Artificial enzyme for detection of analytes

Abstract

Provided herein are artificial enzymes comprising a solid substrate core coated in a shell that is electrochemically inert but conductive. Also provided are electrodes comprising said artificial enzymes and a support structure. Also provided are methods of selectively detecting at least one analyte and / or obtaining the concentration of the analyte in a sample using the electrode. Also provided are methods of preparing the artificial enzymes and the electrode and artificial enzymes and electrodes produced using those methods.

Description

ARTIFICIAL ENZYME FOR DETECTION OF ANALYTESField

[0001] The present invention relates to artificial enzymes. Particularly, artificial enzymes for detecting analytes in samples.Background

[0002] Enzymes are extremely efficient catalysts and have been explored in a plethora of applications, such as glucose meters for diabetes monitoring, remediation devices, labels in immunoassays for the diagnosis of diseases and in cleaning products. The application of enzymes in biomedical devices, however, can be limited by the stability of a given enzyme. This has attracted interest in developing enzyme mimics, that is artificial enzymes that are significantly more stable than their biological inspiration. Mimicking the molecular structure of the enzyme active site alone has resulted in catalysts with exceptional efficiency. The major existing hurdle that still exists is fabricating stable materials that can mimic the substrate selectivity as well as the exceptional activity of enzymes at catalysing reactions in complex biological fluids.

[0003] Here the inventors address this challenge by mimicking not the active site of an enzyme but rather their three-dimensional structure where the active site is located down a nanoconfined channel. Part of the importance of the active site being down a nanochannel in many enzymes is that the solution environment where the reaction proceeds is different to bulk. The alteration of the solution environment not only facilitates achieving high activity but also high selectivity via being able to exclude undesirable species from accessing the nanoconfined space.

[0004] The strategy of mimicking the 3D structure of enzymes has only been made possible through advances in nanomaterial synthesis that have allowed the investigation of nanoconfinement in electrocatalysis. The initial forays into nanoconfinement for electrocatalysis have shown that nanoparticles that mimic the geometric features of enzymes can be used to control the local solution environment, increasing the local concentration of reactants to give a significant higher specific activity than achievable without nanoconfinement, and to facilitate multistep cascade reaction occurring at different catalytic sites. However, thus far, control oflocal environment using nanoconfinement has not been exploited to provide selectivity for a specific reactant within a biological milieu.

[0005] The inventors chose glucose oxidation as a model reaction due to its large industrial importance, and the need for long term enzyme stability when used for continuous blood glucose biosensors. Current efforts to develop non-enzymatic glucose sensors based on direct electrooxidation of glucose have been limited by the low selectivity of such catalysts, and by the fact they rely on alkaline electrolytes for suitable sensitivity. The most successful strategy is to use either gold or platinum as a catalytic surface for glucose oxidation, but this strategy requires both a highly basic pH and the exclusion of chloride to minimise poisoning of the catalyst surface. Therefore, to develop an artificial enzyme three things need to be achieved. These are to 1) exclude chloride or other interferents, 2) generate a desired pH, and 3) exclude fouling by proteins and cells for the catalysts to be used directly in unadulterated biological fluids. Thus, there is a need to develop an artificial enzyme capable of achieving the three requirements above.

[0006] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0007] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.Summary of Invention

[0008] As discussed above, nanoparticles that catalyze biochemically relevant reactions have been promoted as alternative enzymes. However, the application of such artificial enzymes is severely restricted by poor selectivity in biological fluids; mainly because the reactions occur at active sites on the exterior surface of the nanoparticle. Enzymes in contrast typically have their active sites down a nanoconfined substrate channel where the reaction occurs in different solution conditions to bulk solution which aids in achieving selectivity for the substrate.

[0009] To address the disadvantages of presently available nanoparticles, the inventors created an artificial enzyme by mimicking the same three-dimensional structure of enzymes in nanoparticles to allow selective reactions in biological fluids. This is achieved using a core madeof catalytic material (such as a catalytic metal and / or oxide thereof or catalytic nonmetal) coated in a conducting mesoporous shell where isolated nanochannels lead to the surface of the catalytic material. The inventors show that such artificial enzymes can detect analytes (such as glucose, dopamine, and many others) in whole blood with little or no interference from other species. This is achieved by electrochemically pulsing the artificial enzymes to generate the locally required environment (such as alkalinity) for an effective electrocatalytic reaction in the nanochannels, as well as expelling fouling agents that would otherwise passivate the electrocatalytic reaction. The artificial enzymes are shown to be capable of detecting analytes in biological fluids, without loss of signal, for several months. The artificial enzymes are also shown to be capable of detecting an analyte when there are multiple analytes present in the sample (such as detecting glucose when dopamine and other analytes are present and vice versa) by changing the electrochemical pulsing potentials. Thus, nanoconfinement in nanoparticles can be exploited to potentially allow a broad range of species to be selectively detected in biological fluids with stability that can exceed that of enzymes.

[0010] In a first aspect, the present invention provides an artificial enzyme comprising a solid substrate core coated in a shell that is electrochemically inert but conductive, wherein the solid substrate core is made from a catalytic material, wherein the shell comprises a plurality of nanochannels, wherein an internal end of each nanochannel is attached to the surface of the solid substrate core and an external end of the nanochannel extends away from the surface of the solid substrate core, wherein the nanochannel is configured to provide selective access to the surface of the solid substrate core.

[0011] In a second aspect, the present invention provides an electrode comprising a plurality of the artificial enzyme according to the first aspect deposited on a support structure.

[0012] In a third aspect, the present invention provides a method of selectively detecting at least one analyte and / or obtaining the concentration of the analyte in a sample, the method comprising the steps of:A. contacting the electrode according to the second aspect or the artificial enzyme according to the first aspect with the sample;B. applying a first electrochemical pulse to the electrode or the artificial enzyme to:B 1. achieve a pH in the sample within the nanochannel that facilitates electrochemical oxidation or reduction of the analyte andB2. exclude poisoning species and / or fouling species from the nanochannel;C. applying a second electrochemical pulse to the electrode or the artificial enzyme;D. measuring a current produced at step C.;E. repeating steps B. to D. and taking the sum of the current measured on step D.;F. comparing the sum obtained from step E. against a calibration curve to detect the analyte and / or obtain the concentration of the analyte.

[0013] In a fourth aspect, the present invention provides a method of preparing the artificial enzyme according to the first aspect comprising the steps ofA. synthesizing a solid substrate core from a catalytic material;B. synthesizing a plurality of micelles comprising a surfactant template and dopamine, and an additive;C. mixing the solid substrate core from step A. and the plurality of micelles from step B. to induce the formation of a plurality of rod-like micelle formations on the surface of the solid substrate core;D. incubating and stirring the plurality of rod-like micelle formations from step C. to induce self-polymerization of the dopamine thereby obtaining a solid substrate core having polydopamine coating; andE. thermally treating the solid substrate core having polydopamine coating from step D. to remove the surfactant template and transform the polydopamine coating into a shell comprising a plurality of nanochannels thereby obtaining the artificial enzyme.

[0014] In a fifth aspect, the present invention provides a method of preparing the electrode according to the second aspect comprising the steps of:A. preparing the artificial enzyme according to the method of the fourth aspect;B. re-dispersing the artificial enzyme from step A. in water thereby obtaining a suspension;C. drop-casting and drying the suspension from step B. on the surface of a support structure thereby obtaining the electrode

[0015] In a sixth aspect, the present invention provides an electrode produced according to the method of the fifth aspect.

[0016] Numbered statements of invention are as follows:1. An artificial enzyme comprising a solid substrate core coated in a shell that is electrochemically inert but conductive, wherein the solid substrate core is made from a catalytic material, wherein the shell comprises a plurality of nanochannels, wherein an internal end of each nanochannel is attached to the surface of the solid substrate core and an external end of the nanochannel extends away from the surface of the solid substrate core, wherein the nanochannel is configured to provide selective access to the surface of the solid substrate core.2. The artificial enzyme according to statement 1, wherein the nanochannel is configured to provide selective access to the surface of the solid substrate core by control of the diameter and / or length of the nanochannel.3. The artificial enzyme according to statement 1 or statement 2, wherein the nanochannel has a predetermined diameter such that the surface of the solid substrate core is accessible to an analyte.4. The artificial enzyme according to any one of statements 1 to 3, wherein the diameter of the nanochannel is about 2 nm to about 50 nm, preferably about 4 nm to about 20 nm, or more preferably about 6 nm to about 12 nm, or more preferably about 3 nm to about 9 nm.5. The artificial enzyme according to any one of statements 1 to 4, wherein the diameter of the nanochannels is about 8nm to about lOnm or about 6 nm.6. The artificial enzyme according to any one of statements 1 to 5, wherein the nanochannel has a predetermined length such that the surface of the solid substrate core is accessible to an analyte.7. The artificial enzyme according to any one of statements 1 to 6, wherein the length of the nanochannel is about 2 nm to about 200 nm, preferably about 2 nm to about 50 nm, preferably about 2 nm to about 20 nm, more preferably about 4 nm to about 10 nm, or even more preferably about 4 nm to about 7 nm.8. The artificial enzyme according to any one of statements 1 to 7, wherein the length of the nanochannel is about 4.2 nm to about 6.4 nm.9. The artificial enzyme according to any one of statements 1 to 8, wherein the nanochannel is modified with an antifouling agent.10. The artificial enzyme according to statement 9, wherein the antifouling agent is selected from the group comprising phenyl phosphorylcholine (PPC), poly(ethylene) glycol, and poly(ethylene oxide).11. The artificial enzyme according to statement 9 or statement 10, wherein the antifouling agent is phenyl phosphorylcholine (PPC).12. The artificial enzyme according to any one of statements 1 to 11, wherein the plurality of nanochannels are discrete.13. The artificial enzyme according to any one of statements 1 to 11, wherein the plurality of nanochannels are interconnected.14. The artificial enzyme according to any one of statements 1 to 13, wherein the solid substrate core is a solid surface selected from the group comprising a nanoparticle, a plate, a needle, and a wire.15. The artificial enzyme according to any one of statements 1 to 14, wherein the solid substrate core is a nanoparticle core.16. The artificial enzyme according to any one of statements 1 to 15, wherein the catalytic material is a metal and / or oxide thereof, wherein the metal and / or oxide thereof is selected from the group comprising gold, platinum, nickel, silver, copper, bismuth, tungsten, iron, cobalt, zinc, tin, ruthenium, rhodium, palladium, cadmium, iridium and mixtures thereof.17. The artificial enzyme according to statement 16, wherein the metal and / or oxide thereof is gold.18. The artificial enzyme according to any one of statements 1 to 15, wherein the catalytic material is a nonmetal, wherein the nonmetal is glass-like carbon.19. The artificial enzyme according to any one of statements 15 to 17, wherein the nanoparticle core is a gold nanorod.20. The artificial enzyme according to statement 19, wherein the length of the nanoparticle core is about 10 nm to about 500 nm.21. The artificial enzyme according to statement 20, wherein the length of the nanoparticle core is about 20nm to about lOOnm.22. The artificial enzyme according to any one of statements 19 to 21, wherein the diameter of the nanoparticle core is about 10 nm to about 500 nm.23. The artificial enzyme according to statement 22, wherein the diameter of the nanoparticle core is about 20nm to about 50nm.24. The artificial enzyme according to any one of statements 1 to 23, wherein the shell that is electrochemically inert but conductive is a carbon shell.25. The artificial enzyme according to statement 24, wherein the carbon shell has a predetermined thickness such that the surface of the solid substrate core is accessible to an analyte.26. The artificial enzyme according to statement 24 or statement 25, wherein the thickness of the carbon shell is about 2 nm to about 200 nm, preferably about 2 nm to about 50 nm, preferably about 2 nm to about 20 nm, more preferably about 4 nm to about 10 nm, or even more preferably about 4 nm to about 7 nm.27. The artificial enzyme according to any one of statements 24 to 26, wherein the thickness of the carbon shell is about 4.2 nm to about 6.4 nm.28. An electrode comprising a plurality of the artificial enzyme according to any one of statements 15 to 27 deposited on a support structure.29. The electrode according to statement 28, wherein the support structure is a glass-like carbon.30. A method of selectively detecting at least one analyte and / or obtaining the concentration of the analyte in a sample, the method comprising the steps ofA. contacting the electrode according to statement 28 or statement 29 or the artificial enzyme according to any one of statements 1 to 27 with the sample;B. applying a first electrochemical pulse to the electrode or the artificial enzyme to:B 1. achieve a pH in the sample within the nanochannel that facilitates electrochemical oxidation or reduction of the analyte andB2. exclude poisoning species and / or fouling species from the nanochannel;C. applying a second electrochemical pulse to the electrode or the artificial enzyme;D. measuring a current produced at step C.;E. repeating steps B. to D. and taking the sum of the current measured on step D.;F. comparing the sum obtained from step E. against a calibration curve to detect the analyte and / or obtain the concentration of the analyte.31. The method according to statement 30, wherein when the sample further comprises at least one interferent, the first electrochemical pulse in step B does not facilitate electrochemical detection of the interferent.32. The method according to statement 30 or statement 31, wherein the analyte is glucose, dopamine, acetaminophen, oxygen, salicylic acid, ascorbic acid, ascorbate, uric acid, urate, creatinine, maltose, galactose, xylose, phenylalanine, tetracycline, haemoglobin or constituents thereof, lactate, cortisol, antibiotics, other metabolites, other sugars, lipids, other drugs, vitamins, bilirubin, and neurotransmitters including serotonin, norepinephirine, and acetylcholine.33. The method according to statement 32, wherein the analyte is glucose.34. The method according to statement 33, wherein the first electrochemical pulse is a reduction pulse and the second electrochemical pulse is an oxidation pulse.35. The method according to statement 34, wherein the reduction pulse is about -2.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode to about 0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode.36. The method according to statement 35, wherein the reduction pulse is about -1.85 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode.37. The method according to statement 34, wherein the reduction pulse is applied for about 1 ps to about 600 s.38. The method according to statement 37, wherein the reduction pulse is applied for about 0.2 s.39. The method according to statement 34, wherein the oxidation pulse is about -1.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode to about 2.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode.40. The method according to statement 39, wherein the oxidation pulse is about -0.25 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode.41. The method according to statement 34, wherein the oxidation pulse is applied for about 1 ps to about 600 s.42. The method according to statement 41, wherein the oxidation pulse is applied for about 0.1 s.43. The method according to statement 34, wherein steps B to D are repeated from about 1 time to about 100,000 times.44. The method according to statement 43, wherein steps B to D are repeated about 90 times.45. The method according to any one of statements 33 to 44, wherein the interferent is at least one selected from the group comprising dopamine, acetaminophen, oxygen, salicylic acid, ascorbic acid, ascorbate, uric acid, urate, creatinine, maltose, galactose, xylose, phenylalanine, tetracycline, haemoglobin or constituents thereof, lactate, cortisol, antibiotics, other metabolites, other sugars, lipids, other drugs, vitamins, bilirubin, and neurotransmitters including serotonin, norepinephirine, and acetylcholine.46. The method according to statement 45, wherein the interferents are ascorbic acid, ascorbate, uric acid, and urate.47. The method according to statement 45, wherein the interferent is dopamine.48. The method according to statement 32, wherein the analyte is dopamine.49. The method according to statement 48, wherein the first electrochemical pulse is a reduction pulse and the second electrochemical pulse is an oxidation pulse.50. The method according to statement 49, wherein the reduction pulse is about -2.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode to about 0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode.51. The method according to statement 50, wherein the reduction pulse is about -0.7 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode.52. The method according to statement 49, wherein the reduction pulse is applied for about 1 ps to about 600 s.53. The method according to statement 52, wherein the reduction pulse is applied for about 0.2 s.54. The method according to statement 49, wherein the oxidation pulse is about -1.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode to about 2.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode.55. The method according to statement 54, wherein the oxidation pulse is about 0.3 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode.56. The method according to statement 49, wherein the oxidation pulse is applied for about 1 ps to about 600 s.57. The method according to statement 56, wherein the oxidation pulse is applied for about 0.2 s.58. The method according to statement 49, wherein steps B to D are repeated from about 1 time to about 100,000 times.59. The method according to statement 58, wherein steps B to D are repeated about 90 times.60. The method according to any one of statements 48 to 59, wherein the interferent is at least one selected from the group comprising glucose, acetaminophen, oxygen, salicylic acid, ascorbic acid, ascorbate, uric acid, urate, creatinine, maltose, galactose, xylose, phenylalanine, tetracycline, haemoglobin or constituents thereof, lactate, cortisol, antibiotics, other metabolites, other sugars, lipids, other drugs, vitamins, bilirubin, and neurotransmitters including serotonin, norepinephirine, and acetylcholine.61. The method according to statement 60, wherein the interferent is glucose.62. The method according to any one of statements 30 to 61, wherein the poisoning species is at least one selected from the group comprising chloride, citrate, organothiols (such as glutathione), cysteine, and metal ions (such as copper ions).63. The method according to statement 62, wherein the poisoning species is chloride.64. The method according to any one of statements 30 to 61, wherein the fouling species is at least one selected from the group comprising of proteins, cells, lipids, and peptides.65. The method according to statement 64, wherein the protein is at least one selected from the group comprising human serum albumin, other albumin proteins, antibodies, enzymes, haemoglobin, globulin, fibrinogen, regulatory proteins, clotting proteins, transferrin, thrombin, and C-reactive protein.66. The method according to statement 64 or 65, wherein the fouling species is human serum albumin.67. The method according to any one of statements 30 to 66, wherein the sample is a liquid sample.68. The method according to statement 67, wherein the liquid sample is selected from the group comprising biological sample, cell culture media, fermentation tanks media, water, other aqueous solutions, organic solvents, and ionic liquids.69. The method according to statement 68, wherein the biological sample is selected from the group comprising blood, serum, plasma, interstitial fluid, sweat, saliva, sputum, and sebum.70. The method according to statement 69, wherein the biological sample is blood.71. A method of preparing the artificial enzyme according to any one of statements 1 to 27 comprising the steps of:A. synthesizing a solid substrate core from a catalytic material;B. synthesizing a plurality of micelles comprising a surfactant template and dopamine, and an additive;C. mixing the solid substrate core from step A. and the plurality of micelles from step B. to induce the formation of a plurality of rod-like micelle formations on the surface of the solid substrate core;D. incubating and stirring the plurality of rod-like micelle formations from step C. to induce self-polymerization of the dopamine thereby obtaining a solid substrate core having polydopamine coating; andE. thermally treating the solid substrate core having polydopamine coating from step D. to remove the surfactant template and transform the polydopamine coating into a shell comprising a plurality of nanochannels thereby obtaining the artificial enzyme.72. The method according to statement 71, wherein the solid substrate core is a solid surface selected from the group comprising a nanoparticle, a plate, a needle, and a wire.73. The method according to statement 72, wherein the solid substrate core is a nanoparticle core.74. The method according to any one of statements 71 to 73, wherein the catalytic material is a metal and / or oxide thereof, wherein the metal and / or oxide thereof is selected from the group comprising gold, platinum, nickel, silver, copper, bismuth, tungsten, iron, cobalt, zinc, tin, ruthenium, rhodium, palladium, cadmium, iridium and mixtures thereof.75. The method according to statement 74, wherein the catalytic metal and / or oxide thereof is gold.76. The method according to any one of statements 71 to 73, wherein the catalytic material is a nonmetal, wherein the nonmetal is glass-like carbon.77. The method according to any one of statements 73 to 75, wherein the nanoparticle core is a gold nanorod.78. The method according to any one of statements 72 or 76, wherein the solid substrate core is a glass-like carbon solid surface (a glass-like carbon plate).79. The method according to any one of statements 71 to 78, wherein the length of the surfactant template, the additive, and / or the temperature of step D. determines the diameter of the nanochannel.80. The method according to any one of statements 71 to 79, wherein the stirring speed and / or the incubation time in step D. determines the thickness of the shell.81. The method according to any one of statements 71 to 80, wherein the surfactant template is a triblock copolymer.82. The method according to statement 81, wherein the triblock copolymer is Pluronic F127.83. The method according to any one of statements 71 to 82, wherein the additive is selected from the group comprising mesitylene, lipids, hexane, heptane, toluene, aniline, other oils, other aromatic species, and benzene.84. The method according to any one of statements 71 to 83, wherein the additive is mesitylene.85. The method according to any one of statements 71 to 84, wherein the stirring speed in step D. is from about 1 rpm to about 10000 rpm.86. The method according to any one of statements 71 to 85, wherein the stirring speed in step D. is about 375 rpm.87. The method according to any one of statements 71 to 86, wherein the incubation time is from about Is to about 10080 mins.88. The method according to any one of statements 71 to 87, wherein the incubation time is about 30 mins.89. The method according to any one of statements 71 to 88, wherein the temperature of stepD. is from about 0 °C to about 80 °C.90. The method according to statement 89, wherein when the solid substrate core is a gold nanorod, the temperature of step D. is about 25 °C (room temperature).91. The method according to statement 89, wherein when the solid substrate core is a solid metal surface (a solid metal plate) the temperature of step D. is about 40 °C.92. The method according to any one of statements 71 to 91, wherein the temperature of stepE. is from about 100 °C to about 3400 °C and the thermal treatment time is from about 5 mins to about 5760 mins.93. The method according to any one of statements 71 to 92, wherein the temperature of step E. is about 350 °C for about 5 hours, followed by gradual increase from about 350 °C to about 700 °C within about 1 hour, and followed by about 700 °C for about 45 mins.94. An artificial enzyme produced according to the method of any one of statements 71 to 93.95. A method of preparing the electrode according to statement 29 or 29 comprising the steps of:A. preparing the artificial enzyme according to the method of any one of statements 73 to 77, 79 to 90, 92, or 93;B. re-dispersing the artificial enzyme from step A. in water thereby obtaining a suspension;C. drop-casting and drying the suspension from step B. on the surface of a support structure thereby obtaining the electrode.96. The method according to statement 95, wherein the support structure is a glass-like carbon97. The method according to statement 95 or statement 96, wherein the concentration of the suspension is about 0.3 mg / mL to about 5 mg / mL.98. The method according to any one of statements 95 to 97, wherein the concentration of the suspension is about 1.8 mg / mL.99. The method according to any one of statements 95 to 98, wherein the drying temperature is from about 0 °C to about 350 °C and the drying time is from about 1 s to about 10080 mins.100. The method according to any one of statements 95 to 99, wherein the drying temperature is about 120 °C and the drying time is from about 2 to 3 mins.101. An electrode produced according to the method of any one of statements 95 to 100.Brief Description of Drawings

[0017] Preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

[0018] Figure 1 illustrates enzymatic electrochemical sensors having high activity and selectivity, which are related to enzymes having the active sites located down a substrate channel, allowing control of the local environment. Figure 1A shows an artificial enzyme containing the gold active sites located down electrically conducting but catalytic inertnanochannels (cartoons on right hand side). The TEM image (top) shows the gold nanorod coated with a thin carbon shell and the SEM image (bottom) shows the top view of the vertically aligned carbon nanochannels. Figure IB is a graph showing that the electrochemical control of the local environment was achieved by applying 90 consecutive 2-step potential pulses taking a total time of 27 s to collect the data. Figure 1C shows that the reduction potential pulse (-1.85 V for 0.2 s, purple - left) produces OH" locally inside the nanochannels to render the gold highly catalytically active for glucose and removes undesirable species such as chloride ions and electroactive interferents. The combination of the nanochannel structure with the reduction potential pulse also prevents non-specific adsorption of fouling proteins. Glucose oxidation happens during the subsequent oxidation potential pulse (-0.25 V for 0.1 s, green - right). Figure ID is a graph showing that the current at the end of each oxidation pulse was collected for measurements done at different glucose concentrations and summed up to produce the plot in Figure IE. Electrolyte = 0.0302 mol L'1Na2HPO4+ 0.0098 mol L'1NaH2PO4+ 0.1 mol L'1KC1. All potentials are reported against the Ag|AgCl|NaCl 3 mol L'1reference electrode.

[0019] Figure 2A are graphs showing the effect of nanoconfinement and a reduction potential pulse to reduce oxygen and water under formation of hydroxide and increase the pH on the glucose oxidation activity. Figure 2B are graphs showing the effect of nanoconfinement in preventing chloride poisoning. Au nanorods (left) and artificial enzymes (right). Blue circles: - 1.85 V reduction pulsing in chloride containing electrolyte; orange circles: -1.85 V reduction pulsing in chloride-free electrolyte; green circles: -0.5 V reduction pulsing in chloride containing electrolyte. Each point in all plots is the sum of the current at the end of each oxidation potential pulse from the 90 two-step potential pulses; oxidation pulse: -0.25 V for 0.1 s. Electrolyte: phosphate buffer (pH = 7.4, 0.0302 mol L'1Na2HPO4+ 0.0098 mol L'1NaH2PO4with 0.1 mol L'1+ 0.1 mol L'1KC1, except for data in orange where a chloride free electrolyte was used). All potentials are reported against the Ag|AgCl|NaCl 3 mol L'1reference electrode.

[0020] Figure 3A is a graph showing the effect of adding electroactive interferents on the oxidation current. The measurements with 6 mmol L'1glucose were repeated 3 times and the measurements with each interferent (urate and ascorbate) were repeated 2 times each. Figure 3B is a graph showing the effect of adding human serum albumin (HSA - 35 mg mL'1) as the fouling species to the chloride-free phosphate buffer electrolyte with the artificial enzymes (full circles) and uncoated gold nanorods (open circles). Figure 3C is a graph showing the capacitive current subtracted glucose oxidation voltammetric peak without (black) and with (dark red) 35mg ml;1HSA added to the electrolyte; uncoated Au nanorods (top) and artificial enzymes (bottom). Figure 3D is a graph showing the glucose oxidation voltammetric peak of the first 7 cycles with artificial enzymes in electrolyte containing 35 mg mL'1HSA before (top) and after (bottom) capacitive current subtraction. For A and B: the data is the sum of the current of 90 pulses as a function of glucose concentration; reduction potential pulse = -1.85 V for 0.15 s (A) and 0.20 s (B); oxidation potential pulse = -0.25 V for 0.1 s. For C and D: the voltammograms were recorded between -0.1 V and 1.2 V at 50 mV s'1. Electrolyte: phosphate buffer (pH = 7.4, 0.0302 mol L'1Na2HPO4 + 0.0098 mol L'1NaH2PO4) without and with 6 mmol L'1glucose. All potentials are reported against the Ag|AgCl|NaCl 3 mol L'1reference electrode.

[0021] Figure 4 is a bar graph showing the stability of nanoconfmed nanoparticles in electrolyte containing human serum albumin (HSA) with no added glucose (gray), with 6 mmol L'1glucose (orange) and 12 mmol L'1glucose (blue). The electrode was rinsed with HSA free electrolyte and kept dried in between the measurements. Each measurement is the sum of the current at the end of each oxidation potential pulse from the 90 two-step potential pulses; reduction pulse: -1.85 V for 0.2 s and oxidation pulse: -0.25 V for 0.1 s. Electrolyte: phosphate buffer (pH = 7.4, 0.0302 mol L'1Na2HPO4+ 0.0098 mol L'1NaH2PO4with 0.1 mol L'1+ 0.1 mol L'1KC1 + 35 mg mL'1HSA. All potentials are reported against the Ag|AgCl|NaCl 3 mol L'1reference electrode.

[0022] Figure 5A is a graph showing the effect of carbon nanochannels surface modification with PPC (phenyl phosphorylcholine) on background current stability in whole blood without added glucose (full circles = PPC-modified artificial enzymes and open circles = unmodified artificial enzymes). 10 subsequent repetitions of 90 two-step pulses were performed with the sensor being at open circuit potential (OCP) between measurements. The electrode was kept immersed in blood at all times. Figure 5B is a graph showing the glucose detection in whole blood, showing a linear correlation between current and glucose concentration up to 16 mmol L'x; for each glucose concentration, 10 measurements of 90 two-step potentials pulses were made and the data shown is the average of the last 5 measurements. For all measurements, the data is the summation of currents at the end of each oxidation pulse in the series of 90 two-step pulses. Potential pulses conditions: -1.85 V for 0.2 s and -0.25 V for 0.3 s. All potentials are reported against the Ag|AgCl|NaCl 3 mol L'1reference electrode.

[0023] Figure 6 illustrates the morphology of the artificial enzymes. Figure 6A depicts transmission electron microscopy (TEM) images of artificial enzymes. Figure 6B depicts scanning electron microscopy (SEM) images of artificial enzymes. The TEM images (Figure 6A) show the thickness of the carbon shell. A thin carbon shell of (5.3±1.1) nm was deposited over the gold nanorods. The high temperatures required to transform the polydopamine coating into carbon via pyrolysis caused the nanorods to become rounded after the thermal treatment. The SEM images (Figure 6B) show the tridimensional morphology of the artificial nanozymes, where it is possible to observe the top of the vertically aligned carbon nanochannels of around 8- 10 nm diameter.

[0024] Figure 7 is a graph illustrating the overpotential of oxygen and water reduction on gold and carbon surfaces and on the artificial enzymes. Linear sweep voltammetry of artificial enzymes (blue), glass-like carbon (orange), and Au NPs (green) in phosphate buffer (pH = 7.4, 0.0302 mol L'1Na2HPO4 + 0.0098 mol L'1NaHjPCh). Scan rate = 50 mV s'1. The potential was swept in the cathodic direction. The voltammograms show that the overpotential for the water reduction reaction to produce OH' ions on glass-like carbon (orange) is around 300 mV higher than that on Au nanoparticles (green), with the reaction visibly starting at around -1.07 V on the Au NPs and at -1.35 V on glass-like carbon (dash lines). The artificial enzymes (blue) present a water reduction overpotential that is similar to the one on Au nanoparticles, showing that the Au sites inside the carbon nanochannels are electrochemically accessible and that the OH' ions are mostly generated inside the nanochannels. The reduction peaks at -0.82 V (glass-like carbon) and -0.74 V (artificial enzyme) are ascribed to the reduction of oxygen dissolved in the electrolyte.

[0025] Figure 8 illustrates the 2-step potential pulse chronoamperogram with different glucose concentrations. Chronoamperograms showing the last oxidation pulse of a series of 90 two-step potential pulses at different glucose concentrations (left). Zoomed in region of the beginning (top right) and end (middle right) of the oxidation pulse; and zoomed in region of the end of the reduction pulse (bottom right). Reduction potential pulse = -1.85 V for 0.2 s and oxidation potential pulse = -0.25 V for 0.1 s. Electrolyte: 0.0302 mol L'1Na2HPO4 and 0.0098 mol L'1Na^PCU (pH = 7.4). All potentials are reported against the Ag|AgCl|NaCl 3 mol L'1reference electrode.

[0026] Figure 9 illustrates the amount of exposed surface on uncoated Au nanorods and artificial enzymes. Figure 9A is a graph depicting cyclic voltammetry of artificial enzymes. Figure 9B is a graph depicting cyclic voltammetry of gold nanorods. Both performed at 100 mV1in 0.0302 mol L'1Na2HPO4+ 0.0098 mol L'1NaH2PO4(pH = 7.4). The area of the Au oxide reduction peaks (insets) was used as a comparative measure of the area of gold that is electrochemically available. For the measurements, the same mass of gold was deposited on a glass-like carbon electrode for the artificial enzymes and the gold nanorods.

[0027] Figure 10 is a graph illustrating the glucose detection with pulsing at pH=7.4 and no pulsing at pH=13. Sum of the current at the end of each oxidation pulse in a series of 90 two- step pulses as a function of glucose concentration with PPC functionalized artificial enzymes. Electrolyte: phosphate buffer (pH = 7.4, 0.0302 mol L'1Na2HPO4+ 0.0098 mol L'1NaH2PO4). For the data in blue, the pH was adjusted to 13 using KOH. First step: -1.85 V for 0.2 s (green) and -0.5 V for 0.2 s (blue and pink); second step: -0.25 V for 0.1 s.

[0028] Figure 11 depicts graphs illustrating the effect of electroactive interferents on glucose detection. Figure HA provides the sum of the current at the end of each oxidation pulse in a series of 90 two-step pulses as a function of glucose concentration and added interferents (3 mg dL'1urate and 2 mg dL'1ascorbate). Reduction potential pulse = -1.85 V for 0.1 s (blue), 0.15 s (pink), 0.2 s (cyan), 0.3 s (purple); oxidation potential pulse = -0.25 V for 0.1 s. The measurements with 6 mmol L'1glucose were repeated 3 times and the measurements with each interferent were repeated 2 times. Figure 11B provides the cyclic voltammograms at 100 mV s'1of nanozymes in electrolyte without glucose (black), with 6 mmol L'1glucose (blue), with interferent (orange) and with 6 mmol L'1glucose + interferent (green). Top = ascorbate and bottom = urate. Electrolyte = phosphate buffer (pH = 7.4, 0.0302 mol L'1Na2HPO4+ 0.0098 mol L'1NaH2PO4

[0029] Figure 12 illustrates the modification of artificial enzymes with an anti-fouling coating. Figure 12A is a scheme showing the electrode modification with PPC (phenyl phosphorylcholine): the amino group in the PPC is firstly converted to the corresponding diazonium salt by mixing 5 mM PPC in a deaerated aqueous solution containing an equivalent molar amount of NaNO2in 0.1 M HBF4for 10 min, followed by electrochemical reduction of the resulting diazonium group on the surface of the electrode. Figure 12B is a graph showing that the diazonium salt reduction was performed by cyclic voltammetry between 0.2 V and -0.75V, 3 cycles at 50 mV s'1. Figure 12C is a graph showing the sum of the currents at the end of each oxidation pulse in a series of 90 two-step pulses as a function of glucose concentration. Electrolyte: phosphate buffer (pH = 7.4, 0.0302 mol L'1Na2HPO4 + 0.0098 mol L'1NaHjPCh + KC1 0.1 mol L'1with PPC functionalized artificial enzymes (navy) and no-modified artificial enzymes (pink). First step: -1.85 V for 0.2 s; second step: -0.25 V for 0.1 s.

[0030] Figure 13 illustrates complete data of glucose detection in whole blood. Each graph depicts current as a function of time for PPC-modified artificial enzymes in blood with different amounts of added glucose. Each point is the sum of the current at the end of each oxidation potential pulse in a series of 90 two-step potential pulses; 10 subsequent repetitions of the 90 two-step pulses were done in sequence for each glucose concentration. The system was left at OCP between glucose additions. The electrode was kept immersed in blood at all times. Potential pulses conditions: -1.85 V for 0.2 s and -0.25 V for 0.3 s. All potentials are reported against the Ag|AgCl|NaCl 3 mol L'1reference electrode.

[0031] Figure 14 is a calibration curve of the current response pulsing from -0.7 to 0.3 V vs Ag|AgCl during addition of dopamine (10 pmol L'1) showing a linear relationship between dopamine concentration and recorded current. Each reading is the average of the current at the end of the anodic pulse of 90 two-step potential pulses; reduction pulse: -0.7 V for 0.2 s and oxidation pulse: 0.3 V for 0.2 s. Performed in phosphate-buffered saline (pH 7.4, 0.00147 mol L'1KH2PO4, 0.0081 mol L'1Na2HPO4, 0.00268 mol L'1KC1, 0.137 mol L'1NaCl).

[0032] Figure 15 is a calibration curve of the current response pulsing from -0.7 to 0.3 V vs Ag|AgCl during addition of dopamine (1 pmol L'1) showing a linear relationship between dopamine concentration and recorded current. Each reading is the average of the current at the end of the anodic pulse of 90 two-step potential pulses; reduction pulse: -0.7 V for 0.2 s and oxidation pulse: 0.3 V for 0.2 s. Performed in phosphate-buffered saline (pH 7.4, 0.00147 mol L'1KH2PO4, 0.0081 mol L'1Na2HPO4, 0.00268 mol L'1KC1, 0.137 mol L'1NaCl)

[0033] Figure 16 is a calibration curves of the current response pulsing from -0.7 to 0.3 V vs Ag|AgCl during the initial addition of glucose and the subsequent addition of dopamine showing no current response to glucose and a linear relationship between dopamine concentration and recorded current. Each reading is the sum of the current at the end of the anodic pulse of 90 two- step potential pulses; reduction pulse: -0.7 V for 0.2 s and oxidation pulse: 0.3 V for 0.2 s.Performed in phosphate-buffered saline (pH 7.4, 0.00147 mol L'1KH2PO4, 0.0081 mol L'1Na2HPO4, 0.00268 mol L'1KC1, 0.137 mol L'1NaCl) + human serum albumin (HSA - 35 mg ml;1).

[0034] Figure 17 is a calibration curve of the current response pulsing from -1.85 to -0.25 V vs Ag|AgCl during the initial addition of glucose and the subsequent addition of dopamine showing first the linear relationship between glucose concentration and recorded current then the drop in current associated with the addition of dopamine. Each reading is the sum of the current at the end of the anodic pulse of 90 two-step potential pulses; reduction pulse: -0.7 V for 0.2 s and oxidation pulse: 0.3 V for 0.2 s. Performed in phosphate-buffered saline (pH 7.4, 0.00147 mol L'1KH2PO4, 0.0081 mol L'1Na2HPO4, 0.00268 mol L'1KC1, 0.137 mol L'1NaCl) + human serum albumin (HSA - 35 mg mL'1).

[0035] Figure 18 is a graph depicting current response for the initial detection of dopamine pulsing from -0.7 to 0.3 V vs Ag|AgCl during which 60 pmol L'1is added in 10 pmol L'1additions (filled blue dot), subsequent current response for the detection of glucose pulsing from -1.85 to -0.25 V during which 12 mmol L'1is added in 2 mmol L'1additions (filled red dot), after which dopamine detection is repeated pulsing from -0.7 to 0.3 V increasing the dopamine concentration from 60 pmol L'1to 120 pmol L'1(open blue dot). Again, glucose detection is repeated pulsing from -1.85 to -0.25 V with three more glucose additions of 2 mmol L'1resulting in a final electrolyte concentration of 16 mmol L'1of glucose and 120 pmol L’1. Each reading is the sum of the current at the end of the anodic pulse of 90 two-step potential pulses; reduction pulse: -0.7 V for 0.2 s and oxidation pulse: 0.3 V for 0.2 s. Performed in phosphate- buffered saline (pH 7.4, 0.00147 mol L'1KH2PO4, 0.0081 mol L'1Na2HPO4, 0.00268 mol L'1KC1, 0.137 mol L'1NaCl) + human serum albumin (HSA - 35 mg mL'1).Description

[0036] Definitions

[0037] As used in this specification and the appended claims, terms in the singular and the singular forms "a," "an" and "the," for example, optionally include plural referents unless the content clearly dictates otherwise. For example, "an" artificial enzyme includes one artificial enzyme, one or more artificial enzymes and a plurality of artificial enzymes.

[0038] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0039] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0040] The term “about” as used herein contemplates a range of values for a given number of ±25% the magnitude of that number. In other embodiments, the term “about” contemplates a range of values for a given number of ±30%, ±20%, ±15%, ±10%, or ±5% the magnitude of that number. For example, in one embodiment, “about 60 minutes” indicates a value of 54 to 66 minutes (i.e. 60 minutes ±10%), and the like.

[0041] Numeric ranges are inclusive of the numbers defining the range. It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0042] The headings provided herein are not intended to limit the disclosure.

[0043] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristicsmay be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination. Any example or embodiment herein shall be taken to apply mutatis mutandis to any other example or embodiment unless specifically stated otherwise.

[0044] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent methods and systems are clearly within the scope of the disclosure, as described herein.

[0045] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0046] The disclosure is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying drawings. Although the examples herein concern humans and the language is primarily directed to human concerns, the concepts described herein are applicable to other animals. These and other aspects and features of the present disclosure will become more fully apparent from the following description and appended claims, or may be learned by the practice of the disclosure as set forth hereinafter.

[0047] A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

[0048] Overview

[0049] The artificial enzyme of the present invention mimics the architecture of enzymes. Broadly, the artificial enzyme comprises a solid substrate core made from a catalytic material coated in a shell that is electrochemically inert but conductive and that comprises a plurality of nanochannels. In a preferred but non-limiting embodiment, the artificial enzyme comprises ananoparticle with a gold core and a shell made of chemically modified, isolated carbon nanochannels (Fig. 1A). The chemically modified carbon nanochannels prevent fouling species from blocking the access of the reactant to the gold active sites located down the nanochannels whilst ensuring the walls do not react with glucose. An electrochemical potential pulsing protocol has been designed to exclude interferents and poisoning species as well as to alter the solution environment inside the nanochannels to alkaline conditions to facilitate the oxidation of glucose at the gold surface (Fig. IB). In this way, the reaction conditions can be controlled to allow the selective detection of glucose directly in whole blood, which is important for glucose sensors in diabetes monitoring.

[0050] The implications of this invention, however, are much greater than being able to selectively detect glucose in whole blood, as important as that is. The broader implications of this invention are that the inventors show the use of catalytic materials for selectively detecting small molecule species of interest in complex biological media in a way typically only achievable with enzymes. The inventors show how mimicking the geometry of enzymes, where the reaction happens down a nanoconfmed channel such that the solution composition adjacent to the active site is very different to the bulk medium in which the analyte is found, is advantageous as it allows selective detection of species. The inventors also show that by having a nanochannel material being electrochemically inactive for the analyte but electrochemically conducting, provides the basis for the use of electrochemical pulsing to control both the solution conditions inside the nanochannels and the potential at which the detection reaction occurs. This is a capability not achievable with an insulating polypeptide shell in natural enzymes. The inventors show that the artificial enzymes of the present invention, with enzyme like geometry, cannot only achieve selective detection in the way that enzymes do, but that they also have advantages concerning the stability of the recognition elements in a sensor and advantages in how to tune the detection reaction. These principles will be transferrable to other analytes and other artificial enzyme systems.

[0051] A, Artificial Enzyme

[0052] Thus, in one aspect, the present invention provides an artificial enzyme comprising a solid substrate core coated in a shell that is electrochemically inert but conductive, wherein the solid substrate core is made from a catalytic material, wherein the shell comprises a plurality of nanochannels, wherein an internal end of each nanochannel is attached to the surface of thenanoparticle core and an external end of the nanochannel extends away from the surface of the nanoparticle core, wherein the nanochannel is configured to provide selective access to the surface of the nanoparticle core. As used herein “configured to provide selective access” means that the internal end of each nanochannel is open to the surface of the solid substrate core.

[0053] In one embodiment, the nanochannel of the artificial enzyme is configured to provide selective access to the surface of the solid substrate core by control of the diameter of the nanochannel. In another embodiment, the nanochannel of the artificial enzyme is configured to provide selective access to the surface of the solid substrate core by control of the length of the nanochannel. In yet another embodiment, the nanochannel of the artificial enzyme is configured to provide selective access to the surface of the solid substrate core by control of the diameter and the length of the nanochannel.

[0054] A skilled person understood that the nanochannel of the artificial enzyme may adopt any dimensions provided that the surface of the solid substrate core remains accessible to an analyte. In one embodiment, the nanochannel of the artificial enzyme has a predetermined diameter such that the surface of the solid substrate core is accessible to an analyte. In a preferred embodiment, the diameter of the nanochannel of the artificial enzyme is about 2 nm to about 50 nm, preferably about 4 nm to about 20 nm, or more preferably about 6 nm to about 12 nm, or more preferably about 3 nm to about 9 nm. In a more preferred embodiment, the diameter of the nanochannel of the artificial enzyme is about 8 nm to about 10 nm or about 6 nm.

[0055] A skilled person understood that the nanochannel of the artificial enzyme may have any length provided that the surface of the solid substrate core remains accessible to an analyte. In one embodiment, the nanochannel of the artificial enzyme has a predetermined length such that the surface of the solid substrate core is accessible to an analyte. In a preferred embodiment, the length of the nanochannel of the artificial enzyme is about 2 nm to about 200 nm, preferably about 2 nm to about 50 nm, preferably about 2 nm to about 20 nm, more preferably about 4 nm to about 10 nm, or even more preferably about 4 nm to about 7 nm. In a more preferred embodiment, the length of the nanochannel of the artificial enzyme is about 4.2 nm to about 6.4 nm.

[0056] A skilled person understood that the nanochannel of the artificial enzyme may have any type of surface modifications. There can be one or more types of surface modifications on the surface of the nanochannels of the artificial enzyme. Without wishing to be bound by theory, such surface modifications may help in reducing non-specific adsorption of proteins on the surface outside of the nanochannels over time. Thus, in one embodiment, the nanochannel of the artificial enzyme is modified with an antifouling agent. In a preferred embodiment, the antifouling agent for modifying the nanochannels of the artificial enzyme includes but is not limited to phenyl phosphorylcholine (PPC), poly(ethylene) glycol, and polyethylene oxide), and the like. In a more preferred embodiment, the antifouling agent for modifying the nanochannels of the artificial enzyme is phenyl phosphorylcholine (PPC).

[0057] A skilled person understood that the plurality of nanochannels may adopt any types of configuration with respect to each other. Thus, in one embodiment, the plurality of nanochannels of the artificial enzyme are discrete. That is, one nanochannel is not interconnected with another nanochannel. In another embodiment, the plurality of nanochannels of the artificial enzyme are interconnected.

[0058] A skilled person also understood that the solid substrate core of the artificial enzyme may adopt any shape and may be of any size. Thus, the solid substrate core of the artificial enzyme is a solid surface which includes but is not limited to a nanoparticle, a plate, a needle, a wire, and the like. In a preferred embodiment, the solid substrate core of the artificial enzyme is a nanoparticle core.

[0059] A skilled person understood that any types of catalytic material such as catalytic metal, mixture of several catalytic metals, oxides of a catalytic metal, mixture of oxides from multiple catalytic metals, mixture of several catalytic metal and oxides from multiple catalytic metals, or the like may be used for preparing the artificial enzyme. The metal and / or oxide used for preparing the artificial enzyme include but are not limited to gold, platinum, nickel, silver, copper, bismuth, tungsten, iron, cobalt, zinc, tin, ruthenium, rhodium, palladium, cadmium, iridium, mixture thereof, and the like. In one embodiment, the metal and / or oxide used for preparing the artificial enzyme is gold.

[0060] A skilled person also understood that a catalytic material that is nonmetal may be used for preparing the artificial enzyme. The nonmetal used for preparing the artificial enzyme include but is not limited to glass-like carbon.

[0061] In one embodiment, the nanoparticle core of the artificial enzyme is a gold nanorod. In one embodiment, the length of the nanoparticle core of the artificial enzyme is about 10 nm to about 500 nm. In a preferred embodiment, the length of the nanoparticle core of the artificial enzyme is about 20nm to about lOOnm. In one embodiment, the diameter of the nanoparticle core of the artificial enzyme is about 10 nm to about 500 nm. In a preferred embodiment, the diameter of the nanoparticle core of the artificial enzyme is about 20 nm to about 50 nm. In another embodiment, the solid substrate core of the artificial enzyme is a macroscopic gold surface.

[0062] A skilled person understood that the shell of the artificial enzyme may be made from any material provided that the material is electrochemically inert but conductive and is able to form the nanochannels. Such material includes but is not limited to carbon. Thus, in one embodiment, the shell of the artificial enzyme is a carbon shell.

[0063] A skilled person understood that the shell of the artificial enzyme may have any thickness provided that the surface of the solid substrate core remains accessible to an analyte. In one embodiment, the carbon shell of the artificial enzyme has a predetermined thickness such that the surface of the solid substrate core is accessible to an analyte. In a preferred embodiment, the thickness of the carbon shell of the artificial enzyme is about 2 nm to about 200 nm, preferably about 2 nm to about 50 nm, preferably about 2 nm to about 20 nm, more preferably about 4 nm to about 10 nm, or even more preferably about 4 nm to about 7 nm. In a more preferred embodiment, the thickness of the carbon shell of the artificial enzyme is about 4.2 nm to about 6.4 nm.

[0064] B, Electrode comprising Artificial Enzyme

[0065] In another aspect, the present invention provides an electrode comprising a plurality of the artificial enzyme disclosed herein deposited on a support structure. A skilled person understood that deposition of the artificial enzyme on the support structure can be performed using any method known in the art. A skilled person also understood that the support structure can be any support structure suitable for deposition of the artificial enzyme and for preparationof an electrode capable of detecting an analyte. In one embodiment, the support structure of the electrode is a glass-like carbon.

[0066] C. Method of Detecting One or More Analyte using Electrode comprising Artificial Enzyme or using the Artificial Enzyme Itself

[0067] C.1. Detection method when there is at least one analyte of interest in the sample and no interferents.

[0068] The artificial enzyme or the electrode comprising artificial enzyme as disclosed herein can be used to detect and / or obtain the concentration of one or more analyte in the sample when there are no interferents within the sample. Thus, in yet another aspect, the present invention provides a method of selectively detecting at least one analyte and / or obtaining the concentration of the analyte in a sample, the method comprising the steps of:A. contacting the electrode or the artificial enzyme as disclosed herein with the sample;B. applying a first electrochemical pulse to the electrode or the artificial enzyme to:B 1. achieve a pH in the sample within the nanochannel that facilitates electrochemical oxidation or reduction of the analyte andB2. exclude poisoning species and / or fouling species from the nanochannel;C. applying a second electrochemical pulse to the electrode or the artificial enzyme;D. measuring a current produced at step C.;E. repeating steps B. to D. and taking the sum of the current measured on step D.;F. comparing the sum obtained from step E. against a calibration curve to detect the analyte and / or obtain the concentration of the analyte.

[0069] A skilled person understood that the first and second electrical pulses in the method of the preceding paragraph are calibrated such that combinations of both pulses enable detection of the analyte. The pulses can be any type of electrochemical pulses. Accordingly, in one embodiment, the first and second electrochemical pulses can both be oxidation pulses. In another embodiment, the first and second electrochemical pulses can both be reduction pulses. In yet another embodiment, the first electrochemical pulse can be an oxidation pulse and the second electrochemical pulse can be a reduction pulse. In yet another embodiment, the first electrochemical pulse can be a reduction pulse and the second electrochemical pulse can be a oxidation pulse.

[0070] C.2. Detection method when there is more than at least one analyte of interest in the sample and there is at least one interferent.

[0071] The electrode comprising the artificial enzyme as disclosed herein can also be used to detect and / or obtain the concentration of an analyte in the sample when there is more than one analyte of interest in the sample. When the other analyte of interest is not being detected, it is considered as an interferent. For example, when a sample comprises both glucose and dopamine and glucose is to be detected, dopamine is considered as an interferent and vice versa. Accordingly, in one embodiment, the present invention provides a method of selectively detecting at least one analyte and / or obtaining the concentration of the analyte in a sample, wherein when the sample further comprises at least one interferent, the method comprising the steps ofA. contacting the electrode or the artificial enzyme as disclosed herein with the sample;B. applying a first electrochemical pulse to the electrode or the artificial enzyme to:B 1. achieve a pH in the sample within the nanochannel that facilitates electrochemical oxidation or reduction of the analyte and does not facilitate electrochemical detection of the interferent andB2. exclude poisoning species and / or fouling species from the nanochannel;C. applying a second electrochemical pulse to the electrode or the artificial enzyme;D. measuring a current produced at step C.;E. repeating steps B. to D. and taking the sum of the current measured on step D.;F. comparing the sum obtained from step E. against a calibration curve to detect the analyte and / or obtain the concentration of the analyte.

[0072] In other words, the present invention also provides a method of selectively detecting an analyte and / or obtaining the concentration of the analyte in a sample, wherein when the sample further comprises at least one interferent, by ensuring that the first electrochemical pulse in step B does not facilitate electrochemical detection of the interferent.

[0073] C.3. Detection of Analytes

[0074] The analytes that can be detected using the two detection methods as described in sections C. l. and C.2. above includes but are not limited to glucose, dopamine, acetaminophen, oxygen, salicylic acid, ascorbic acid, ascorbate, uric acid, urate, creatinine, maltose, galactose, xylose, phenylalanine, tetracycline, haemoglobin or constituents thereof, lactate, cortisol, antibiotics, other metabolites, other sugars, lipids, other drugs, vitamins, bilirubin, neurotransmitters including serotonin, norepinephirine, and acetylcholine, and the like.

[0075] C.4. Detection of Glucose

[0076] In one embodiment, the analyte that can be detected using the two detection methods as described in sections C.l. and C.2. above is glucose. When glucose is to be detected, the following parameters are applied. The first electrochemical pulse used in the method is a reduction pulse and the second electrochemical pulse used in the method is an oxidation pulse. The reduction pulse used in the methods is about -2.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode to about 0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode. In a preferred embodiment, the reduction pulse used in the methods is about -1.85 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode. Said reduction pulse is applied for about 1 ps to about 600 s. In a preferred embodiment, said reduction pulse is applied for about 0.2 s. The oxidation pulse used in the methods is about -1.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode to about 2.0 V vsAg|AgCl|NaCl 3 mol L'1reference electrode. In a preferred embodiment, the oxidation pulse used in the methods is about -0.25 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode. Said oxidation pulse is applied for about 1 ps to about 600 s. In a preferred embodiment, said oxidation pulse is applied for about 0.1 s. Steps B to D of the methods may be repeated from about 1 time to about 100,000 times. In a preferred embodiment, steps B to D of the methods are repeated about 90 times.

[0077] As stated in section C.2, the electrode comprising artificial enzyme as disclosed herein can also be used to detect and / or obtain the concentration of an analyte in the sample when there is one or more other analytes of interest in the sample. Those other analytes of interest are referred as interferents in this paragraph. Accordingly, when glucose in the presence of interferents is to be detected using the method described in section C.2, the same parameters as described in the paragraph directly above are applied. In one embodiment, when glucose is to be detected using the method described in section C.2, the interferent includes but is not limited to dopamine, acetaminophen, oxygen, salicylic acid, ascorbic acid, ascorbate, uric acid, urate, creatinine, maltose, galactose, xylose, phenylalanine, tetracycline, haemoglobin or constituents thereof, lactate, cortisol, antibiotics, other metabolites, other sugars, lipids, other drugs, vitamins, bilirubin, neurotransmitters including serotonin, norepinephirine, and acetylcholine, and the like. In a preferred embodiment, when glucose is to be detected using the method described in section C.2, the interferents are ascorbic acid, ascorbate, uric acid, and urate. In another preferred embodiment, when glucose is to be detected using the method described in section C.2, the interferent is dopamine.

[0078] C.5. Detection of Dopamine

[0079] In one embodiment, the analyte that can be detected using the two detection methods as described in sections C.l. and C.2. above is dopamine. When dopamine is to be detected, the following parameters are applied. The first electrochemical pulse used in the method is a reduction pulse and the second electrochemical pulse used in the method is an oxidation pulse. The reduction pulse used in the methods is about -2.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode to about 0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode. In a preferred embodiment, the reduction pulse used in the methods is about -0.7 V vs Ag|AgCl|NaCl 3 mol L"1reference electrode. Said reduction pulse is applied for about 1 ps to about 600 s. In a preferred embodiment, said reduction pulse is applied for about 0.2 s. The oxidation pulse used in themethods is about -1.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode to about 2.0 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode. In a preferred embodiment, the oxidation pulse used in the methods is about 0.3 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode. Said oxidation pulse is applied for about 1 ps to about 600 s. In a preferred embodiment, said oxidation pulse is applied for about 0.2 s. Steps B to D of the methods may be repeated from about 1 time to about 100,000 times. In a preferred embodiment, steps B to D of the methods are repeated about 90 times.

[0080] As stated in section C.2, the electrode comprising artificial enzyme as disclosed herein can also be used to detect and / or obtain the concentration of an analyte in the sample when there is one or more other analytes of interest in the sample. Those other analytes of interest are referred as interferents in this paragraph. Accordingly, when dopamine in the presence of interferents is to be detected using the method described in section C.2, the same parameters as described in the paragraph directly above are applied. In one embodiment, when dopamine is to be detected using the method described in section C.2, the interferent includes but is not limited to glucose, acetaminophen, oxygen, salicylic acid, ascorbic acid, ascorbate, uric acid, urate, creatinine, maltose, galactose, xylose, phenylalanine, tetracycline, haemoglobin or constituents thereof, lactate, cortisol, antibiotics, other metabolites, other sugars, lipids, other drugs, vitamins, bilirubin, neurotransmitters including serotonin, norepinephirine, and acetylcholine, and the like. In a preferred embodiment, when dopamine is to be detected using the method described in section C.2, the interferent is glucose.

[0081] C.6. Description of Poisonins and Foulins Species

[0082] In any of the detection methods as described in sections C. l. to C.5., the first electrochemical pulse is applied to also exclude various poisoning species and / or fouling species from the nanochannel of the artificial enzyme itself or the artificial enzyme deposited within the electrode used in the method. Example of poisoning species excluded from the nanochannel of the artificial enzyme includes but is not limited to chloride, citrate, organothiols (such as glutathione), cysteine, metal ions (such as copper ions), and the like. In one embodiment, the poisoning species excluded from the nanochannel of the artificial enzyme is chloride. Example of fouling species excluded from the nanochannel of the artificial enzyme includes but is not limited to proteins, cells, lipids, peptides, and the like. In one embodiment, wherein when the fouling species is a protein, the protein excluded from the nanochannel of the artificial enzymeincludes but is not limited to human serum albumin, other albumin proteins, antibodies, enzymes, haemoglobin, globulin, fibrinogen, regulatory proteins, clotting proteins, transferrin, thrombin, C-reactive protein, and the like. In a preferred embodiment, wherein when the fouling species is a protein, the protein excluded from the nanochannel of the artificial enzyme is a human serum albumin.

[0083] C. 7. Description of Samples Tested

[0084] A skilled person appreciates that various types of samples can be tested using the detection methods as described in sections C.l. to C.6. In one embodiment, the sample tested using the detection methods described herein is a liquid sample. The liquid sample tested using the detection method described herein includes but is not limited to biological sample, cell culture media, fermentation tanks media, water, other aqueous solutions, organic solvents, ionic liquids, and the like. In a preferred embodiment, the liquid sample tested using the detection methods described herein is a biological sample. Examples of such biological samples include but are not limited to blood, serum, plasma, interstitial fluid, sweat, saliva, sputum, sebum, and the like. In a more preferred embodiment, the biological sample tested using the detection methods described herein is blood.

[0085] D, Method of Preparing the Artificial Enzyme

[0086] In yet another aspect, the present invention provides a method of preparing the artificial enzyme as described herein comprising the steps ofA. synthesizing a solid substrate core from a catalytic material;B. synthesizing a plurality of micelles comprising a surfactant template and dopamine, and an additive;C. mixing the solid substrate core from step A. and the plurality of micelles from step B. to induce the formation of a plurality of rod-like micelle formations on the surface of the solid substrate core;D. incubating and stirring the plurality of rod-like micelle formations from step C. to induce self-polymerization of the dopamine thereby obtaining a solid substrate core having polydopamine coating; andE. thermally treating the solid substrate core having polydopamine coating from step D. to remove the surfactant template and transform the polydopamine coating into a shell comprising a plurality of nanochannels thereby obtaining the artificial enzyme.

[0087] A skilled person understood that solid substrate having any shape and of any size may be used for preparing the artificial enzyme according to the method in preceding paragraph. Thus, the solid substrate core used in the artificial enzyme preparation method is a solid surface which includes but is not limited to a nanoparticle, a plate, a needle, a wire, and the like. In a preferred embodiment, the solid substrate core of the artificial enzyme is a nanoparticle core.

[0088] A skilled person understood that any types of catalytic material such as catalytic metal, mixture of several catalytic metals, oxides of a catalytic metal, mixture of oxides from multiple catalytic metals, or mixture of several catalytic metal and oxides from multiple catalytic metals may be used for preparing the artificial enzyme according to the method in preceding paragraph. The metal and / or oxide used for preparing the artificial enzyme according to the method in preceding paragraph includes but are not limited to gold, platinum, nickel, silver, copper, bismuth, tungsten, iron, cobalt, zinc, tin, ruthenium, rhodium, palladium, cadmium, iridium, mixture thereof, and the like. In one embodiment, the metal and / or oxide used for preparing the artificial enzyme according to the method in preceding paragraph is gold. In one embodiment, the solid substrate core of the artificial enzyme is a gold nanorod. In another embodiment, the solid substrate core of the artificial enzyme is a macroscopic gold surface.

[0089] A skilled person also understood that a catalytic material that is nonmetal may be used for preparing the artificial enzyme according to the method in preceding paragraph. The nonmetal used for preparing the artificial enzyme according to the method in preceding paragraph include but is not limited to glass-like carbon.

[0090] In preparing the artificial enzyme, having control of the diameter of the nanochannels within the artificial enzyme and / or the thickness of the artificial enzyme’s carbon shell are crucial. The inventors surprisingly found that the diameter of the nanochannels within the artificial enzyme is determined by the length of the surfactant template used in the artificialenzyme preparation method, the additive(s) used in the artificial enzyme preparation method, and the temperature of step D. in the artificial enzyme preparation method. Each of these parameters affect the diameter of the nanochannels within the artificial enzyme either individually or in combination. The inventors also surprisingly found that the thickness of the artificial enzyme’s shell is determined by the stirring speed of step D and the incubation time in step D of the artificial enzyme preparation method. Each of these parameters affect the thickness of the artificial enzyme’s shell either individually or in combination.

[0091] As stated above, the surfactant template and / or the additive used in the artificial enzyme preparation method determines the diameter of the nanochannels within the artificial enzyme. A skilled person will be able to choose suitable surfactants for obtaining certain diameter of nanochannels within the artificial enzyme. In one embodiment, the surfactant template used in the artificial enzyme preparation method is a triblock copolymer. Examples of said triblock copolymer includes but is not limited to Pluronic F127 and the like. In a preferred embodiment, the surfactant used for the artificial enzyme preparation method is Pluronic F 127. Similarly, a skilled person will also be able to choose suitable additives for obtaining certain diameter of nanochannels within the artificial enzyme. Examples of said additives includes but is not limited to mesitylene, lipids, hexane, heptane, toluene, aniline, other oils, other aromatic species, benzene, and the like. In a preferred embodiment, the additive used for the artificial enzyme preparation method is mesitylene.

[0092] As also stated above, the stirring speed of step D. and the incubation time in step D. of the artificial enzyme preparation method determines the thickness of the artificial enzyme’s carbon shell. A skilled person will be able to determine suitable stirring speed for obtaining certain thickness of the artificial enzyme’s carbon shell. In one embodiment, the stirring speed in step D. of the artificial enzyme preparation method is from about 1 rpm to about 10000 rpm. In a preferred embodiment, the stirring speed in step C of the artificial enzyme preparation method is about 375 rpm. Similarly, a skilled person will also be able to determine suitable incubation time for obtaining certain thickness of the artificial enzyme’s carbon shell. In one embodiment, the incubation time of step D. of the artificial enzyme preparation method is from about 1 s to about 10080 mins. In a preferred embodiment, the incubation time of step D. of the artificial enzyme preparation method is from about Is to about 1440 mins (i.e., 24 hours). Without wishing to be bound by this limitation, the inventors observed that the polymer does not grow further after 24 hours incubation. In another embodiment, the incubation time of step C. ofthe artificial enzyme preparation method is about 30 mins and the incubation time of step D. of the artificial enzyme preparation method is about 15 mins.

[0093] A skilled person will be able to determine the suitable temperature for step D. of the artificial enzyme preparation method described herein. In one embodiment, the temperature of step D. of the artificial enzyme preparation method is from about 0 °C to about 80 °C. The inventors have observed that different temperatures may be required when the artificial enzymes are prepared using different types of solid substrate core. In one embodiment, wherein when the solid substrate core is a gold nanorod, the temperature of step D. of the artificial enzyme preparation method is about 25 °C (room temperature). In another embodiment, wherein when the solid substrate core is a solid metal surface (a solid metal plate or a gold plate or a macroscopic gold surface), the temperature of step D. of the artificial enzyme preparation method is from about 20 °C to about 50 °C. Preferably, wherein when the solid substrate core is a solid metal surface (a solid metal plate or a gold plate or a macroscopic gold surface), the temperature of step D. of the artificial enzyme preparation method is about 40 °C.

[0094] A skilled person will be able to determine the suitable temperature and the length of thermal treatment time for step E. of the artificial enzyme preparation method described herein. Without wishing to be bound by theory, the pyrolysis step (i.e., step E) may be conducted for a prolonged time provided that the artificial enzyme is not exposed to air. In one embodiment, the temperature of step E. of the artificial enzyme preparation method is from about 100 °C to about 3400 °C and the thermal treatment time of the artificial enzyme preparation method is from about 5 mins to about 5760 mins. In a preferred embodiment, the temperature of step E. of the artificial enzyme preparation method is about 350 °C for about 5 hours, followed by gradual increase from about 350 °C to about 700 °C within about 1 hour, and followed by about 700 °C for about 45 mins.

[0095] In yet another aspect, the present invention provides an artificial enzyme produced according to the method described herein. In one embodiment, the present invention provides an artificial enzyme produced according to the method as described in section D. above.

[0096] E, Method of Preparing the Electrode comprising the Artificial Enzyme

[0097] In yet another aspect, the present invention provides a method of preparing the electrode as describe herein comprising the steps of:A. preparing the artificial enzyme according to the method as described herein;B. re-dispersing the artificial enzyme from step A. in water thereby obtaining a suspension;C. drop-casting and drying the suspension from step B. on the surface of a support structure thereby obtaining the electrode.

[0098] A skilled person will be able to determine the suitable concentration of the suspension for obtaining an electrode using the electrode preparation method described herein. In one embodiment, the concentration of the suspension for obtaining said electrode is from about 0.3 mg / mL to about 5 mg / mL. Without wishing to be bound by this limitation, the inventors have found that if the suspension for obtaining said electrode is too diluted, the surface of the electrode will not be uniform. In a preferred embodiment, the suspension for obtaining said electrode is from about 0.9 mg / mL to about 3.6 mg / mL. In a more preferred embodiment, the concentration of the suspension for obtaining said electrode is about 1.8 mg / mL Similarly, a skilled person will also be able to determine the suitable drying temperature and drying time from step C. of the electrode preparation method described herein. In one embodiment, the drying temperature for obtaining said electrode is from about 0 °C to about 350 °C and the drying time for obtaining said electrode is from about 1 s to about 10080 mins. In a preferred embodiment, the drying temperature for obtaining said electrode is about 120 °C and the drying time for obtaining said electrode is from about 2 to 3 mins.

[0099] In yet another aspect, the present invention provides an electrode produced according to the method described herein. In one embodiment, the present invention provides an electrode produced according to the method as described in section E. above.[000100] Examples[000101] 1 , Example 1 - Preparation of the Artificial Enzymes and Electrodes comprising theArtificial Enzymes and Electrochemical Measurements[000102] 1, 1. Synthesis of the Artificial Enzyme (Au Core-Carbon Nanochannels Shell Nanoparticles)[000103] 1, 1.1. Synthesis of Gold (Au) Nanorods[000104] Au nanorods were synthesized using a seed growth procedure as described by Ye, et al. (Ye, X.; Zheng, C.; Chen, J.; Gao, Y.; Murray, C. B., Using binary surfactant mixtures to simultaneously improve the dimensional tunability and monodispersity in the seeded growth of gold nanorods. Nano Lett 2013, 13 (2), 765-71). Firstly, Au seeds were obtained by adding HAuCl4(50 mmol L’1, 50 pL) to CTAB (0.1 mol L’1, 10 mL). NaBH4(50 mmol L’1, 0.6 mL, freshly prepared and kept in an ice bath) was then added under vigorous stirring over 2 min. The seed solution was left undisturbed for 30 min before using it. For the nanorods, cetyltrimethylammonium bromide (CTAB - 7 g) and sodium oleate (NaOL - 1.234 g) were dissolved in 250 mL of water in a 1 L conical flask by stirring at 50 °C. The mixture was then cooled to room temperature and AgNO, (10 mmol L’1, 7.2 mL) was added. After leaving the mixture for 15 min, 245 mL of water and HAUC14(50 mmol L’1, 5 mL) were added and the yellow mixture was stirred for 90 min, becoming transparent within the first 20-30 min of stirring. HC1 (32 %, 1.5 mL) was added after the 90 min and the mixture was stirred for another 15 min. Ascorbic acid (64 mmol L’1, 1.25 mL) was added followed by vigorous stirring for 30 s. Au seeds (0.4 mL) were added and the mixture was stirred for another 1 min before being left undisturbed overnight in the dark, when the mixture became dark red.[000105] 1, 1.2. Preparation of the Carbon Coating[000106] The carbon coating procedure was adapted from a nano-emulsion assembly approach to produce mesoporous carbon nanospheres as described by Peng et al. (Peng, L.; Hung, C. T.; Wang, S.; Zhang, X.; Zhu, X.; Zhao, Z.; Wang, C.; Tang, Y.; Li, W .; Zhao, D., Versatile Nanoemulsion Assembly Approach to Synthesize Functional Mesoporous Carbon Nanospheres with Tunable Pore Sizes and Architectures. J Am Chem Soc 2019, 141 (17), 7073-7080.). Pluronic F127 (96 mg) and dopamine hydrochloride (48 mg) were dissolved in a mixture of water and ethanol (4.8 mL of each) in a 15 mL glass vial. Then, mesitylene (195 pL) was added dropwise using a syringe with the needle immersed inside the solution and under stirring at 375 rpm for 30 min. Au nanorods (60 mL in 4x15 mL centrifuge tubes - centrifuged and washed lx with water at 6500 rpm for 25 min) were added by taking ~1 mL of the emulsion to disperse the nanorods and transfer them from the centrifuge tube to the reaction vial. NH40H (495 pL) was then quickly added into the reaction and the emulsion was stirred for further 15 min after which the reaction mixture was immediately transferred to 2x5 mL centrifuge tubes and centrifuged at8000 rpm for 25 min, following by washing with water + ethanol (1 : 1 v:v, 3x, 8000 rpm, 8 min) and dispersed in ethanol.[000107] Thermal treatment was performed to firstly remove the surfactant and then to transform the polydopamine coating into carbon. The polydopamine coated Au nanorods, dispersed in ethanol, were transferred to a silica combustion boat and the ethanol was evaporated at 120 °C. The boat was transferred to a tube furnace and heated under argon flow from room temperature to 350 °C in 5 h to remove the surfactant, then heated up to 700 °C in an hour and maintained at 700 °C for another 45 min to convert the polydopamine to carbon by pyrolysis. After cooling down to room temperature under argon, the obtained nanoparticles were retrieved from the boat by scratching them into a small amount of added isopropanol using a plastic tip. The morphology of the obtained artificial enzymes was analysed by transmission electron microscopy (TEM) using a FEI Tecnai G2 20 TEM microscope and by scanning electron microscopy (SEM) using a JEOL JSM-IT800SHL Ultra High-Resolution FE SEM microscope.[000108] 1,2, Electrochemical Measurements[000109] 1,2.1. Electrode Preparation[000110] The artificial enzymes dispersed in isopropanol were centrifuged and re-dispersed in water (150 pL) to form an ink of which 7.5 pL (3x2.5 pL) were drop-casted onto a 0.07 cm2glass-like carbon surface and dried at 120 °C for 2-3 min after each 2.5 pL addition to give a uniform coating. For the glucose oxidation measurements in blood, a glass-like carbon plate was used (0.5 cm'2) with 45 pL of the artificial enzymes ink added on each side of the plate. The functionalization of the artificial enzymes with phenyl phosphorylcholine (PPC) was performed by cyclic voltammetry between 0.2 V and -0.75 V (vs. Ag|AgCl|NaCl 3 mol L'1) at 50 mV s'1in PPC 5 mmol L'1dissolved in HBF4 0.1 mol / L where an equimolar amount of NaNCh was added under argon purging, which was maintained for 15 min before the measurements. The electrodes were rinsed with MilliQ water after the functionalization.[000111] 1,2.2. Electrochemical Setup[000112] The measurements in phosphate buffer (pH = 7.4, 0.0302 mol L'1Na2HPO4 + 0.0098 mol L'1Na^PCU) in the absence and in the presence of chloride (KC1 100 mmol L'1), humanserum albumin (35 mg mL'1) and interferents (ascorbic acid 0.11 mmol L'1and uric acid 0.18 mmol L'1) were conducted in a 12 mm diameter wide glass vial containing 1 mL of electrolyte. The measurements in blood were conducted in a 25 mm diameter wide glass vial containing 5 mL of electrolyte. In both setups, a Ag| AgCl wire reference and a Pt plate counter electrode were used. The electrolyte was stirred in between measurements using a 3 mm stirring bar placed at the bottom of the vials. Different aliquots of glucose were added using a glass syringe from a 1 mol L'1stock solution prepared 24 h prior and kept in dark at 4 °C.[000113] 1,2.3. Electrochemical Pulsing Protocol[000114] In phosphate buffer, a series of 90 two-step potential pulses, consisting of a reduction pulse (-1.85 V for 0.2 s) and an oxidation pulse (-0.25 V for 0.1 s) were firstly performed in the absence of glucose until the oxidation current was stabilized (typically 6-10 repetitions). A 2 pL aliquot (= 2 mmol L’1, up to 12 pL = 12 mmol L'1) of a 1 mol L'1glucose solution was added into the electrolyte which was stirred for ~30 s before starting the measurement. The current at the end of each oxidation pulse was collected and summed up to produce the current versus glucose concentration plots. In whole blood, each measurement consisted of 10 repetitions of the series of 90 pulses without opening the circuit and the time of oxidation pulse was increased to 0.3 s.[000115] 2, Example 2 - Optimization of the Electrode comprising the Artificial Enzyme and Detection of Glucose using said Electrode[000116] 2, 1, Experimental Results[000117] The use of gold and other metallic nanoparticles for non-enzymatic glucose detection has been extensively reported but these nanoparticles cannot be used directly in whole blood. This is because not only do these nanoparticles require alkaline pH to be catalytically active for glucose oxidation, but they are also poisoned by chloride ions and fouled by proteins, both present in high concentrations in whole blood. To overcome these challenges, the inventors designed gold nanoparticles coated with carbon shell consisting of isolated mesopores (nanochannels) that extended all the way through to the underlying gold surface to allow the exclusion of undesirable species and to electrochemically generate the alkaline pH directly within the nanochannels for subsequent glucose oxidation (Fig. 1A and Fig. 6). This was achieved by applying a series of 90 two-step potential pulses (Fig. IB) taking a total time of 27s. The first potential step to -1.85 V for 0.2 s (reduction - purple shaded in top left of Fig. 1C, all potentials are versus Ag|AgCl|NaCl 3mol L'1) generates hydroxide ions via electrochemical reduction of oxygen and water. The local alkaline environment then facilitates the electrochemical glucose oxidation during the subsequent potential step at -0.25 V for 0.1 s (oxidation - green shaded in top right of Fig. 1C). Note, the overpotential for the water reduction reaction is around 300 mV more positive on Au than on carbon, which means that the hydroxide ions are predominantly generated at the Au surface at the bottom of the nanochannels (Fig. 7). This can be seen in Fig. 7 where it is evident that on gold as the potential was scanned negatively first oxygen in the nanochannels was reduced at potentials around -0.6 V versus Ag|AgCl|NaCl 3mol L-1and then water at potentials more negative than -1.15 V. The currents at the end of each oxidation step of each of the 90 pulses, performed in phosphate buffer electrolyte (0.0302 mol L'1Na2HPO4+ 0.0098 mol L'1NaH2PO4+ 0.1 mol L'1KC1; pH = 7.4), were collected (Fig. ID see Fig. 8 for full chronoamperograms) and summed up to produce the glucose calibration plots (Fig. IE).[000118] As shown on Fig. 8, during the reduction pulse at -1.85 V, OH" is electrochemically produced from the reduction of water. The local alkaline environment promotes the oxidation of glucose during the subsequent oxidation pulse at -0.25 V. The oxidation currents are always lower in the absence of glucose than in the presence of glucose for the whole duration of the oxidation pulse. With the addition of glucose, the increase in current is only observable after a current cross-over at 0.03-0.04 s. This can be attributed to the fact that the reduction of water during the reduction pulse does not only generate OH" but also H2which is readily oxidized at shorter times during the anodic pulse.[000119] The first step of dehydrogenation is the following: H2O + e" — H* + OH" (Volmer step). HER can proceed via Heyrovsky or Tafel pathways as follows: H* + H2O + e" — H2+ OH" (Heyrovsky) or 2H* — H2(Tafel). And glucose reduction produces sorbitol (using H* from dehydrogenation of water) as follows: glucose* + 2H* — sorbitol.[000120] Electrochemical glucose oxidation on gold at physiological pH was possible due to a combination of the nanoconfmed environment where the gold active sites are located and the electrochemical pulsing protocol to generate OH" locally. No glucose detection was observed when the reduction potential pulse was not sufficiently negative to produce OH" on either gold nanorods (no confinement - green circles of left-hand side plot in Fig. 2A) or with the artificialenzymes (green circles of right-hand side plot in Fig. 2A). With electrochemical pulsing, a glucose oxidation current was observed with the uncoated nanorods (blue circles of left-hand side plot in Fig. 2A) but it was approximately five times lower than the magnitude observed with the artificial enzymes (blue circles of right-hand side plot in Fig. 2A), even though the area of exposed gold of the artificial enzymes was approximately eight times smaller with the artificial enzymes relative to the uncoated nanoparticles (Fig. 9). The increased catalytic activity of the artificial enzymes shows that the nanoconfmed environment provides a much higher local concentration of hydroxide ions than in absence of nanoconfinement.[000121] In addition to providing the local alkaline environment, the reduction potential pulse in nanoconfinement also prevented electrode surface poisoning by chloride ions, present in high concentrations in blood and other biological fluids. Whilst the glucose oxidation current with the uncoated nanorods was significantly lower in the electrolyte with chloride than in the electrolyte without chloride (blue and orange plots respectively of left-hand side plot in Fig. 2B), with the artificial enzyme the oxidation currents were in fact marginally higher in the presence of chloride than in absence of chloride (blue and orange plots respectively of right-hand side plot in Fig. 2B). The higher catalytic activity with the artificial enzymes in the chloride containing electrolyte is possibly due to increased ionic conductivity of the bulk electrolyte when the ionic strength is higher (0.1004 mol L'1and 0.2004 mol L'1for chloride free and chloride containing electrolytes, respectively). Although higher ionic conductivity can influence mass transport with the uncoated nanorods, any advantage was offset by significant chloride adsorption on the gold surface in absence of nanoconfinement.[000122] The high activity in the nanoconfmed volumes resulting from the local alkaline environment and absence of chloride poisoning, allowed glucose to be oxidized at a potential as low as -0.25 V, a much less positive potential than is usually possible even in the highly alkaline solutions adopted as electrolytes in non-enzymatic systems (Fig. 10). The very low oxidation potential also means that the system is selective against other common electroactive species in blood, such as uric acid and ascorbic acid as seen in Fig. 3A where no interfering currents were observed when physiological concentrations of the electroactive interfering species were added in the electrolyte (more details in Fig. 11).[000123] As urate and ascorbate are both negatively charged at pH = 7 and above, it is expected that they will be removed from the nanochannels during the reduction potential step. The effectof the reduction pulse on the species interference is dependent on the duration of the pulse. When the duration of the reduction potential pulse is 0.1 s, the current slightly increases when urate is added into the electrolyte, but it does not change when ascorbate is added into the electrolyte (Fig. 11A - blue). This means that the duration of the reduction pulse of 0.1 s is sufficient to remove the interference of ascorbate, but not of urate. By increasing the duration of the reduction pulse to 0.15 s (Fig. 11A - pink) and 0.2 s (Fig. 11A - green), no change in the glucose response is observed in the presence of either urate or ascorbate, indicating that these reduction pulse durations are sufficiently long to eliminate the interference of both species. When the duration of the reduction pulse is increased to 0.3 s (Fig. 11A - purple), a decrease in the glucose response is observed in presence of urate. This can be ascribed to the reduction of urate to xanthine suppressing OH" production. Therefore, there is a compromise between how fast urate can be removed from the pores when the reduction potential is applied before it is electrochemically reduced.[000124] The effect of the interferents is also evident from the voltammograms in Fig. 11B. While the reduction current in the water reduction potential range (from -1.25 V in the cathodic direction) are not affected by the presence of ascorbate, it decreases in the presence of urate (black and blue voltammograms without interferent and orange and green voltammograms with interferent, top = ascorbate and bottom = urate). The glucose oxidation current in the presence of urate is lower and the peak is shifted to more positive potentials, which is due to a lower amount of OH" locally produced as the water reduction current is lower. From the voltammograms in electrolyte with the interferent only (orange), ascorbate oxidation starts from around -0.2 V and urate oxidation starts from around 0.1 V.[000125] The influence of the electrochemical pulsing in nanoconfinement on preventing protein fouling was also assessed. Human serum albumin (HSA), a protein present in blood at concentrations close to saturation (30-35 mg mL'1), accounts for ~60 % of the total protein present in blood and is the main species causing electrode fouling. Although a decrease in the oxidation currents was observed in the electrolyte containing 35 mg mL'1HSA compared to the electrolyte in absence of HSA, glucose was detected in the presence of the fouling protein with the artificial enzymes (Fig. 3B, filled circles), whilst no glucose detection was observed with the uncoated nanorods (Fig. 3B, open circles). The ability of the artificial enzymes to resist fouling is attributed to the reduction pulses repelling the negatively charged protein (the isoelectric point of HSA is 4.8) and the nanoconfmed channels acting as a filter that prevents protein adsorptionon the Au sites. Note the 8 nm channels are smaller than the HSA molecules (around 15 nm long). The decrease in oxidation current when HSA is added in the electrolyte is then mostly attributed to partial blocking of the nanochannels entrance by the fouling proteins as they adsorb on the carbon nanochannel surface rather than HSA adsorption directly on the gold surface.[000126] The adsorption of HSA on the carbon nanochannels surface was evident from cyclic voltammetry measurements performed in electrolyte containing HSA (Fig. 3C). A glucose oxidation peak current was observable in presence of HSA with the artificial enzymes (dark red line in Fig. 3C - bottom) although the peak was smaller than in absence of HSA (black line in Fig. 3C - bottom). No oxidation peak was observed with the uncoated nanorods in the electrolyte containing HSA (Fig. 3C - top). The cyclic voltammograms enable us to deconvolute the effect of fouling on the faradaic current arising from glucose oxidation and the capacitive current arising from surface adsorption of charged species on the carbon surface. Background capacitance subtraction of the CVs shows that the decrease in current with repeated voltammetric sweeps in the electrolyte containing HSA is due to a decrease in the background capacitive current caused by non-specific adsorption of HSA on the carbon surface, (compare Fig. 3D - top and bottom) as no significant change in the faradaic current arising from glucose oxidation was observed with cycling after subtraction of the background capacitance.stability of the artificial enzymes during electrochemical measurements was assessed (Fig. 4). The measurements were performed in electrolyte containing physiological levels of both chloride ions and HSA in absence of glucose and with 6 mmol L'1and 12 mmol L'1of glucose added into the electrolyte. The electrode was rinsed with buffer and kept dried in between measurements. The electrode was stable for at least 80 days, with a distinct increase in current observed when increasing concentrations of glucose were added into the electrolyte. These results show that the physical barrier provided by the nanochannels combined with the potential pulsing protocol to expel the negatively charged species were able to maintain the active sites available for glucose oxidation in the presence of both poisoning chloride and fouling protein species over a prolonged period of time.[000128] The results in HSA in Fig. 4 show that the electrochemical pulsing protocol in nanoconfmed volumes allows glucose detection in the presence of fouling proteins and other species that are in whole blood. Measurements were next performed in whole blood without added glucose, where a significant current decrease was observed overtime, with 70 % currentretention after 2 h decreasing to 30% after 7 h (shaded area in Fig. 5A - open circles). The decrease in current is attributed to non-specific adsorption of proteins on the surface outside of the nanochannels over time. This protein adsorption decreases the background (capacitive) current as no faradaic reaction occurs in the absence of glucose and in accordance with the voltammetry measurements performed in electrolyte containing HSA (Fig. 3D).[000129] To prevent the decrease in background current over time, the surface of the carbon nanochannels of the artificial enzymes was then chemically modified with a low-impedance zwitterionic phenyl layer (phenyl phosphorylcholine - PPC) as an anti-fouling agent (see Fig. 12 for more details, including a scheme of the surface chemistry). The voltammogram in Fig. 12A shows that two reduction peaks at -0.15 V and -0.45 V, corresponding to aryl diazonium grafting on the artificial enzymes surface, are present in the first cycle and they progressively disappear in the subsequent cycles. The voltammogram also show that the current in the hydrogen evolution reaction (HER) region decreases with cycling, suggesting that the PPC is partially depositing on the Au surface which is corroborated by the decrease in glucose detection currents after PPC functionalization, as shown in Fig. 12B.[000130] The resulting PPC-modified artificial enzymes retained the background current during the oxidation potential pulses in whole blood for at least 42 h, with just a 5 % current decrease observed within the first hour (Fig. 5A - full circles). The highly stable background current in whole blood obtained with the PPC-modified artificial enzymes is attributed to a combination of the hydrophilic surface provided by the PPC layer and the electrochemical pulsing protocol. The hydrophilic surface mitigates against the proteins strongly adsorbing onto the carbon surface; the loosely bound proteins are expected to be easily expelled from the electrode surface during the reduction potential pulses. The role of the potential pulses in removing weakly adsorbed proteins from the electrode surface was evident when the electrode containing the PPC-modified artificial enzymes was left immersed in blood without applying any potential (i.e., at open circuit potential - OCP) for a time interval in between the measurements. When the electrode was left immersed in blood at OCP, a decrease in the background current was observed that became greater with time. This decrease in the background current when PPC was employed, was recovered with repeated potential pulsing irrespectively of the time at OCP (Fig. 5A - full circles). Note with the unmodified artificial enzymes, the contribution of the potential pulses to remove the fouling proteins was minimal, being only observed in the first pulses but becomingrapidly overcome by the irreversible protein adsorption on the electrode surface (Fig. 5A - open circles).[000131] Finally, the capability of the electrochemical potential pulsing protocol and the nanoconfinement in these artificial enzymes to electrochemically detect glucose in whole blood was tested using the PPC-modified artificial enzymes. A linear increase of the oxidation current with increasing concentration of glucose added into whole blood was observed between 4 mmol L'1and 16 mmol L'1as shown in Fig. 5B. The data is the average of the oxidation currents of the last 5 out of 10 subsequent series of two-step pulses, showing the stability of the currents after each glucose addition. The last 5 out of a sequence of 10 measurements were used to ensure that the background current was recovered before collecting the data, since the electrode was maintained at OCP while glucose was added into the electrolyte, causing the background current to decrease due to protein adsorption (Fig. 13 for all data points). These results show that the potential pulsing protocol, combined with nanoconfinement, can control the local nanoconfmed environment at the gold surface located down the carbon nanochannels, allowing selective glucose detection in a complex biological fluid such as whole blood.[000132] 2,2, Discussion[000133] Herein it is shown how isolated mesoporous carbon channels around a catalytic gold nanorod allow the detection of glucose at the gold surface in unadulterated whole blood. This is achieved by exploiting a combination of nanoconfinement in the channel which means the solution conditions where the reaction is occurring is different to the bulk blood and electrochemical potential pulsing to manipulate the solution conditions inside the nanochannels to facilitate the electrocatalytic reaction. The direct oxidation of glucose on gold surfaces is most effective at highly basic pH values and with chloride ions, that poison the electrode surface, excluded. In whole blood the exclusion of proteins that foul the electrode and other electroactive species that might contribute a current is also required for selective measurement of glucose. To overcome these challenges, the inventors designed a catalytic gold nanorod particle with a mesoporous carbon coating that is conducting but not active for glucose oxidation. Applying a potential to an underlying carbon electrode will then influence the electrochemical reactions occurring inside the nanochannels and hence influence the solution conditions inside these channels above the catalytic gold surface. To achieve the detection of glucose in unadulterated whole blood, the potential is first pulsed at -1.85 V to generate hydroxide via the reduction ofwater, to achieve alkaline pH values, and simultaneously expel chloride and other negatively charges species form the channel. The potential was then pulsed back to -0.25 V to allow the oxidation of glucose. The high pH in the nanoconfmed channels facilitates a highly catalytically active surface for glucose whilst the very low oxidation potential for glucose of -0.25 V, a much less positive potential than is usually possible even in the highly alkaline solutions, means that the system is selective against other electroactive species in blood. The nanoparticles themselves are highly stable over long time periods and the inventors show this allows them to continue to record reliable glucose measurements in blood over many hours and continue to show no signal change over 80 days (Fig. 4). This makes these artificial enzymes ideal for both continuous glucose monitoring and for classical glucose finger prick devices.[000134] 3, Example 3 - Detection of Dopamine using the Electrode comprising the Artificial Enzyme[000135] The inventors demonstrate that a reductive pulse of -0.7 V for 0.2 s followed by an oxidative pulse of 0.3 V for 0.2 s allows the artificial enzyme to detect dopamine in lx PBS at a concentration of between 0 to 60 pmol L'1(as shown on Fig. 14) and between 0 to 6 pmol L'1(as shown on Fig. 15). All potentials are reported against the Ag|AgCl|NaCl 3 mol L'1reference electrode.[000136] 4, Example 4 - Detection of Glucose and Dopamine using the Electrode comprising the Artificial Enzyme[000137] Fig. 16 demonstrates that when the potential pulses as described in Example 3 are performed, detection of dopamine is not affected by changing concentrations of glucose. In Fig. 17, the inventors demonstrates that when the potential pulses for glucose (as discussed in Example 2) are performed, changing concentrations of dopamine slightly affects the detection of glucose.[000138] Furthermore, the inventors also show in Fig. 18 the simultaneous detection of dopamine and glucose in the same solution. The experiment starts with detecting dopamine with the previously described potentials optimised for dopamine (-0.7 to 0.3 V) while adding dopamine. Then switching to the potential pulses optimised for the detection of glucose. (-1.85 to -0.25) while adding glucose. The potential pulse is then reverted back to those that detect dopamine and more dopamine added. Finally, the potential applied is again changed back tothose that detect glucose and glucose added. This demonstrates the ability to switch back and forth between detection of two different small molecules by only changing to potentials applied to the artificial enzyme.[000139] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall within the spirit and scope of the present invention.

Claims

CLAIMS1. An artificial enzyme comprising a solid substrate core coated in a shell that is electrochemically inert but conductive, wherein the solid substrate core is made from a catalytic material, wherein the shell comprises a plurality of nanochannels, wherein an internal end of each nanochannel is attached to the surface of the solid substrate core and an external end of the nanochannel extends away from the surface of the solid substrate core, wherein the nanochannel is configured to provide selective access to the surface of the solid substrate core.

2. The artificial enzyme according to claim 1, wherein the nanochannel is configured to provide selective access to the surface of the solid substrate core by control of the diameter and / or length of the nanochannel.

3. The artificial enzyme according to claim 1 or claim 2, wherein the nanochannel has a predetermined diameter and / or length such that the surface of the solid substrate core is accessible to an analyte.

4. The artificial enzyme according to any one of claims 1 to 3, wherein the diameter of the nanochannels is about 8nm to about lOnm or about 6 nm and / or the length of the nanochannel is about 4.2 nm to about 6.4 nm.

5. The artificial enzyme according to any one of claims 1 to 4, wherein the nanochannel is modified with an antifouling agent, preferably wherein the antifouling agent is phenyl phosphorylcholine (PPC).

6. The artificial enzyme according to any one of claims 1 to 5, wherein the plurality of nanochannels are discrete or interconnected.

7. The artificial enzyme according to any one of claims 1 to 6, wherein the solid substrate core is a macroscopic gold surface.

8. The artificial enzyme according to any one of claims 1 to 6, wherein the solid substrate core is a gold nanorod.

9. The artificial enzyme according to claim 8, wherein the length of the gold nanorod is about 20nm to about lOOnm, and / or the diameter of the gold nanorod is about 20nm to about 50nm.

10. The artificial enzyme according to any one of claims 1 to 9, wherein the shell that is electrochemically inert but conductive is a carbon shell and wherein the carbon shell has a predetermined thickness such that the surface of the solid substrate core is accessible to an analyte.

11. The artificial enzyme according to claim 10, wherein the thickness of the carbon shell is about 4.2 nm to about 6.4 nm.

12. An electrode comprising a plurality of the artificial enzyme according to any one of claims 8 to 11 deposited on a support structure.

13. A method of selectively detecting at least one analyte and / or obtaining the concentration of the analyte in a sample, the method comprising the steps ofA. contacting the electrode according to claim 12 or the artificial enzyme according to any one of claims 1 to 11 with the sample;B. applying a first electrochemical pulse to the electrode or the artificial enzyme to:B 1. achieve a pH in the sample within the nanochannel that facilitates electrochemical oxidation or reduction of the analyte andB2. exclude poisoning species and / or fouling species from the nanochannel;C. applying a second electrochemical pulse to the electrode or the artificial enzyme;D. measuring a current produced at step C.;E. repeating steps B. to D. and taking the sum of the current measured on step D.;F. comparing the sum obtained from step E. against a calibration curve to detect the analyte and / or obtain the concentration of the analyte.

14. The method according to claim 13, wherein when the sample further comprises at least one interferent, the first electrochemical pulse in step B does not facilitate electrochemical detection of the interferent.

15. The method according to claim 13 or 14, wherein when the analyte is glucose, the first electrochemical pulse is a reduction pulse and the second electrochemical pulse is an oxidation pulse, wherein the reduction pulse is about -1.85 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode and is applied for about 0.2 s, wherein the oxidation pulse is about -0.25 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode and is applied for about 0.1 s, and wherein steps B to D are repeated about 90 times.

16. The method according to claim 15, wherein the interferent is at least one selected from the group comprising ascorbic acid, ascorbate, uric acid, urate, and dopamine.

17. The method according to claim 13 or 14, wherein when the analyte is dopamine, the first electrochemical pulse is a reduction pulse and the second electrochemical pulse is an oxidation pulse,wherein the reduction pulse is about -0.7 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode and is applied for about 0.2 s, wherein the oxidation pulse is about 0.3 V vs Ag|AgCl|NaCl 3 mol L'1reference electrode and is applied for about 0.2 s, and wherein steps B to D are repeated about 90 times.

18. The method according to claim 17, wherein the interferent is glucose.

19. The method according to any one of claims 13 to 18, wherein the poisoning species is chloride and / or wherein the fouling species is human serum albumin.

20. The method according to any one of claims 13 to 19, wherein the sample is a liquid sample, preferably wherein the liquid sample is blood.

21. A method of preparing the artificial enzyme according to any one of claims 1 to 11 comprising the steps of:A. synthesizing a solid substrate core from a catalytic material;B. synthesizing a plurality of micelles comprising a surfactant template and dopamine, and an additive;C. mixing the solid substrate core from step A. and the plurality of micelles from step B. to induce the formation of a plurality of rod-like micelle formations on the surface of the solid substrate core;D. incubating and stirring the plurality of rod-like micelle formations from step C. to induce self-polymerization of the dopamine thereby obtaining a solid substrate core having polydopamine coating; andE. thermally treating the solid substrate core having polydopamine coating from step D. to remove the surfactant template and transform the polydopamine coating into a shell comprising a plurality of nanochannels thereby obtaining the artificial enzyme.

22. The method according to claim 21, wherein the solid substrate core is a gold nanorod or is a macroscopic gold surface.

23. The method according to claim 21 or 22, wherein the length of the surfactant template, the additive, and / or the temperature of step D. determines the diameter of the nanochannel, and / or wherein the stirring speed and / or the incubation time in step D. determines the thickness of the shell.

24. The method according to any one of claims 21 to 23, wherein the surfactant template is Pluronic F127, and / or wherein the additive is mesitylene.

25. The method according to any one of claims 21 to 24, wherein the stirring speed in step D. is about 375 rpm and / or the incubation time is about 30 mins and / or wherein when the solid substrate core is a gold nanorod the temperature of step D. is about 25 °C and / or wherein when the solid substrate core is a macroscopic gold surface the temperature of step D. is about 40 °C.

26. The method according to any one of claims 21 to 25, wherein the temperature of step E. is about 350 °C for about 5 hours, followed by gradual increase from about 350 °C to about 700 °C within about 1 hour, and followed by about 700 °C for about 45 mins.

27. An artificial enzyme produced according to the method of any one of claims 21 to 26.

28. A method of preparing the electrode according to claim 12 comprising the steps of:A. preparing the artificial enzyme according to the method of any one of claims 21 to 26;B. re-dispersing the artificial enzyme from step A. in water thereby obtaining a suspension;C. drop-casting and drying the suspension from step B. on the surface of a support structure thereby obtaining the electrode.

29. The method according to claim 28, wherein the support structure is a glass-like carbon30. The method according to claim 28 or 29, wherein the concentration of the suspension is about 1.8 mg / mL, and / or wherein the drying temperature is about 120 °C and the drying time is from about 2 to 3 mins.

31. An electrode produced according to the method of any one of claims 28 to 30.

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