阅读说明：本技术 一种基于ZnO纳米材料的电化学生物传感器及其进行葡萄糖浓度检测的方法 (Electrochemical biosensor based on ZnO nano material and method for detecting glucose concentration by using electrochemical biosensor ) 是由 杨娟 杨占军 陈建忠 平文卉 顾俊 陈�峰 杨清华 戴志英 杨梅桂 张颖茜 施逸 于 2020-11-30 设计创作，主要内容包括：本发明涉及一种基于ZnO纳米材料的葡萄糖电化学生物传感器及其进行葡萄糖检测的方法,首先将ZnO纳米材料与葡萄糖氧化酶共同修饰到玻碳电极表面得到葡萄糖电化学生物传感器；采用该葡萄糖电化学生物传感器进行葡萄糖浓度的电化学检测,方法为：在检测池中以PBS缓冲溶液为基液,以上述葡萄糖电化学生物传感器为工作电极,连续滴加不同浓度的葡萄糖溶液得到电流–时间曲线；再拟合上述检测条件下葡萄糖浓度与电流的标准曲线；再按上述电流–时间曲线法,在检测池,同样的检测池中加入一定体积的葡萄糖待检测溶液,读取工作电极的响应电流A值；最后根据上述标准曲线上描点确定检测池中葡萄糖的浓度,再换算得到第三步中葡萄糖待检测溶液的浓度。(The invention relates to a glucose electrochemical biosensor based on ZnO nano material and a method for detecting glucose by the same, firstly, the ZnO nano material and glucose oxidase are jointly modified on the surface of a glassy carbon electrode to obtain the glucose electrochemical biosensor; the electrochemical detection of the glucose concentration by adopting the glucose electrochemical biosensor comprises the following steps: continuously dripping glucose solutions with different concentrations in a detection cell by taking PBS buffer solution as base solution and the glucose electrochemical biosensor as a working electrode to obtain a current-time curve; fitting a standard curve of the glucose concentration and the current under the detection condition; adding a certain volume of glucose solution to be detected into a detection cell according to the current-time curve method, and reading the response current A value of the working electrode; and finally, determining the concentration of the glucose in the detection cell according to the points on the standard curve, and then converting to obtain the concentration of the glucose solution to be detected in the third step.)

1. An electrochemical biosensor based on a ZnO nano material is characterized in that the ZnO nano material and glucose oxidase are jointly modified on the surface of a glassy carbon electrode to obtain the glucose electrochemical biosensor.

2. The electrochemical biosensor based on ZnO nanomaterial of claim 1, which is prepared by the following method:

step 1, dispersing a ZnO nano material in deionized water, uniformly mixing by ultrasonic oscillation to prepare a dispersion liquid with the dispersion concentration of 1-2 mg/mL, and adding glucose oxidase to ensure that the mass of the glucose oxidase in the solution is 5 times of that of ZnO; ultrasonic oscillation is carried out until the materials are completely dissolved and uniformly mixed, so that glucose oxidase is uniformly loaded on the surface of the ZnO nano material;

step 2, polishing the glassy carbon electrode by using 0.3 mu m and 0.5 mu m of alumina powder in sequence, washing away residual alumina powder by using deionized water, placing the polished glassy carbon electrode into a dilute nitric acid aqueous solution for ultrasonic cleaning, and finally, cleaning the glassy carbon electrode by using ethanol and secondary distilled water in sequence, and drying the glassy carbon electrode at low temperature or drying the glassy carbon electrode by using nitrogen;

step 3, dripping the mixed solution obtained in the step 1 on the surface of the glassy carbon electrode dried after the treatment in the step 2, and then drying the glassy carbon electrode at low temperature;

and 4, modifying a naphthol film on the surface of the glassy carbon electrode dried in the step 3 to obtain the ZnO nano material-based glucose electrochemical biosensor, and storing the electrochemical biosensor at a constant temperature of 4 ℃.

3. The electrochemical biosensor based on ZnO nanomaterial of claim 1, wherein in step 4, the method for modifying a layer of naphthol film on the surface of the glassy carbon electrode comprises: and dripping 0.5 mass percent naphthol water solution on the surface of the glassy carbon electrode, and drying the glassy carbon electrode at low temperature.

4. The electrochemical biosensor based on ZnO nanomaterial as defined in claim 2 or 3, wherein the glassy carbon electrode is dried at constant temperature of 4 ℃ for 10-16 hours.

5. The electrochemical biosensor based on ZnO nano-material of claim 1, wherein the preparation method of the ZnO nano-material comprises: and (3) mixing the raw materials in a molar ratio of 0.02-0.06: 2, mixing the zinc dichloride, the urea and the deionized water in a container with a polytetrafluoroethylene lining to ensure that the mixing concentration of the zinc dichloride in the mixed solution is 0.02-0.06 mol L⁻¹The mixed concentration of urea is 2 mol L⁻¹Uniformly stirring the mixed solution, adjusting the pH value to 3.5-4.5 by using dilute hydrochloric acid, then transferring the mixed solution and a container into a high-pressure reaction kettle, heating for 10-12 h at 80-90 ℃, naturally cooling a reaction system to room temperature in the reaction kettle, respectively washing white precipitates obtained by filtering the reaction system for 5 times by using absolute ethyl alcohol and water, drying for 10-12 h at 60 ℃ in a vacuum drying box, grinding to the particle size of 10-20 nm or less, and calcining the ground powder for 30-40 min at 300-350 ℃ to obtain the ZnO nano material.

6. A method for detecting a glucose concentration using the electrochemical biosensor according to any one of claims 1 to 4,

first, a current-time curve of the glucose solution is prepared: in a detection pool, continuously stirring, air-saturating, 0.1M PBS solution with pH of 7.0 is used as a test base solution, an electrochemical biosensor is used as a working electrode, a saturated calomel electrode is used as an auxiliary electrode, a platinum sheet electrode is used as a counter electrode, glucose test solution with known concentration is sequentially added into the detection pool in a definite volume amount, an electrochemical signal of the working electrode is detected, and simultaneously, a current response value and an integral current response curve of the glucose test solution with known concentration which is quantitatively added each time are output;

secondly, taking the concentration value of the glucose in the detection pool after adding the glucose into the volume-fixed amount in the first step as an abscissa and taking the corresponding current response value as an ordinate, and tracing and fitting a standard curve of the glucose concentration and the response current;

thirdly, according to the method for manufacturing the current-time curve in the first step, in a detection pool, a PBS solution which is continuously stirred and has an air saturation pH value of 7.0 is used as a test base solution, an electrochemical biosensor is used as a working electrode, a saturated calomel electrode is used as an auxiliary electrode, a platinum sheet electrode is used as a counter electrode, a certain volume of glucose solution to be detected is added into the detection pool, and the response current A value of the working electrode is read;

and fourthly, drawing points on the response current A value read in the third step on the standard curve in the second step to determine the concentration of the glucose in the detection cell, and then converting to obtain the concentration of the glucose solution to be detected in the third step.

7. The method for detecting glucose concentration according to claim 5, wherein the concentration of the PBS solution in the first step is 0.1 mol/L.

Technical Field

The invention relates to the technical field of electrochemical analysis and detection, in particular to a ZnO nano material-based glucose electrochemical biosensor and a method for detecting glucose by using the same.

Background

Diabetes can cause various complications of the body, has great harm to the body, and has no method or medicine for thoroughly curing the diabetes at present, so the early diagnosis of the diabetes and the daily monitoring of the blood sugar of a human body are particularly important. The health condition of people can be indirectly known by detecting the concentration of glucose in the blood of a human body, and a means is provided for the diagnosis of diabetes.

Currently, methods for detecting the glucose concentration include an electrochemical sensor method, a photochemical method (i.e., a biochemical analyzer), an optoacoustic spectroscopy, and a raman spectroscopy. The photochemical method has harsh measurement conditions, consumes time and requires a large amount of blood; the photoacoustic spectrometry detection method has high sensitivity but has high requirements on environmental conditions so as to influence the detection result; raman spectrometry measurement disadvantages: because the detection part is the anterior chamber of the eye, the light intensity cannot be increased, the detection signal is weak, and the signal processing is difficult; the electrochemical biosensor method has the advantages of high detection accuracy, simple operation, high analysis speed, small size, portability, low instrument price and the like.

At present, researchers are continuously striving for high sensitivity and high accuracy of detection when using electrochemical analysis techniques for glucose concentration.

Disclosure of Invention

The invention provides an electrochemical biosensor for detecting the ZnO nano material, aiming at solving the problems existing in the glucose concentration detection technology in the prior art, and the electrochemical biosensor for detecting the ZnO nano material can realize the high-sensitivity detection of the glucose concentration.

The invention aims to realize the electrochemical biosensor based on the ZnO nano material, and is characterized in that the ZnO nano material and glucose oxidase are jointly modified on the surface of a glassy carbon electrode to obtain the glucose electrochemical biosensor.

The nanometer ZnO particles of the glucose electrochemical biosensor have a flower structure which is wrapped in a stacked mode, so that a better biocompatibility microenvironment can be provided for glucose oxidase, the glucose oxidase can be fixed by utilizing the nanometer ZnO particles, the biological activity of the enzyme can be kept, the linear range of detection is widened, the electron transfer rate during detection is improved, and the detection sensitivity is improved; and the glucose oxidase modified on the surface of the ZnO particles is utilized to realize the quantitative detection of glucose, and the method has the characteristics of good photochemical stability, electronic communication performance, strong adsorption capacity, good surface activity and high catalytic efficiency.

Further, the electrochemical biosensor of the present invention is prepared by the following method:

step 1, dispersing a ZnO nano material in deionized water, uniformly mixing by ultrasonic oscillation to prepare a dispersion liquid with the dispersion concentration of 1-2 mg/mL, and adding glucose oxidase to ensure that the mass of the glucose oxidase in the solution is 5 times of that of ZnO; ultrasonic oscillation is carried out until the materials are completely dissolved and uniformly mixed, so that glucose oxidase is uniformly loaded on the surface of the ZnO nano material;

step 2, polishing the glassy carbon electrode by using 0.3 mu m and 0.5 mu m of alumina powder in sequence, washing away residual alumina powder by using deionized water, placing the polished glassy carbon electrode into a dilute nitric acid aqueous solution for ultrasonic cleaning, and finally, cleaning the glassy carbon electrode by using ethanol and secondary distilled water in sequence, and drying the glassy carbon electrode at low temperature or drying the glassy carbon electrode by using nitrogen;

step 3, dripping the mixed solution obtained in the step 1 on the surface of the glassy carbon electrode dried after the treatment in the step 2, and then drying the glassy carbon electrode at low temperature;

and 4, modifying a naphthol film on the surface of the glassy carbon electrode dried in the step 3 to obtain the ZnO nano material-based glucose electrochemical biosensor, and storing the electrochemical biosensor at a constant temperature of 4 ℃.

Further, in the step 4, the method for modifying the surface of the glassy carbon electrode with a layer of naphthol film comprises the following steps: and dripping 0.5 mass percent naphthol water solution on the surface of the glassy carbon electrode, and drying the glassy carbon electrode at low temperature.

Furthermore, the glassy carbon electrode is dried at a constant temperature of 4 ℃ for 10-16 hours at a low temperature.

Further, the preparation method of the ZnO nano material comprises the following steps: and (3) mixing the raw materials in a molar ratio of 0.02-0.06: 2, mixing the zinc dichloride, the urea and the deionized water in a container with a polytetrafluoroethylene lining to ensure that the mixing concentration of the zinc dichloride in the mixed solution is 0.02-0.06 mol L⁻¹The mixed concentration of urea is 2 mol L⁻¹Uniformly stirring the mixed solution, adjusting the pH value to 3.5-4.5 by using dilute hydrochloric acid, then transferring the mixed solution and a container into a high-pressure reaction kettle, heating for 10-12 h at 80-90 ℃, naturally cooling a reaction system to room temperature in the reaction kettle, respectively washing white precipitates obtained by filtering the reaction system for 5 times by using absolute ethyl alcohol and water, drying for 10-12 h at 60 ℃ in a vacuum drying box, grinding to the particle size of 10-20 nm or less, and calcining the ground powder for 30-40 min at 300-350 ℃ to obtain the ZnO nano material.

The invention also provides a method for detecting the glucose concentration by adopting the electrochemical biosensor based on the ZnO nano material, which comprises the following steps:

first, a current-time curve of the glucose solution is prepared: in a detection pool, continuously stirring, air-saturating, 0.1M PBS solution with pH of 7.0 is used as a test base solution, an electrochemical biosensor is used as a working electrode, a saturated calomel electrode is used as an auxiliary electrode, a platinum sheet electrode is used as a counter electrode, glucose test solution with known concentration is sequentially added into the detection pool in a definite volume amount, an electrochemical signal of the working electrode is detected, and simultaneously, a current response value and an integral current response curve of the glucose test solution with known concentration which is quantitatively added each time are output;

secondly, taking the concentration value of the glucose in the detection pool after adding the glucose into the volume-fixed amount in the first step as an abscissa and taking the corresponding current response value as an ordinate, and tracing and fitting a standard curve of the glucose concentration and the response current;

thirdly, according to the method for manufacturing the current-time curve in the first step, in a detection pool, a PBS solution which is continuously stirred and has an air saturation pH value of 7.0 is used as a test base solution, an electrochemical biosensor is used as a working electrode, a saturated calomel electrode is used as an auxiliary electrode, a platinum sheet electrode is used as a counter electrode, a certain volume of glucose solution to be detected is added into the detection pool, and the response current A value of the working electrode is read;

and fourthly, drawing points on the response current A value read in the third step on the standard curve in the second step to determine the concentration of the glucose in the detection cell, and then converting to obtain the concentration of the glucose solution to be detected in the third step.

The method can detect the concentration of glucose, provides a new electrochemical detection method for the detection of human blood sugar and the early diagnosis of diabetes, and has the advantages of simple and rapid operation, low cost, high sensitivity, good reproducibility and stability based on the excellent characteristics of the electrochemical biosensor.

In the first step, the concentration of the PBS solution was 0.1 mol/L.

Drawings

Fig. 1a and 1b are scanning electron micrographs of ZnO nanomaterial prepared in example 1 and ZnO loaded with glucose oxidase, respectively.

Fig. 2 is an XRD pattern of zinc oxide.

FIG. 3 is a comparison graph of a current response curve a of a method for detecting glucose concentration by using the GOx/ZnO/Nafion/GCE electrochemical biosensor in example 2 and a current response curve b of a method for detecting glucose concentration by using the GOx/Nafion/GCE electrochemical biosensor modified by glucose oxidase alone.

FIG. 4 is a standard curve of glucose concentration versus plateau response current obtained by point-wise fitting in the method for detecting glucose concentration according to the present invention.

Detailed Description

Example 1

(1) Firstly, preparing a flower-shaped ZnO nano material:

0.2726 g (0.05 mol L) were taken⁻¹) Zinc dichloride (ZnCl)₂，AR)、4.8048 g（2 mol L⁻¹) Urea (CON)₂H₄AR) and 35 ml of deionized water are placed in a polytetrafluoroethylene inner cup, the mixture is stirred for 5 min, then the pH value is adjusted to 4 by using dilute hydrochloric acid, the mixture is stirred for 10 min, the polytetrafluoroethylene inner cup is transferred into a stainless steel high-pressure reaction kettle and sealed, the stainless steel high-pressure reaction kettle is placed in an oven to be heated for 12 h at 90 ℃, when the system is naturally cooled to room temperature, white precipitates in the reaction system are obtained through separation and are centrifuged, the white precipitates are respectively washed for 5 times by using absolute ethyl alcohol and water, the anhydrous ethyl alcohol and the water are dried for 12 h at 60 ℃ in a vacuum drying oven, the materials are taken out and ground into granules with the granularity of less than 10-20 nm, and the ground granules are calcined for 30 min at 300 ℃ to obtain; the ZnO nano material is shown in figure 1a by a scanning electron microscope, the ZnO nano material is in a flower-shaped structure wrapped layer by layer, the grain size is about 12 microns, namely the ZnO nano material is composed of nano flaky zinc oxide, the thickness of the ZnO nano material is about 20 nanometers, and the flower structure wrapped layer by layer has a large specific surface area, so that a reliable surface area is provided for a large amount of loads of subsequent glucose oxidase;

(2) under the ultrasonic condition, uniformly dispersing the flower-shaped ZnO nano material prepared in the previous step into deionized water to form an aqueous solution with the ZnO content of 2 mg/mL;

(3) using 0.3 and 0.5 mu m Al for the glassy carbon electrode GCE in sequence₂O₃Polishing, washing with deionized water to remove residual alumina powder, and standingUltrasonic cleaning in dilute nitric acid solution, and finally repeatedly cleaning the surface of the glassy carbon electrode for 2 times by using ethanol and secondary distilled water in sequence to finish the cleaning pretreatment of the glassy carbon electrode;

(4) adding glucose oxidase (Gox) into the aqueous solution obtained in the step (2) according to the mass volume ratio of 10mg/mL, uniformly mixing, dropwise coating the mixed solution on the surface of the glassy carbon electrode subjected to cleaning pretreatment in the step (3), drying the electrode in a constant-temperature refrigerator at 4 ℃ for 16 hours, dropwise coating naphthol with the mass concentration of 0.5% on the surface of the electrode to obtain the glucose oxidase electrochemical biosensor (GOx/ZnO/Nafion/GCE), and storing the glucose oxidase electrochemical biosensor in the refrigerator at 4 ℃ for later use.

And (3) preparing the electrochemical biosensor (GOx/Nafion/GCE) without the ZnO nano material as a sensor for a subsequent comparative experiment according to the steps (3) and (4).

As shown in figure 1b, a scanning electron microscope image of ZnO surface modified with glucose oxidase shows that glucose oxidase is successfully loaded on the surface of flower-shaped zinc oxide, and compared with figure 1a, the flower-shaped zinc oxide has obviously different structural morphology, and a large amount of glucose oxidase is uniformly loaded on the surface of the petal of ZnO.

As shown in fig. 2, which is an XRD pattern of zinc oxide, the X-ray diffraction peak of pure zinc oxide is consistent with the diffraction peak of the standard hexagonal wurtzite ZnO structure. The diffraction peak of the synthesized product in the figure corresponds to the diffraction peak of the hexagonal wurtzite ZnO structure, no other impurity peak appears, and the product is pure zinc oxide according to an XRD (X-ray diffraction) diagram.

Example 2

In this embodiment, the GOx/ZnO/Nafion/GCE sensor and the GOx/Nafion/GCE sensor prepared in the above embodiment 1 are used to detect the glucose concentration. The specific detection method comprises the following steps:

firstly, a method for detecting the concentration of glucose by adopting a GOx/ZnO/Nafion/GCE sensor,

firstly, adding 10ml of continuously stirred, air-saturated and 0.1M-concentration PBS solution with the pH value of 7.0 as a test base solution, a GOx/ZnO/Nafion/GCE sensor as a working electrode, a saturated calomel electrode as an auxiliary electrode and a platinum sheet electrode as a counter electrode into a detection pool by a cyclic voltammetry at an electrochemical workstation, sequentially adding a glucose solution with the molar volume concentration of 0.1mol/L into the detection pool according to the volume of the table 1, detecting the current response value of the working electrode, and outputting a current response time curve of the glucose test solution with the known concentration quantitatively added each time, wherein the current response time curve is shown as a curve a in figure 3;

TABLE 1

；

Secondly, taking the concentration value of the glucose in the detection pool after adding the glucose into the volume-determined amount in the first step as an abscissa and the corresponding current response value as an ordinate, and performing point tracing to fit a standard curve of the glucose concentration and the response current, such as a curve a' shown in fig. 4;

thirdly, when the concentration of the glucose solution with unknown concentration is detected, adding 10ml of PBS solution which is continuously stirred, is saturated by air and has the concentration of 0.1M and the pH value of 7.0 into a detection pool as a test base solution according to the cyclic voltammetry of the first step, taking a GOx/ZnO/Nafion/GCE sensor as a working electrode, a saturated calomel electrode as an auxiliary electrode and a platinum sheet electrode as a counter electrode, adding a certain volume of glucose solution to be detected into the detection, and reading the response current A value of the working electrode;

and fourthly, the concentration of the glucose in the detection cell is determined by dotting the response current A value read in the third step on the standard curve a in the second step, and the concentration of the glucose solution to be detected in the third step is obtained through conversion according to the volume of the glucose solution added in the third step and the volume of the PBS solution.

A comparative example test was performed simultaneously: using the GOx/Nafion/GCE sensor without ZnO as a working electrode, sequentially adding glucose solutions with the same concentration according to the volume in the table 1 by the methods of the first step and the second step, sequentially detecting the current response of the working electrode, and finally outputting a response curve b shown in the figure 3; the standard curve is further point-fitted to the curve b' shown in fig. 4.

As can be seen from the standard curve in FIG. 4, the current response of the working electrode increases linearly with the glucose concentration, and the linear response range of GOx/ZnO/Nafion/GCE is wider than that of GOx/Nafion/GCE. These results indicate that the ZnO-immobilized glucose oxidase GOx has a high affinity for glucose, and that GOx/ZnO/Nafion/GCE-immobilized GOx still has a high activity and a good linear response range for glucose.

That is, by using the electrochemical detection method for glucose using the GOx/ZnO/Nafion/GCE sensor as the working electrode in this embodiment, an electrochemical detection method with a wider detection range, higher accuracy and sensitivity is provided for clinical detection of human blood glucose and early diagnosis of diabetes, and the method is simple and rapid to operate and has low cost. Good reproducibility and stability.

Example 3

In this embodiment, according to the electrochemical detection method of the first step in embodiment 2, a GOx/ZnO/Nafion/GCE sensor is used as a working electrode, 10 microliters of glucose solution with a concentration of 0.1mol/L is continuously and parallelly added into a detection cell to perform parallel detection for 6 times, and the obtained current response value of the working electrode has a relative standard deviation of 3.5%, which indicates that the sensor has high measurement precision.

Meanwhile, 3 GOx/ZnO/Nafion/GCE sensors and 3 detection cells with the same conditions are taken, 10 microliters of glucose solution with the concentration of 0.1mol/L is added respectively to carry out 6 times of repeated measurement according to the electrochemical detection method of the step 1 in the step 2 in the embodiment, and the obtained current has the relative standard deviation of 6.5 percent, which indicates that the detection result of the sensor has better reproducibility. After 6 times of continuous measurement of 3 sensors, the current response signals can reach 93% of the original response value, which shows that Nafion/GOx/ZnO/GCE can effectively maintain the activity of glucose oxidase and can prevent enzyme from falling off.

In addition, after the detection is finished, the Nafion/GOx/ZnO/GCE sensor is cleaned by deionized water, naturally dried and stored in a storage box 4^oC, stored in a refrigerator, and repeatedly examined after two weeks, no decrease in the current response of the enzyme electrode to glucose was observed.

Therefore, the electrochemical analysis method for the glucose concentration by adopting the GOx/ZnO/Nafion/GCE sensor is simple, rapid, low in cost, high in sensitivity, good in reproducibility and stability.