CN114894865A - High-precision glucose sensor and preparation method thereof - Google Patents
High-precision glucose sensor and preparation method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/38—Cleaning of electrodes
Abstract
The invention discloses a high-precision glucose sensor and a preparation method thereof. The method comprises the following steps: (1) activating the electrode surface of the three-electrode system, and then adsorbing the osmium redox polymer coupled with chitosan to the surface of a working electrode; (2) and (3) coating the enzyme solution containing the glucose oxidase on a reaction area of the working electrode, crosslinking at room temperature, and then cleaning and drying. The biosensor prepared by the invention has the service life of 14 days at 4 ℃, and has reversible electrochemical characteristics, low working potential and strong anti-interference capability.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a high-precision glucose sensor and a preparation method thereof.
Background
Diabetes has become one of the diseases threatening human health. Blood glucose monitoring in diabetics is important to control the condition of the disease. For the invasive tests generally adopted at present, patients mostly have fear and discomfort. Therefore, the research and development of noninvasive glucometers are the key subjects of the medical community of all countries. Among them, a reverse iontophoresis technique is used to obtain subcutaneous interstitial sugar molecules, and an electrochemical biosensor is used to test subcutaneous exuded glucose, and related studies have been carried out abroad. Compared with direct blood sampling for detecting blood sugar (mmol/L magnitude) of a human body, the concentration of subcutaneous exuded glucose is very low (mu mol/L magnitude), the traditional blood sugar biosensor can hardly meet the requirements on detection range and sensitivity, and a sensor capable of detecting low-concentration glucose can not be put into practical use due to the limitation of electrode manufacturing. Noninvasive detection of subcutaneous blood glucose was achieved by using a hydrogen peroxide sensor at 0.42V operating voltage. However, higher operating potentials tend to cause other electroactive species in the body fluid to interfere with the test.
To avoid the disadvantages of enzymes, enzyme-free glucose sensors are receiving increasing attention. Noble metals, such as platinum, gold and palladium, have been used in the development of enzyme-free glucose sensors due to their oxidation catalytic activity that is stable towards glucose. But the glucose oxidation catalysis kinetics is poor, the reaction time is long, the sensitivity is low, and reaction intermediates and Cl in electrolyte - Is toxic to noble metal electrodes. In addition, the electrochemical potential for glucose oxidation is typically high enough to catalyze some of the human body's own physiological substances, resulting in poor interference resistance.
In order to improve the sensitivity and selectivity, surface modification technologies such as electroplating, anodic oxidation and vapor deposition are studied to carry out surface modification on the sensing electrode. These techniques can increase the surface roughness of the electrode, accelerate the transfer of electrons from glucose to the electrode, and improve the sensitivity of the sensor. Furthermore, on noble metal electrodes, oxidation of common physiological interfering substances, such as Ascorbic Acid (AA) and Uric Acid (UA), is a diffusion-controlled process, whereas glucose oxidation is controlled by surface reactions. The increase in surface roughness reduces the diffusion of interfering substances, but does not affect the reaction rate of glucose, thereby improving selectivity. While in theory it is possible to increase the selectivity and sensitivity of the sensor by increasing the electrode surface roughness, in practice, this approach has limited improvement in performance.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a high-precision glucose sensor and a preparation method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a preparation method of a high-precision glucose sensor comprises the following steps:
(1) activating the electrode surface of the three-electrode system, and then adsorbing the osmium redox polymer coupled with chitosan to the surface of a working electrode;
(2) and (3) coating the enzyme solution containing the glucose oxidase on a reaction area of the working electrode, crosslinking at room temperature, and then cleaning and drying.
Furthermore, the three-electrode system consists of a glassy carbon working electrode, a glassy carbon counter electrode and an Ag/AgCl reference electrode.
Further, the process of the activation treatment is as follows:
and cleaning the surface of the electrode by sequentially adopting 50% nitric acid solution, absolute ethyl alcohol and ultrapure water.
Further, the osmium redox polymer coupled with chitosan was adsorbed to the surface of the working electrode at room temperature in the dark.
Further, glucose oxidase and osmium are crosslinked for 3-5 days in a vacuum environment through glutaraldehyde to form an osmium redox polymer, wherein the mass concentration of glutaraldehyde is 2-5%.
Furthermore, the concentration of glucose oxidase in the enzyme solution is 2-5U/mu L, and the adding amount of BSA is 1.5-2% of the volume of the enzyme solution.
Further, the cross-linking agent used in the cross-linking in the step (2) is glutaraldehyde, and the addition amount of the glutaraldehyde in the enzyme solution is 2-5% of the volume of the enzyme solution.
Further, the crosslinking time in the step (2) is 3-5 h.
Furthermore, the area of the sensitive part of the working electrode is 0.85 cm.
The high-precision glucose sensor prepared by the method.
When the sensor prepared by the invention is used, two sensors with the same shape are matched together to complete the functions of extraction and detection of the sensors. Wherein, W1 and W2 are thin film glassy carbon working electrodes, C1 and C2 are glassy carbon counter electrodes, W and R form an extraction electrode loop, and R1 and R2 are screen printing Ag/AgCl reference electrodes for providing a stable reference voltage during sensor test. (see FIG. 1)
The invention has the beneficial effects that:
1. the method adopts the osmium redox polymer coupled with chitosan as an electron mediator to modify a thin film glassy carbon electrode, so that the osmium redox polymer has reversible electrochemical properties; and because osmium can carry out rapid electron transfer between the electrode and the enzyme, dependence on the oxygen concentration in the bulk solution is eliminated.
2. The method adopts a glutaraldehyde method to fix the glucolase molecules to prepare the novel biosensor, thereby effectively reducing the working voltage of the sensor; meanwhile, the cross-linked enzyme solution has low working potential and can obviously improve the anti-interference capability.
3. The preparation method of the material adopted by the invention is carried out in aqueous solution, organic solvent is not needed, the production process is green and environment-friendly, and meanwhile, organic solvent residue is avoided, thereby being beneficial to expanding the application range.
4. The invention has cheap raw materials and simple synthesis path, can quickly prepare a large amount of conductive materials, and is beneficial to the mass production and commercial application of novel flexible bioelectronic devices.
5. The sensor prepared by the invention has sensitivity of 250.55 nA/(mu mol. L) within the standard concentration range of glucose of 5.0-25.0mmol/L -1 ) The lowest detection limit is 0.018 mu mol/L, and the correlation coefficient is 0.999; in the standard subcutaneous glucose concentration range of 5.0-30.0mmol/L, the extracted glucose current response value has a linear relation with the subcutaneous glucose concentration, the linear correlation coefficient is 0.993, and the sensitivity is 343.21 nA/(mmol.L) -1 ) (ii) a The accuracy of 10mmol/L glucose detection by a single sensor is 1.34% (n is 10), the accuracy of 20mmol/L glucose measurement between different sensors is 2.22% (n is 10), and the service life of the sensor can reach 14 days under the condition of 4 ℃.
Drawings
FIG. 1 is a three electrode architecture diagram of a sensor;
FIG. 2 is a graph of the i-t response of a sensor to different glucose concentrations;
FIG. 3 is a linear plot of sensor versus glucose concentration;
FIG. 4 is a graph of long term stability of the sensor.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1
A preparation method of a high-precision glucose sensor comprises the following specific steps:
(1) forming a three-electrode system by adopting a glassy carbon working electrode, a glassy carbon counter electrode and an Ag/AgCl reference electrode; wherein the area of the reaction area of the working electrode is 0.85 cm;
(2) cleaning the surface of the electrode by sequentially adopting 50% nitric acid solution, absolute ethyl alcohol and ultrapure water to complete the activation treatment of the electrode;
(3) glutaraldehyde is used as a cross-linking agent, glucose oxidase and osmium are cross-linked for 3 days in a vacuum environment to form an osmium redox polymer, then chitosan and the osmium redox polymer are coupled, 6 mu L of coupled product is absorbed and is dripped to the surface of a working electrode, and the working electrode is kept out of the sun overnight at room temperature, so that the polymer is firmly adsorbed on the surface of the working electrode;
(4) sucking 20 mu L of enzyme solution, uniformly coating the enzyme solution on a reaction area of a working electrode, wherein the content of glucose oxidase in the enzyme solution is 2U/mu L, the weight of BSA is 1.5 percent of the volume of the enzyme solution, the content of glutaraldehyde is 2.7 percent of the volume of the enzyme solution, and then crosslinking for 3 hours at room temperature;
(5) washing the product obtained in the step (4) by using deionized water and PBS, washing free substances, drying in the air, and storing in a refrigerator at 4 ℃ for later use.
Example 2
A preparation method of a high-precision glucose sensor comprises the following specific steps:
(1) forming a three-electrode system by adopting a glassy carbon working electrode, a glassy carbon counter electrode and an Ag/AgCl reference electrode; wherein the area of the reaction area of the working electrode is 0.85 cm;
(2) cleaning the surface of the electrode by sequentially adopting 50% nitric acid solution, absolute ethyl alcohol and ultrapure water to complete the activation treatment of the electrode;
(3) glutaraldehyde is used as a cross-linking agent, glucose oxidase and osmium are cross-linked for 3 days in a vacuum environment to form an osmium redox polymer, then chitosan and the osmium redox polymer are coupled, 6 mu L of coupled product is absorbed and is dripped to the surface of a working electrode, and the working electrode is kept out of the sun overnight at room temperature, so that the polymer is firmly adsorbed on the surface of the working electrode;
(4) sucking 20 mu L of enzyme solution, uniformly coating the enzyme solution on a reaction area of a working electrode, wherein the content of glucose oxidase in the enzyme solution is 3U/mu L, the weight of BSA is 2% of the volume of the enzyme solution, the content of glutaraldehyde is 2.5% of the volume of the enzyme solution, and then crosslinking for 3 hours at room temperature;
(5) washing the product obtained in the step (4) by using deionized water and PBS, washing free substances, drying in the air, and storing in a refrigerator at 4 ℃ for later use.
Example 3
A preparation method of a high-precision glucose sensor comprises the following specific steps:
(1) forming a three-electrode system by adopting a glassy carbon working electrode, a glassy carbon counter electrode and an Ag/AgCl reference electrode; wherein the area of the reaction area of the working electrode is 0.85 cm;
(2) cleaning the surface of the electrode by sequentially adopting 50% nitric acid solution, absolute ethyl alcohol and ultrapure water to complete the activation treatment of the electrode;
(3) glutaraldehyde is used as a cross-linking agent, glucose oxidase and osmium are cross-linked for 3 days in a vacuum environment to form an osmium redox polymer, then chitosan and the osmium redox polymer are coupled, 6 mu L of coupled product is absorbed and is dripped to the surface of a working electrode, and the working electrode is kept out of the sun overnight at room temperature, so that the polymer is firmly adsorbed on the surface of the working electrode;
(4) sucking 20 mu L of enzyme solution, uniformly coating the enzyme solution on a reaction area of a working electrode, wherein the content of glucose oxidase in the enzyme solution is 4U/mu L, the weight of BSA is 1.8 percent of the volume of the enzyme solution, the content of glutaraldehyde is 3 percent of the volume of the enzyme solution, and then crosslinking for 3 hours at room temperature;
(5) washing the product obtained in the step (4) by using deionized water and PBS, washing free substances, drying in the air, and storing in a refrigerator at 4 ℃ for later use.
Examples of the experiments
1. Electrochemical characteristics of working electrode
And 8 mu L of osmium polymer is adopted to modify the thin film glassy carbon working electrode. At different scan rates, the change in the cathode and anode peak currents of the redox polymer increases with increasing scan rate. The scan rates were 5,10,20,50,100,200,500 mV/s: oxidation peak current (I) thereof pa ) And reduction peak current (I) pc ) The ratio of (A) to (B) is I pa /I pc 1 and the peak current is linear with the square root of the scan rate, indicating that the osmium redox polymer has reversible electrochemical properties.
2. Current response detection of sensor to standard glucose
Fig. 2 and 3 show the current response curves of the sensor to standard glucose. The measurement was repeated 3 times for each sample at a working voltage of 0.21V. As can be seen from the figure: after the glucose solution is added, the current response of the sensor is gradually increased along with the increase of the glucose concentration, the increase of the current value and the increase of the glucose concentration are in a linear relation, and the current response signal quickly tends to be stable. Meanwhile, the current response signal of the glucose solution has a very good linear relation in the concentration range of 5.0-25 mmol/L. Linear correction equation I for sensor in 5.0-25mmol/L range p The detection limit is 0.018 mu mol/L, the correlation coefficient R is 0.99937, and the sensitivity of the sensor is 343.21 nA/(mu mol. L) -1 ). The results show that: the use of osmium polymers effectively reduces the operating voltage of the sensor.
3. Current response of sensor to subcutaneous glucose
Experiments of percutaneous extraction and detection of subcutaneous glucose were performed using the reverse iontophoresis technique. The process of subcutaneous glucose extraction is a routine procedure in the art, and specific procedures can be referred to the open literature. Wherein, the concentration ranges of subcutaneous glucose are respectively 0,3,7,10,14 and 19mmol/L, the sensor is pasted on the surface of the rat skin, the extraction detection period of the sensor is 10min, the working voltage is 0.21V, and the current response value of the sensor for 100s is taken as the detection result. The sensor carries out percutaneous extraction and detection experiments of subcutaneous glucose by the reverse iontophoresis technology of the current meeting of the extracted glucose and the subcutaneous glucose. The results show that the current encounter of the sensor to the extracted glucose is comparable to subcutaneous glucose.
4. Sensor repeatability detection
The accuracy of repeated tests on 10mmol/L glucose among single biosensors in the same batch and different biosensors in batches is researched, and the current response results obtained by 10 times of repeated tests on 10mmol/L glucose at the response time of 100s are obtained for the biosensors. The intra-batch accuracy refers to the current response results of 1 sensor repeating 10 times to test 10mmol/L glucose. The inter-batch accuracy refers to the current response results of 10mmol/L glucose measured with 10 sensors, respectively. As shown in fig. 4, the sensor was shown to have an intra-and inter-lot accuracy of 4.07% and 3.22%, respectively. The average value is less than 5 percent, which shows that the prepared sensor has better repeatability and consistency.
5. Sensor stability detection
The conventional life test evaluates the performance of a sensor by measuring how long it has been stored at 4 ℃. In physicochemical terms, the rate of a chemical reaction increases with increasing reaction temperature. Biological reagents are stored at different temperatures, and the 'rate' of reagent failure and the temperature and time of storage also conform to the physicochemical reaction rate relationship. The arrhenius empirical formula summarizes this physicochemical rule. Arrhenius describes the effect of temperature on reaction rate empirically as follows: lnk ═ E a RT + B, wherein k is the reaction rate; t is the absolute temperature of the reaction. Experiments were designed to perform a number of storage times at which the agent failed, the reciprocal of which (days or hours) is the rate at which the agent deteriorates at that temperature. The test temperature was converted into the absolute temperature T and the reciprocal of the retention time to form respective data, which were plotted on semilogarithmic paperLet lnk be Y and 1/T be X. An optimal fit line is plotted on the graph, which is extrapolated back to the logarithm of the corresponding reaction rate at T of 277K (4 ℃). The logarithm (exponential) is solved by the logarithm value, and then the reciprocal of the logarithm value is solved, namely the estimated stable time. The glucose biosensor was observed for life stability performance according to the above principle. The tests were carried out at 20,35,55 ℃. The response of the biosensor at 4 ℃ is used as the detection result of the control product, and the detection result is invalid when 90% or less of the response result of the biosensor at 4 ℃ is used as the failure result.
The paired results of X (1/T) and Y [ lg (1/d) ] are plotted or subjected to a linear regression process (in a linear relationship on semi-logarithmic paper). The number of days expected to be stable at 277K absolute temperature of 4 ℃ was estimated to be 14d by regression. Therefore, the biosensor prepared was stable at 4 ℃ for about 14 days as predicted by the test.
Claims (9)
1. A preparation method of a high-precision glucose sensor is characterized by comprising the following steps:
(1) activating the electrode surface of the three-electrode system, and then adsorbing the osmium redox polymer coupled with chitosan to the surface of a working electrode;
(2) and (3) coating the enzyme solution containing the glucose oxidase on a reaction area of the working electrode, crosslinking and fixing at room temperature, and then cleaning and drying.
2. The method of claim 1, wherein the three-electrode system consists of a glassy carbon working electrode, a glassy carbon counter electrode, and an Ag/AgCl reference electrode.
3. The method according to claim 1, wherein the activation treatment is performed by:
and cleaning the surface of the electrode by sequentially adopting 50% nitric acid solution, absolute ethyl alcohol and ultrapure water.
4. The method of claim 1, wherein the osmium redox polymer to which chitosan is coupled is adsorbed to the surface of the working electrode at room temperature in the absence of light.
5. The method of claim 1 or 4, wherein the osmium redox polymer is formed by cross-linking glucose oxidase with osmium via glutaraldehyde.
6. The method according to claim 1, wherein the concentration of glucose oxidase in the enzyme solution is 2 to 5U/. mu.L, and the amount of BSA added is 1.5 to 2% by volume of the enzyme solution.
7. The preparation method according to claim 1, wherein the crosslinking agent used in the step (2) is glutaraldehyde, and the addition amount of the glutaraldehyde in the enzyme solution is 2-5% by volume of the enzyme solution.
8. The method according to claim 1, wherein the crosslinking time in step (2) is 3 to 5 hours.
9. A high-precision glucose sensor produced by the method according to any one of claims 1 to 8.
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