ELECTROCHEMICAL SENSOR

Abstract

The application provides a method of detecting an analyte in a sample. The method comprises disposing a binding agent in in an electrochemical compartment. The binding agent is configured to bind to an interfering species. The method further comprises disposing a solution comprising a sample in the electrochemical compartment. The sample comprises an analyte and the interfering species. The method then comprises applying a voltage across first and second spaced apart electrodes disposed in the solution, and thereby causing a current to flow through the solution between the electrodes. Finally, the method comprises measuring the current and/or voltage and thereby detecting the analyte.

Claims

1. A method of detecting an analyte in a sample, the method comprising: disposing a binding agent in an electrochemical compartment, wherein the binding agent is configured to bind to an interfering species; disposing a solution comprising a sample in the electrochemical compartment, wherein the sample comprises an analyte and the interfering species; applying a voltage across first and second spaced apart electrodes disposed in the solution, and thereby causing a current to flow through the solution between the electrodes; and measuring the current and/or voltage and thereby detecting the analyte.

2. The method according to claim 1, wherein the method comprises measuring the current and thereby detecting the concentration of the analyte in the solution.

3. The method according to claim 1, wherein the binding agent comprises a molecularly imprinted polymer (MIP), an antibody or a fragment thereof, an aptamer, an affimer, a lectin, a peptide, a protein, a macrocyclic ligand or an organic molecule.

4. The method according to claim 3, wherein the binding agent comprises a MIP, and wherein the MIP comprises a film, a microparticle, a nanoparticle, and/or a linear MIP.

5. The method according to claim 1, wherein the interfering species and the analyte are redox active molecules, and wherein the interfering species has a similar redox profile to the analyte.

6. The method according to claim 1, wherein the interfering species is an acid, an organometallic complex, a metal ion complex, an organic redox active molecule, an electroconductive polymer, a metal ion, a nanoparticle containing a redox active centre and/or a gas.

7. The method according to claim 6, wherein the interfering species is ascorbic acid, ferrocene or a derivative thereof, flavin or a derivative thereof, dopamine, humic acid, hydrogen peroxide and/or oxygen.

8. The method according to claim 1, wherein the interfering species and the analyte are not redox active molecules, and wherein the interfering species has a similar structure and chemical properties to the analyte.

9. The method according to claim 8, wherein the method comprises disposing a mediating agent in the electrochemical compartment, and wherein the mediating agent is configured to react with the analyte and thereby produces a redox active molecule.

10. The method according to claim 9, wherein the mediating agent comprises an enzyme and a molecule configured to donate or receive electrons.

11. The method according to claim 1, wherein the binding agent has a dissociation constant (K.sub.D) of less than 10 mM for the interfering species.

12. The method according to claim 1, wherein the binding agent is disposed in the solution.

13. The according to claim 12, wherein the concentration of the binding agent in the solution is the same as or higher than the concentration of the interfering species in the solution.

14. The method according to claim 1, wherein the sample is a biological sample, a food or drink sample or an environmental sample.

15. A kit for detecting an analyte in a sample, the kit comprising: an electrochemical cell comprising an electrochemical compartment configured to receive a solution comprising a sample to be tested, wherein the sample comprises an analyte and an interfering species, and first and second spaced apart electrodes configured to be disposed in the solution; and a binding agent configured to bind to the interfering species, wherein the electrochemical cell is configured to apply a voltage across the first and second electrodes thereby causing a current to flow through the first and second electrodes and the solution disposed in the electrochemical compartment and wherein the electrochemical cell is further configured to measure the current and/or voltage, and thereby detect the analyte.

16. The kit according to claim 15, wherein the kit further comprises a power supply configured to apply a voltage across the first and second electrodes.

17. The kit according to claim 15, wherein the kit further comprises an ammeter configured to measure a current flowing through the first and second electrodes and the solution disposed in the electrochemical compartment.

18. The kit according to claim 17, wherein the kit further comprises a signal processor that is configured to measure, filter or compress the signals produced by the ammeter.

19. The kit according to claims 15, wherein the kit further comprises a potentiostat.

Description

[0054] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

[0055] FIG. 1 is a graph showing recorded chrono-amperograms. Current values are recorded continuously and plotted against time. The ascorbic acid (AA) concentration is highlighted after every addition. i values refer to the difference i.sub.xi.sub.blank where x is the concentration of AA in the sample solution after each addition and i.sub.blank refers to the current value recorded when no AA is present;

[0056] FIG. 2 is a graph showing how the recorded current values change as the concentration of ascorbic acid (AA) varies in sample solutions. i values refer to the difference i.sub.xi.sub.blank where x is the concentration of AA in the sample solution after each addition and i.sub.blank refers to the current value recorded when no AA is present;

[0057] FIG. 3 is a graph showing the % suppression of AA by the nanoMlPs;

[0058] FIG. 4 is a graph showing how the recorded current values change as the concentration of dopamine varies in sample solutions. i values refer to the difference i.sub.xi.sub.blank where x is the concentration of dopamine in the sample solution after each addition and i.sub.blank refers to the current value recorded when no dopamine is present;

[0059] FIG. 5 is a graph showing how the recorded current values change as the concentration of dopamine varies in sample solutions. i values refer to the difference i.sub.xi.sub.blank where x is the concentration of dopamine in the sample solution after each addition and i.sub.blank refers to the current value recorded when no dopamine is present.

EXAMPLES

Methods

[0060] The protocol for the preparation of ascorbic acid modified solid-phase was adapted from Canfarotta et al. Briefly, glass beads were activated by boiling them in NaOH (4 M) for 10 min and washed with DI water and acetone. After drying in the oven at 80 C. overnight, the beads were incubated in a 2% v/v solution of GOPTS (3-glycidyloxypropyl trimethoxysilane) in anhydrous toluene, containing also N-ethyldiisopropylamine 2 mg/mL, at 55 C. for 5 h. Afterwards, the glass beads were poured into a Buchner funnel, rinsed with toluene twice, then with acetone (800 mL) and eventually dried under vacuum. These epoxy-functionalised beads were then incubated in a solution of ascorbic acid 2 mg/mL in PBS 0.01 M pH 7.2 overnight. Finally, the glass beads were filtered and rinsed with water (800 mL water for 150 g beads), dried under vacuum and stored under inert atmosphere (N.sub.2) at 4 C. until use.

[0061] To synthesise nanoMlPs imprinted against ascorbic acid, a polymerisation in PBS was performed. In particular, N-isopropylacrylamide (Sigma-Aldrich) (39 mg), N-tert-butylacrylamide (Sigma-Aldrich) (33 mg), N-(3-aminopropyl)methacrylamide hydrochloride (Polysciences, Inc.) (5.8 mg), acrylic acid (Sigma-Aldrich) (2.2 L), and N,N-methylenebisacrylamide (Sigma-Aldrich) (2 mg) were dissolved in 100 ml PBS. Prior to initiating the polymerisation process, the mixture was purged with N.sub.2 and sonicated for 30 min. At the same time, functionalised beads were purged with N.sub.2 for 20 min. The beads were then added to the polymerisation solution and purged with N.sub.2 for 5 min. The mixture of beads and polymerisation solution was swirled gently and then the polymerisation was started by adding ammonium persulfate (APS) (60 mg/ml) and N,N,N,N-tetramethylethylenediamine (TEMED) (22 L) as catalysts. The polymerisation was performed overnight at room temperature, after flushing the headspace of the bottle used for the reaction with N.sub.2 for 30 seconds. The following day, the beads were washed with deionised water, following the steps described by Canfarotta et al., by using a 60 mL solid-phase extraction (SPE) cartridge fitted with a frit of 20 m porosity. Afterwards, the high-affinity nanoMlPs were eluted at 65 C. with deionised water, until a total volume of 100 mL of solution was obtained.

[0062] Typically, the nanoMlPs produced using the above method have a dissociation constant in the nanomolar range e.g. between 0.1 and 1000 nM.

Multi-Pulsed Amperometry (MPA)

[0063] Cyclic Voltammetry (CV) was performed to obtain the value of oxidation potentials of the chosen electroactive molecules, setting the following parameters: scan range from 0.6 V to +0.5 V, step potential of 0.025 V, scan rate 0.1 V/s. Measurements were performed using the screen printed cells as a drop on cell, placing 50 L of solution onto the electrodes surfaces and performing the scans.

[0064] Multi-pulsed Amperometry (MPA) was performed adapting the procedure described by Taktsy et al. by setting the following parameters: [0065] number of pulses 2, [0066] E.sub.pulse1 0.250 V, [0067] E.sub.pulse2 0 V, [0068] t.sub.pulse1 0.2 sec, and [0069] t.sub.pulse2 1.5 sec.

[0070] Current was recorded during the application of E.sub.pulse1. Measurements were performed immersing the screen printed cell into a 5 mL sample solution under stirring.

[0071] All reported potentials refer to the pseudo-reference silver electrode and the measurements were performed at room temperature.

[0072] Amperometric measurements based on the oxidation of electroactive targets at the electrode surface were carried out continuously. Volumes of ascorbic acid and dopamine solutions were directly added from buffered 100 82 M stock solutions to the 5 mL volume sample solution kept under stirring. Total volume variation was neglected.

[0073] Tested concentrations were within 50-550 nM and 12-35 M for ascorbic acid (AA) and 100-500 nM for dopamine (DA). Current increases after each addition were taken as analytical signal, reporting (i-i.sub.o) versus analyte concentration. All measurements were carried out in PBS pH=7.2 buffered solutions or PBS pH=7.2 diluted matrices.

[0074] Electrochemical measurements carried out for ascorbic acid and dopamine were repeated in PBS pH=7 .2 buffered solutions containing the synthesised nanoMlPs, in order to investigate their ability to selectively suppress ascorbic acid oxidation signal. Current increases after each addition were considered as analytical signal, reporting (i.sub.MIP-i.sub.oMIP) versus analyte concentration and comparing them to those previously obtained in PBS pH=7.2 buffered solutions without nanoMlPs. Per cent decrease of current increases after every analyte addition was also used to evaluate signal suppression.

[0075] For all of the experiments described, the concentration of the nanoMlPs in solution was 0.1 mg/ml. However, it will be appreciated that other concentrations could be used.

Example 1

Quantitative Electrochemical Suppression of Ascorbic Acid Oxidation Current

[0076] For proof of concept purposes, the inventors explored the possibility of applying the proposed method for suppressing interfering currents in sensing devices. To this end, analysis of ascorbic acid was performed in the nanomolar range, from 50 nM to 550 nM, see FIGS. 1 and 2. Current response of tested AA concentrations in PBS buffered solution was compared to measurements carried out in nanoMlPs solution, obtaining a quantitative current suppression for AA concentrations close to the lower limit of 50 nM.

[0077] The amount the current reading was suppressed by using the nanoMlPs was calculated as a percentage. Values of 90% were achieved for AA concentrations lower than 100 nM and values of 90% were achieved for all other AA concentrations, see FIG. 3.

[0078] The inventors believe that higher concentrations of MIPs could further suppress the signal caused by AA.

Example 2

Dopamine Measurements

[0079] Measurements of dopamine (DA) were carried out in order to assess whether DA could be detected even in the presence of an interfering concentration of ascorbic acid, avoiding any sample pretreatment. Current response to subsequent DA additions in PBS solution was analyzed in the range 100-500 nM and then repeated in the presence of 50 nM and 100 nM AA. The experiment was then repeated in a nanoMlP solution.

[0080] The results, shown in FIG. 4 clearly show that the synthesized nanoMlPs were able to selectively suppress AA interfering current in an electrochemical detection of DA in PBS. The maximum AA concentration that could be quantitatively sequestered with the described setup was 50 nM, allowing the inventors to correctly determining DA, without any other sample pretreatment. Again, the inventors believe that higher concentrations of MIPs could suppress the signal caused by AA at a concentration of greater than 50 nM.

Example 3

Real Samples

[0081] The proposed method was then applied to analyze DA in real samples. Measurements were carried out in diluted human serum samples, applying the same experimental setup already described, and the results are showed in FIG. 5.

[0082] In a complex matrix, interfering current generated by the presence of a fixed concentration of AA is still capable of affecting measurements, leading to errors in DA detection. However, as shown in FIG. 5, nanoMlPs were able to suppress interfering current, thus proving their suitability as novel analytical tools for the electrochemical suppression of potential interfering species.

[0083] Conclusion

[0084] The inventors have shown that they can use nanoMlPs to suppress signals from interfering species, and allow analytes to be accurately detected using amperometry.

REFERENCES

[0085] [1] Molecularly imprinted polymer sensors (WO2015130529 A3). Belbruno, 2015. [0086] [2] Molecular imprinted nanosensors (U.S. Pat. No. 9,134,305 B2). Cai et al., 2012. [0087] [3] Methods and apparatus for the fabrication and use of graphene petal nanosheet structures (CA2845539 A1). Claussen et al., 2012. [0088] [4] Molecular recognition sensor system (U.S. Pat. No. 6,807,842 B). Williams et al., 2003. [0089] [5] Small Volume In Vitro Analyte Sensor (U.S. Pat. No. 9,234,864 B2). Heller et al., 2016. [0090] [6] Disposable sensor for liquid samples (US2009050477 A). Catt et al., 2009. [0091] [7] J. Wang, Chemical Reviews, 108 (2), 814-825, 2008. [0092] [8] T. G. Drummond, M. G. Hill, J.K. Barton. Nature Biotechnology, 21(10), 1192-1199, 2003. [0093] [9] K. Cammann, W. Kleibhmer, E. Mussenbrock, B. RoB, F. Zuther. Fresenius' Journal of Analytical Chemistry, 349 (5), 338-345, 994 [0094] [10] I. Volov, O. Mann, Y. Hoenersch, B. Wahl, A.C. West. Journal of Separation Science, 34 (18), 2385-2390, 2011. [0095] [11] S. Koide, Y. Kinoshita, N. Ito, J. Kimura, K. Yokoyama, I. Karube. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 878 (23), 2163-2167, 2010. [0096] [12] E. Paleek, M. Fojta. Talanta 74 (3), 276-290, 2007. [0097] [13] D. J. Daly, C. K. O'Sullivan, G. G. Guilbault. Talanta 49, 667-678, 1999. [0098] [14] F. Canfarotta, A. Poma, A. Guerreiro, S. Piletsky, Nat. Protocols, 11: 443-455, 2016.