KR102337896B1 - Electrode of Biosensor for Glucose Detection and Method thereof - Google Patents

Abstract

The present invention relates to an electrode used in a biosensor capable of electrochemically measuring the concentration of glucose in a body fluid, a method for manufacturing the same, and a glucose detection sensor comprising the electrode, wherein the electrode for glucose detection is formed on a nanofiber having excellent electrical conductivity. Since the metal nanoparticle and enzyme nanoparticle complex required for glucose detection is located, selectivity and sensitivity are high, and thus it can be usefully used in a glucose detection sensor that detects a trace amount of glucose in a body fluid.

Description

Electrode of biosensor for glucose detection and manufacturing method thereof

The present invention relates to an electrode used in a biosensor capable of electrochemically measuring the concentration of glucose in a body fluid, a method for manufacturing the same, and a glucose detection sensor including the electrode.

Glucose plays an essential role in numerous metabolic pathways in humans that produce adenosine triphosphate (ATP). However, abnormally high glucose levels in body fluids lead to several serious complications such as blindness, heart disease, high blood pressure, and kidney failure. Therefore, monitoring glucose levels in body fluids shows the importance of biomedical research to prevent and control diabetes. Monitoring of glucose levels in body fluids using glucose oxidase has attracted much attention due to several advantages such as selectivity, ease and speed. In this respect, glucose oxidase is a quantum dot-based fluorescence sensor, an electrochemical sensor, an optical sensor, a chemiluminescence sensor, and a surface plasmon for glucose detection in biological samples. It has been used in the development of various analytical approaches, including surface plasmon resonance. Among these technologies, the electrochemical sensor has surprising advantages such as high sensitivity, rapidity, cost-effcitiveness, and selectivity to detect glucose in various sample matrices. It also indicates that the portable electrochemical device is designed for bioassays so that it can be operated by unskilled persons with minimal samples. Importantly, electrochemical biosensors based on glucose enzymatic reactions played a major role in the continuous monitoring of glucose levels in personal glucometers.

Enzymatic electrochemical sensors have been shown to be promising tools for the analysis of clinically important molecules due to their high sensitivity, good selectivity, water solubility and low toxicity. To improve electrocatalytic activity and selectivity, many studies have used enzyme-coated electrochemical sensors to detect a wide variety of molecules from complex samples [Adv. Funct. Mater. 18 (2008) 591-599]. At the same time, the free enzyme has limitations in producing an electrochemical sensor with poor reproducibility, low operational stability, and unstable reliability under specific conditions such as pH and temperature. However, this limitation is successfully overcome by immobilizing the enzyme on a solid support, which offers several advantages such as high enzyme-to-substrate ratio, large enzyme stability, reusability and good diagnostics, and remarkably It also made it possible to reduce the diagnosis time. Recently, enzyme immobilization in various nanostructured materials (nanoparticles, organic or inorganic composites and three-dimensional materials) has received considerable attention in electrochemical sensors because it improves selectivity and enzyme activity for selected analytes of interest. Furthermore, protein-inorganic hybrid nanoflowers have been shown to be efficient electrochemical sensors for the sensitive analysis of various molecules [ACS Appl. Mater. Interfaces 6 (2014) 10775-10782]. Enzyme immobilization in nanoflowers markedly exhibits advantages such as improved enzyme activity, stability, durability and reusability with the large surface-to-volume ratio of nanoflowers. Importantly, the enzyme-immobilized nanomaterial-based electrochemical sensor showed better analytical properties than the pure enzyme-based electrochemical sensor [Nanoscale 6 (2014) 255-262; Nanoscale 9 (2017) 5658-5663]. Therefore, the integration of novel organic-inorganic nanostructured materials and electrodes may provide the simplest way to establish biosensors with significant analytical characteristics.

Electrospinning is a simple and novel technique for fabricating fibrous nanostructured materials in the nanometer to micrometer range from various polymers [Anal. Methods 2 (2010) 202-211]. It is a simple process to form inorganic nanomaterials that yield yields by changing the shape and surface area for special bioapplications. In recent years, because nanofibers (NFs) have long storage stability, flexibility and large surface area, fluorescence, electrochemical devices, and UV-visible spectroscopy (UV-visible) have been used for qualitative and quantitative analysis of various target analytes. It has attracted attention as a promising material that can be integrated with various analytical techniques such as spectrometry) and drug delivery/tracking. Among many polymers, poly(vinyl alcohol) (PVA) is a water-soluble polymer, which is widely used in various industries due to its non-toxic, low-cost, eco-friendly, chemical resistance and biological compatibility exhibited by a hydroxyl group. have. To prepare NFs using PVA, various crosslinking methods including irradiation, heating and chemical treatment were used to prepare insoluble NFs. However, because of their low electrical conductivity, they can be difficult to use in developing electrochemical sensors. Recently, PVA has been combined with chitosan and graphene oxide, and its biocompatibility for ATDC5 cells has been studied [Sci. Eng. C 79 (2017) 697-701].

Accordingly, the present inventors developed an electrode for glucose detection by forming nanofibers composed of a conductive material and a polymer as a support using electrospinning, and placing an enzyme nanoparticle complex composed of metal nanoparticles and an enzyme required for glucose measurement thereon did The present invention was completed by confirming that such an electrode for glucose detection sensed with high sensitivity a change in current generated by glucose and glucose oxidase and peroxidase.

One aspect of the present invention aims to provide an electrode for glucose detection in which a metal chip, a nanofiber, a metal nanoparticle, and an enzyme nanoparticle complex are sequentially stacked.

Another object of the present invention is to provide a glucose detection sensor including the electrode for glucose detection.

In addition, another aspect of the present invention comprises the steps of: a) forming a nanofiber on a metal chip using electrospinning; b) coating the metal nanoparticles on the nanofibers; And c) it aims to provide a method of manufacturing an electrode for glucose detection comprising the step of dripping the enzyme nanoparticle complex on the metal nanoparticles.

<Electrode for glucose detection>

According to one embodiment, the present invention with reference to FIG. 1 , a metal chip 10 , a nanofiber 20 , a metal nanoparticle 30 and an enzyme nanoparticle complex 40 are sequentially stacked electrode for glucose detection (100) is given.

The metal chip may be made of a metal having excellent durability, and for example, may be a noble metal such as platinum (Pt), gold (Au), silver (Ag), or palladium (Pd).

The nanofiber is positioned on a metal chip, and serves as a support for fixing the metal nanoparticle and enzyme nanoparticle complex used for glucose detection. These nanofibers are insoluble nanofibers in which a polymer and a conductive material having excellent electrical conductivity are crosslinked. In this case, in order to exhibit excellent electrical conductivity of the nanofiber, a conductive material and a polymer may be included in a weight ratio of 1:1 to 30. The conductive material may be graphene oxide or an ionic material, and the polymer may be polyvinyl chloride, polyvinyl alcohol, polyether imide, polyethylene glycol, or the like.

The metal nanoparticles (NP) catalyze a reaction for oxidizing a substance, and serves to oxidize gluconic acid, an oxide of glucose, to decompose it into hydrogen peroxide. In this case, the metal nanoparticles may be platinum (Pt), gold (Au), silver (Ag), copper (Cu), or the like. These metal nanoparticles can be bonded to the surface of the nanofiber through electrostatic attraction or covalent bonding, and thus the conductivity of the nanofiber and the sensitivity of glucose detection can be improved. Therefore, nanofibers coated with metal nanoparticles have improved thermal resistance, mechanical strength and electrical properties, and are applicable to biosensors.

The enzyme nanoparticle complex is uniformly positioned in the form of droplets on the nanofiber coated with metal nanoparticles, and is a nanoparticle containing an enzyme necessary for detecting glucose. Such an enzyme nanoparticle complex may include enzymes such as glucose oxidase that oxidizes glucose to produce gluconic acid, and peroxidase that dehydrogenates a substrate using hydrogen peroxide, and a metal such as copper It may be in the form of a flower in which enzymes are bound with the nucleus.

Such an electrode for glucose detection has a structure in which metal chips, nanofibers, and metal nanoparticles are sequentially stacked, and an enzyme nanoparticle complex is uniformly distributed on the metal nanoparticles, in which case the metal chip and The metal nanoparticles and the nanofibers coated with the enzyme nanoparticle complex may be chemically bonded using a binder.

<Glucose detection sensor>

According to another embodiment, the present invention provides a glucose detection sensor including the electrode for glucose detection.

The glucose detection sensor includes an electrode for glucose detection (copper-nanoflower@AuNPs-GO NFs) in which graphene oxide nanofibers coated with an enzyme nanoparticle complex and gold nanoparticles are formed on an Au chip with reference to FIG. 2 . can do. When glucose is injected into the glucose detection sensor, it _{reacts with oxygen (O 2 ) at the glucose detection electrode to generate hydrogen peroxide (H 2} O ₂ ) through an enzyme-catalyzed reaction, and the electric flow increases. As a result, since the electrode for glucose detection exhibits an electrochemical reaction with respect to the glucose concentration, a glucose detection sensor including the same has high selectivity and sensitivity, and can electrochemically detect glucose in a body fluid.

<Method for manufacturing an electrode for glucose detection>

According to another embodiment, the present invention with reference to Figure 3, a) forming a nanofiber on a metal chip using electrospinning; b) coating the metal nanoparticles on the nanofibers; And c) provides a method of manufacturing an electrode for glucose detection comprising the step of dripping the enzyme nanoparticle complex on the metal nanoparticles.

Steps a) to c) will be described in detail as follows.

Step a) is a process of forming a nanofiber as a support on a metal chip by using electrospinning.

The metal chip may be made of a noble metal such as platinum (Pt), gold (Au), silver (Ag), or palladium (Pd) having excellent durability.

The nanofiber may be prepared by mixing a polymer forming the backbone of the nanofiber and a conductive material capable of improving the conductivity of the nanofiber. The conductive material may be graphene oxide or an ionic material, preferably graphene oxide. The polymer may be polyvinyl chloride, polyvinyl alcohol, polyetherimide, polyethylene glycol, or the like, preferably polyvinyl alcohol. These polymers or conductive materials can be dissolved in a solvent and electrospun. In this case, the mixing ratio of the conductive material and the polymer may be a weight ratio of 1:1 to 30, preferably 1:5 to 15 by weight.

For example, a spray solution is prepared by mixing a polyvinyl alcohol (PVA) solution and a graphene oxide (GO) solution in a ratio of 1:1 ~ 30 (v/v), and then a pressure of 10 ~ 30 kV, 0.5 ~ Insoluble graphene oxide nanofibers (GO NFs) can be prepared on the Au chip by electrospinning on the Au chip at a syringe pump flow rate of 3.0 mL/h and firing in a drying oven at 150 ~ 200 °C.

Step b) is a process of coating the metal nanoparticles on the metal chip on which the nanofibers are formed.

The metal nanoparticles serve as catalysts for oxidizing a material, and may be nanoparticles of platinum (Pt), gold (Au), silver (Ag), copper (Cu), or the like.

For example, gold nanoparticles (AuNPs) solution may be dropped on graphene oxide nanofibers and dried to prepare graphene oxide nanofibers (AuNP-GO NFs) coated with gold nanoparticles. In this case, the gold nanoparticles may be attached to the surface of the graphene oxide nanofiber by electrostatic bonding.

Step c) is a process of coating the enzyme nanoparticle complex used for glucose detection.

The enzyme nanoparticle complex is a nanoparticle containing enzymes such as glucose oxidase and peroxidase, and since it has a large surface area, it is possible to improve the reaction between glucose and enzymes. Such an enzyme nanoparticle complex can be uniformly positioned in the form of droplets on the surface of the metal nanoparticle.

For example, a glucose oxidase solution, a peroxidase solution, and a copper-containing solution are mixed and reacted to form a flower-shaped enzyme nanoparticle complex (copper-nanoflower), which is then mixed with graphene coated with gold nanoparticles. It can be dried by dripping onto the oxide nanofiber. In this case, the enzyme nanoparticle complex may be located on the surface of the gold nanoparticles.

In addition, after step c), the metal chip and the nanofibers coated with the metal nanoparticles and the enzyme nanoparticle complex formed on the surface may be fixed to each other using a chemical binder.

The electrode for glucose detection according to the present invention has high selectivity and sensitivity as metal nanoparticles and enzyme nanoparticle complexes required for glucose detection are located on nanofibers with excellent electrical conductivity, so it is suitable for a glucose detection sensor that detects a small amount of glucose in body fluid. It can be useful.

1 is a side view of an electrode for electrochemical glucose detection; 10: metal chip, 20: nanofibers, 30: metal nanoparticles, 40: enzyme nanoparticle complex, 100: biosensor electrode.
2 is a method for detecting glucose in body fluid using an electrochemical sensor based on copper-nanoflower@AuNPs-GO NF.
Fig. 3 is a manufacturing flow diagram of an electrode for electrochemical glucose detection based on copper-nanoflower@AuNPs-GO NF.
Figure 4 is a graph and images for the characterization of GO NFs; (a) Electrochemical impedance values of PVA NFs for various GO concentrations (0 to 2.0 mg/mL). (b) shows the results of FT-IR spectroscopy of GO NFs and PVA NFs. Characteristics of calcined GO NFs. These are FE-SEM images before and after calcination (c) and (d).
Figure 5 is an FE-SEM image of AuNPs-GO NFs for various reaction times; The reaction times are (a) 10 minutes, (b) 30 minutes, (c) 60 minutes, (d) 90 minutes, (e) 120 minutes and (f) 150 minutes.
6 is an image confirming the optimal conditions for copper-nanoflower at various GOx concentrations; GOx concentrations are (a) 0.3 mg/mL, (b) 0.5 mg/mL, (c) 0.7 mg/mL and (d) 1.0 mg/mL. Each nanoflower was synthesized under the same conditions except for the concentration of GOx (concentrations of Cu ²⁺ ions and HRP were 120 mM and 0.05 mg/mL, respectively).
7 is a graph confirming the glucose detection conditions of copper-nanoflower@AuNPs-GO NFs; (a) Cyclic voltages for GO NFs, Cu-nanoflower, Cu-nanoflower@GO NF and copper-nanoflower@AuNPs-GO NF electrodes measured at a detection rate of 20 mV/s and a glucose condition of 30 μM. is the current curve. As a result of the optimization of the working electrode for copper-nanoflower@AuNPs-GO NFs, (b) copper-nanoflower at various concentrations (0 to 60 µg/mL) or (c) at various pH (pH 5 to 9) environments. The current of the enzyme electrode in 50 μM glucose was measured.
8 is a graph confirming the glucose detection performance of copper-nanoflower@AuNPs-GO NFs; (a) is the measured CV current at various glucose concentrations (0 ~ 0.1 mM), and (b) is a calibration curve. (c) Current-dependent current response of copper-nanoflower@AuNPs-GO NFs to 50 μM glucose and various interferences. (d) is the relative activity value measured for 20 days for the stability test.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

1. Materials and Methods

1-1. matter

Polyvinyl alcohol [(-CH ₃ CHOH-)n, MW 22,000], D(+)-glucose (98%) and sucrose (C ₁₂ H ₂₂ O ₁₁ ) were purchased from Junsei (Japan). Copper (II) sulfate pentahydrate (CuSO ₄ 5H ₂ O, 99.9%), calcium chloride (CaC _l2 , anhydrous, 96.0%), L(+)-ascorbic acid, D(+)-sucrose Ross), urea and hydrogen peroxide (H ₂ O ₂ ) were obtained from Samcheon (Korea). Potassium chloride (KCl) and sodium chloride (NaCl) were obtained from Deoksan (Korea). HRP (horseradish peroxidase), gold (III) chloride trihydrate (HAuCl _{4 ·} 3H ₂ O), permanganate (KMnO ₄ ), potassium _{persulfide (K 2} S ₂ O ₈ ), phosphorus pentoxide (P ₂ O) ₅ ), concentrated sulfuric acid (Conc. H ₂ SO ₄ ), cysteamine hydrochloride, and glucose oxidase derived from Aspergillus niger were purchased from Sigma-Aldrich (USA) without further purification. was used. Sodium borohydride (NaBH ₄ ) was purchased from Daejeong Chemical (Korea). Bovine serum albumin (BSA) was purchased from GenDEPOT (USA) and fetal bovine serum (FBS) was purchased from Gibco (USA). Graphite powder was purchased from Kanto Chemical (Japan).

1-2. Preparation of AuNPs-GO NFs

To prepare a 25 wt% polyvinyl alcohol (PVA) solution, PVA (25 g) was dissolved in ultrapure purified water (DI water) (100 mL) at 80° C. for 6 hours under continuous stirring. Graphene oxide (GO) (1 mg) was added to purified water (1 mL) and sonicated for 30 min to homogenize the GO solution. Thereafter, a GO solution of 1.0 mg/mL at a ratio of 1:10 (v/v) was added to a 25 wt% PVA solution, and stirred at room temperature for 3 hours. For loading on Au chips, PVA/GO solution was sprayed with a 22-G needle via electrospinning using a syringe pump flow rate of 1.0 mL/h under high pressure (15 kV) conditions. At this time, the distance between the needle tip and the collector (needle tip-to-collector) was 15 cm, and GO NF was electrospun on the Au chip for 10 minutes. After electrospinning, graphene oxide nanofibers (GO NF) were calcined in a drying oven at 180° C. for 1 hour to make NF insoluble. Then, 200 μL of cysteamine-AuNP solution (9 nM) was dropped on the surface of GO NFs and left at room temperature for 1 hour. The prepared AuNPs-GO NFs were washed 3 times with purified water to remove unattached AuNPs, and dried at 60°C.

1-3. Preparation of Cu-nanoflower@AuNPs-GO NFs (Cu-nanoflower@AuNPs-GO NFs)

Copper-nanoflowers were synthesized according to the following procedure. Briefly, 1.0 mg/mL of glucose oxidase (GOx) and 0.05 mg/mL of HRP were added to PBS (phosphate-buffered saline). 120 mM CuSO ₄ solution (20 μL) was added thereto and stirred at room temperature for 3 hours. Then, the solution was reacted at room temperature for 72 hours and washed 5 times with PBS. The synthesized copper-nanoflowers were stored at 4°C for next use.

To prepare the electrochemical biosensor, copper-nanoflower@AuNPs-GO NFs, 40 μg/mL nanoflower solution (10 μL) was dropped on AuNP-GO NFs and dried at room temperature. Copper-nanoflower@AuNPs-GO NFs used as working electrodes for glucose sensing and Au chips were bound using 1% nafion (3 μL) in 2-propanol as the binding agent.

1-4. Electrochemical glucose detection using copper-nanoflower@AuNPs-GO NFs

The copper-nanoflower@AuNPs-GO NF-based electrochemical biosensor was identified according to the following procedure. A small amount (usually 50 μL) of glucose solution (0.001 to 0.1 mM) was added to an electrochemical cell containing copper-nanoflower@AuNPs-GO NF Au-chip electrodes and 0.1 M PBS (pH 5.0) as working electrolyte. It was injected into 5 mL, and electrochemical measurements were performed through a CHI 750E instrument (CH Instruments, Austin, TX) (USA) using a conventional 3-electrode cell. The reference electrode was Ag/AgCl, and the counter electrode was platinum (Pt). Chronoamperometry and CV current measurement were performed in 0.1 M PBS solution (pH 5.0). CV was recorded from -0.8 V to +0.6 V at a sweep speed of 0.02 V/s. Chronoamperometric measurements were recorded at an applied potential of −0.25 V (vs. Ag/AgCl). In addition, redundancy measurements and calibrations were performed by continuously injecting various concentrations of glucose, and the total addition amount did not exceed 500 μL to avoid large changes in the electrolyte composition.

1-5. Characterization

UV-visible absorption spectroscopy was measured with a JASCO 670 spectrophotometer (Japan). Field emission scanning electron microscope (FE-SEM) images were acquired using a SIGMA instrument (Carl Zeiss) (UK) or JEM-F200 (JEOL) (Japan). FT-IR (fourier transform infrared) spectroscopic analysis was recorded with JASCO 6660FV (Japan). The XRD (X-ray diffraction) pattern of the sample was measured with a D8-Advance equipment (Bruker AXS) (Germany) that irradiates Cu Kα at room temperature (40 kV, 40 mA). Raman spectra analysis was performed with a Kaiser optical system (Raman Rxn 1) (USA). Electrospinning treatment was performed with an electrospinning system (NNC-ESDR) (Korea).

2. Results

2-1. Optimization and Characterization of GO NFs

The impedance value of GO NF according to the concentration of GO was confirmed. As a result, when the GO concentration was increased to a maximum of 1.0 mg/mL, the impedance value decreased and entered a plateau, and it was found that GO significantly improved the electrical conductivity of the PVA-based NF (Fig. 4a). ). Therefore, 1.0 mg/mL was selected as the optimal concentration of GO to prepare GO NFs.

FT-IR spectroscopy was performed on PVA NF and GO NF. As a result, while no peak appeared in PVA NF, peaks for functional groups such as hydroxy, carboxy and epoxide appeared in GO NF, confirming the formation of GO NF (Fig. 4b).

Since PVA is a water-soluble polymer, an electrospinning crosslinking process is required to develop a GO NF-based electrochemical sensor with water stability. To prepare water-resistant GO NFs, GO NFs were calcinated at 180 °C for 1 h. As a result, the PVA chain was rearranged after firing to increase the crystallinity of PVA. Calcined PVA has better mechanical properties in dry and wet conditions compared to chemically crosslinked PVA. As a result, water-insoluble GO NFs were obtained without morphological changes (Fig. 4c, d).

2-2. Structure of GO NFs with AuNPs

To increase the sensitivity for glucose sensing, AuNPs were deposited on the surface of GO NFs. Since GO NFs have negative charges and AuNPs have positive charges, a strong electrostatic force is generated and AuNPs are located on the surface of GO NFs.

Doping of AuNPs was performed by dropping a cysteamine-AuNP solution on the GO NF surface at various time intervals (10, 30, 60, 90, 120, 150 min). To find the optimal time for doping AuNPs on GO NFs, FE-SEM was performed. As a result, cysteamine-AuNPs were well doped on the surface of GO NFs through electrostatic force (Fig. 5).

2-3. Preparation of copper-nanoflower@AuNPs-GO NFs

The formation mechanism of copper-nanoflower follows the nuclear growth process. In the initial stage, Cu ²⁺ ions interact with phosphate anions in PBS solution to form copper phosphate crystals, and the amide backbone in proteins (GOx and HRP) organizes with copper phosphate crystals to form copper phosphate crystals. -forms protein complexes The complex acts as the seed of the nanoflower, and using it as the nucleus, it grows according to the reaction time to form the petals of the nanoflower. According to this sequential reaction, a multi-layered structure of nanoflowers can be formed.

The formation of copper-nanoflowers according to the GOx concentration was observed through FE-SEM. As a result, when GOx was 1.0 mg/mL, it was found that copper-nanoflowers with a uniform size were prepared ( FIG. 6 ).

2-4. Electrochemical glucose detection using copper-nanoflower@AuNPs-GO NFs

To confirm that the prepared copper-nanoflower@AuNPs-GO NFs showed better performance compared to copper-nanoflowers, each electrode was analyzed in the presence of 30 μM glucose by cyclic voltammetry technique. . As a result, the voltammogram for all electrodes showed that the redox peak (peak I/peak II) related to Au redox was -0.26/-0.028 V. On the other hand, it is related to ^{the formation of a preliminary monolayer consisting of initial hydrous gold oxides [Au +} (H ₂ O)n] _ads and the reduction of Au(I) functional oxides for Au adsorption atoms (adatom). A peak (peak I'/peak II") appeared except for AuNPs-GO NF due to the absence of glucose oxidase (Fig. 7a). Copper-nanoflower showed a small amount of redox peak in response to glucose, AuNPs-GO NFs did not show a clear response to glucose In the synthesis of copper-nanoflower@GO NFs, GO NFs could be combined with Au chips and copper-nanoflowers to further enhance the signal of copper-nanoflowers. Furthermore, AuNPs with copper-nanoflower@GO NFs enhance the redox peak of copper-nanoflower@GO NFs through peroxidase-induced activity.Copper-nanoflowers respond to glucose bound to GO NFs. Thus, AuNPs show more synergistic effects. In addition, stability was enhanced by the support GO NF. By expanding the surface area of AuNPs, which are 3D structures, the endogenous peroxidase-like activity of HRP to enhance electrochemical activity. Among the electrodes, copper-nanoflower@AuNPs-GO NF exhibited the highest cathodic current at the same amount of glucose compared to other samples.

Before measuring the electrochemical performance of copper-nanoflower@AuNPs-GO NFs on glucose, the following experimental conditions were checked. First, the effect of copper-nanoflower concentration in AuNPs-GO NFs was confirmed using a fixed amount of glucose (50 μM). As a result, as the concentration of copper-nanoflower increased up to 40 μg/mL, the current gradually increased and then decreased due to the insulating effect. Although copper-nanoflower is a key material for recognizing glucose, since it contains a biomaterial that acts as an insulator, the concentration of copper-nanoflower was selected to be 40 μg/mL ( FIG. 7b ).

Since enzyme activity is greatly affected by the observation environment, current values under various pH conditions were first observed. As a result, GOx shows the best performance under weakly acidic conditions (~ pH 5.0), and hydrogen peroxide, which generates electrons by HRP, is more stable at low pH because deprotonation occurs under basic conditions, and through this It was found that the current increased at low pH (Fig. 7c). Therefore, when using copper-nanoflower@AuNPs-GO NFs as electrochemical sensors, pH 5.0 was chosen as the optimal pH for the electrochemical sensing of glucose.

Under optimal conditions, the prepared glucose sensor was evaluated by electrochemical analysis to judge the assay performance. As a result, since the cathodic current is related to the amount of electrons generated by the reaction between Cu-nanoflower@AuNPs-GO NFs and glucose, the electrochemical signal (peak II) intensity gradually increased with the increase of the amount of glucose. (Fig. 8a). The linear regression equation in the low concentration range (0 ~ 0.01 mM) is μA = 3656.4933x + 0.6867, with a correlation coefficient of 0.9618 (n=3), and the linear regression equation in the high concentration range (0.03 ~ 0.1 mM) is μA = 636.3007x + 19.9981, and the correlation coefficient is 0.9903 (n=3). The detection limit was 0.018 μM calculated by 3σ-rule (Fig. 8b).

To verify the selectivity of the prepared copper-nanoflower@AuNPs-GO NF-based glucose sensor, chronoamperometric analysis was performed under various component conditions that could interfere with the interaction of the target and the sensor. As a result, it was found that the current did not change even when other interfering substances such as ascorbic acid, saccharose, urea, NaCl, KCl and BSA were added ( FIG. 8c ). On the other hand, the current was significantly increased only when glucose was injected, indicating that copper-nanoflower@AuNPs-GO NFs acted as a selective glucose sensor for trace glucose.

To evaluate the time-dependent stability of copper-nanoflower@AuNPs-GO NF electrodes, a 30 μM glucose solution was analyzed for 20 days. As a result, compared with the first day, the current value was maintained at 95% until the 16th day, and showed more than 91% even after 20 days (FIG. 8d).

10: metal chip
20: nanofiber
30: metal nanoparticles
40: enzyme nanoparticle complex
100: biosensor electrode

Claims (13)

An electrode for glucose detection, comprising:
The electrode is a metal chip, nanofiber, metal nanoparticles and enzyme nanoparticle complex are sequentially stacked,
The metal chip is made of gold (Au),
The nanofiber is composed of a conductive material and a polymer, and is insoluble through firing,
The enzyme nanoparticle complex will include glucose oxidase, peroxidase and copper, the electrode.
delete The method according to claim 1,
The nanofiber is an electrode comprising a conductive material and a polymer in a volume ratio of 1:1 to 30.
The method according to claim 1,
The conductive material is graphene oxide and/or an ionic material, the electrode.
The method according to claim 1,
The polymer is one or more selected from the group consisting of polyvinyl chloride, polyvinyl alcohol, polyetherimide and polyethylene glycol, the electrode.
The method according to claim 1,
The metal nanoparticles are one or more selected from the group consisting of platinum (Pt), gold (Au), silver (Ag) and copper (Cu), the electrode.
The method according to claim 1,
The metal nanoparticles are bonded to the surface of the nanofiber by electrostatic attraction or covalent bonding, the electrode.
delete A glucose detection sensor comprising the electrode for glucose detection of any one of claims 1, 3 to 7.
a) forming and firing nanofibers on a metal chip using electrospinning;
b) coating the metal nanoparticles on the fired nanofibers; and
c) dripping the enzyme nanoparticle complex on the coated metal nanoparticles
includes,
The metal chip of step a) is made of gold (Au),
The nanofiber of step b) is composed of a conductive material and a polymer, and is insoluble through firing,
The method for manufacturing an electrode for glucose detection, wherein the enzyme nanoparticle complex comprises glucose oxidase, peroxidase and copper.
delete 11. The method of claim 10,
In the nanofiber of step a), the mixing ratio of the conductive material and the polymer is a volume ratio of 1:1 to 30, the method.
delete

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