CN110887884A

CN110887884A - Flexible electrochemical glucose sensor and preparation method thereof

Info

Publication number: CN110887884A
Application number: CN201911333165.XA
Authority: CN
Inventors: 王云兵; 胡雪丰; 张婕妤; 梁震
Original assignee: Sichuan University
Current assignee: Jiangsu Yuekai Biotechnology Co ltd; Jiangsu Yuyue Kailite Biotechnology Co ltd; Zhejiang Poctech Corp
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2020-03-17
Anticipated expiration: 2039-12-23
Also published as: CN110887884B

Abstract

The invention discloses a flexible electrochemical glucose sensor and a preparation method thereof, and belongs to the technical field of sensors. CeO according to the invention₂/MnO₂The hollow nanospheres are used as an electro-catalysis medium, and the quaternary ammonium salt chitosan is used as a hydrogel matrix material. For CeO₂/MnO₂The surface of the hollow nanosphere is subjected to carboxyl modification, the hollow nanosphere is connected to self-healing conductive hydrogel through a covalent bond, GOx is loaded into the hydrogel through electrostatic interaction, and finally a layer of polymer is wrapped outside the hydrogel. By the method, leakage of GOx and an electrocatalysis medium can be effectively prevented, the prepared hydrogel sensor has the characteristics of wide linear range, quick response and high sensitivity to glucose detection, and the problems of easy loss of a coating body, compact coating, poor adhesion between the coating and electrodes and the like of the conventional glucose sensor are solved.

Description

Flexible electrochemical glucose sensor and preparation method thereof

Technical Field

The invention relates to the technical field of sensors, in particular to a flexible electrochemical glucose sensor and a preparation method thereof.

Background

In recent decades, the number of global diabetic and impaired glucose tolerance patients has increased dramatically year by year, and the international diabetes association (IDF) has predicted that by 2045 years, the number of global diabetic patients will increase to 6.29 billion. Conventional treatments for diabetes are oral or subcutaneous injections of hypoglycemic agents, which may not match the patient's immediate blood glucose level. Overdose or underdose may lead to fatal hypoglycemia or ketoacidosis. Therefore, it is necessary for serious diabetes patients to accurately monitor their blood glucose levels in real time by using a continuous blood glucose monitoring system (CGMS) so as to accurately administer insulin.

A key component of CGMS is the glucose sensor. Although the current glucose measurement methods are various, electrochemical analysis is the most widely used method due to the characteristics of high response speed, wide detection range, quantitative analysis and the like. Current glucose electrochemical sensors are prepared by coating glucose oxidase (GOx) and an electrocatalytic medium on the surface of an electrode. The glucose electrochemical sensor prepared by the method has compact coating, and the sensing reaction mainly occurs on the outer surface of the coating, thereby reducing the efficiency of the sensor. In addition, since GOx and the electrocatalytic medium are physically fixed on the electrode surface, they are easy to exude during use, and the coating is very brittle, and may be broken during use, which may result in failure of the sensor. Because the sensing coating is fixed on the electrode surface by physical action (such as van der waals force and electrostatic force), the interaction force is weak and the coating is unstable.

Disclosure of Invention

The invention aims to provide a flexible electrochemical glucose sensor and a preparation method thereof, and aims to solve the key problems that glucose oxidase and an electrocatalytic medium in the coating of the conventional glucose sensor are easy to consume and bleed out, the monitoring efficiency of the sensor is low due to the compact coating, the adhesion force between the coating and electrodes is poor, and the like.

For simplicity of description, the following terms are abbreviated herein:

GOx: a glucose oxidase;

cbs: carbon nanoparticles (carbon spheres);

MOx: carboxylated CeO₂/MnO₂Hollow nanospheres;

OD: oxidizing dextran;

QCS: quaternary ammonium salt chitosan.

The technical scheme for solving the technical problems is as follows:

flexible electrochemical glucose sensorThe preparation method comprises the following steps: preparation of CeO₂/MnO₂Subjecting the hollow nanospheres to carboxylation treatment to obtain carboxylated CeO₂/MnO₂And (3) reacting the hollow nanospheres with oxidized dextran and quaternary ammonium salt chitosan to prepare a pre-gel solution, adding glucose oxidase into the pre-gel solution, and coating the pre-gel solution on the surface of the treated electrode to prepare the flexible electrochemical glucose sensor.

The invention adopts CeO₂/MnO₂The hollow nanospheres are used as electrocatalytic media, and the quaternary ammonium salt chitosan is used as a hydrogel matrix material to prepare hydrogel. The hydrogel has a hydrophilic three-dimensional network structure, is beneficial to the permeation and enrichment of glucose, can enhance the contact between the glucose and GOx, broadens the detection range and improves the sensitivity and the response speed. Meanwhile, the self-healing performance of the hydrogel is utilized to avoid the failure of the sensor due to physical damage. Inventive CeO₂/MnO₂The surface of the hollow nanosphere is modified by carboxyl, and is arranged in an electrocatalytic medium (CeO)₂/MnO₂Hollow nanospheres) surface-COOH is introduced to covalently cross-link with hydrogel, preventing its exudation. GOx is grafted on CeO through strong electrostatic force₂/MnO₂The GOx can keep good bioactivity in the hydrogel on the surface of the nano-sphere due to no covalent crosslinking effect. Furthermore, prepared CeO₂/MnO₂The nanospheres are hollow, have large specific surface area, provide more attachment sites for enzyme and improve the overall performance of the sensor. Meanwhile, the hydrogel sensing layer is combined with the electrode material through covalent bonds, so that the separation of the hydrogel sensing layer and the electrode in the use process can be effectively prevented.

By the preparation method, leakage of GOx and an electrocatalytic medium can be effectively prevented, the prepared hydrogel sensor has the characteristics of wide linear range, quick response and high sensitivity on glucose detection, and the problems of easy loss of a coating body, compact coating, poor adhesion between the coating and electrodes and the like of the conventional glucose sensor are solved.

Further, in a preferred embodiment of the present invention, the preparation method further includes: after applying the pre-gel solution containing glucose oxidase, an encapsulant is applied to the electrode surface.

By coating a layer of packaging agent with good hydrophilicity and without influencing the permeation of glucose molecules outside the hydrogel, the permeation of the glucose molecules is ensured, and GOx and CeO are prevented₂/MnO₂And (4) seepage of the nanospheres.

Further, in a preferred embodiment of the present invention, CeO is prepared₂/MnO₂The hollow nanosphere comprises: preparing carbon nano particles with the particle size of 500-1000nm by adopting a hydrothermal method at the temperature of 150-200 ℃ by taking glucose as a carbon source, and then taking the carbon nano particles as a template and Ce as³⁺Compound and MnO₄ ^-CeO is obtained by depositing compound on carbon nano-particles₂And MnO with MnO₂Then sintering at above 250 ℃ to obtain CeO₂/MnO₂Hollow nanospheres.

Further, in the preferred embodiment of the present invention, CeO is used₂/MnO₂The compound of the hollow nanosphere subjected to carboxylation treatment is a compound with two ends respectively containing carboxyl and bonding groups.

Further, in a preferred embodiment of the present invention, the oxidized dextran is obtained by oxidizing dextran with periodate, and the quaternary ammonium salt chitosan is obtained by introducing quaternary ammonium salt ions into chitosan molecules with 2, 3-epoxypropyltrimethylammonium chloride.

Further, in a preferred embodiment of the present invention, the preparation process of the pre-gel solution comprises: using N-hydroxysuccinimide and 1-ethyl- (3-dimethyl aminopropyl) carbonyl diimine as cross-linking agent to make carboxyl CeO₂/MnO₂The hollow nanospheres and the quaternary ammonium salt chitosan solution are rapidly stirred for 10-14h at room temperature, and then the oxidized dextran solution is added to prepare the chitosan nano-composite material.

Using N-hydroxysuccinimide and 1-ethyl- (3-dimethyl aminopropyl) carbonyl diimine as cross-linking agent to make carboxylated CeO₂/MnO₂The hollow nanospheres are covalently connected to a quaternary ammonium salt chitosan molecular chain, and meanwhile, the oxidized glucan and the chitosan react through Schiff base to form reversible imine bonds.

Further, in a preferred embodiment of the present invention, the electrode surface is processed by: the electrode surface was plasma treated and then treated with triethoxysilane containing amino groups.

The surface of the electrode is treated by plasma to introduce hydroxyl, and then treated by amino-containing triethoxysilane, amino is introduced to react with carboxyl in hydrogel, the hydrogel is connected to the electrode in a covalent bond manner, the hydrogel layer is prevented from falling off from the electrode, and the amino-containing triethoxysilane is used with amino as a terminal group. The amino-containing triethoxysilane includes 3-aminopropyltriethoxysilane, 3-aminopropyl (diethoxy) methylsilane. Suitable electrode materials include Indium Tin Oxide (ITO) and metal electrodes.

Further, in a preferred embodiment of the present invention, the preparation method comprises the following specific steps:

(1) preparation of CeO₂/MnO₂Hollow nanosphere

Reacting the glucose aqueous solution in a high-pressure kettle at the temperature of 160-200 ℃ for 10-14h to obtain carbon nano particles; adding Ce after the carbon nano particles are dispersed in ethanol by ultrasonic³⁺Stirring the compound and an aqueous solution of polyvinylpyrrolidone for 1.5 to 2.5 hours at room temperature, and then reacting for 3.5 to 4.5 hours at 70 to 80 ℃ to obtain C @ CeO₂Nanospheres;

the invention adopts specific 160-200 ℃ to prepare the carbon nano particles, thereby avoiding that the glucose can not be decomposed and recrystallized due to the low temperature; addition of Ce³⁺The compound and the aqueous solution of polyvinylpyrrolidone are selected to react at 70-80 ℃ so as to lead Ce to react³⁺Oxidized and to nanoparticles of suitable size.

Adding C @ CeO₂Nanospheres to MnO₄ ^-Ultrasonically dispersing in compound solution, reacting at 70-90 deg.C for 3.5-4.5h, cooling to room temperature, vacuum filtering, cleaning, drying, and annealing at 280 deg.C for 1.5-2.5h to obtain CeO₂/MnO₂Hollow nanospheres;

the invention can disperse at a specific temperature of 70-90 ℃ to ensure the full reaction and obtain a proper deposition speed, so that the deposition layer is moderate; and, atThe annealing treatment is carried out at a specific temperature of 270 ℃ and 280 ℃, and CeO can be induced₂Crystallizing to form a crystal area, and ablating the carbon nanosphere template in the middle to form a hollow structure.

(2) Carboxylation treatment

Adding CeO₂/MnO₂Adding the hollow nanospheres and the compound with carboxyl and bonding groups at two ends into PBS buffer solution, adjusting pH to 7-8, introducing nitrogen at room temperature, stirring for 5-8h, centrifuging, and washing to obtain carboxylated CeO₂/MnO₂Hollow nanospheres;

(3) preparation of oxidized dextran and Quaternary ammonium Chitosan

Stirring periodate aqueous solution and dextran aqueous solution for 3.5-4.5h under the conditions of keeping out of the sun and room temperature, then adding ethylene glycol, stirring for 1.5-2.5h, dialyzing and freeze-drying to obtain oxidized dextran;

dissolving chitosan in acetic acid solution, adding 2, 3-epoxypropyltrimethylammonium chloride, reacting for 16-20h at 50-60 ℃, then centrifuging the reaction product at the rotation speed of 4000-;

(4) preparation of the Pre-gel solution

By carboxylating CeO₂/MnO₂Mixing the hollow nanospheres with a quaternary ammonium salt chitosan solution containing 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, rapidly stirring at room temperature for 10-14h, and adding an oxidized dextran solution to obtain a pre-gel solution;

(5) application of a Pre-gel solution

Ultrasonically cleaning an electrode in ethanol and deionized water in turn, and then activating the surface of the electrode for 3-6min by using plasma in the air at the power of 25-35W and the pressure of 180-; soaking the surface-activated electrode in an ethanol solution of triethoxysilane containing amino at 60-80 deg.C for 50-70min, cleaning with ethanol, and blow-drying with nitrogen;

and coating the pre-gel solution on the surface of the electrode, coating glucose oxidase and an encapsulant solution, and irradiating under ultraviolet light to obtain the flexible electrochemical glucose sensor.

Further, in a preferred embodiment of the present invention, the Ce is³⁺The compound is: CeCl₃、CeBr₃、Ce(NO₃)₃Or hydrates thereof, MnO₄ ^-The compound is: KMnO₄、NaMnO₄Or Mg (MnO)₄)₂；

The compound with two ends respectively containing carboxyl and bonding groups is as follows: 3, 4-dihydroxybenzoic acid, 3, 4-dihydroxyphenylacetic acid or 3, 4-dihydroxyphenylpropionic acid;

amino-containing triethoxysilanes are: 3-aminopropyltriethoxysilane or 3-aminopropyl (diethoxy) methylsilane;

the encapsulant is: pyrocatechol, cellulose acetate, PVA-SBQ or perfluorosulfonic acid.

The flexible electrochemical glucose sensor prepared by the preparation method.

The invention has the following beneficial effects:

the invention adopts CeO₂/MnO₂The hollow nanospheres are used as an electro-catalysis medium, and the quaternary ammonium salt chitosan is used as a hydrogel matrix material. For CeO₂/MnO₂The surface of the hollow nanosphere is subjected to carboxyl modification, the hollow nanosphere is connected to self-healing conductive hydrogel through a covalent bond, GOx is loaded into the hydrogel through electrostatic interaction, and finally a layer of polymer is wrapped outside the hydrogel. By the method, leakage of GOx and an electrocatalysis medium can be effectively prevented, the prepared hydrogel sensor has the characteristics of wide linear range, quick response and high sensitivity to glucose detection, and the problems of easy loss of a coating body, compact coating, poor adhesion between the coating and electrodes and the like of the conventional glucose sensor are solved.

The raw materials used in the invention are all common commercial products, the sources are easy to obtain, and the production cost is reduced. The method has the advantages of simple process, easy operation, no use of organic solvent and green and environment-friendly process flow. The sensor gel prepared by the invention has a self-healing function, and can avoid sensor failure caused by gel breakage in the using process. The invention adopts multiple packaging and cross-linking technology, and can effectively prevent leakage of GOx and electrocatalysis medium.

The flexible electrochemical glucose sensor prepared by the invention has a hydrophilic three-dimensional network structure, is beneficial to the permeation and enrichment of glucose, can enhance the contact between the glucose and GOx, and simultaneously uses the hollow nanospheres as an electro-catalysis medium to obviously improve the electron conduction efficiency, thereby widening the detection range and improving the sensitivity and the response speed.

Drawings

FIG. 1 shows synthesized CeO₂/MnO₂Schematic representation of hollow nanospheres and corresponding FESEM and TEM photographs.

FIG. 2 is a schematic diagram of the synthesis of QCS-MOx-OD self-healing hydrogel.

FIG. 3 is a schematic structural diagram of a flexible PET/QCS-MOx-OD/GOx/PVA-SBQ glucose electrochemical sensor.

FIG. 4 is a diagram of a flexible PET/QCS-MOx-OD/GOx/PVA-SBQ glucose electrochemical sensor.

Fig. 5 shows the self-healing performance of the QCS-MOx-OD hydrogel, the left graph shows the original state of the QCS-MOx-OD hydrogel, the middle graph shows the QCS-MOx-OD hydrogel being cut into two sections, and the right graph shows the QCS-MOx-OD hydrogel completely self-healing after 2 hours at room temperature.

FIG. 6 is a turbidity chart of hydrogel leaching solution prepared by different methods.

In fig. 7:

a) cyclic voltammograms for PET/QCS-MOx-OD/GOx electrodes after addition of 1mM glucose at different scan rates (50-500mV s-1) in 0.1M PBS (pH 7.4);

b) corresponding calibration curves for the PET/QCS-MOx-OD/GOx electrodes used for glucose detection;

c) is measured at 100mV s in 0.1M PBS (pH 7.4)^-1After glucose with different concentrations is added at the scanning speed, a cyclic voltammogram of a PET/QCS-MOx-OD/GOx electrode;

d) the peak current of the anode was linearly dependent on the glucose concentration at 0.6V.

In fig. 8:

a) for the current profile of the PET/QCS-MOx-OD/GOx electrode, glucose (0.05 μ M-20mM) was added continuously under constant stirring in 0.1M PBS (pH 7.4) at an applied potential of 0.6V.

b) A plot of current obtained from PET/QCS-MOx-OD/GOx electrodes versus glucose concentration is shown. The inset shows a linear calibration curve with glucose concentrations ranging from 1-111 mM.

FIG. 9 shows the Amperometric response of PET/QCS-MOx-OD/GOx electrodes at 0.6V with different analytes (1mM glucose, 0.1mM UA, 0.1mM AA, 0.1mM NaCl and 0.1mM DA) added in 0.1M PBS (pH 7.4).

In fig. 10:

a) a schematic of a glucose sensor built on a screen printed circuit.

b) And c) is a photograph of the constructed glucose sensor.

d) The response of the flexible sensor to the addition of 4mM and 8mM glucose to 0.6V electrolyte was repeated.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1:

a method of making a flexible electrochemical glucose sensor, comprising the steps of:

carbon spheres (Cbs) were prepared by reacting 0.56mM/mL of an aqueous glucose solution in a Teflon-lined stainless steel autoclave at 180 ℃ for 12 h;

0.1g of Cbs was dispersed in 15mL of ethanol and after 1h of sonication 25mL of Ce (NO) was added₃)₃·6H₂O (12mM) and polyvinylpyrrolidone (PVP, 3.6mg/mL) in water were stirred at room temperature for 2h, and reacted at 75 ℃ for 4h to prepare C @ CeO₂Nanospheres;

0.1g of C @ CeO₂The nanospheres were loaded with 25mL of KMnO₄(32mM) in the solution, uniformly dispersed by ultrasonic treatment for 30min, and then reacted at 80 ℃ for 4 h. Cooling to room temperatureThen, nanospheres are obtained by suction filtration and separation, washed by ethanol and deionized water respectively, dried for 1h at 60 ℃, and then annealed for 2h in air at 275 ℃ to prepare CeO₂/MnO₂Hollow nanospheres (as shown in figure 1);

add 12mL NaIO₄Dropping the aqueous solution (72mg/mL) into 10mL of dextran aqueous solution (10 wt%), stirring at room temperature for 4h in the dark, adding 223 μ L of ethylene glycol, stirring for 2h, dialyzing, and lyophilizing to obtain OD;

1g of chitosan was dissolved in 36mL of acetic acid solution (1 wt%), and 1.41g of glycidyltrimethylammonium chloride (GTMAC) was added and reacted at 55 ℃ for 18 hours. Then, the reaction product was centrifuged at 4200rpm for 30 minutes, and the supernatant was subjected to filtration under reduced pressure. Dialyzing and freeze-drying the filtrate to obtain QCS;

10mg of CeO₂/MnO₂Nanospheres and 60mg of 3, 4-dihydroxyphenylacetic acid were added to 10mL of PBS buffer (pH 7.4), and the pH of the solution was adjusted to 7-8 with 1M NaOH. Then, nitrogen is introduced into the solution at room temperature, the solution is stirred for 6 hours, solid precipitate is obtained by centrifugation, and MOx is obtained after the solid precipitate is washed by ethanol and deionized water. Then, 10. mu.L of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC, 7.2mM), 10. mu.L of N-hydroxysuccinimide (NHS, 28.8mM), 150. mu.L of QCS solution were added and the mixture was rapidly stirred at room temperature for 12 h. Finally, 30. mu.L of OD solution was added to form a pre-gel of QCS-MOx-OD hydrogel (as shown in FIG. 2);

the PET electrode (0.6cm x 4cm) was ultrasonically cleaned in ethanol and deionized water in sequence, and then the electrode surface was subjected to activation treatment with plasma in air at a power of 30W and a pressure of 200mtorr for 5 min. Thereafter, the surface-activated electrode was soaked in a 2% ethanol solution of (3-aminopropyl) triethoxysilane (APTES) at 70 ℃ for 60 min. The resulting PET/APTES electrode surface was rinsed with ethanol to remove unbound APTES and blown dry with nitrogen. Then, 20. mu.L of the pre-gel of QCS-MOx-OD hydrogel was coated on the surface of the PET/APTES electrode, 5. mu.L of PVA-SBQ and 5. mu.L of GOx (10mg/mL) solution were coated on the pre-gel of QCS-MOx-OD hydrogel, and after 1min of irradiation under 365nm ultraviolet light, a flexible PET/QCS-MOx-OD/GOx/PVA-SBQ glucose electrochemical sensor (as shown in FIGS. 3 and 4) was obtained.

As shown in FIG. 1, the Cbs cores produced by hydrothermal methods are uniform in size and about 700nm in diameter. Deposition of CeO on negatively charged Cbs₂After, Cbs @ CeO₂The surface of the ball becomes rough and porous, which makes KMnO₄Diffusion of the solution into CeO₂In the layer, a hollow core-shell structure of Cbs @ CeO is formed by the Kirkendall effect₂/MnO₂Nanospheres. After annealing to remove Cbs, CeO₂/MnO₂The size of the hollow nanospheres is 500 nm.

The QCS-MOx-OD hydrogel prepared has good self-healing performance, and can completely heal after being cut into two sections and contacted for 2h at room temperature as shown in figure 5. Therefore, the flexible PET/QCS-MOx-OD/GOx/PVA-SBQ glucose electrochemical sensor prepared based on the QCS-MOx-OD hydrogel has certain self-repairing performance, and is not easy to be mechanically damaged in use. In addition, the QCS-MOx-OD hydrogel is formed by amino on QCS and an electrocatalytic medium CeO₂/MnO₂Covalent crosslinking of carboxyl groups on nanospheres to CeO₂/MnO₂The nanospheres are immobilized in a hydrogel. The stability test result shows that compared with simple physical embedding, the electrocatalytic medium CeO in the invention₂/MnO₂The nanospheres are more stable in hydrogel and are not easy to leak. The QCS-MOx-OD hydrogel (covalently bound) was almost free of CeO after 30 days of soaking in PBS₂/MnO₂The nanospheres leaked, and when the same amount of MOx was physically incorporated into the hydrogel and soaked in PBS for more than 7 days, a large amount of MOx was released from the PBS, so that the haze value was significantly increased (fig. 6).

The hydrogel on the prepared flexible PET/QCS-MOx-OD/GOx/PVA-SBQ glucose electrochemical sensor has a hydrophilic three-dimensional network structure, is beneficial to permeation and enrichment of glucose, can enhance contact between the glucose and the GOx, widens the detection range, and improves sensitivity and response speed. Electrocatalytic medium CeO on sensor₂/MnO₂The nanospheres are hollow, have large specific surface area, provide more attachment sites for enzyme and improve the overall performance of the sensor. Prepared PET/QCS-MOThe anodic current of the x-OD/GOx glucose sensor increased linearly with increasing scan rate (fig. 7a, b) and the current of the glucose sensor was in a good linear relationship with the glucose concentration (R2 ═ 0.9931) in a linear range of 1mM to 111mM at a fixed scan rate of 100mV s-1 (fig. 7c, d).

The i-t curves in FIG. 8 show that the oxidation current of the PET/QCS-MOx-OD/GOx sensor increases and reaches steady state within 3 seconds after glucose addition. The peak current showed good linearity when the glucose concentration added to the electrolyte was varied between 1mM and 20mM (R2 ═ 0.9929). As shown in the inset of FIG. 8b, the linear range of electrode current for glucose detection is 1mM to 111 mM. LOD for glucose assay was calculated as 32.4 μ M (S/N-3). As shown in table 1, the PET/QCS-MOx-OD/GOx electrodes of the present application have a wider linear range, lower LOD and higher sensitivity than most sensors reported in the literature. The enormous performance of the electrodes of the present application is due to the unique structure of CeO2/MnO2 hollow nanospheres, the mild immobilization strategy of GOx and the permeability of the hydrogel as described above. In addition, the interferents vitamin c (aa), Dopamine (DA), Urea (UA) and NaCl failed to induce a significant response from the PET/QCS-MOx-OD/GOx electrode, indicating a strong anti-interference ability for the detection of glucose (fig. 9).

Example 2:

0.1g of C @ CeO₂The nanospheres were loaded with 25mL of KMnO₄(32mM) in the solution, uniformly dispersed by ultrasonic treatment for 30min, and then reacted at 80 ℃ for 4 h. Cooling to room temperature, separating by suction filtration to obtain nanospheres, and adding BWashing with alcohol and deionized water, drying at 60 deg.C for 1 hr, and annealing at 275 deg.C in air for 2 hr to obtain CeO₂/MnO₂Hollow nanospheres (as shown in figure 1);

the gold electrode (three-electrode printing chip) is cleaned by ultrasonic in ethanol and deionized water in sequence, and then the surface of the electrode is activated for 5min by plasma in air at the power of 30W and the pressure of 200 mtorr. Thereafter, the surface-activated electrode was soaked in a 2% ethanol solution of (3-aminopropyl) triethoxysilane (APTES) at 70 ℃ for 60 min. The resulting PET/APTES electrode surface was rinsed with ethanol to remove unbound APTES and blown dry with nitrogen. Then, pre-gel of 20 μ L QCS-MOx-OD hydrogel was coated on the PET/APTES electrode surface, and then 5 μ L PVA-SBQ and 5 μ L GOx (10mg/mL) solution were coated on the pre-gel of QCS-MOx-OD hydrogel, and after 1min of UV irradiation at 365nm, a flexible PET/QCS-MOx-OD/GOx/PVA-SBQ glucose electrochemical sensor (as shown in FIGS. 3 and 4) was obtained.

In addition to being built on PET/ITO electrodes, QCS-MOx-OD hydrogels could also be attached on gold surfaces using the same surface modification strategy, thus the present application assembled glucose sensors on flexible three-electrode screen-printed chips (fig. 10a, b). The sensor may be connected to an electrochemical workstation (fig. 10c) via a signal conversion connector and used for current-current response measurement of glucose. As shown in fig. 10d, when 4mM (normal blood glucose level) and 8mM (hyperglycemia) glucose solutions were sequentially added to the electrolyte, the anode current was increased accordingly and assumed a stepped shape. After the glucose was used up, the continuous addition of the 4mM and 8mM glucose solutions again resulted in another round of step signal, indicating good reproducibility in the glucose assay. Since the chip sensor is similar in structure and morphology to commercial CGMS sensors, the results show potential for clinical applications.

A comparison of the analytical performance of the sensors made in the examples of the present invention and the previously reported sensing systems for glucose detection is shown in table 1.

TABLE 1

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method of making a flexible electrochemical glucose sensor, comprising: preparation of CeO₂/MnO₂Subjecting the hollow nanospheres to carboxylation treatment to obtain carboxylated CeO₂/MnO₂And (3) reacting the hollow nanospheres with oxidized dextran and quaternary ammonium salt chitosan to prepare a pre-gel solution, adding glucose oxidase into the pre-gel solution, and coating the pre-gel solution on the surface of the treated electrode to prepare the flexible electrochemical glucose sensor.

2. The method of making a flexible electrochemical glucose sensor of claim 1, further comprising: after applying the pre-gel solution containing glucose oxidase, an encapsulant is applied to the electrode surface.

3. Method for preparing a flexible electrochemical glucose sensor according to claim 1 or 2, characterized in that CeO is prepared₂/MnO₂The hollow nanosphere comprises:

preparing carbon nano particles with the particle size of 500-1000nm by adopting a hydrothermal method at the temperature of 150-200 ℃ by taking glucose as a carbon source, and then taking the carbon nano particles as a template and Ce as³⁺Compound and MnO₄ ^-CeO is obtained by depositing compound on the carbon nano-particles₂And MnO with MnO₂Then sintering at above 250 ℃ to obtain CeO₂/MnO₂Hollow nanospheres.

4. The method of claim 1 or 2, wherein the CeO is applied to the flexible electrochemical glucose sensor₂/MnO₂The compound of the hollow nanosphere subjected to carboxylation treatment is a compound with two ends respectively containing carboxyl and bonding groups.

5. The method of claim 1 or 2, wherein said oxidized dextran is obtained by periodate oxidation of dextran, and said quaternary ammonium salt chitosan is obtained by introducing quaternary ammonium salt ions on chitosan molecules with 2, 3-epoxypropyltrimethylammonium chloride.

6. The method of preparing a flexible electrochemical glucose sensor according to claim 1 or 2, wherein the pre-gel solution is prepared by a specific process comprising:

using N-hydroxysuccinimide and 1-ethyl- (3-dimethyl aminopropyl) carbonyl diimine as cross-linking agent to make carboxyl CeO₂/MnO₂Hollow nanosphere and quaternary ammonium salt chitosanThe solution is rapidly stirred for 10-14h at room temperature, and then oxidized dextran solution is added.

7. The method of claim 1 or 2, wherein the electrode surface is treated by: the electrode surface was plasma treated and then treated with triethoxysilane containing amino groups.

8. The method of making a flexible electrochemical glucose sensor of claim 1, comprising the specific steps of:

(1) preparation of CeO₂/MnO₂Hollow nanosphere

Reacting the glucose aqueous solution in a high-pressure kettle at the temperature of 160-200 ℃ for 10-14h to obtain carbon nano particles; adding Ce after the carbon nano particles are ultrasonically dispersed in ethanol³⁺Stirring the compound and an aqueous solution of polyvinylpyrrolidone for 1.5-2.5h at room temperature, and then reacting for 3.5-4.5h at 70-80 ℃ to obtain C @ CeO2 nanospheres;

adding the C @ CeO2 nanospheres to MnO₄ ^-Ultrasonically dispersing in compound solution, reacting at 70-90 deg.C for 3.5-4.5h, cooling to room temperature, vacuum filtering, cleaning, drying, and annealing at 280 deg.C for 1.5-2.5h to obtain CeO₂/MnO₂Hollow nanospheres;

(2) carboxylation treatment

Subjecting the CeO to₂/MnO₂Adding the hollow nanospheres and the compound with carboxyl and bonding groups at two ends into PBS buffer solution, adjusting pH to 7-8, introducing nitrogen at room temperature, stirring for 5-8h, centrifuging, and washing to obtain carboxylated CeO₂/MnO₂Hollow nanospheres;

(3) preparation of oxidized dextran and Quaternary ammonium Chitosan

(4) preparation of the Pre-gel solution

Subjecting the carboxylated CeO₂/MnO₂Mixing the hollow nanospheres with a quaternary ammonium salt chitosan solution containing 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide, rapidly stirring at room temperature for 10-14h, and adding an oxidized dextran solution to obtain a pre-gel solution;

(5) application of a Pre-gel solution

and coating the pre-gel solution on the surface of an electrode, coating glucose oxidase and an encapsulant solution, and irradiating under ultraviolet light to obtain the flexible electrochemical glucose sensor.

9. The method of claim 8, wherein the electrochemical glucose sensor is a flexible electrochemical glucose sensor,

the Ce³⁺The compound is: CeCl₃、CeBr₃、Ce(NO₃)₃Or a hydrate thereof, said MnO₄ ^-The compound is: KMnO₄、NaMnO₄Or Mg (MnO)₄)₂；

the amino-containing triethoxysilane is: 3-aminopropyltriethoxysilane or 3-aminopropyl (diethoxy) methylsilane;

the packaging agent is as follows: pyrocatechol, cellulose acetate, PVA-SBQ or perfluorosulfonic acid.

10. A flexible electrochemical glucose sensor prepared by the method of any one of claims 1 to 9.