Low-trigger-potential electrochemiluminescence enzyme biosensor for detecting glucose as well as construction method and application of low-trigger-potential electrochemiluminescence enzyme biosensor

Abstract

The invention discloses a low-trigger-potential electrochemical luminescence enzyme biosensor for detecting glucose and a construction method and application thereof, the sensor comprises a three-electrode system formed by a working electrode, a reference electrode and a counter electrode, and a liquid to be detected contains luminol; the working electrode is a VMSF modified electrode of confined gold-palladium nanoparticles and is obtained by covalently immobilizing glucose oxidase. Luminol and glucose oxidase are respectively used as an electrochemical luminescence signal molecule and a specific recognition element; by combining the efficient and specific catalytic activity of natural enzyme, the efficient catalytic activity of gold-palladium nanoparticles on luminol electrochemical luminescence and the anti-pollution capacity of a vertical ordered mesoporous silica film, the low-trigger-potential electrochemical luminescence enzyme biosensor without an exogenous co-reactant is constructed; the method has the characteristics of high sensitivity, wide detection linear range and low detection limit, and has great application potential in constructing a portable glucose sensor for diagnosing and monitoring diabetes mellitus.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose, and a construction method and application thereof. Background Art

[0002] Glucose is a colorless monosaccharide that is widely distributed in nature and has important biological and industrial value. Glucose is the main energy source for organisms. In the human body, glucose can quickly replenish energy, promote liver detoxification, and enhance memory. The concentration of glucose has a great influence on physiological activities. If the concentration is too low, people will feel palpitations, cramps, or even coma, or cause stroke or other vascular diseases; if the concentration is too high, it will lead to obesity, diabetes, kidney disease, heart disease and nerve damage. Long-term high blood sugar can even cause lesions in various tissues and organs throughout the body, leading to various acute and chronic complications, such as decreased resistance, impaired renal function, neuropathy and cardiovascular and cerebrovascular diseases. Therefore, the development of excellent glucose sensors is crucial for the diagnosis and monitoring of diabetes.

[0003] At present, the most widely used glucose detection methods are high performance liquid chromatography, gas chromatography, fluorescence spectroscopy, ultraviolet spectroscopy, etc. These methods can obtain satisfactory analysis results, but the sample pretreatment is complicated and requires expensive large instruments. Therefore, it is very important to establish a low-cost, high-efficiency, high-sensitivity and accurate analysis method to achieve quantitative detection of glucose in biological samples.

[0004] Electrochemiluminescence (ECL) enzyme sensor is a branch of biosensor, which has the advantages of ECL and biosensor technology, such as wide linear range, high sensitivity, specific recognition, etc. The basic principle of ECL enzyme biosensor detection is to use enzymes as biological recognition elements to catalyze the target analyte to form one or more products through enzymatic reactions, which have a significant impact on the ECL signal, thereby achieving quantitative analysis of the target analyte. For example, alkaline phosphatase can catalyze the substrate disodium phenyl phosphate (DPP) to produce phenol, and phenol is oxidized to form benzoquinone during the test process, thereby quenching the ECL signal of terpyridine ruthenium. In the presence of substrate glucose and oxygen, glucose oxidase (GOD) can catalyze the production of hydrogen peroxide (H2O2), and the reactive oxygen species produced by H2O2 during the test process have the effect of enhancing the ECL signal of the electrochemiluminescent molecule luminol.

[0005] Luminol is increasingly being used in the construction of enzyme ECL sensors due to its advantages such as good water solubility, high quantum yield and low excitation potential. With the continuous advancement of point-of-care testing (POCT) technology, the requirements for sensor performance are becoming increasingly higher. POCT technology emphasizes immediacy, rapidity, portability and user-friendliness, which require innovation and optimization in the design of luminol ECL enzyme biosensors. Although luminol has been widely used in the field of ECL due to its unique properties, its quantum yield in aqueous solution is relatively low. In the absence of a co-reactant, the ECL signal of luminol is weak, which affects the sensitivity of the detection to a certain extent. In order to increase the ECL signal intensity of luminol, it is usually necessary to introduce additional co-reactants. However, this step not only increases the complexity of the detection process, but also contradicts the principle of simplicity and rapidity pursued by POCT. In addition, reducing the ECL trigger potential of luminol can avoid damage to biomolecules at higher potentials and enhance the stability and repeatability of enzyme sensors.

[0006] Vertically ordered silica nanofilm (VMSF) is composed of countless silica nanochannels perpendicular to the electrode substrate, with adjustable pore size of about 2 to 11.8 nm. The ultra-small pore size of VMSF and the abundant silanol groups on the surface of the pores bring it a series of unique functional properties, such as antifouling performance, size selectivity, charge selectivity, etc. The abundant silanol groups on the inner wall of the VMSF pores provide VMSF with good hydrophilicity, which helps to reduce the nonspecific adsorption of biomacromolecules such as proteins, thereby showing excellent antifouling ability. Secondly, the deprotonation of silanol groups under specific pH conditions carries a positive charge, which gives VMSF significant charge selectivity. For example, under pH = 7.4, the degree of deprotonation of silanol groups is high, so that the surface of the VMSF pores carries a negative charge, thereby showing selective electrostatic attraction or repulsion to cations or anions. Finally, the ultra-small pore size of VMSF selectively screens the entry of molecules or particles of different sizes, and can achieve size-based exclusion separation. At present, there is no report on the construction of low-trigger potential electrochemiluminescent enzyme biosensors without the need for external co-reactants in combination with VMSF. Summary of the invention

[0007] The present invention solves the problem that the detection sensitivity of glucose in biological samples is low and the detection difficulty is great. A low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose is provided. The sensor is used to detect glucose in sweat and has the advantages of high detection sensitivity and specificity, good selectivity and low cost, and the detection process does not require a complicated pretreatment process.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose, the low trigger potential electrochemiluminescent enzyme biosensor comprising a three-electrode system formed by a working electrode, a reference electrode and a counter electrode, and the liquid to be detected contains luminol;

[0010] The working electrode is a VMSF modified electrode of confined gold-palladium nanoparticles, which is obtained by covalently fixing glucose oxidase.

[0011] In the present invention, luminol and glucose oxidase are used as electrochemiluminescent signal molecules and specific recognition elements respectively; the enzymatic reaction between glucose oxidase and glucose substrate generates H2O2 in situ, which promotes the ECL signal amplification of luminol, and the electrochemiluminescent signal enhancement effect of gold palladium nanoparticles synthesized by vertical ordered mesoporous silica film confinement on luminol is combined to construct an immobilized enzyme biosensor for high-sensitivity detection of glucose in samples such as sweat and blood, with low detection limit, wide detection range, and detection does not require complex pretreatment and separation processes, reducing detection costs, simple operation, and suitable for large-scale applications. Compared with traditional ECL enzyme sensors, the electrochemiluminescent enzyme sensor constructed by the present invention eliminates the step of adding a co-reactant and simplifies the detection process; greatly reduces the ECL trigger potential, avoids damage to biological molecules under high potential, enhances the stability and reusability of the sensor, and provides an efficient and economical solution for accurate detection of glucose.

[0012] The working electrode preparation comprises the steps of:

[0013] Step 1, immersing the de-micelle VMSF modified electrode in a solution containing a gold precursor and a palladium precursor, and electrodepositing to obtain a confined AuPd NPs@VMSF electrode;

[0014] Step 2, placing the confined AuPd NPs@VMSF electrode into a silane solution containing an epoxy group, incubating it statically, and then taking it out to obtain an epoxy-functionalized confined AuPd NPs@VMSF electrode;

[0015] Step 3: drip a buffer solution of glucose oxidase on the epoxy-functionalized confined AuPd NPs@VMSF electrode for incubation to obtain the working electrode.

[0016] The VMSF modified working electrode is prepared by growing a nanometer-sized porous silica film on the electrode surface;

[0017] The preparation method of the VMSF modified working electrode comprises an electrochemical assisted method, Any one of solution growth method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent-induced self-assembly method;

[0018] The electrode in the VMSF modified working electrode includes any one of an indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode.

[0019] The gold precursor is any one of chloroauric acid, gold nitrate, gold cyanide, and a gold complex; the palladium precursor is any one of sodium tetrachloropalladate, palladium chloride, palladium nitrate, an aminopalladium compound, and a palladium complex;

[0020] The solution containing the gold precursor and the palladium precursor has a gold precursor concentration of 0.01-1 mmol / L and a palladium precursor concentration of 0.05-2 mmol / L;

[0021] The solvent used in the solution containing the gold precursor and the palladium precursor includes any one or more of potassium nitrate aqueous solution, sodium chloride aqueous solution, potassium chloride aqueous solution or phosphate buffer solution;

[0022] In step 1, the electrodeposition potential is -1.0 to 0 V, the electrodeposition time is 1 to 10 seconds, and the temperature is room temperature. When the deposition time is short, the amount of deposited silver nanoparticles is small, resulting in a weak electrochemical luminescence intensity of luminol, thereby affecting the detection sensitivity. When the deposition time is too long, the amount of deposited gold palladium nanoparticles is too large, thereby blocking the pores and affecting the mass transfer process, thereby causing the luminol electrochemical luminescence signal to decrease.

[0023] The epoxysilane includes any one or more of (3-glycidyl propoxy) trimethoxysilane, 3-(2,3-epoxypropoxy) propyl trimethoxysilane, and 2-(3,4-epoxycyclohexyl) ethyl triethoxysilane;

[0024] The solvent used in the epoxysilane solution includes any one or more of acetone, ethanol, methanol, and cyclohexane;

[0025] The volume proportion of epoxysilane in the epoxysilane solution is 0.05% to 1%; the standing time is 0.2-5h, and the temperature is room temperature. The introduction of epoxy groups enables glucose oxidase to be covalently fixed through the ring-opening reaction between its own nucleophilic amino residue and epoxy groups. If the amount of epoxy groups introduced is too small, the amount of covalently bound enzyme is too small, reducing the reaction efficiency; while too many epoxy groups will affect the mass transfer at the electrode interface and have low economic benefits.

[0026] The concentration of glucose oxidase in step 3 affects the overall reaction rate. A reasonable concentration range will achieve a moderate reaction rate and higher economic benefits. The concentration of glucose oxidase in the glucose oxidase-containing buffer solution is 1 to 50 mg / mL, more preferably 8 to 12 mg / mL;

[0027] The buffer solution in step 3 includes any one or more of a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution;

[0028] In step 3, the incubation condition is 0-5°C for 1-8 hours. Sufficient incubation time can make glucose react more fully with the enzyme and improve the detection sensitivity.

[0029] Preferably, the concentration of luminol in the test solution is 5 to 500 μmol / L;

[0030] The pH of the liquid to be tested is 6-8. Too strong acidity will inhibit the ionization of silanol and the activity of natural enzymes, thereby weakening the catalytic performance and stability of VMSF to gold palladium nanoparticles inside the pores, as well as the catalytic activity to the substrate, so the signal is low in a low pH environment. An overly high pH environment is prone to corroding silica, which is not conducive to the long-term stability of VMSF. Preferably, the pH of the liquid to be tested is 6.5-8, and more preferably, the pH is 6.5-7.5.

[0031] The reference electrode includes any one or more of an Ag / AgCl electrode (saturated KCl), a saturated calomel electrode, and a Hg / HgO electrode, and the counter electrode includes any one or more of a platinum wire and a platinum sheet.

[0032] The present invention also provides a method for constructing the low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose, comprising:

[0033] Step 1, immersing the de-micelle VMSF modified electrode in a solution containing a gold precursor and a palladium precursor, and electrodepositing to obtain a confined AuPd NPs@VMSF electrode;

[0034] Step 2, placing the confined AuPd NPs@VMSF electrode into a silane solution containing an epoxy group, incubating it statically, and then taking it out to obtain an epoxy-functionalized confined AuPd NPs@VMSF electrode;

[0035] Step 3: drip a buffer solution of glucose oxidase on the epoxy-functionalized confined AuPd NPs@VMSF electrode for incubation to obtain the working electrode.

[0036] Step 4, using the three-electrode system formed by the working electrode, the reference electrode and the counter electrode, luminol is added to the sample to be detected to form a test solution, thereby constructing a low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose content.

[0037] The present invention also provides a low trigger potential electrochemiluminescence detection method for glucose, comprising the steps of: adding luminol to a sample to be detected to form a liquid to be detected, incubating the working electrode in the liquid to be detected to obtain an electrode to be detected; and performing electrochemiluminescence signal detection using a three-electrode system formed by the electrode to be detected, a reference electrode, and a counter electrode.

[0038] The detection principle is as follows: the in-situ synthesis of gold-palladium nanoparticles using the nanochannels of the vertically ordered mesoporous silica film as a nanoreactor can enhance the low trigger potential electrochemiluminescence of the luminol-H2O2 system. When glucose is present in samples such as sweat and blood to be detected, the substrate glucose and oxygen participate in the reaction under the catalysis of glucose oxidase, effectively generating hydrogen peroxide (co-reactant of the luminol electrochemiluminescent molecule) in situ, enhancing the luminescence signal. The increase in the electrochemiluminescence signal is associated with the amount of glucose in the test object, so the electrochemiluminescence detection of glucose can be achieved according to the degree of increase in the electrochemiluminescence signal.

[0039] In order to solve the problem of high glucose concentration in samples such as sweat or blood, electrolyte solution can be used to dilute them during testing, such as diluting them 10 times, to reduce the glucose concentration.

[0040] The sample to be detected may contain one or more interfering molecules such as fructose, lactose, maltose, ascorbic acid, glycine, sodium chloride, urea, creatinine, uric acid and glucose, and the concentration of the interfering molecules is below 50 μmol / L.

[0041] The sensor of the present invention has a linear relationship with the logarithmic value of the detection concentration of glucose in the range of 10 nmol / L to 20 μmol / L, and the detection limit is 2.6 nmol / L. The detection method has a wide linear detection range, high selectivity, high detection sensitivity, and low detection limit. In addition, the detection does not require complex pretreatment and separation processes, which reduces the detection cost, is easy to operate, and is suitable for large-scale applications. Compared with the traditional ECL enzyme sensor, the electrochemiluminescent enzyme sensor constructed by the present invention eliminates the step of adding a co-reactant, simplifies the detection process, greatly reduces the ECL trigger potential, avoids damage to biological molecules under high potential, enhances the stability and reusability of the sensor, and provides an efficient and economical solution for the accurate detection of glucose.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The preparation method of the low trigger potential electrochemiluminescent enzyme biosensor of the present invention is simple, does not require a complicated electrode modification process, and adopts an immobilized enzyme strategy, which reduces the amount of enzyme used compared to the free enzyme system and reduces the economic cost; and the in situ generation of hydrogen peroxide on the outer surface of the electrode can effectively avoid its ineffective diffusion, increase its local concentration, and thus improve the reaction efficiency.

[0044] (2) Compared with traditional electrochemiluminescent enzyme sensors, the low trigger potential electrochemiluminescent enzyme biosensor of the present invention greatly reduces the ECL trigger potential, avoids damage to biological molecules under high potential, and enhances the stability and reusability of the sensor; at the same time, it eliminates the step of adding a co-reactant and simplifies the detection process.

[0045] (3) The present invention uses a vertically ordered mesoporous silica film to confine gold-palladium nanoparticles, thereby preventing interfering substances in complex samples from damaging the active centers of the gold-palladium nanoparticles and maintaining their high catalytic activity and stability.

[0046] (4) The detection method of the present invention is to measure the electrochemiluminescence signal of the sample to be tested after incubating it with the enzyme electrode. There is no need for separation and purification steps, the operation is simpler, the cost is lower, and the detection concentration is linearly related to glucose. The detection method has the advantages of a wide linear detection range, high accuracy, high detection sensitivity, and a low detection limit.

[0047] (5) The silica nanoporous membrane of the present invention has ultra-small nanochannels (2 to 3 nm) and the surface of the pores is rich in silanol groups with a pKa of about 2. Ionization in a conventional solution makes the membrane negatively charged. It can hinder the influence of large-sized negatively charged pollutants (such as cells, proteins and other biological macromolecules) in complex samples on the substrate electrode through electrostatic repulsion, hydrophilic effect and size screening effect, and has good anti-fouling and anti-interference capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic diagram of the structure of an indium tin oxide (ITO) electrode modified with a vertically ordered mesoporous silica film (VMSF) having covalently modified glucose oxidase on the outer surface prepared in Example 1 and confined growth of gold palladium nanoparticles (AuPd NPs) in the pores.

[0049] Figure 2 The AuPd NPs@VMSF / ITO electrode in Example 1, the VMSF / ITO electrode with epoxy groups functionalized on the outer surface and gold palladium nanoparticles confined in the pores (AuPd NPs@O-VMSF / ITO), and the VMSF / ITO electrode with glucose oxidase covalently modified on the outer surface and gold palladium nanoparticles confined in the pores (GOD / AuPd NPs@O-VMSF / ITO) were heated to 0.5 mM [Fe(CN)6]3– Cyclic voltammetry (CV) curves in solution and in 0.5 mM [Fe(CN)6] 3– / 4– Electrochemical impedance spectroscopy (EIS) obtained in solution.

[0050] Figure 3 Figure 1 shows the ECL response of AuPd NPs@VMSF / ITO to different concentrations of H2O2 in phosphate buffer solution (PBS (0.01 M, pH = 7.4)) containing luminol (100 μM) (A) and the calibration curve of ECL intensity versus the logarithm of H2O2 concentration (B). The ECL-time curve reveals the changes over time (C).

[0051] Figure 4 ECL responses of AuPd NPs@VMSF / ITO, AuPd NPs@O-VMSF / ITO and GOD / AuPd NPs@O-VMSF / ITO electrodes in PBS (0.01M, pH=7.4) containing 100μM luminol and 10μM glucose in Application Example 2 (A). (B) ECL-potential curve of GOD / AuPd NPs@O-VMSF / ITO electrode in PBS (0.01M, pH=7.4) containing 100μM luminol and 10μM glucose. (C) ECL-time curve of GOD / AuPd NPs@O-VMSF / ITO electrode in PBS (0.01M, pH=7.4) containing 100μM luminol and 10μM glucose.

[0052] Figure 5 The figure is an optimization diagram of the glucose oxidase immobilization amount (A) and the water bath incubation time (B) in Application Example 4. The detection solution contains 100 μM luminol and 10 μM glucose.

[0053] Figure 6 The ECL responses (A) of the GOD / AuPd NPs@O-VMSF / ITO electrode in Application Example 3 to different concentrations of glucose in PBS (0.01 M, pH = 7.4) containing luminol (100 μM) and the calibration curve (B) corresponding to the logarithm of the ECL intensity and the glucose concentration.

[0054] Figure 7The ECL intensity changes of the GOD / AuPd NPs@O-VMSF / ITO electrode in Application Example 4 before and after incubation with fructose, lactose, maltose, ascorbic acid, glycine, sodium chloride, urea, creatinine, uric acid, glucose (interfering substance concentration and target concentration are 50μM and 10μM, respectively) and their mixtures (A); (B) Repeatability study of GOD / AuPd NPs@O-VMSF / ITO enzyme sensor. The concentration of glucose is 10μM. DETAILED DESCRIPTION

[0055] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical scheme of the present invention without departing from the spirit and scope of the technical scheme of the present invention, and all should be included in the protection scope of the present invention.

[0056] Unless otherwise specified, the raw materials used in the following specific embodiments were purchased from commercial sources and used directly without special treatment. The main raw materials used in the examples are as follows:

[0057] Potassium ferrocyanide: Shanghai Aladdin Biotechnology Co., Ltd.;

[0058] Luminol: Shanghai Aladdin Biotechnology Co., Ltd.;

[0059] Cetyltrimethylammonium bromide (CTAB): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0060] Tetraethoxysilane (TEOS): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0061] 3-Glycidyloxypropyltrimethoxysilane (GPTMS): Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0062] Potassium hydrogen phthalate (KHP): Shanghai Aladdin Biotechnology Co., Ltd.;

[0063] Sodium dihydrogen phosphate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0064] Sodium hydrogen phosphate hydrate: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0065] Hexaammineruthenium(III) trichloride: Shanghai MacLean Biochemical Technology Co., Ltd.;

[0066] Glucose oxidase (GOD): Shanghai McLean Biochemical Technology Co., Ltd.

[0067] Example 1

[0068] A low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose comprises the following steps:

[0069] (1) Adoption VMSF was grown on an ITO substrate using the solution growth method. The specific steps are as follows: H2O (70 mL), ethanol (30 mL), CTAB (160 mg), ammonia water (100 μL) and TEOS (80 μL) were prepared into a precursor solution. The cleaned ITO electrode was immersed in the above precursor solution and incubated in a constant temperature water bath at 60°C for 24 hours. After the growth is completed, the electrode is removed from the precursor solution and quickly rinsed with a large amount of clean water. After rinsing, the electrode was blown dry with nitrogen and aged at 120°C for 12 hours. The obtained electrode was stirred in a 0.1 M ethanol-hydrochloric acid mixed solution for 5 minutes to remove the micelles in the pores, and an electrode with open pores was obtained, which is called a VMSF / ITO electrode;

[0070] (2) The open-pore electrode VMSF / ITO electrode obtained in (1) was immersed in a solution containing a gold precursor and a palladium precursor (containing 0.25 mM chloroauric acid and 0.75 mM sodium tetrachloropalladate, and the supporting electrolyte was 0.1 M potassium nitrate), and a constant voltage of -0.2 V was applied for 2 seconds. The electrode was taken out, washed with a large amount of flowing ultrapure water, and dried with nitrogen gas to obtain an ITO modified with a vertically ordered mesoporous silica film of confined gold palladium nanoparticles (AuPd NPs@VMSF / ITO);

[0071] (3) The AuPd NPs@VMSF / ITO electrode obtained in step (2) was immersed in a 2.26 mM GPTMS-ethanol solution and allowed to react at room temperature for 1 h. After the reaction was completed, the electrode was removed from the GPTMS-ethanol solution and gently rinsed with deionized water to wash away the unbound GPTMS remaining on the electrode, and then gently blown dry with nitrogen to obtain the AuPd NPs@O-VMSF / ITO electrode. Subsequently, PBS (0.01 M, pH = 7.4) containing 10 mg / mL GOD was drop-coated on the AuPd NPs@O-VMSF / ITO electrode and incubated at 4°C for 3 h. After the incubation was completed, the electrode was rinsed with PBS (0.01 M, pH = 7.4) to remove the unbound GOD. The electrochemiluminescent enzyme sensor finally obtained was named GOD / AuPd NPs@O-VMSF / ITO electrode, which was subsequently used as a working electrode. Its structural schematic diagram is shown in the figure. Figure 1 As shown;

[0072] (4) Using the GOD / AuPd NPs@O-VMSF / ITO prepared in step (3) as a working electrode, a reference electrode, and a counter electrode to form a three-electrode system, luminol is added to the sample to be detected to form a test solution, and a low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose content is constructed.

[0073] Application Example 1

[0074] The outer surface of Example 1 is covalently modified with glucose oxidase, and the vertical ordered mesoporous silica film of gold palladium nanoparticles is confined inside the pores to modify the ITO electrode GOD / AuPd NPs@O-VMSF / ITO, and the process of detecting glucose in artificial sweat by electrochemiluminescence method is as follows:

[0075] Artificial sweat was diluted 10 times with phosphate buffer (0.01 mol / L, pH=7), and a certain amount of glucose was added in proportion. The GOD / AuPd NPs@O-VMSF / ITO electrode was placed in artificial sweat containing 100 μM luminol and different concentrations of glucose. After incubation in a 37°C water bath for 15 minutes, the electrode to be tested was obtained. The schematic diagram of the electrode structure to be tested is shown in FIG. Figure 1 shown.

[0076] The electrochemiluminescence signal was detected by using a three-electrode system consisting of the electrode to be tested and a silver / silver chloride electrode (the solution was saturated potassium chloride) as a reference electrode and a platinum wire as a counter electrode. The electrochemiluminescence test was performed in a potential range of -0.2V to 0.6V.

[0077] Performance observation test

[0078] 1. Characterization of gold-palladium nanoparticles

[0079] The AuPd NPs@VMSF / ITO, AuPd NPs@O-VMSF / ITO and GOD / AuPdNPs@O-VMSF / ITO prepared in Example 1 were used as working electrodes in a solution containing 0.5 mmol / L [Fe(CN)6] 3– Cyclic voltammetry was performed in the solution, where the electrolyte solution was potassium hydrogen phthalate (0.05 mol / L, pH = 7.4). Figure 2 , Figure 2 The AuPd NPs@VMSF / ITO, AuPd NPs@O-VMSF / ITO, and GOD / AuPd NPs@O-VMSF / ITO electrodes were shown to have good thermal conductivity and good thermal conductivity. 3–The cyclic voltammetric response obtained in the experiment. With the gradual introduction of epoxy groups and glucose oxidase, the electrochemical signal gradually decreased. This is because the non-conductive biomacromolecules hindered the mass transfer process and electron transfer of the luminescent probe and the co-reactant to a certain extent. The gradual increase in electrochemical impedance further proved the successful modification of the enzyme sensor.

[0080] 2. Mechanism exploration

[0081] The different electrodes were placed in a water bath containing 100 μM luminol and 10 μM glucose for 15 min. The ECL curves obtained were as shown in Figure 3 (A) As shown. By comparing different modified electrodes, it was found that only the electrode immobilized with GOD had an obvious cathode ECL response at around 0V ( Figure 3 (B)). This is because GOD and the substrate glucose produce H2O2 through an enzymatic reaction. In this study, H2O2 is not only an intermediate product of the reaction, but also acts as a co-reactant for the luminescent probe luminol, greatly enhancing the ECL signal.

[0082] like Figure 3 The ECL-time curve shown in (C) reveals that the ECL emission intensity gradually decays over time. This phenomenon may be because the concentration of H2O2 generated in situ by the catalytic action of GOD on the VMSF surface reaches the highest. As the ECL reaction continues, H2O2 begins to diffuse into the bulk of the solution, resulting in a decrease in its concentration on the VMSF surface. At the same time, considering that the detection potential (–0.2-0.3V) of the low-trigger potential ECL enzyme sensor constructed in this study is much lower than the luminol emission potential, the oxidation of luminol mainly depends on the oxidative reactive oxygen species, which are generated by AuPdNPs confined in the VMSF pores. In order to achieve the low-trigger potential ECL of luminol, a large amount of reactive oxygen species needs to be consumed. It can be inferred that the reason for the signal decline may be that the consumption rate of H2O2 is greater than its generation rate, so the maximum emission intensity of ECL appears in the first cycle.

[0083] During the determination of glucose concentration, the enzymatic reaction catalyzes the conversion of glucose and generates H2O2 as an intermediate product in the process, which enhances the ECL of luminol. Therefore, the amount of intermediate H2O2 generated is positively correlated with the concentration of glucose. Before detecting glucose, we used AuPd NPs@VMSF / ITO as a working electrode to detect a series of H2O2 concentrations. Figure 4 As shown in (A), as the H2O2 concentration increases, the ECL signal also increases, and shows a good linear relationship between the two ranges of 10nM to 1μM and 1μM to 100μM ( Figure 4(B)), the calculated detection limit LOD was 3.2 nM. The above results prove that GOD on the VMSF surface catalyzes H2O2 generated by glucose, which enhances the electrochemiluminescence intensity of luminol, thereby achieving quantitative analysis of glucose.

[0084] Application Example 2: Optimization of detection conditions

[0085] The optimization of the amount of glucose oxidase immobilized on the electrode and the water bath incubation time of the in situ enzymatic reaction were investigated. First, the concentration of glucose oxidase was fixed, and the amount of glucose oxidase immobilized on the electrode was controlled by changing the incubation time of glucose oxidase on the electrode. Figure 5 (A), under the same substrate concentration and water bath incubation time, the incubation time in step (3) of Example 1 was changed to 0.5h, 1h, 2h, 3h and 4h. When the glucose oxidase incubation time was increased to 4h, the ECL signal decreased, so 3h was selected as the optimal incubation time for glucose oxidase.

[0086] Then, the water bath incubation time in step (4) of Example 1 was changed to 0 min, 5 min, 10 min, 15 min, 30 min and 45 min. Figure 5 As shown in (B), the signal growth rate remains almost unchanged when the incubation time exceeds 15 min. Considering the time cost, 15 min was finally selected as the optimal water bath incubation time. Application Example 3: Electrochemiluminescence Detection of Glucose

[0087] Figure 6 (A) shows the electrochemiluminescence signal of the GOD / AuPd NPs@O-VMSF / ITO electrode under the optimal experimental conditions for detecting different concentrations of glucose. As the glucose concentration increases, the electrochemiluminescence signal increases; the electrochemiluminescence intensity is the ordinate and the glucose concentration (C glucose ) logarithm (lgC glucose ) as the horizontal axis, such as Figure 6 (B). The electrochemiluminescence detection solution is a phosphate buffer solution (0.01 mol / L, pH = 7.4) containing 100 μM luminol. The electrochemiluminescence intensity and the logarithmic concentration of glucose show a good linear relationship in the range of 10 nmol / L–1 μmol / L and 1 μmol / L–20 μmol / L, with a detection limit of 2.6 nmol / L, which has a low detection limit and high sensitivity.

[0088] Application Example 4: Sensor Selectivity

[0089] Figure 7(A) is the selectivity and anti-interference data of the constructed low trigger potential electrochemiluminescent enzyme biosensor. Fructose, lactose, maltose, ascorbic acid, glycine, sodium chloride, urea, creatinine, and uric acid are used as interfering substances (the added amount is 50μmol / L). The above interfering substances and 10μmol / L glucose are added to a phosphate buffer solution containing luminol, incubated in a 37°C water bath for 15 minutes, and then detected. The electrochemiluminescence intensity of the sensor after incubation with different interfering substances or targets is recorded. Wherein mixture refers to a mixed solution of the above fructose, lactose, maltose, ascorbic acid, glycine, sodium chloride, urea, creatinine, uric acid and glucose.

[0090] The results showed that 5 times the concentration of interfering substances had almost no effect on the ECL signal, and only when the target glucose was present would it cause a significant change in the ECL signal. This was attributed to the specific selection of glucose by glucose oxidase itself. Only when glucose was present could glucose oxidase catalyze the substrate to produce H2O2 to produce a strong ECL signal. This proved the excellent selectivity of the proposed low trigger potential ECL luminescent enzyme sensor for glucose.

[0091] In order to further evaluate the reproducibility of the designed GOD / AuPd NPs@O-VMSF / ITO enzyme sensor, five prepared electrodes were selected to detect 10 μM glucose, e.g. Figure 7 (B) There was no obvious fluctuation in the ECL signal (relative standard deviation was 3.3%), which indicated that the developed enzyme sensor had good reproducibility.

[0092] The above analysis results prove the efficient enhancement effect of gold-palladium nanoparticles on the electrochemiluminescence of luminol, the specificity of the enzymatic reaction, and the anti-fouling effect of the vertically ordered mesoporous silica film. A low-trigger potential electrochemical enzyme sensor without the need for a reagent has been developed, which achieves highly sensitive detection of trace glucose in biological samples such as artificial sweat.

Claims

1. A low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose, characterized in that: The low trigger potential electrochemiluminescent enzyme biosensor comprises a three-electrode system formed by a working electrode, a reference electrode and a counter electrode, and the liquid to be detected contains luminol; The working electrode is a VMSF modified electrode of confined gold-palladium nanoparticles, which is obtained by covalently fixing glucose oxidase.

2. The low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose according to claim 1, characterized in that: The working electrode preparation comprises the steps of: Step 1, immersing the de-micelle VMSF modified electrode in a solution containing a gold precursor and a palladium precursor, and electrodepositing to obtain a confined AuPd NPs@VMSF electrode; Step 2, placing the confined AuPd NPs@VMSF electrode into a silane solution containing an epoxy group, incubating it statically, and then taking it out to obtain an epoxy-functionalized confined AuPd NPs@VMSF electrode; Step 3: drip a buffer solution of glucose oxidase on the epoxy-functionalized confined AuPd NPs@VMSF electrode for incubation to obtain the working electrode.

3. The low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose according to claim 2, characterized in that: The VMSF modified working electrode is prepared by growing a nano-sized silica porous film on the electrode surface; The preparation method of the VMSF modified working electrode comprises an electrochemical assisted method, Any one of solution growth method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent-induced self-assembly method; The electrode in the VMSF modified working electrode includes any one of an indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode.

4. The low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose according to claim 2, characterized in that: The gold precursor is any one of chloroauric acid, gold nitrate, gold cyanide, and a gold complex; the palladium precursor is any one of sodium tetrachloropalladate, palladium chloride, palladium nitrate, an aminopalladium compound, and a palladium complex; The solution containing the gold precursor and the palladium precursor has a gold precursor concentration of 0.01-1 mmol / L and a palladium precursor concentration of 0.05-2 mmol / L; The solvent used in the solution containing the gold precursor and the palladium precursor includes any one or more of potassium nitrate aqueous solution, sodium chloride aqueous solution, potassium chloride aqueous solution or phosphate buffer solution; In step 1, the electrodeposition potential is -1.0 to 0 V, the electrodeposition time is 1 to 10 s, and the temperature is room temperature.

5. The low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose according to claim 2, characterized in that: The epoxysilane includes any one or more of (3-glycidyl propoxy) trimethoxysilane, 3-(2,3-epoxypropoxy) propyl trimethoxysilane, and 2-(3,4-epoxycyclohexyl) ethyl triethoxysilane; And / or, the solvent used in the epoxysilane solution includes any one or more of acetone, ethanol, methanol, and cyclohexane; And / or, the volume proportion of epoxysilane in the epoxysilane-containing solution is 0.05% to 1%; And / or, the standing time is 0.2-5h and the temperature is room temperature.

6. The low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose according to claim 2, characterized in that: The concentration of glucose oxidase in the glucose oxidase-containing buffer solution in step 3 is 1 to 50 mg / mL; And / or, the buffer solution in step 3 includes any one or more of a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution; And / or, in step 3, the incubation condition is 0-5° C. and the incubation time is 1-8 h.

7. The low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose according to claim 1, characterized in that: The concentration of luminol in the test solution is 5-500 μmol / L; and / or the pH of the test solution is 6-8.

8. The method for constructing a low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose according to any one of claims 1 to 7, characterized in that: include: Step 1, immersing the de-micelle VMSF modified electrode in a solution containing a gold precursor and a palladium precursor, and electrodepositing to obtain a confined AuPd NPs@VMSF electrode; Step 2, placing the confined AuPd NPs@VMSF electrode into a silane solution containing an epoxy group, incubating it statically, and then taking it out to obtain an epoxy-functionalized confined AuPd NPs@VMSF electrode; Step 3, drop-coating a buffer solution of glucose oxidase on the epoxy-functionalized confined AuPd NPs@VMSF electrode for incubation to obtain the working electrode; Step 4, using the three-electrode system formed by the working electrode, the reference electrode and the counter electrode, luminol is added to the sample to be detected to form a test solution, thereby constructing a low trigger potential electrochemiluminescent enzyme biosensor for detecting glucose content.

9. A low trigger potential electrochemiluminescence detection method for glucose, characterized in that: The method comprises the following steps: adding luminol to a sample to be detected to form a liquid to be detected, incubating the working electrode in the liquid to be detected to obtain an electrode to be detected; and performing electrochemical luminescence signal detection using a three-electrode system formed by the electrode to be detected, a reference electrode and a counter electrode.

10. The low trigger potential electrochemiluminescence detection method for glucose according to claim 9, characterized in that: The sample to be detected may contain one or more interfering molecules including fructose, lactose, maltose, ascorbic acid, glycine, sodium chloride, urea, creatinine, uric acid and glucose.

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