BIOSENSOR SYSTEM FOR MULTIPLEXED DETECTION OF BIOMARKERS

Abstract

The invention refers to a biosensor system for quick and multiplexed detection of biomarkers present in biological fluids. The biosensor system comprises a reusable array of at least two individual electrochemical cells (1a,1b,1c,1d,1e) coupled to a disposable fluidic component. Each cell can be addressed individually. The array includes a set of working electrodes (2a,2b,2c,2d) and at least one shared counter/reference electrode (5) in common for all the electrochemical cells, such that each electrochemical cell includes one working electrode and the shared counter/reference electrode. Preferably, the system includes a disposable paper component (8) having a reactive microfluidic component distributed in fluidic channels (9), isolated by hydrophobic barriers (10). The paper component (8) is operatively aligned with the array of electrochemical cells for the electrochemical detection by means of a polymeric cartridge. The multiplexed biosensor system features a reduced size, and that allows reduction of analysis costs and material waste.

Claims

1. A biosensor system for multiplexed detection of biomarkers, the system comprising: an array of at least two electrochemical cells (1a,1b,1c,1d,1e), adapted to be individually addressable, a working electrode (2a,3b,2c,2d,2e) in each electrochemical cell (1a, 1b, 1c, 1d, 1e) and one counter/reference electrode (5) shared by all the electrochemical cells (1a,1b,1c,1d,1e) of the array, a fluidic component (8) made of a porous material for driving fluids by capillary action towards the electrochemical cells (1a,1b,1c,1d,1e), and a cartridge (19) configured to align and put in contact the array and the fluidic component (8) for the electrochemical detection of biological samples.

2. System according to claim 1, wherein the fluidic component (8) is made of paper having microfluidic channels (9) including biocomponents, wherein the microfluidic channels (9) are isolated from each other, and wherein the fluidic component (8) is operatively couplable with the array of electrochemical cells (1a,1b,1c,1d,1e) such that each microfluidic channel (9) is placed over an electrochemical cell to carry out the electrochemical detection.

3. System according to claim 1 or 2, wherein the working electrodes (2a,3b,2c,2d,2e) are aligned in a longitudinal straight direction, and the shared counter/reference electrode (5) is a straight track, and wherein the working electrodes are adjacent to one side of the shared counter/reference electrode (5).

4. System according to claim 1, further comprising a shared counter electrode (6) and a shared reference electrode (7), wherein the shared counter and reference electrodes (6,7) are common electrodes for all the electrochemical cells.

5. System according to claim 4, wherein the shared counter electrode (6) and the shared reference electrode (7) are straight tracks parallel to each other, and parallel to the longitudinal alignment direction of the working electrodes (2a,3b,2c,2d,2e), and wherein the working electrodes (2a,3b,2c,2d,2e) are aligned and arranged in between the shared counter electrode and a shared reference electrode.

6. System according to any of the preceding claims, further comprising a set of connection pads (3), and a set of connection tracks (4) connecting the working electrodes (2a,3b,2c,2d,2e), the shared counter/reference electrode (5), or shared counter electrode and a shared reference electrode (6,7), with the connection pads (3), wherein a part of each connection tracks (4) is parallel to the shared counter and shared reference electrodes.

7. System according to claim 1 or 4, further comprising a chip substrate (30) enclosed inside the cartridge (19), and wherein the counter (6), reference (7), or shared counter/reference (5), and working electrodes (2a,3b,2c,2d,2e) are formed on one side of the chip substrate (30).

8. System according to claims 6 and 7, wherein the chip substrate (30) is rectangular and the connection pads (3) are grouped in a reticular matrix arrangement and formed adjacent to one short side of the substrate, and wherein the system further comprises spring-loaded contacts (16) connected with the connection pads (3).

9. System according to any of the claims 2 to 8, wherein the microfluidic channels (9) have the form of straight strips, such that when the fluidic component (8) is coupled with the electrochemical cells (1a,1b,1c,1d,1e), the microfluid channels (9) are transversally arranged with respect to the shared counter and reference electrodes (6,7).

10. System according to any of the preceding claims, wherein the cartridge (19) has top and bottom parts (20,21) and clamping structures (22,23,24,25) adapted to attach and press the top and bottom parts (20,21) of the cartridge (19) to keep the array and the fluidic component in close and stable contact.

11. System according to any of the claims 2 to 10, further comprising an absorbent pad (11) in contact and partially overlapping the microfluidic channels (9) of the fluidic component (8).

12. System according to claim 11, wherein the top part (20) of the cartridge (19) has an evaporation window (20a) that is placed over the absorbent pad (11), and serves as sink area evaporation window, and a sample access window (26) that is placed right over a first window (17) of the fluidic component (8), and a connector window (27) for receiving the spring-loaded contacts (16).

13. System according to any of the claims 2 to 12, wherein the microfluidic channels (9) are provided with antibodies or DNA strands to carry out affinity assays.

14. System according to any of the claims 2 to 13, wherein functionalized magnetic particles are applied to the microfluidic component (8) for the biological reaction.

15. System according to claim 14, further comprising magnets arranged for trapping magnetic nanoparticles in the microfluidic channels (9).

16. System according to any of the preceding claims, wherein the fluidic component (8) is made of cellulose or nitrocellulose.

17. System according to claim 2, wherein the microfluidic channels (9) are isolated from each other by means of hydrophobic barriers (10).

18. System according to any of the preceding claims, comprising instrumentation to carry out the electrochemical detection by chronoamperometry of the multiplexed biosensor.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Preferred embodiments of the invention are henceforth described with reference to the accompanying drawings, wherein:

[0029] FIG. 1.—shows a top plan view of a first implementation of the array of electrochemical cells according to the invention, that includes a layout of a two-electrode configuration.

[0030] FIG. 2.—shows a top plan view of a second implementation of the array of electrochemical cells, that includes a layout of three-electrode configuration.

[0031] FIG. 3.—shows an exploded view of a polymeric cartridge defining liquids wells.,

[0032] FIG. 4.—shows in a top plan view, a fluidic component (Figure A), and the arrangement of the fluidic component coupled with the array of electrochemical cells (Figure B) and an absorbent pad overlapping the channels of the fluidic component.

[0033] FIG. 5.—shows in plan view the layout of the component once coated with vinyl layers for better alignment with the array of electrochemical cells. Figure A is a plan view of the upper side, Figure B is a plan view of the bottom side, and Figure C is a top plan view of the component coupled with the array of electrochemical cells.

[0034] FIG. 6.—shows in Figure A a top plan view of the layout of a device packaging to easily couple and align the fluidic component with the array of electrochemical cells, and in Figure B an exploded view of the packaging components including the clamping structures used por applying a constant pressure between the top and bottom parts of the cartridge.

[0035] FIG. 7.—shows a graph of a cyclic voltammogram recorded with one electrochemical cell in a 0.1 M TRIS.Math.HCl buffer solution pH 9.8 containing 1 mM BQ electroactive species. Scan rate=50 mV/s.

[0036] FIG. 8.—shows in FIGS. 8A-8C graphs of the calibration curves for the three biomarkers with ferrocene-methanol as redox mediator.

[0037] FIG. 9.—shows the results of the measurement of the three biomarkers in human sputum samples.

PREFERRED EMBODIMENT OF THE INVENTION

[0038] FIG. 1 shows a first preferred implementation of the layout of the electrodes, connection pads (3) and connection tracks formed on a surface of a substrate (30) to produce an array of electrochemical cells (1a,1b,1c,1d,1e) in a two-electrode configuration. A plurality of working electrodes (2a,2b,2c,2d,2e) are aligned along a straight longitudinal direction, and one counter/reference electrode (5) is provided common for all the working electrodes (2a,2b,2c,2d,2e). The counter/reference electrode (5) is a straight track and the working electrodes are arranged adjacent to one side of the shared counter/reference electrode (5), in a way that the longitudinal direction of alignment of the working electrodes, is parallel to the counter/reference electrode (5).

[0039] The substrate (30) is rectangular and includes a set of connection pads (3) that are placed adjacent to one short side of the substrate (30) as shown in FIG. 1. The connection pads (3) are grouped in a reticular matrix arrangement for their connection with a group of spring-loaded connectors, as it will be explained later on.

[0040] A plurality of the connection tracks (4) connect individually each working electrode (2a,2b,2c,2d,2e) and the counter/reference electrode (5), each with one connection pad (3). As represented in FIG. 1, the connection tracks run longitudinally in the substrate (30), in a way that a part of each connection track is parallel to the shared counter/reference electrode (5).

[0041] As it can be noted in FIG. 1, with this layout, where the counter/reference electrode is common to all the working electrodes, the number of connection tracks and connection pads, is significantly reduced so that the substrate (30) features a compact design.

[0042] Each electrochemical cell (1a,1b,1c,1d,1e) comprises one working electrode (2a,2b,2c,2d,2e) and the shared counter/reference electrode (5), thus, in the embodiment of FIG. 1 there are five electrochemical cells that could be integrated in a single device.

[0043] FIG. 2 shows an alternative electrode layout on the substrate (30) for a three-electrode cell configuration. In this case, there are four working electrodes (2a,2b,2c,2d), a reference electrode (6), a counter electrode (7), a group of connection pads (3), and a plurality of connection tracks (4) connecting individually the working electrodes with the connection pads (3). The reference and the counter electrodes (6,7), are common for all the working electrodes, so they are shared by all the electrochemical cells (1a,1b,1c,1d,1e).

[0044] The working electrodes (2a,2b,2c,2d) are also aligned, and the reference and counter electrodes (6,7) are straight tracks parallel to each other, and parallel to the longitudinal alignment direction of the working electrodes. The working electrodes are arranged in between the reference and the counter electrodes (6,7). Several connection tracks (4) connect individually all the electrodes (2,6,7) with the connection pads, and have a part parallel to the reference and the counter electrodes (6,7).

[0045] In this case, each electrochemical cell (1a,1b,1c,1d,1e) includes a working electrode (2a,2b,2c,2d) and a defined area of the reference and counter electrodes (6,7).

[0046] The embodiment of FIG. 2 includes four electrochemical cells (1b,1c,1d,1e) in a three-electrode configuration for the simultaneous measurement of biomarkers, and an additional cell (1a) including only two electrodes (the counter and the reference electrodes), which is used as reference channel.

[0047] In both implementations described above, all the electrodes of the electrochemical cells can be manufactured by standard photolithographic techniques, by patterning gold or platinum electrodes on silicon substrates.

[0048] As it can be observed in FIGS. 1 and 2, the fact that all electrochemical cells share counter/reference electrode (two-electrode cells) or a common counter and reference electrodes (three-electrode cells), has the advantage that only six contact pads are required, which greatly reduce the number of contact pads for connecting externally all the cells.

[0049] As represented in FIG. 3, the array of electrochemical cells (1a,1b,1c,1d,1e) can be individually characterized in a cartridge (12) having top and bottom parts (12a,12b) couplable to each other, for example by means of screws (13) or other type of clamping means, so that the array is enclosed between the top and bottom parts (12a,12b). The top part (12a) is a substrate having several windows that configure isolated liquid wells (14), that are distributed such that each well (14) is placed right on top of an electrochemical cell.

[0050] In a practical implementation of the invention, the capacity of each well (14) is around 75-μL.

[0051] In the preferred embodiment represented in the FIG. 3, the wells (14) are aligned in a straight longitudinal direction, that is parallel to the shared reference and counter electrodes, and parallel to the longitudinal alignment direction of the working electrodes.

[0052] The top part (12a) has an additional window (15) for the insertion of a set of spring-loaded connectors (16), located right on the connection pads (3), in a way that when the cartridge is assembled, each connector (16) would be connected to a connection pad (3). The connectors (16) serve to connect the electrochemical cells with an external measurement device for the electrochemical cell characterization.

[0053] As shown in FIG. 4A, the biosensor system includes a disposable paper component (8) in the form of a substrate being a reactive fluidic component comprising fluidic channels (9) arranged in a way that the channels (9) are isolated by hydrophobic barriers (10), that can be realized as printed hydrophobic walls.

[0054] In this preferred embodiment, the fluidic channels (9) have the form of straight strips. However, in other preferred embodiment, the fluidic channels (9) might have other configurations suitable for each application.

[0055] The two implementations of FIGS. 1 and 2 are designed such that they could be easily coupled to the paper microfluidic component (8). In addition, the design in both implementations features a reduced size as the overall dimensions of the device are 25×8 mm.sup.2.

[0056] As represented in FIG. 4B, the paper component (8) is operatively couplable with the array of electrochemical cells (1a,1b,1c,1d,1e) formed on the substrate (30) for the electrochemical detection, such that, each channel (9) is aligned over an electrochemical cell (1a,1b,1c,1d,1e), that is, on a working electrode (2a,2b,2c,2d,2e) and on a part of the counter/reference electrode (5).

[0057] When the disposable paper component (8) is coupled with the electrochemical cell array, the fluidic channels (9) are transversally arranged, preferably orthogonally, with respect to the counter/reference electrode (5), and with respect to the alignment of the working electrodes (2a,2b,2c,2d,2e).

[0058] The system includes an absorbent pad (11) overlapped and in contact with the channels (9) of the paper component, for increasing capillary force in the disposable paper component (8) while driving the fluid towards the electrochemical cells (1a,1b,1c,1d,1e).

[0059] Having a very lost-cost easy-to-manufacture disposable paper component (8), reduces cost per analysis required for Point-Of-Care devices.

[0060] FIG. 5 shows the layout of the paper component (8) sandwiched between vinyl layers (31,31″) for its easy alignment with the array of electrochemical cells. The vinyl layers (31,31″) are patterned using a blade plotter, so that several areas of the paper component (8) are exposed.

[0061] As shown in FIG. 5A a first window (17) is opened at a top surface for receiving samples, that leaves open the initial part of the paper channels for the sample addition. A second window (28) is opened at the sink area to allow liquid evaporation once it has reached the absorbent pad (11). A third window (18) is opened at the back side of the paper component (8) to provide access to the sensing areas of the same (FIG. 5B), so that the paper component and the electrochemical cells can get in contact.

[0062] On both vinyl layers (31,31″), additional windows (29,29″) are opened for the connector of the electrode array to be placed over the electrode contact pads. FIG. 5C shows the relative positions of all the above-described windows.

[0063] In practice, the coupling of the array of electrochemical cells and the disposable paper fluidic component (8) is done by means of a packaging cartridge (19) including top and bottom parts (20.21) and pressed together with clamping means, as shown in FIG. 6.

[0064] FIG. 6 illustrates the packaging cartridge (19) that integrates the packaged paper fluidic component (8) and the array of electrochemical cells (1a,1b,1c,1d,1e). The packaging cartridge (19) comprises top and bottom parts (20,21), and clamping means configured in this example as first and second large lateral walls (22,23), and first and second short lateral walls (24,25), such that, the lateral walls retain top and bottom parts (20,21) pressed against each other and keep both, the paper component and electrochemical cell array, robustly aligned during measurements.

[0065] The top part (20) has several windows to provide access to the packaging cartridge (19) interior, in particular it has: an evaporation window (20a) that is placed over the absorbent pad (11), and serves as sink area evaporation window, a sample access window (26) that is placed right over the first window (17) of the paper component (8), and a connector window (27) for receiving the spring-loaded connectors (16).

[0066] The bottom part (21) can integrate five 2-mm diameter Nd magnets (not shown) located right before the detection area of the electrode array, and aligned with the fluidic channels (9) so that, when working with magnetic nanoparticles, these flow on the paper and can be trapped and accumulated before carrying out the measurements.

[0067] As shown specially in FIG. 6B, top and bottom parts (20,21) are flat and generally rectangular and have cut-out corners, that conform protruding four edges in both covers.

[0068] Clamping means are provided for package assembly, wherein the clamping means are adapted to attach and press together top and bottom parts (20,21). In this exemplary embodiment, the clamping means are embodied as first and second large lateral walls (22,23) having respective windows (22a,23a), and first and second short lateral walls (24,25) have respective windows (24a,25a). The large lateral edges of the top and bottom covers (20,21) can be inserted respectively in the windows (22a,23a) of the large lateral walls, and the short edges of the top and bottom covers (20,21) can be inserted in the windows (24a,25a) of the two lateral short walls (24,25), conforming thereby a packaging cartridge (19) as the one shown in FIG. 6A.

[0069] Since the lateral walls are used as clamping means, the packaging cartridge (19) can be easily assembled and disassembled to get access to both the electrochemical cell array and the paper component so that the paper component can be easily removed after one measurement and replace it for a new one and the reusable array of electrochemical cells can be cleaned if necessary and replaced once its life cycle finishes. The packaging cartridge (19) can be manufactured at a low cost, because it does not require mechanical fastening means for its assembly. The package can be made for example by laser cutting of polymethylmethacrylate (PMMA) plates. Other polymeric materials can be used and patterned by injection molding or 3D printing.

[0070] In addition, the encapsulation of the paper component (8) with the vinyl layers (31,31′) achieves a reproducible alignment between the electrochemical cells and the paper channel that does not rely on double-sided adhesives to bond the paper fluid component to the electrochemical cells as in prior art solutions. The packaging cartridge (19) applies an even pressure on the paper component (8), and ensures a proper contact between the paper channels and all the electrochemical cells in the array.

[0071] For the analytical assessment of the proposed electrochemical cell array, several experiments have been carried out with the three-electrode cell array in solutions containing ferrocyanide/ferricyanide redox pair by cyclic voltammetry. It was seen that the electrochemical response of each cell in the array was very similar to the one of an individual cell. Because of the cell configuration and in order to carry out the quasi-simultaneous measurements in all cells of the array, the two species of the redox pair should be present in solution. Working in this way, calibration curves for ferricyanide could be plotted for all cells in the array. The potential difference between the counter and reference electrode was kept very low (below 40 mV) during the timescale of the experiments. Thus, the use of a single counter/reference electrode was feasible and the two-electrode cell array was then characterised.

[0072] Similar experiments to the ones carried out with the previous array, have been performed and all the two-electrode cells in the array could be calibrated. As an example, the array was characterized by cyclic voltammetry (CV) in solutions containing ferrocyanide/ferricyanide or hydroquinone/benzoquinone (HQ/BQ) reversible redox pairs, as represented in FIG. 7. One of the species of the redox pair was measured with a linear response in individual cells of the array in the presence of an excess concentration of the redox counterpart.

[0073] Using the ferrocyanide/ferricyanide redox pair and as a first approach for measuring the activity of an enzyme label, some tests with Horseradish Peroxidase (HRP) have been carried out using ferrocyanide as the enzyme mediator. This enzyme converted ferrocyanide into ferricyanide product, which was measured by chronoamperometry using the two-electrode cell array. A similar experiment was performed with myeloperoxidase (MPO) but using ferrocene-methanol as a mediator as well as with alkaline phosphatase (ALP) using hydroquinone diphosphate (HQDP) as substrate.

[0074] In a practical application of the invention, a silicon chip of 8×25 mm.sup.2 is manufactured containing five electrochemical cells, with five working electrodes and a shared counter/pseudoreference electrode for all the cells. Since the array of electrochemical cells is reusable, the electrodes are made of gold.

[0075] The paper component of the Whatman Grade 1 Chromatographic type has 5 different channels. Together with the PMMA packaging, the determination of the concentration cytokine interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α), together with the enzyme myeloperoxidase (MPO) in sputum samples has been addressed by a sandwich type magneto immunoassay. All three analytes are biomarkers of Chronic Obstructive Pulmonary Disease (COPD) and other inflammatory diseases of the respiratory system.

[0076] The electrochemical detection scheme of the device for the three biomarkers is as follows: [0077] the two-electrode cell array was implemented and the electrochemical transduction mode is chronoamperometry. [0078] MPO activity could be detected directly by using ferrocene-methanol redox substrate. [0079] HRP label together with ferrocene-methanol substrate was the alternative chosen for carrying out the electrochemical detection of TNF-α and IL-β.

[0080] Magnetic nanoparticles functionalized with primary antibodies are incubated with the sample, and after recognition, a second incubation is performed with a secondary antibody labeled with horseradish peroxidase (HRP) enzyme. The conjugated nanoparticles can then be concentrated, conditioned in a suitable solution and run through the paper component. A magnet embedded in the bottom part of the PMMA cartridge is capable of trapping them, and finally carrying out the electrochemical detection by adding a solution containing with the enzyme substrates to the paper component.

[0081] It was shown that both species could be easily detected by chronoamperometry and that just 5 μL of solutions were necessary for carrying out the measurements on each channel.

[0082] Then, measurements for the three biomarkers (TNF-α, IL-8 and MPO) were carried out in standard buffered solutions. These involved the following steps: Incubation of the antibody-coated magnetic nanoparticles in solutions containing different biomarker concentrations; washing; incubation of the magnetic nanoparticles in solutions containing the corresponding detection antibodies labelled with HRP for TNF-α and IL-8 assay; washing; casting of 5 μL of the MNP suspension on the paper channels; 2 rinsing steps, each of them with 5 μL rinsing solution; casting of 5 μL of enzyme substrate solutions and incubation for 3 min; final chronoamperometric measurement. The overall assay time is under 90 min. The calibration curves obtained for the three biomarkers in buffered solutions are represented in FIG. 8.

[0083] The same procedure was applied for the detection of MPO, TNF-α and IL-8 in human sputum samples. Five healthy individuals and five patients with COPD was used for the assay. The results shown a significant difference between the two groups of samples, as can be seen in FIG. 9.

[0084] This same procedure can be performed with different types of analytes, for example proteins, enzymes, viruses or bacteria, as well as DNA or RNA sequences, provided that we have the antibodies to perform the affinity assays or the necessary DNA strands.

[0085] Alternatively, the two incubations could be done directly in the device, running the functionalized magnetic nanoparticles on the paper while reacting with labelled antibodies or DNA strands previously deposited on the paper fluidic channels.

[0086] The biosensor system of the invention, can be marketed as a point-of-care or point-of-need device.

[0087] Other preferred embodiments of the present invention are described in the appended dependent claims and the multiple combinations of those claims.