Glucose electrochemical sensor based on protein passivated graphene as well as preparation method and application of glucose electrochemical sensor

Abstract

The invention discloses a glucose electrochemical sensor based on protein passivated graphene as well as a preparation method and application of the glucose electrochemical sensor. The glucose electrochemical sensor comprises a glass substrate, a working electrode, a reference electrode, a counter electrode, Ag / AgCl ink coated on the surface of the reference electrode, and a composite structure formed on the surface of the working electrode, and the composite structure is composed of Prussian blue, graphene, a protein film and glucose oxidase which are sequentially modified on the surface of the working electrode. According to the present invention, the Prussian blue, the graphene, the protein film and the glucose oxidase are sequentially modified on the surface of the working electrode to construct the glucose electrochemical sensor based on the protein passivated graphene, and the glucose electrochemical sensor has characteristics of good specificity, good reproducibility and good long-term stability, therefore, the method has a good application prospect in glucose detection.

Description

Technical Field

[0001] The present invention relates to the field of sensors and preparation thereof, and more specifically to a glucose electrochemical sensor based on protein passivated graphene and a preparation method and application thereof. Background Art

[0002] Electrochemical biosensors based on glucose oxidase (GOx) are highly favored due to their high selectivity, high sensitivity, low cost and fast response time. At present, the main methods for fixing enzymes in glucose sensors are drop coating, embedding and covalent crosslinking. The drop coating method mainly binds the enzyme to the electrode surface by physical adsorption. This method is simple but easy to fall off. The use of Nafion membrane can protect the enzyme to a certain extent, but studies have found that its performance will be greatly reduced in complex biological fluid environments. In addition, the Nafion layer modified on the planar electrode needs further research to prove its feasibility. The embedding method is to embed the enzyme in a gel or polymer matrix. This method can protect the activity of the enzyme, but may affect the diffusion of the substrate. The covalent crosslinking method is to couple the enzyme to the carrier through a chemical reaction to achieve stable binding. Obviously, these methods can greatly improve the stability of the enzyme and the reusability of this electrochemical sensor. Some covalently coupled electrochemical structures based on three-dimensional structures show particularly good electrochemical detection performance, but most covalent coupling methods involve relatively complex material layers or material synthesis methods, and basically do not consider the stability of Prussian blue (PB).

[0003] In the enzymatic reaction, GOx catalyzes the oxidation of glucose to produce gluconolactone and hydrogen peroxide. PB is a complex hexacyanoferrate iron (HCF), known as an "artificial peroxidase", which consists of metal centers Fe(II) and Fe(III). The iron(II) ion is surrounded by carbon atoms and the Fe(III) ion is surrounded by nitrogen atoms. It is oxidized to H 2 O 2 The low reduction potential and high selectivity allow for interference-free H 2 O 2 Detection. Therefore, PB is widely used as an electrocatalyst to promote the transfer of ions to electrons. PB complexes can be obtained by chemical and electrochemical routes. Among them, electrochemical synthesis is the most widely used method in the development of PB-modified electrochemical biosensors.

[0004] Although electrochemical biosensors based on Prussian blue (PB) show excellent performance, their catalytic activity decreases over time in the presence of alkali metal ions. Moreover, the effect of pH is crucial: in neutral or alkaline solutions, the loss of catalytic activity increases, mainly due to the interaction of hydroxide ions with Fe 3+Currently, most of the liquids used for glucose detection, such as blood, sweat, and cell culture medium, are neutral or weakly alkaline. This change may further affect the performance and stability of the sensor. Therefore, evaluating the reproducibility and stability of biosensors is a necessary condition to ensure the reliability of measurement results.

[0005] Considering the above problems, it is of great significance to study new glucose sensors to achieve stable and repeatable glucose measurements. Summary of the invention

[0006] The purpose of the present invention is to provide a glucose electrochemical sensor based on protein passivated graphene and a preparation method thereof, so as to solve the problems of poor reproducibility and low stability of electrochemical biosensors in the prior art.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] According to a first aspect of the present invention, a glucose electrochemical sensor based on protein-passivated graphene is provided, comprising: a glass substrate, a working electrode, a reference electrode, a counter electrode, Ag / AgCl ink coated on the surface of the reference electrode, and a composite structure formed on the surface of the working electrode, wherein the composite structure is composed of Prussian blue, graphene, a protein film and glucose oxidase sequentially modified on the surface of the working electrode.

[0009] Preferably, the protein film is a BSA film.

[0010] Preferably, the graphene is single-layer graphene.

[0011] Preferably, the working electrode, the reference electrode and the counter electrode are all made of ITO (indium tin oxide) material. It should be understood that ITO is an inorganic composite material, mainly composed of 90% indium oxide (In 2 O 3 ) and 10% tin oxide (SnO 2 ) with high light transmittance and excellent conductivity. Due to its unique electronic structure and crystal structure, ITO has a light transmittance of 85% to 95% in the visible light range.

[0012] According to a second aspect of the present invention, a method for preparing a glucose electrochemical sensor based on protein passivated graphene is provided, comprising the following steps: 1) providing a glass substrate having an ITO layer; 2) performing laser etching patterning on the ITO layer to form a working electrode, a counter electrode and a reference electrode respectively; 3) applying Ag / AgCl ink on the reference electrode and heating it in an oven until it is completely dry; 4) synthesizing Prussian blue by an electrochemical method and depositing it on the surface of the working electrode, repeatedly washing the electrode surface with deionized water to remove the incompletely deposited Prussian blue, and blowing the surface dry with nitrogen; 5) using a wet transfer strategy to transfer the Prussian blue to the working electrode surface; The copper-based single-layer graphene is slightly transferred to the working electrode; 6) 50-200 mg / mL of protein film solution is dripped on the working electrode and heated in a 70-90°C oven for 2-5 minutes to form a uniform film; 7) The working electrode is protected by an overlay process, and the residual graphene and protein film outside the working electrode are removed under plasma cleaning; 8) Glucose oxidase solution is dripped on the surface of the working electrode and placed in a 30-40°C oven for incubation for 1-2 hours. After the incubation, the electrode surface is cleaned with PBS to obtain a glucose electrochemical sensor based on protein passivated graphene.

[0013] Preferably, step 1) comprises: using a glass substrate having an ITO layer of 185 nm and a square resistance of 6-8Ω formed by magnetron sputtering.

[0014] Preferably, step 4) comprises: immersing the working electrode in a solution containing 2.5 mmol L -1 FeCl 3 , 2.5mmol L -1 K 3 [Fe(CN) 6 ]、100mmol L -1 KCl and 1 mmol L -1 In a solution composed of HCl, cyclic voltammetry was performed in the range of -0.15-0.35 V at 50 mV s -1 The scan rate was cycled for 30 cycles to deposit Prussian blue on the surface of the working electrode.

[0015] Preferably, step 6) comprises: dropping 100 mg / mL BSA solution on the working electrode and heating in an oven at 80° C. for 3 minutes to form a uniform BSA film.

[0016] Preferably, step 8) comprises: mixing EDC, NHS and glucose oxidase solution in a volume ratio of 1:1:1, dropping the mixture on the surface of the working electrode, and incubating the mixture in an oven at 30-40°C for 1-2 hours, wherein the contents of EDC, NHS and glucose oxidase solution are 100-500 mg / mL, 20-100 mg / mL and 0.2-2 mg / mL, respectively.

[0017] According to a preferred embodiment of the present invention, the contents of EDC, NHS and Gox solution are 500 mg / mL, 100 mg / mL and 2 mg / mL respectively.

[0018] It should be understood that step 8) is not limited to these three reagents, wherein the glucose oxidase solution can be replaced by a lactate oxidase solution, thereby preparing a lactate electrochemical sensor for detecting the lactate content.

[0019] According to a third aspect of the present invention, there is provided a use of the above-described protein-passivated graphene-based glucose electrochemical sensor in glucose detection.

[0020] The main inventive point of the present invention is that a composite structure is formed by sequentially modified Prussian blue, graphene, protein film and glucose oxidase on the surface of the working electrode. In the preparation method of the glucose sensor, the copper-based monolayer graphene is transferred to the surface of the Prussian blue by a wet transfer strategy, and the protein film is prepared on the monolayer graphene to couple the glucose oxidase in a covalently bonded manner, which is first proposed and realized by the present invention. The present invention also proves through experiments that the presence of graphene can protect the Prussian blue, prevent the corrosion of alkali metal ions in the reaction process, and enhance the stability of the sensor in repeated measurements. The preparation of protein film-coupled glucose oxidase on graphene is also one of the inventive points of the present invention, because most sensors based on glucose oxidase use physical adsorption to fix the enzyme, while the present invention uses chemical covalent bonding, which has stronger bonding and achieves better stability, thereby effectively avoiding the shedding phenomenon of glucose oxidase during the test process.

[0021] A glucose electrochemical sensor based on protein-passivated graphene and a preparation method and application thereof provided by the present invention have the following significant advantages over the prior art:

[0022] After lactic acid, creatinine and uric acid were added to the low concentration glucose solution in sequence, the measurement results showed that the current increased only when glucose was added, while the current response remained almost unchanged when interfering substances were added. Therefore, the glucose electrochemical sensor has good specificity for glucose detection.

[0023] After 10 measurements, the glucose electrochemical sensor retained 95% of the initial current response, so the glucose electrochemical sensor has good reproducibility;

[0024] After being stored in PBS at 4°C for 7 days, the original detection performance can still be maintained, so the glucose electrochemical sensor also has long-term stability;

[0025] By replacing the glucose oxidase solution in the construction process with a lactate oxidase solution, it is also possible to prepare a lactate electrochemical sensor for detecting lactate content.

[0026] In summary, according to the present invention, a composite structure is obtained by sequentially modifying the surface of a working electrode with Prussian blue, graphene, a protein film and glucose oxidase, thereby constructing a glucose electrochemical sensor based on protein-passivated graphene. The glucose electrochemical sensor has good specificity, reproducibility and long-term stability, and therefore has good application prospects in glucose detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The overall structure of a glucose electrochemical sensor provided according to a preferred embodiment of the present invention is shown;

[0028] Figure 2 The construction process of the glucose electrochemical sensor is shown;

[0029] Figure 3 The XPS (X-ray photoelectron spectroscopy) spectra of the four elements of carbon, nitrogen, oxygen and sulfur at different stages in the construction process of the glucose electrochemical sensor are shown;

[0030] Figure 4 CV (cyclic voltammetry) characterization of the modification process of the glucose electrochemical sensor in 0.1 M KCl;

[0031] Figure 5 The EIS (electrochemical impedance spectroscopy) characterization of the modification process of the glucose electrochemical sensor in 0.1 M KCl;

[0032] Figure 6 The electrochemical glucose sensor showed that the glucose -1 The current response of glucose solution;

[0033] Figure 7 The specificity test results of the glucose electrochemical sensor are shown;

[0034] Figure 8 The reproducibility test results of the glucose electrochemical sensor are shown;

[0035] Fig. 9 The long-term stability test results of the glucose electrochemical sensor are shown;

[0036] Fig.10 The working principle of the reagents in step 8) of the preparation method of the glucose electrochemical sensor is shown. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the instrument and equipment manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0038] Example 1 Preparation of Glucose Electrochemical Sensor Based on Protein Passivated Graphene

[0039] According to a preferred embodiment of the present invention, a glucose electrochemical sensor is provided, the overall structure of which is as follows: Figure 1 As shown, it includes: a glass substrate 1, an ITO working electrode 2, an ITO reference electrode 3, an Ag / AgCl ink 4 coated on the ITO reference electrode 3, and a composite structure 5 constructed on the surface of the ITO working electrode 2. The composite structure 5 is formed by sequentially modifying the surface of the ITO working electrode 2 with Prussian blue, graphene, a protein film and glucose oxidase.

[0040] like Figure 2 As shown, the preparation method of the glucose electrochemical sensor includes the following steps:

[0041] 1) This sensor adopts a standard electrochemical three-electrode structure. The substrate of the entire electrochemical electrode is a glass substrate with an ITO layer of 185nm and a square resistance of 6-8Ω formed by magnetron sputtering.

[0042] 2) The ITO was patterned by laser etching to form a working electrode (WE), a counter electrode (CE) and a reference electrode (RE).

[0043] 3) One of the electrodes was used as a reference electrode and coated with Ag / AgCl ink, and heated in an oven at 120°C for 5 minutes until completely dry. The other two electrodes were the working electrode and the counter electrode, respectively.

[0044] 4) Prussian blue was synthesized by electrochemical method and deposited on the ITO electrode. Specifically, the working electrode was immersed in 2.5 mmol L -1 FeCl 3 , 2.5mmol L -1 K 3[Fe(CN) 6 ]、100mmol L -1 KCl and 1 mmol L -1 In a solution composed of HCl, cyclic voltammetry was performed in the range of -0.15-0.35 V at 50 mV s -1 The scan rate was cycled for 30 cycles to deposit Prussian blue on the surface of the working electrode. The electrode surface was then repeatedly washed with deionized water to remove the incompletely deposited Prussian blue, and the surface was blown dry with nitrogen.

[0045] 5) As a protective layer of Prussian blue, the copper-based single-layer graphene was transferred to the working electrode using a traditional wet transfer strategy.

[0046] 6) Add 100 mg / mL BSA solution onto the electrode and heat it in an oven at 80°C for three minutes to form a uniform BSA film.

[0047] 7) Protect the working electrode by overlay process, and remove the residual graphene and BSA film outside the working electrode under plasma cleaning. Specifically, AZ5214 positive photoresist is first spin-coated on the previously modified electrode, pre-baked at 100 degrees for 1 minute, and then the electrode is photolithographically processed in a laser direct writing lithography machine with an exposure dose of 400mj / cm 2 After the photolithography is completed, the electrode is developed in a developer (the developer is a mixture of AZ400k and deionized water in a volume ratio of 1:3) for 10-20 seconds. After the development is completed, only the photoresist of the specified shape will remain at the working electrode position, and the rest of the positions will be dissolved.

[0048] 8) EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide), NHS (N-hydroxysuccinimide) and GOx (glucose oxidase) solutions were mixed at a volume ratio of 1:1:1 and placed in a 37°C oven for 1 hour. The contents of EDC, NHS and Gox solutions were 500 mg / mL, 100 mg / mL, and 2 mg / mL, respectively. After the incubation, the electrode surface was washed with PBS to remove unbound and physically adsorbed GOx.

[0049] It should be understood that in step 8), the working principle of the reagent is as follows Fig.10 As shown, the R1 group in (1) represents the dBSA film, and the R2 group in (3) represents Gox, and coupling is performed through the reaction process shown in the figure.

[0050] like Figure 3As shown, the XPS (X-ray photoelectron spectroscopy) spectra of the four elements of carbon, nitrogen, oxygen, and sulfur at different stages in the construction process of the electrochemical sensor are shown. In the figure, PB represents Prussian blue, Gr represents graphene, dBSA represents protein membrane, and GOx represents glucose oxidase. Different characteristic peaks prove the success of sensor modification. Among them, PB, Gr, dBSA and GOx are marked on the left side of each small figure, representing four modification processes, and ABCD respectively represent the XPS spectra of the corresponding elements in each modification process (carbon, oxygen, nitrogen and sulfur four elements).

[0051] Figure 4 and Figure 5 The modification process of the sensor was characterized by CV (cyclic voltammetry) and EIS (electrochemical impedance spectroscopy) in 0.1M KCl. The CV results showed that the presence of graphene and dBSA film did not affect the redox characteristics of PB. In addition, the EIS test results showed that the presence of dBSA film led to an increase in low-frequency impedance, while the presence of glucose oxidase promoted charge transfer on the electrode surface.

[0052] Example 2 Glucose Detection Ability Test

[0053] Glucose was added to the sugar-free medium to prepare 0.05-50 mmol L -1 The current response of the sensor in this range was tested by glucose solution. Figure 6 ). Within this range, the current signal has a nonlinear relationship with the glucose concentration, which can be well fitted by the Hill model:

[0054] Among them, I min is the current of the sensor at the lowest concentration. I max is the current of the sensor at the highest concentration. k is the glucose concentration at which the half-maximum current response is achieved, which reflects the sensitivity of the sensor to glucose. n is the Hill coefficient, which describes the cooperative nature of the sensor's response to changes in glucose concentration. Figure 6 The fitting curve in I min ,I max , k and n are 0.457, 22.133, 2.147 and 1.413 respectively.

[0055] Example 3 Sensor specificity and stability test

[0056] The influence of interfering electrochemical active substances on the test signal and the repeatability of the analytical signal response are important characteristics of biosensors. The anti-interference ability of the sensor was tested by adding metabolites that may be produced during cell culture. 1 mmol L -1After adding lactate, creatinine and uric acid, the current response of the sensor was measured ( Figure 7 ). The current increased only when glucose was added, while the current response remained almost unchanged or slightly decreased when interfering substances were added, which may be due to the decrease in glucose concentration. Therefore, under specific experimental conditions, the developed biosensor has good specificity for the detection of glucose.

[0057] In the reproducibility study, 1 mmol L -1 The glucose concentration was determined as the current response measurement value, and the reproducibility of the glucose biosensor was evaluated by continuous measurements (n = 10). Figure 8 After 10 measurements, the sensor retained 95% of the initial current response. However, the sensor without the graphene layer, that is, the ITO / PB / dBSA / Gox sensor without the graphene layer, only had 85% of the initial current response. This shows that the protein-passivated Gr layer has a significant effect on improving the stability of the sensor (as shown in Figure 1). Figure 8 (as shown in B in the figure).

[0058] The detection after storage in a liquid environment is also an important indicator for measuring the performance of the sensor. In order to test the long-term stability of the glucose sensor, we immersed the electrochemical biosensor prepared in Example 1 in a 4°C PBS solution. First, the sensor was recorded for the initial measurement of 1 mmol L -1 The current response to glucose solution was then tested again at specific times over the next 14 days ( Fig. 9 ). On the 7th day, about 94% of the current response remained unchanged. However, on the 14th day, it was only 72% of the initial value, which may be related to the shedding and decreased activity of glucose oxidase. Therefore, it can be considered that the glucose sensor based on this structure can be stored in PBS at 4°C for 7 days and maintain the original detection performance.

[0059] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A glucose electrochemical sensor based on protein-passivated graphene, characterized in that: include: A glass substrate, a working electrode, a reference electrode, a counter electrode, Ag / AgCl ink coated on the surface of the reference electrode, and a composite structure formed on the surface of the working electrode, wherein the composite structure is composed of Prussian blue, graphene, a protein film and glucose oxidase sequentially modified on the surface of the working electrode.

2. The glucose electrochemical sensor according to claim 1, characterized in that: The protein film is a BSA film.

3. The glucose electrochemical sensor according to claim 1, characterized in that: The graphene is single-layer graphene.

4. The glucose electrochemical sensor according to claim 1, characterized in that: The working electrode, reference electrode and counter electrode are all made of ITO material.

5. A method for preparing a glucose electrochemical sensor based on protein-passivated graphene according to any one of claims 1 to 4, characterized in that: The following steps are involved: 1) providing a glass substrate having an ITO layer; 2) performing laser etching and patterning on the ITO layer to form a working electrode, a counter electrode and a reference electrode respectively; 3) applying Ag / AgCl ink on the reference electrode and heating it in an oven until it is completely dry; 4) synthesizing Prussian blue by an electrochemical method and depositing it on the surface of the working electrode, repeatedly washing the electrode surface with deionized water to remove the incompletely deposited Prussian blue, and blowing the surface dry with nitrogen; 5) transferring the copper-based single-layer graphene to the working electrode using a wet transfer strategy; 6) adding 50-200 mg / mL of protein film solution to the working electrode and heating in an oven at 70-90° C. for 2-5 minutes to form a uniform film; 7) protecting the working electrode by an overlay process, and removing the graphene and protein film remaining outside the working electrode under plasma cleaning; 8) dripping glucose oxidase solution onto the surface of the working electrode, and incubating in an oven at 30-40° C. for 1-2 hours. After the incubation, washing the electrode surface with PBS, thereby obtaining a glucose electrochemical sensor based on protein-passivated graphene.

6. The preparation method according to claim 5, characterized in that: Step 1) comprises: using a glass substrate having an ITO layer of 185 nm and a square resistance of 6-8Ω formed by magnetron sputtering.

7. The preparation method according to claim 5, characterized in that: Step 4) comprises: immersing the working electrode in 2.5 mmol L -1 FeCl3, 2.5mmol L -1 K3[Fe(CN)6], 100mmol L -1 KCl and 1 mmol L -1 In a solution composed of HCl, cyclic voltammetry was performed in the range of -0.15-0.35 V at 50 mV s -1 The scan rate was cycled for 30 cycles to deposit Prussian blue on the surface of the working electrode.

8. The preparation method according to claim 5, characterized in that: Step 6) includes: dropping 100 mg / mL BSA solution on the working electrode and heating in an oven at 80° C. for 3 minutes to form a uniform BSA film.

9. The preparation method according to claim 5, characterized in that: Step 8) comprises: mixing EDC, NHS and glucose oxidase solution in a volume ratio of 1:1:1, dropping on the surface of the working electrode, and incubating in a 30-40°C oven for 1-2 hours, wherein the contents of EDC, NHS and glucose oxidase solution are 100-500 mg / mL, 20-100 mg / mL and 0.2-2 mg / mL, respectively.

10. Use of the glucose electrochemical sensor based on protein-passivated graphene according to any one of claims 1 to 4 in glucose detection.

Images