the substrate supporting the microstructure and in which under-etching around the mask is performed such that the electrically conductive means can be in contact with the solution to be present in the microfluidic device.
A third aspect of the present invention provides the use of the electrochemical microfluidic device according to claim 49.
The device and method of the invention can advantageously be used in electrochemical sensor applications, and more particularly in chemical and/or biological analysis for instance physicochemical characterisation of compounds or analytical testing e.g. immunological, enzymatic, ion, DNA, peptide, oligonucleotide or cellular assays. The invention can find many applications in medical diagnostics, veterinary testing, environmental or water analysis, quality control, industrial control, pharmaceutical research, detection of warfare agents, monitoring of production processes, etc.
The present invention thus provides a microfluidic device comprising one or a plurality of electrically conductive means allowing minimised ohmic resistance within a microstructure (generally a microchannel or a network of microchannels). The microfluidic device of this invention also comprises one or a plurality of working electrode(s) (preferably micro-electrode(s)) in addition to said electrically conductive means. Both the working electrode(s) and the electrically conductive means may be integrated in wall portions of the microstructure in such a manner that they face each other, so as to minimise the distance between each individual working electrode(s) and the electrically conductive means. Generally, the reference or pseudo-reference electrode is also part of the microfluidic device (preferably placed at the inlet and/or outlet of the microstructure when it is a microchannel), and one or a series of counter-electrode(s) can also be part of the microfluidic device in order to enable electrochemical detection in a three-electrode mode.
There is no restriction in the size and shape of the microfluidic device or of the microstructure, which can be fabricated by any means (for instance, but not limited to, injection moulding, embossing, polymer casting, silicon etching, UV Liga, wet etching or dry etching) and in any electrically insulating material (for instance glass, quartz, ceramic, polymer or combination thereof). In an embodiment, the microfluidic device is composed of an assembly of materials and solid structures: for instance in a microfluidic sensor made of a polymer foil serving as microstructure support in which the various electrodes, the electrically conductive means and the connection pads and tracks can be present (as in a printed circuit board system), as well as a cover layer e.g of a polymer or glass which serves to seal or cover the microstructure in order to enable microfluidic manipulations. An additional part can be made of another polymer material and may for instance comprise access hole(s) to the inlet(s) and/or outlet(s) and additional reservoirs, enabling sample and reagent introduction or withdrawal and/or connection to fluidic control unit(s), which can also provide rigidity to the entire sensor device or which can also enable microfluidic sensor cartridges of relatively large size compared to the microstructure itself, so as to facilitate the handling of the sensor. Such a multi-structure and multi-material device can advantageously be fabricated by a pick-and-place approach where the microstructure support with its cover layer is cut from a panel or board comprising a series of microstructures before being precisely assembled (e.g. by gluing) to an additional part (e.g. an injection-moulded structure) having access holes for fluidic and/or electrical connection and optionally sample or reagent reservoirs (see for instance the example of
The working electrodes are adapted to control, monitor and/or measure one or several electrochemical property(ies) of the fluid present in said microfluidic device. In particular, these electrodes are adapted to perform amperometric, cyclic voltammetric, chrono-amperometric and/or impedance measurements, and the device of the invention can be advantageously used in chemical and/or biological applications such as but not limited to immunological, enzymatic, affinity, ion, peptide, DNA, oligonucleotide or cellular assays, as well as in physico-chemical tests, for instance solubility, lipophilicity or permeability assays or determination of redox properties. Depending on the applications, the microstructure can also advantageously be functionalised with chemical and/or biological compound(s). To this end, functional groups can be created (e.g. by chemical or physical means) on the inner surface of the microstructure. For instance carboxylic, amino, thiol or phenol groups can be integrated by chemical reaction with the material(s) constituting the microstructure surface or with that serving as electrode(s) or electrically conductive means. Chemical and/or biological compounds can also advantageously be reversibly or irreversibly immobilised in at least one portion of the microstructure, for instance but not limited to adsorption, ionic bonding or covalent binding. The chemical and/or biological compound(s) can be immobilised on at least one part of the microstructure walls and/or on the integrated working electrode(s) and electrically conductive means. In one embodiment, the device of the invention can be adapted in order to keep only the integrated working electrode(s) without immobilised compound(s), that is to say that the device can be adapted to allow immobilisation of chemical and/or biological compound(s) on the walls of the microstructure, but without touching the working electrode(s). To this end, the microfluidic device can advantageously be fabricated in such a manner that the working electrode(s) is(are) recessed with respect to the microstructure. Such recess(es) can be made hydrophobic and/or have a shape appropriate to let an hydrophilic solution flowing through the microstructure pass over this(these) recess(es) without touching the working electrode(s), thereby preventing its(their) functionalisation with a chemical or biological material. In such microfluidic devices, multiple-step assays can for instance be run in such a manner that the solutions (e.g. sample, buffer, washing medium, revelation of captured molecules) do not enter into contact with the working electrode(s) as long as a solution capable of wetting the working electrode(s) has not been introduced in the microstructure. When the microstructure has been filled with such a wetting solution, as can for instance be achieved with a surfactant as Tween buffer in polyimide microchannels having recessed gold working electrodes), the hydrophobicity of the recess is reduced, so that the filling of the microstructure with other, even hydrophilic solutions, will still wet the working electrode(s). The introduction of the wetting solution can be carried out at any step of an assay, depending when it is desired that the working electrode(s) are in contact with the solution present in the microstructure. In multi-step assays such as immunological tests, it can indeed be advantageous to run all steps of the assay (capture of the desired analyte, washing, incubation of the secondary antibody and additional washing) without having any contact between these various solutions and the working electrode(s) and to add a wetting solution (which can for instance comprise the enzymatic substrate serving to reveal the captured analytes) just before the detection.
In another embodiment, the device of this invention can also be manufactured in such a way that only the working electrode(s) is(are) functionalised with a chemical or biological material. This can for instance be achieved by deposition directly on the working electrode(s) only. Such a process can be used to functionalise the working electrode(s) with for instance oligonucleotide(s), DNA strain(s) or cell(s).
In some embodiments, a dried reagent can also be used to functionalise the microfluidic device, and functionalisation can also be achieved by use of beads, membrane(s) or filter(s) comprising the desired chemical and/or biological entity(ies).
In one embodiment, the microfluidic device of the present invention is a two-electrode system comprising at least one working electrode (or array of working electrodes) that is integrated in at least one wall portion of the microstructure and one pseudo-reference electrode (i.e. an electrode playing the roles of both the reference and the counter electrodes); in such a 2-electrode configuration, the electrically conductive means is present in addition to the working and pseudo-reference electrodes but is not connected so that it is not part of the 2-electrode system serving for the electrochemical detection; in this case, the pseudo-reference electrode can advantageously be placed outside the micro-structure, close to the inlet and/or outlet and in such a manner as to be in contact with the solution to probe. Due to the nature of the electrically conductive means, the resistance is dramatically decreased along the microstructure(s), thereby enabling optimal electrochemical manipulation and detection; the fact that the electrically conductive means does not need to be connected as a counter-electrode was not expected, but constitutes a supplementary advantage of the present invention.
In another embodiment, the device of the present invention constitutes a three-electrode system comprising at least one working electrode (or array of working electrodes), at least one reference electrode and at least one electrically conductive means. In one embodiment, the electrically conductive means can be adapted to directly serve as counter electrode. In another embodiment, the electrically conductive means is not part of the three-electrode system, it is not connected to the electrodes, and the device further comprises at least one additional electrode serving as counter-electrode.
In an embodiment, the microfluidic device of the invention comprises an electrically conductive means along the entire length of the microstructure, and the electrically conductive means can advantageously surround the microstructure and form a frieze of conducting material in contact with the solution present in the microstructure.
In a further embodiment, the electrically conductive means and the reference or pseudo-reference electrode can also be short-circuited. This can for instance be achieved by providing a microfluidic device where the electrically conductive means is a conducting pad forming a frieze around the microstructure which encompasses the inlet and/or outlet and where the reference or pseudo-reference electrode is simply deposited on this conducting pad on the external side of the inlet and/or outlet but a such a manner that is is in contact with the solution.
In some applications of the present invention, the counter-electrode can be used to re-generate the product of the reaction taking place at the working electrode(s), thereby increasing the measured signal and hence improving the analytical sensitivity of the device. The electrically conductive means can advantageously be used for this purpose; in such a case, the electrically conductive means would play both the roles of counter-electrode, of re-generation of the compound to detect and of minimizing the resistance along the microstructure.
In a further embodiment of the present invention, the microfluidic device may comprise at least one biological and/or chemical entity. Such a biological or chemical moiety can be immobilized either by physisorption, covalent binding, ionic bonding or simply dried on at least one portion of at least one wall of the microstructure(s). In another embodiment, the microfluidic device may also comprise beads and/or a membrane (which can be placed for instance at the inlet and/or outlet of the microstructure) so as to capture one or a plurality of target sample molecules or to wash or desalt a sample. Such beads or membrane may also comprise one or a plurality of biological and/or chemical entity(ies), which can be immobilized on these types of supports.
The microstructure walls and/or the integrated electrode(s) or electrically conductive means can also be partially coated by an organic phase (solidified or not) that can for instance be used as a protective layer for the electrode(s) or as a phase immiscible with the sample solution and can be set-up for measurements of ion transfer reactions at the interface between two immiscible solutions and can for instance be used for the dosage of ions.
The microfluidic device can be surrounded by a supplementary layer which can be used as a solidifier and/or comprise reservoirs (e.g. for stocking of reagents and/or washing solution), as well as access holes for fluidic and/or electrical connection, or guides for interfacing with other instruments.
In the microfluidic device of this invention, there is no limitation in the chemical and/or physical nature of the microstructure substrate or of the cover layer. Each of the substrate and cover layer can for instance be made of a polymer (like but not limited to polyimide, polystyrene, polycarbonate, polyethylene, polyethylene terephthalate, liquid crystal polymer), glass, quartz, a ceramic, etc. In the present invention, the term “substrate” actually refers to any material in which the microstructure can be fabricated. One substrate is a polymer foil, having a thickness smaller than 1 mm. In one embodiment, the cover layer serves to seal the microstructure so as to enable microfluidic manipulations (such as with microchannels), and this cover layer may also comprise a microstructure and/or conductive pads in which one or a plurality of electrode(s) can be fabricated. Similarly, there is no limitation in the materials and/or nature of the microfluidic device of the present invention, nor in the shape and size of the microstructures, provided that a “microstructure” generally has at least one dimension smaller than 1 mm. In a preferred embodiment, the microfluidic device is yet a multilayer body constituted by at least an assembly of a microstructure substrate, electrically conducting tracks to form the electrode(s) and/or the electrically conductive means serving to reduce the ohmic resistance of the device, as well as a cover layer generally serving to seal the microstructure (thereby enabling microfluidic manipulations). In a further preferred embodiment, the cover layer is a polymer layer that is laminated or glued on the microstructure substrate.
The electrically conductive means as well as the electrode(s) can be made of any electrically conductive material such as but not limited to a metal (e.g. gold, silver, platinum or any inert metal) or a metal assembly (e.g. for instance copper coated (e.g. by electroplating) with gold, silver, platinum or the like), an electrically conductive ink (e.g. Ag/AgCl ink) or gel (e.g. ion permeable gels).
In the present method of fabricating a microfluidic system, the ohmic resistance is reduced due to the presence of one or a plurality of electrically conductive means. In one embodiment, the invention provides a particular arrangement in a multilayer body comprising a substrate having one or a plurality of microstructure(s) (preferably microchannel(s)), one or a plurality of electrically conductive means defining at least one wall portion of the microstructure(s) and a cover layer in order to cover and/or seal said microstructure(s). In certain microfluidic devices of the invention, the or the plurality of electrically conductive means comprise(s) one or a plurality of grooves or holes which is(are) part of the microstructure(s) when said microstructures is(are) covered or sealed on the side of the electrically conductive means (see
In another embodiment, the invention provides a method of producing electrochemical microfluidic devices which comprise an electrically conductive means comprising one or a plurality of through-holes along at least one portion of the microstructure(s); by covering or sealing of the microstructure by application of a cover layer, these holes can be sealed on their side opposite to the microstructure(s), so that they become part of the microstructure(s), which has then at least one wall portion made of an assembly of different materials (namely part of the microstructure substrate, part of the material of the electrically conductive means and part of the cover layer, as illustrated below in the cross-section of
After fabrication of said microstructure, undesirable electrically conductive parts (generally used to protect the substrate portions that should not be exposed during the etching step of the microfabrication process) can be removed (e.g. by chemical etching) so as to give the desired design to the electrically conducting means and to the pads and tracks connecting the other electrodes. The electrodes can indeed also be integrated during this process, before adding the cover layer serving to close or seal the microstructure(s). One or a plurality of working electrode(s) for instance can be integrated in wall portions of the microstracture(s), and preferably from the side of the microstructure substrate which is opposite to the electrically conductive means. Such a disposition can indeed be chosen to facilitate the fabrication of the entire microfluidic device, but also to minimise the distance between the integrated working electrode(s) and the electrically conductive means, thereby reducing the ohmic resistance to a minimum, providing small diffusion distances when the electrically conductive means is used as counter-electrodes and serves to regenerate the analyte to detect.
When the microstructure has to be functionalised (e.g. by immobilisation of biological and/or chemical material), it may be advantageous to wash or modify at least one portion of the microstructure walls by physical (e.g. by exposition to a plasma) and/or chemical means (acidic or basic treatment). Similarly, functionalisation of the microstructure(s) may be advantageously carried out before addition of the cover layer.
Specific embodiments of the invention will now be described through reference to the accompanying drawings (but without being restricted to the features shown therein), in which:
In a preferred embodiment described in
In this embodiment of the microfluidic electrochemical sensor of this invention, the pseudo-reference electrode (when working in 2-electrode mode) is placed outside the microstructure, close to the inlet and/or outlet but adapted to be in contact with the solution to probe, so as to simplify the manufacturing process. In this case, the distance between the working electrode(s) integrated within the microstructure and the pseudo-reference electrode is rather large, which, in addition to the small section of the microstructure, tends to result in relatively large resistance and hence in perturbation of the electrochemical response. The integration of the electrically conductive means allows one to minimise the resistance between the working and the pseudo-reference electrode. As can be deduced from the schematic illustration of
In the case where there is no electrically conductive means integrated along the microchannel, the iR drop would be given by the resistance of the current flowing over the 0.5 cm separating the pseudo-reference electrode and the working electrode (in the case where the working electrode is placed in the middle of the microchannel length), so that the ratio L/A would be 0.5 cm * (50 μm)2=2*106 m−1. In this case, the global resistance of the device would be ·106Ω (which is thus about which is thus 5000 times larger than in the same microfluidic device incorporating an electrically conductive means), and a current of 100 nA would thus generate an iR drop of ˜100 mV, which represents an important shift of potential which disturbs electrochemical measurement.
It should be noted here that, when the electrically conductive means is connected as counter-electrode, the resistance path is the same as in the above described case (where the electrically conductive means is present but not connected) so that the ohmic resistance is also minimised as well as the iR drop.
The configuration shown in
One advantageous optional feature of the method of the present invention consists in using a metallic mask to create the microstructure(s) in a substrate before being structured itself in such a way that it may serve as electrically conductive means. An isotropic etching process can be used to microstructure the substrate and one advantage of such a method relies on the fact that the electrically conducting means first serves as mask for the creation of the microstructure(s), which, during the isotropic etching step, results in the removal of substrate material not only on the open part of the mask but also beneath the mask due to under-etching (10), thereby providing a large surface of contact even after covering the microstructure(s) with a cover layer, which ensures a solution present in the microstructure(s) to be in physical contact with the electrically conductive means, thereby enabling to decrease the ohmic resistance along the microstructure.
The present embodiments do not limit the possible techniques used to fabricate the device of the present invention. Technologies such as but not limited to embossing, injection moulding, polymer casting, silicon etching or UV Liga can be used to fabricate the microstructure.
Each of these microfluidic devices comprises a 1 cm long microchannel (2), a gold-coated copper support (5) which contains 24 working electrodes of 50 μm diameter exhibiting a recess of about 15 μm with respect to the microchannel wall, gold-coated copper pads (6) for the reference and/or counter-electrode (an Ag/AgCl dot is used but not shown here), access holes (9) serving as inlet and outlet, and electrical tracks (11) and pads (12) serving for connection to an external electrical meter such as a potentiostat. On the opposite side of the polyimide support (3), each device also comprises an electrically conductive means (7) made of copper coated with gold as well as electrical tracks (11′) and pads (12′) serving for connection that can for instance be used in order to connect the electrically conductive means as counter-electrode. In the present example, the electrically conductive means surrounds the microchannel (2) because it first served as a mask to etch the microstructure. On its side comprising the electrically conductive means, the polyimide support is covered by a polyethylene/polyethylene terephthalate layer of ˜32 μm thickness which cannot be seen in
As shortly mentioned above in relation with
It should also be noted here that the presence of the electrically conductive means on the side of the chip substrate used to close the microstructure can also facilitate the application of the cover layer serving for the sealing. It has indeed been empirically shown with the fabrication of the microfluidic devices shown in
The object of the present invention has been demonstrated by the fabrication of a polymer microfluidic device (1) where the conductive means (7) is a gold coated copper layer surrounding a microstructure (2), which, in this particular case, is a microchannel of 1 cm in length and about 100 micrometer in diameter produced by isotropic etching of a polyimide foil coated on both sides with copper. The working electrode (4) and pseudo-reference electrode (6) are gold-coated copper electrodes that are connected to a potentiostat due to their respective pads (12). The working electrode array (4) is composed of 24 microelectrodes of 50 μm in diameter. The present microstructures are sealed by lamination of a 32 μm thick layer made of polyethylene and polyethylene terephthalate. Detection of 0.5 mM ferrocene carboxylic acid without the conductive means (7) is presented in
In one embodiment of this invention, the conductive means (7) can be connected as the counter electrode or not, depending on the counter reaction that takes place. For instance, when the counter reaction may pollute the working electrode (4), it may be better to make this reaction occur outside the microstructure (2) at the pseudo-reference electrode (6) placed in contact with the solution at the microstructure inlet and/or outlet, with a potentiostat working in 2-electrode mode. The system is still working satisfactorily, and the electrically conductive means (7) still avoids large iR drops, even though it is not connected. In the presence of a reversible or pseudo-reversible reaction the connection of the electrically conductive means (7) as a counter electrode enables a regeneration of at least some of the molecules detected at the working electode (4) as shown in
In some cases, for instance when an enzymatic reaction takes place in a microstructure, the concentration of the analyte to detect can increase with time, so that it can reach a concentration that would generate a current intensity which is difficult to pass through the chip without iR drop. As a consequence, the potential-current response shifts and the current at a given potential reaches a plateau due to ohmic (iR) drop. This case is shown in
In a further embodiment, this enzymatic reaction can be detected with a microfluidic device (1) containing a conductive means (7) with and without being connected as counter electrode. In this case, the product of the enzyme hydrolysis (here p-aminophenol) can be oxidised into quinone imide. This oxidation reaction is reversible, so that quinone imide can be reduced back into p-aminophenol at a counter-electrode or by inverting the potential applied at the working electrode(s) so as to regenerate the analyte molecules to detect. In the case where the conductive means is connected as the counter electrode, the reduction of quinone imide is done at the counter electrode and the concentration of p-aminophenol is larger, thereby giving a larger current response during the reaction. This case is shown in
As another example of applications of the device of the present invention,
In order to determine TSH concentration in plasma, the microfluidic devices is first coated with anti-TSH and blocked against non-specific adsorption using a calf serum solution. After incubation of TSH samples having a known concentration of 56.1 μUI/mL, the microchannels are filled with a solution of anti-TSH conjugate labelled with alkaline phosphatase (ALP). Detection is then performed by amperometric measurements at different times upon application of 200 mV vs Ag/AgCl and with p-aminophenyl phosphate (PAPP) as enzymatic substrate. The assay is repeated twice with both types of microchips, and the time evolution of the measured current is shown in
It should be stressed here that the size of the electrodes is not exactly the same in the devices used to generate the data shown in
In a further embodiment, the microfluidic device of the present invention comprises a top layer (10), preferably composed of a polymer, in order to solidify and stabilise the entire assembly. This top layer (30) is illustrated in
The microfluidic device of the present invention can advantageously be used in analytical applications, such as but not limited to electrochemical sensors, sampling by ion spray ionisation or as a detector in capillary electrophoresis.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/12112 | 11/11/2005 | WO | 5/7/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60627055 | Nov 2004 | US |
