Electrochemical biosensor using dual electrode pair

Abstract

An electrochemical biosensor using a sensing system includes a working electrode including an active surface modified through a linker; and an auxiliary electrode. The sensor has a high current value compared with an existing sensor and retains excellent stability and sensitivity, and thus can be expected to be easily used for sensing various kinds of biomaterials.

Claims

1. A method for determining a-concentration of glucose in a fluid using an electrochemical sensor including a substrate; a working electrode formed on the substrate and having glucose oxidase attached thereon by a linker; and an auxiliary electrode formed to be separated apart from the working electrode in a horizontal direction of the substrate; wherein the working electrode and the auxiliary electrode are interdigitated array electrodes, the method comprising: contacting the glucose in the fluid to the working electrode and the auxiliary electrode of the electrochemical sensor in a presence of redox species which is recycled, between the working electrode and the auxiliary electrode, measuring a current of the working electrode and the auxiliary electrode, and determining the concentration of the glucose in the fluid using the measured current, wherein the measured current is a sum of a current collected by the working electrode resulted from direct electron transfer between the working electrode and the enzyme and collected by the working electrode, a reduction current of the redox species collected by the working electrode, and an oxidation current of the redox species collected b the auxiliary electrode.

2. The method of claim 1, wherein the linker is attached on nanoparticles directly formed on the electrode.

3. The method of claim 2, wherein the nanoparticles are selected from the group consisting of gold, platinum and palladium.

4. The method of claim 1, wherein the redox species are selected from ruthenium, hexamine complex, ferricyanide, ferrocenemethanol and ferrocenemonocarboxylic acid.

5. The method of claim 1, wherein a gap between the working electrode and the auxiliary electrode is 10 nm to 10 μm.

6. The method of claim 1, wherein the auxiliary electrode is a non-coated electrode.

7. A method for determining a concentration of glucose in a fluid using an electrochemical sensor including a substrate, a working electrode having glucose oxidase attached thereon by a linker is formed to be separated apart from the substrate in a vertical direction, and has a mesh shape, and an auxiliary electrode formed under the working electrode while having a space therebetween, wherein the working electrode and the auxiliary electrode are interdigitated array electrodes, the method comprising: contacting the glucose in the fluid to the working, electrode and the auxiliary electrode of the electrochemical sensor in a presence of redox species which is recycled between the working electrode and the auxiliary electrode, measuring an oxidation current of the working electrode and the auxiliary electrode, and determining the concentration of the glucose in the fluid using the oxidation current, wherein the oxidation current is a sum of a current collected by the working electrode resulted from direct electron transfer between the working electrode and the enzyme and collected the working electrode, a reduction current of the redox species collected by the working electrode, and an oxidation current of the redox species collected by the auxiliary electrode.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic view of glucose sensing using a single electrode functionalized by glucose oxidase.

(2) FIG. 2 is a schematic view of redox cycling in (a) IDA electrodes having a thin band shape, and (b) IDA electrodes having a high aspect ratio.

(3) FIG. 3 is a schematic view of glucose sensing using IDA electrodes selectively functionalized by glucose oxidase.

(4) FIG. 4 is enzyme immobilization using a diazonium-modified electrode.

(5) FIG. 5 is a schematic view for selective immobilization of biomolecules using a diazonium salt.

(6) FIG. 6 is a schematic process chart of selective electrode functionalization.

(7) FIG. 7 is a graph of cyclic voltammogram curves of carbon IDA nanoelectrodes (comb 1) in a solution of 1 mM 4-nitrophenyl diazonium tetrafluoroborate (4-NP) and 0.1 M tetrabutylammonium tetrafluoroborate/acetonitrile at a scan speed of 200 mV/sec.

(8) FIG. 8 is a graph of cyclic voltammogram curves for reducing a nitro group of 4-NP to an amino group in 0.1 M KCl+deionized water/ethanol (9:1) at a scanning speed of 100 mV/sec.

(9) FIG. 9 is a drawing representing oxidation current values collected at IDA electrodes depending on glucose concentrations using carbon IDA nanoelectrodes.

(10) FIG. 10 is a schematic view representing immobilization of biomolecules on IDA nanoelectrodes modified with metal nanoparticles, wherein (A) both electrodes are coated with nanoparticles, and (B) only one electrode (comb) is coated with nanoparticles.

(11) FIG. 11 is SEM images of a stacked polymer setbefore pyrolysis (a, b), and the stacked carbon electrode set after pyrolysis (c, d).

(12) FIG. 12 is a schematic view of glucose sensing using a stacked electrode set selectively modified with enzymes.

BEST MODE

(13) Hereinafter, the embodiment of the present invention will be described in detail with reference to the following examples and accompanying drawings. However, they are for describing the embodiment of the present invention in more detail, and the scope of the embodiment of the present invention is not limited thereto.

(14) (Chemical Materials)

(15) Acetonitrile (Fisher Scientific), tetrabutylammonium tetrafluoroborate (NBu.sub.4BF.sub.4, Fluka), 4-nitrophenyl diazonium tetrafluoroborate (4-NP), potassium ferricyanide, potassium ferrocyanide, glutaraldehyde, potassium chloride, sodium cyanoborohydride, Aspergillus niger-derived glucose oxidase (Type X-S, 100,000-250,000 units/g solid, Sigma Aldrich), and phosphate buffer (PBS, pH 7.4; Life Technologies).

(Preparation Example 1) Electrode Manufacturing

(16) A carbon IDA nanoelectrode was manufactured on a 6 inch (100) silicon wafer (Si wafer).

(17) First, a 700-nm-thick silicon dioxide (SiO.sub.2) layer was deposited on the silicon wafer by thermal oxidation. An SU-8 negative photoresist IDA structures were patterned using photolithography. In order to convert the photoresist structures into carbon electrodes, the predefined photoresist IDA patterns were pyrolyzed at 900° C. in a vacuum condition. During the pyrolysis, the size of the IDA structure was reduced by 60% in width, and by 90% in height. Finally, the carbon electrodes were passivated except for the interdigitated electrode area.

(Preparation Example 2) Selective Surface Functionalization

(18) As in Example 6, the immobilization of an enzyme may include three steps. 4-nitrophenyl diazonium tetrafluoroborate (4-NP) was used as a base. In order to link the enzyme to diazonium, the functional group of diazonium was converted into an amine group, and glutaraldehyde was used between the amine group of diazonium and the enzyme.

(19) Electrochemical Adsorption of 4-NP

(20) The electrochemical modification of the carbon electrode was measured by scanning electric potential of the electrode from 0.5 to −0.7 V at a scan rate of 200 mV/s vs a Ag/AgCl reference electrode, in acetonitrile containing 1 mM 4-NP in 0.1 M NBu.sub.4BF.sub.4. Before modification, impurities were removed from the solvent using argon gas for 30 minutes. After modification, the electrodes were washed with deionized water (DI water) for 30 minutes.

(21) Reduction of Nitro Group to Amine Group

(22) For production of an amine group by reducing a nitro group, a protic solvent containing 0.1 M potassium chloride and water/ethanol (90:10, v/v) was used. The potential of the electrode was measured by scanning the electric potential of the electrode from 0 to 0.8 V at a scan rate of 100 mV/s vs a Ag/AgCl reference electrode.

(23) Enzyme Immobilization

(24) For enzyme immobilization via a linker, glutaraldehyde was used as a bifunctional crosslinking agent. After converting the amine group, the carbon IDA electrodes were soaked in 200 μL of 0.1% sodium cyanoborohydride and 2.5 wt % glutaraldehyde solution for 2 hours, and then taken out.

(25) Thereafter, the electrodes were cleaned with deionized water, and dried using nitrogen gas. In order to couple a glucose oxidase enzyme to an aldehyde group produced on a carbon electrode, the electrodes were incubated in a buffer solution containing 0.1% sodium cyanoborohydride and 10 mg/mL glucose oxidase in a 50 mM PBS buffer (pH 7.4) at 4° C. overnight.

(Preparation Example 3) Electrochemical Characterization of Electrodes

(26) All electrodes were characterized using cyclic voltammetry by scanning electric potential of the electrodes from 0 to 0.6 V at a scan rate of 50 mV/s to a Ag/AgCl reference electrode in 0.5 M potassium chloride and 10 mM [Fe(CN).sub.6].sup.4− in deionized water. Glucose sensing was carried out using 10 mM [Fe(CN).sub.6].sup.3− as a redox mediator in 0.5 mM PBS (pH 7.4).

(27) A glucose solution was prepared at a concentration of 0.1 M in 100 mM PBS, and subjected to mutarotation at room temperature for 24 hours so as to reach anomeric equilibrium. All solutions for glucose sensing were cleaned by emitting argon gas to the solutions for at least 30 minutes before carrying out an electrochemical test. As a counter electrode, platinum wire was used. Electrochemical detection was carried out using a multi-potentiostat(CHI 1020; CH Instrument Inc., USA).

(Example 1) Surface-Modification of Carbon IDA Nanoelectrode

(28) Carbon IDA nanoelectrodes were manufactured by the procedure of above Preparation Example 1. As illustrated in FIG. 3, the surface of the carbon IDA nanoelectrode was functionalized using a linker and an enzyme. One comb of the carbon IDA nanoelectrodes was functionalized with glucose oxidase (GOx), while the other adjacent comb was used for collecting oxidation current of a ferrocyanide redox species.

(29) FIG. 7 illustrates a cyclic voltammogram in 1 mM 4-NP and 0.1 M NBu.sub.4BF.sub.4/acetonitrile solution in one comb. The irreversible reduction curve at −0.05 V in the first cycle contributed to the formation of 4-nitrophenyl radical from a diazonium salt derivative. The fact that the first irreversible curve disappears in the second scan means that the nitrophenyl group binded to the carbon surface blocks electron transfer. From the last reversible curve, it is shown that a radical anion formed by dissociation is reduced to an aryl anion on the carbon electrode surface.

(30) The result of reducing a nitro group to an amine group in Preparation Example 2 is illustrated in FIG. 8. The increase in reduction current allows the nitro group to be converted to the amine group.

(31) The amine group activating the carbon electrode was incubated with glucose oxidase using 2.5% glutaraldehyde. Finally, one of the carbon IDA nanoelectrodes was functionalized with glucose oxidase using the method of Preparation Example 2, and the adjacent electrode was used for collecting oxidation current of ferrocyanide.

(Example 2) Glucose Sensing

(32) Glucose sensing was carried out in the presence of a ferricyanide redox mediator having rapid electron transfer kinetics and a stable oxidation/reduction form.

(33) During enzyme reaction, glucose molecules were oxidized by the flavin-adenine dinucleotide (FAD) redox key element of the glucose oxidase enzyme. In the oxidation process, FAD was reduced to FADH.sub.2(GOx.sub.red) as described in the following Reaction Formula 4. In this system, oxygen in the reaction of the Reaction Formula 5 may be replaced with ferricyanide as an alternative electron receptor. Thereafter, FADH.sub.2 was oxidized back to FAD, as [Fe(CN).sub.6].sup.3− was reduced to [Fe(CN).sub.6].sup.4− as in Reaction Formulae 6 and 7. The reaction occurred at the carbon electrode (comb 1) functionalized with glucose oxidase, whereas [Fe(CN).sub.6].sup.4− was oxidized back to [Fe(CN).sub.6].sup.3− in the electrode (comb 1) and an adjacent carbon electrode (comb 2), as illustrated in FIG. 3. The reaction in the final step produced measurable current in direct proportion to glucose concentration as the oxidation form of the mediator was regenerated.
Glucose+FAD.fwdarw.Gluconic acid+FADH.sub.2+2H.sup.+  [Reaction Formula 4]
FADH.sub.2+O.sub.2.fwdarw.FAD+H.sub.2O.sub.2  [Reaction Formula 5]
FADH.sub.2+[Fe(CN).sub.6].sup.3−.fwdarw.FADH+[Fe(CN).sub.6].sup.4−+H.sup.+  [Reaction Formula 6]
FADH.sub.2+[Fe(CN).sub.6].sup.3−.fwdarw.FADH+[Fe(CN).sub.6].sup.4−  [Reaction Formula 7]

(34) From the above results, redox cycling occurred between the enzyme and the two carbon IDA nanoelectrode combs. In the carbon electrode (comb 1) modified with glucose oxidase, an enzyme reaction including glucose oxidation and [Fe(CN).sub.6].sup.3− reduction occurred. However, in the modified carbon electrode (comb 1) and the non-modified adjacent carbon electrode (comb 2), [Fe(CN).sub.6].sup.4− oxidation reaction occurred. Therefore, it is recognized that both electrodes in the IDA nanoelectrodes participate in the redox cycling of ferricyanide, which contributes to impart high sensitivity in glucose detection.

(35) In FIG. 9, it is shown that as the glucose concentration increases at the two electrodes of the IDA nanoelectrodes, current increases linearly. The current in comb 2 is higher than that in comb 1, since though the distance between enzyme sites and the electrode surface area in comb 2 are longer and larger than those in comb 1, the non-modified surface in comb 2 has higher surface reactivity than that in comb 1 which is modified with multiple molecules. Therefore, it was possible to collect more current using the IDA nanoelectrodes using the carbon electrode not further modified.

(Preparation Example 4) Biosensor Using IDA (Interdigitated Array) Nanoelectrodes Modified with Metal Nanoparticles

(36) Similar to the manufacturing method of the electrodes of Preparation Examples 1 and 2, nanoparticles such as gold, platinum or palladium were coated on a carbon electrode before attaching a linker using a diazonium salt thereto as in FIG. 10, thereby manufacturing a biosensor in which biomolecules containing glucose oxidase linked to the electrode through the nanoparticles.

(Preparation Example 5) Glucose Sensing Using Stacked Carbon Electrode Set

(37) As in FIG. 11, a stacked electron set including a suspended carbon mesh and a substrate-bound plane electrode was manufactured as a replacement for the IDA electrodes for using as a biosensor platform similar to the method described in Preparation Examples 1 and 2. For the redox mechanism and manufacturing process for the biosensing, the methods described in Preparation Examples 1 to 3 were used.

(38) It was confirmed that the stacked electrode set manufactured as above showed good efficiency in the redox cycling, as compared with that using the IDA nanoelectrode.

(39) For glucose sensing, the selective diazonium modification of the suspended nanomesh was carried out by the electrochemical reductive adsorption of 4-NP as described in Preparation Example 2. Thereafter, glucose oxidase was immobilized using glutaraldehyde. As in (a) of FIG. 12, the plane electrode was left without any modification for redox cycling of the ferricyanide/ferrocyanide redox couple.

(40) As a different method from that of the electrode manufactured above, as illustrated in (b) of FIG. 12, a method of attaching and modifying the substrate-bound plane electrode as in the method of Preparation Example 2 was used for enzyme immobilization, thereby manufacturing the electrode.

(41) For each of (a) and (b) in FIG. 12, the redox couple was recycled between the enzyme and the electrode surface, and oxidation current on the electrode surface was measured, thereby measuring glucose concentration.