Electrode for biosensor for NADH measurement and manufacturing method therefor

Abstract

The present invention relates to an electrode for a biosensor for NADH measurement and a manufacturing method therefor. An electrode manufactured by the method according to the present invention enjoys the advantages of stabilizing current flow during an electric polymerization reaction, making the contact angle of the modified material remarkably small to increase the efficiency of surface modification, and being reusable several times. In addition, when applied to a biosensor for NADH measurement, the electrode of the present invention maintains sensitivity and selectivity at a high level without interference and thus easily measures a target of interest even in blood or serum that necessarily requires a pretreatment process due to the existence of a trace amount of a material to be measured. In addition, when applied to a biosensor for NADH measurement, the electrode can measure cell viability in a continuous manner and in real time, which leads to the application thereof to the cell toxicity assay field, and enables the measurement of cell viability in apoptotic cells lacking the mitochondrial function.

Claims

1. A method for manufacturing an electrode for a biosensor, comprising: a) washing the electrode with sulfuric acid; b) after the electrode of said step a) is placed in 4-aminothiophenol (4-ATP) and cultured, immersing the electrode in a first solution and then applying a voltage; and c) immersing the electrode of said step b) in a second solution and then applying a voltage; wherein the first solution in said step b) is a phosphate buffer solution of a molar concentration of 90 mM to 100 mM; and wherein the second solution in said step c) is a phosphate buffer solution of a molar concentration of 5 mM to 15 mM.

2. The method for manufacturing an electrode of claim 1, wherein the voltage in said steps b) and c) is applied by cyclic voltammetry.

3. The method for manufacturing an electrode of claim 2, wherein in said steps b) and c), said cyclic voltammetry is to sweep a potential from 0.8 to −0.4 V.

4. The method for manufacturing an electrode of claim 1, wherein the electrode for a biosensor is for measuring NADH (reduced form of nicotinamide adenine dinucleotide).

5. The method for manufacturing an electrode of claim 1, wherein the electrode comprises one or more kinds selected from a group consisting of gold, aluminum, platinum, nickel, graphene, silver nanowire films, metal grids, carbon, and indium tin oxide.

6. An electrode for a biosensor for NADH measurement by the method for manufacturing of claim 1.

7. A biosensor comprising the electrode of claim 1.

8. An electrode for a biosensor for NADH measurement by the method for manufacturing of claim 2.

9. An electrode for a biosensor for NADH measurement by the method for manufacturing of claim 3.

10. An electrode for a biosensor for NADH measurement by the method for manufacturing of claim 4.

11. An electrode for a biosensor for NADH measurement by the method for manufacturing of claim 5.

12. A biosensor comprising the electrode of claim 2.

13. A biosensor comprising the electrode of claim 3.

14. A biosensor comprising the electrode of claim 4.

15. A biosensor comprising the electrode of claim 5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic diagram of the modified surface of an electrode and results of the measurements of the degree of hydrophilicity, in accordance with an embodiment of the present invention;

(2) FIG. 2 shows a graph of the measurements of the contact angle of imine that appears upon surface modification, in accordance with an embodiment of the present invention;

(3) FIG. 3 shows the results of the measurements of the cyclic voltage-current by the concentration of a phosphate buffer solution, in accordance with an embodiment of the present invention;

(4) FIG. 4 shows the results of performing image analysis by an SEM on the surface of an electrode, in accordance with an embodiment of the present invention;

(5) FIG. 5 shows the results of performing image analysis by an SEM on the surface of an electrode, in accordance with an embodiment of the present invention;

(6) FIG. 6 shows NADH measurement values by concentration, using an electrode in accordance with an embodiment of the present invention; and

(7) FIGS. 7(a) and (b) show schematic diagrams of an NADH measurement process using a biosensor for NADH measurement in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS

(8) Hereinafter, the present invention will be described in more detail by way of embodiments. These embodiments are merely for describing the present invention in greater detail, and it will be apparent to those having ordinary skill in the art that the scope of the present invention is not limited by these embodiments in accordance with the subject matter of the present invention.

Embodiments

Production Example

Production of Electrode for Biosensor for NADH Measurement

(9) An electrode suitable for a biosensor for NADH measurement was produced by performing the following steps.

(10) A gold (Au) electrode was washed with sulfuric acid (H.sub.2SO.sub.4) of a molar concentration of 10 mM. Then, in order to form a self-assembled monolayer, the electrode was immersed in 4-aminothiophenol prepared at a molar concentration of 10 mM, and then cultured for 2 hours. Thereafter, N-phenylquinone diimine (hereinafter, referred to as ‘NPQD’) was formed through a process in which the electrode was immersed in a phosphate buffer solution of a molar concentration of 100 mM (high concentration) and then a voltage was swept for a potential between 0.8 V and −0.4 V in cyclic voltammetry. After immersing the electrode having said NPQD formed thereon in a phosphate buffer solution of a molar concentration of 10 mM (low concentration), a process of sweeping a voltage is performed once again by applying the same voltage as the step of forming NPQD in the phosphate buffer solution of 100 mM, to finally produce an electrode in accordance with the present invention.

Comparative Example

Production of Electrode

(11) For comparison with Production Example above, a process of modifying the surface of an electrode was performed using only a phosphate buffer solution of high concentration. Specifically, a gold (Au) electrode was washed with sulfuric acid (H.sub.2SO.sub.4) of a molar concentration of 10 mM. Then, in order to form a self-assembled monolayer, the electrode was immersed in 4-aminothiophenol prepared at a molar concentration of 10 mM, and then cultured for 2 hours. Thereafter, NPQD was formed through a process in which the electrode was immersed in a phosphate buffer solution of a molar concentration of 100 mM (high concentration) and then a voltage was swept for a potential between 0.8 V and −0.4 V in cyclic voltammetry.

[Embodiment 1] Measurement of Contact Angle of 4-aminothiophenol (4-ATP)

(12) Changes in the contact angle of NPQD, formed on the surfaces of the electrodes according to Production Example above, were checked. Specifically, after dropping 10 μl of distilled (DI) water on the electrodes of Production Example above at room temperature and humidity condition of 46%, photographs of the respective electrodes were taken, and the measurement and analysis of the contact angle were performed using the method provided by the manufacturer through IMAGE J software, thereby showing the results in FIGS. 1 and 2.

(13) As shown in FIGS. 1 and 2, NPQD bonded to the surface of Production Example was able to be bonded to the surface in a larger amount, and through this, it can be seen that the surface of the electrode was converted to be more hydrophilic. In particular, as shown in FIG. 2, Comparative Example showed little change in its contact angle compared with the case of bonding only 4-aminothiophenol, whereas in the case of Production Example, the contact angle was significantly lowered from 55° to 39°.

(14) From the above results, it can be seen that Production Example in accordance with the present invention can significantly lower the contact angle of NPQD, thereby making the surface hydrophilic, through the two steps of reactions with a high concentration and low concentration of a phosphate buffer solution.

[Embodiment 2] Measurement and Comparison of Stabilization of Electric Current Values

(15) In order to compare whether the phosphate buffer solution of a high concentration (100 mM) and the phosphate buffer solution of a low concentration (10 mM) stabilize electric current values, an electrochemical analysis was performed according to the protocol provided by the manufacturer using a multi-potentiostat device of the CH1040C series, thereby showing the results in FIG. 3.

(16) As shown in FIG. 3, the measured electric current values were relatively unstable in the phosphate buffer solution of high concentration, whereas the electric current values exhibited a very stable graph in the phosphate buffer solution of 10 mM, corresponding to a low concentration.

(17) From the above results, it can be seen that performing polymerization reactions in different steps with the conditions of high and low concentrations in producing Production Example in accordance with the present invention leads to the stabilization of electric current values.

[Embodiment 3] Measurement of Electrode Surface

(18) When NADH was measured several times in a biosensor comprising Production Example and Comparative Example, changes in the electrode surface were measured with a scanning electron microscope (SEM), and the results are shown in FIGS. 4 and 5. Here, the electrodes were subjected to a sputtering process to a thickness of 10 nm for scanning electron microscopy.

(19) In addition, the measurement of NADH using the biosensor was performed through a process of inserting a sample containing NADH into the biosensor, followed by measuring the final value of the current generated by applying a voltage of −600 mV for 10 seconds.

(20) As shown in FIG. 4, Comparative Example shows that NPQD was well produced in both the reference electrode (RE) and the working electrode (WE) sites; however, in the case of measuring after 20 reuses, the surface was rapidly deteriorated after it was used for NADH measurement multiple times in both the reference electrode and the working electrode sites.

(21) On the other hand, as shown in FIG. 5, not only was NPQD well produced in both the reference electrode and the working electrode sites, but the degree of deterioration of the surface was also significantly lower even after 20 reuses, in the case of Production Example.

(22) From the above results, it can be seen that the electrode of Production Example in accordance with the present invention can be reused a number of times in measurement when applied to a biosensor.

[Embodiment 4] Results of NADH Sensitivity Measurements

(23) In order to compare the sensitivity of the NADH measurement between Production Example and Comparative Example, NADH in the sample was measured through the same method as in Embodiment 3 described above, and the results are shown in FIG. 6.

(24) As shown in FIG. 6, the NADH measurement value of 100 μM corresponded to about 140% for Production Example, whereas a value corresponding to 110% was measured in Comparative Example, and the NADH measurement value of 140 μM corresponded to about 155% for Production Example, whereas it was measured at a significantly lower level of about 120% in the case of Comparative Example.

(25) From the above results, it can be seen that when measuring NADH using Production Example in accordance with the present invention as an electrode, the sensitivity is significantly higher than that of Comparative Example.

(26) While the present invention has been described in detail above, the scope of the present invention is not limited thereto, and it will be apparent to those having ordinary skill in the art that various modifications and changes can be made without departing from the spirit of the present invention as set forth in the claims.