阅读说明：本技术 一种谷氨酸生物传感器的制备方法 (Preparation method of glutamic acid biosensor ) 是由 谢颖 储震宇 金万勤 于 2021-08-19 设计创作，主要内容包括：本发明属于谷氨酸检测技术领域,涉及一种谷氨酸生物传感器的制备方法。使用金纳米颗粒/巯基化合物保护的银纳米立方颗粒涂覆于电极材料上,所制备的谷氨酸生物传感器适用于食品工程、发酵领域以及临床医学中谷氨酸的检测。传感材料制备简单并可批量生产,以谷氨酸氧化酶作为生物识别元件,对谷氨酸检测线性范围宽,可实现对发酵液的实时监测。(The invention belongs to the technical field of glutamic acid detection, and relates to a preparation method of a glutamic acid biosensor. The prepared glutamic acid biosensor is suitable for glutamic acid detection in the fields of food engineering, fermentation and clinical medicine by coating the gold nanoparticles/silver cubic nanoparticles protected by sulfhydryl compounds on an electrode material. The sensing material is simple to prepare and can be produced in batch, glutamate oxidase is used as a biological identification element, the linear range of glutamic acid detection is wide, and the real-time monitoring of fermentation liquor can be realized.)

1. A preparation method of a glutamic acid biosensor is characterized by comprising the following steps:

(1) preparing an ethylene glycol solution A containing polyvinylpyrrolidone, preparing an ethylene glycol solution B containing chloride, and preparing an ethylene glycol solution C containing silver nitrate; uniformly mixing the solution A and the solution B, adding the solution C for reaction, and centrifuging after reaction to obtain silver nanoparticles with a regular cubic structure;

(2) adding silver nanoparticles into the dispersion liquid, mixing the silver nanoparticles with a mercapto compound solution to obtain a solution D, and centrifuging after oscillation reaction to obtain a silver nanocube protected by the mercapto compound;

(3) immersing the silver nanocube protected by the sulfhydryl compound into a mixed solution of a chloroauric acid solution and an acid solution for reaction, centrifuging, cleaning with ethanol and drying after the reaction is finished to obtain silver nanocube powder protected by gold nanoparticles/sulfhydryl compound;

(4) adding carbon printing ink into silver nano cubic powder protected by gold nanoparticles/mercapto compounds, uniformly mixing, and preparing a working electrode on a support body by a screen printing technology;

(5) and preparing a glutamate oxidase mixed solution containing glutaraldehyde, uniformly coating the glutamate oxidase mixed solution on a working electrode, and drying at low temperature to obtain the biosensor chip for detecting the glutamic acid.

2. The method for preparing the glutamic acid biosensor as claimed in claim 1, wherein the chloride is NaCl, KCl or CuCl in the step (1)₂Any one of the above; the concentration of the solution A is 0.001-0.5 mM; the concentration of the solution B is 0.01-0.5 mM; the concentration of the solution C is 0.001-0.01M; the volume ratio of the solution A to the solution B is 50:1-100: 1; the volume ratio of the solution A to the solution C is 4:1-10: 1; the reaction temperature is 150-; the centrifugal speed is 5000 plus 10000rpm, and the centrifugal time is 10-40 min.

3. The method for preparing the glutamic acid biosensor according to claim 1, wherein in the step (2), the thiol compound is at least one of poly-2-mercaptoaniline, poly-3-mercaptoaniline, poly-4-mercaptoaniline, and 1, 4-benzenedithiol; the dispersion liquid is one of ethanol, methanol and dimethyl sulfoxide, the mass fraction of the silver nanoparticles in the dispersion liquid is 0.01-0.1%, the concentration of the sulfhydryl compound solution is 1-10 mM, the solvent is one of ethanol, methanol and dimethyl sulfoxide, the volume ratio of the dispersion liquid to the sulfhydryl compound solution is 1:1-1:3, the oscillation reaction time is 1-5h, and the oscillation reaction temperature is 10-40 ℃.

4. The method for preparing the glutamic acid biosensor as claimed in claim 1, wherein the acid solution in the step (3) is one of citric acid, toluenesulfonic acid and nitric acid, and the volume ratio of the chloroauric acid to the acid solution is 20:1-40: 1; the concentration of the chloroauric acid solution is 10-30 mM, the concentration of the acid solution is 0.1-0.5 mM, the mass of the silver nanocube protected by the sulfhydryl compound is 0.001-0.01g, the immersion reaction time is 5-60 min, the centrifugal rotation speed is 3000-8000rpm, the centrifugal time is 5-50min, the volume of ethanol used for cleaning is 10-30ml, the drying temperature is 30-60 ℃, and the drying time is 2-5 h.

5. The method for preparing the glutamic acid biosensor as claimed in claim 1, wherein the support in the step (4) is at least one of PVC, PET and alumina, and the mass ratio of the gold nanoparticle/mercapto compound-protected silver nano cubic powder to the carbon ink is 1: 10-1:50.

6. The preparation method of the glutamic acid biosensor according to claim 1, wherein the concentration of the glutamate oxidase solution in the step (5) is 0.05-1.5U/μ L, the volume percentage of the glutaraldehyde in the mixed enzyme solution is 0.1% -10%, the mixed enzyme solution coated on the working electrode is 0.5-10 μ L, the low-temperature drying temperature is 0-15 ℃, and the drying time is 5-10 hours.

7. Use of the biosensor obtained by the method according to any one of claims 1 to 6 for detecting glutamic acid.

Technical Field

The invention belongs to the technical field of glutamic acid detection, and relates to a preparation method of a glutamic acid biosensor.

Background

Glutamic acid is a well-known taste enhancer, naturally occurring in some food products, and although glutamic acid is generally regarded as safe, its use in food products remains controversial. Meanwhile, glutamate, as an excitatory neurotransmitter in the central nervous system, plays a crucial role in memory, synaptic plasticity, learning, motor function and neurotransmission. In addition, glutamate has been proposed to modulate nerve regeneration, tumor development, synapse formation, neurogenesis and apoptosis. Long-term high levels of extracellular glutamate are excitotoxic and are prone to cause neuronal cell death, and high fluctuations in glutamate levels are associated with many neurodegenerative diseases, such as parkinson's disease and alzheimer's disease, amyotrophic lateral sclerosis and ischemic stroke. Therefore, the method has important significance for real-time monitoring of glutamic acid.

In recent years, nanomaterials have been successfully used in the fields of medicine, pharmacy, chemistry, biology, and the like. But precise control of nanostructures is always a challenge due to their susceptibility to natural aggregation. Electrochemical biosensors have attracted much attention because of their advantages of easy operation, low cost, stable performance, high accuracy, and the like. The core of the method lies in a sensing electrode, including the development of high-performance electrode materials and the preparation of a sensing chip. The development of biosensors is practically accompanied by the development of electrode materials, thereby continuously realizing more stable and shorter electron transfer paths. In this way, a significant amplification of the electrical signal of the enzymatic reaction is achieved, resulting in high biosensing performance. In the construction of biosensors, non-uniform distribution and irregular nanostructures will increase electron transfer resistance and decrease enzyme-loaded active sites, resulting in decrease of conductivity and electrocatalytic effect. Therefore, it is necessary to develop a nano material with regular geometric shape and uniformly distributed on the electrode to better realize the application of biosensing. The method for detecting the glutamic acid by using an electrochemical method mainly depends on the catalytic oxidation of the glutamic acid by an electrode material under a certain voltage and the generation of current, and the content of the glutamic acid is measured by the current reaction. The silver nanocube wrapped by the organic matter with sulfydryl has good stability, can be effectively combined with gold nanoparticles, so that the conductivity of the material is obviously improved, and the detection of glutamic acid can be effectively realized.

Disclosure of Invention

The invention provides a preparation method and application of a novel glutamic acid biosensor, aiming at the problems in the traditional glutamic acid real-time detection.

In order to achieve the purpose, the invention is realized by adopting the following technical scheme:

the invention relates to a simple preparation method of a glutamic acid biosensor, which is characterized in that gold nanoparticles/silver cubic nanoparticles protected by mercapto compounds are coated on an electrode material, and the prepared glutamic acid biosensor is suitable for glutamic acid detection in the fields of food engineering and fermentation and clinical medicine.

A preparation method of a glutamic acid biosensor comprises the following steps:

(1) preparing an ethylene glycol solution A containing polyvinylpyrrolidone, preparing an ethylene glycol solution B containing chloride, and preparing an ethylene glycol solution C containing silver nitrate; uniformly mixing the solution A and the solution B, adding the solution C for reaction, and centrifuging after reaction to obtain silver nanoparticles with a regular cubic structure;

(2) adding silver nanoparticles into the dispersion liquid, mixing the silver nanoparticles with a mercapto compound solution to obtain a solution D, and centrifuging after oscillation reaction to obtain a silver nanocube protected by the mercapto compound;

(3) immersing the silver nano cubic solution protected by the mercapto compound into a mixed solution of a chloroauric acid solution and an acid solution for reaction, centrifuging, cleaning with ethanol and drying after the reaction is finished to obtain gold nano particles/silver nano cubic powder protected by the mercapto compound;

(4) adding carbon printing ink into silver nano cubic powder protected by gold nanoparticles/mercapto compounds, uniformly mixing, and preparing a working electrode on a support body by a screen printing technology;

(5) and preparing a glutamate oxidase mixed solution containing glutaraldehyde, uniformly coating the glutamate oxidase mixed solution on a working electrode, and drying at low temperature to obtain the biosensor chip for detecting the glutamic acid.

Preferably, in step (1), the chloride is NaCl, KCl or CuCl₂Any one of the above; the concentration of the solution A is 0.001-0.5 mM; the concentration of the solution B is 0.01-0.5 mM; the concentration of the solution C is 0.001-0.01M;the volume ratio of the solution A to the solution B is 50:1-100: 1; the volume ratio of the solution A to the solution C is 4:1-10: 1; the reaction temperature is 150-; the centrifugal speed is 5000 plus 10000rpm, and the centrifugal time is 10-40 min.

Preferably, in the step (2), the mercapto compound is at least one of poly-2-mercaptoaniline, poly-3-mercaptoaniline, poly-4-mercaptoaniline, and 1, 4-benzenedithiol; the dispersion liquid is one of ethanol, methanol and dimethyl sulfoxide, the mass fraction of the silver nanoparticles in the dispersion liquid is 0.01-0.1%, the concentration of the sulfhydryl compound solution is 1-10 mM, the solvent is one of ethanol, methanol and dimethyl sulfoxide, the volume ratio of the dispersion liquid to the sulfhydryl compound solution is 1:1-1:3, the oscillation reaction time is 1-5h, and the oscillation reaction temperature is 10-40 ℃.

Preferably, the acid solution in the step (3) is one of citric acid, toluenesulfonic acid and nitric acid, and the volume ratio of the chloroauric acid to the acid solution is 20:1-40: 1; the concentration of the chloroauric acid solution is 10-30 mM, the concentration of the acid solution is 0.1-0.5 mM, the mass of the silver nanocube protected by the sulfhydryl compound is 0.001-0.01g, the immersion reaction time is 5-60 min, the centrifugal rotation speed is 3000-8000rpm, the centrifugal time is 5-50min, the volume of ethanol used for cleaning is 10-30ml, the drying temperature is 30-60 ℃, and the drying time is 2-5 h.

Preferably, in the step (4), the support is at least one of PVC, PET and alumina, and the mass ratio of the gold nanoparticle/mercapto compound-protected silver nano cubic powder to the carbon ink is 1: 10-1:50.

Preferably, in the step (5), the concentration of the glutamate oxidase solution is 0.05-1.5U/muL, the volume percentage of the glutaraldehyde in the mixed enzyme solution is 0.1-10%, the mixed enzyme solution coated on the working electrode is 0.5-10 muL, the low-temperature drying temperature is 0-15 ℃, and the drying time is 5-10 h.

The biosensor prepared by the preparation method is applied to glutamic acid detection.

Compared with the prior art, the invention has the advantages and positive effects that:

the glutamic acid biosensor is prepared based on an electrochemical sensing technology, the sensing material is simple to prepare and can be produced in batches, glutamate oxidase is used as a biological recognition element, the linear range of glutamic acid detection is wide, and the real-time monitoring of fermentation liquor can be realized.

Drawings

FIG. 1 is a scanning electron microscope image of the prepared silver nanocube.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.

Example 1

1) 40ml of a polyvinyl pyrrolidone-containing ethylene glycol solution having a concentration of 0.002 mM was prepared, and 0.4ml of a NaCl-containing ethylene glycol solution having a concentration of 0.01 mM was added thereto. AgNO with concentration of 0.002M is prepared₃And adding 10ml of ethylene glycol solution into the mixed solution obtained in the previous step for a plurality of times in small amount for synthesis reaction. Controlling the reaction temperature at 160 ℃, controlling the synthesis time at 40min, and centrifuging after the reaction is finished, wherein the centrifugation speed is 5000 r/min, and the centrifugation time is 10 min. Dispersing the silver nanocube obtained by centrifugation (microscopic silver nanocube shows that the particles are uniform in size in figure 1) in 10ml of ethanol (mass fraction is 0.01%), and mixing and oscillating the ethanol solution with 1mM of 1, 4-benzenedithiol (30 ml) for reaction; controlling the reaction temperature at 25 ℃, controlling the oscillation time at 2h, and centrifuging after the reaction is finished, wherein the centrifugation speed is 5000 r/min, and the centrifugation time is 40 min. To obtain silver nano cube protected by sulfhydryl compound.

2) 1 ml of 25 mM HAuCl was prepared₄Immersing 0.01g of silver nano cube protected by sulfhydryl compound into the mixed solution, reacting at room temperature for 5min, and mixing the mixed solution with 25 muL of 0.25 mM citric acid solutionCentrifuging at the speed of 8000r/min for 5min, washing with 20ml of ethanol, and drying at 30 ℃ for 5h to obtain the silver nanocube protected by gold nanoparticles/mercapto compounds. Adding carbon ink into silver nano cubic powder protected by gold nanoparticles/mercapto compounds, mixing uniformly (mass ratio is 1: 10), and preparing a working electrode on pvc by using the mixed ink through a screen printing technology.

3) Preparing 0.5U/mu L glutamate oxidase solution, adding 1% glutaraldehyde by volume, placing 5 mu L on a working electrode, and drying at 0 deg.C for 5h to obtain glutamic acid biosensor chip.

In this example, the performance of the obtained glutamic acid biosensor chip is basically maintained after 10 times of parallel repetition, which shows that the glutamic acid biosensor chip obtained by the method provided by the present invention has stable performance and is suitable for mass production.

Example 2

This example is not particularly described, and is the same as example 1, as follows.

1) 100ml of a polyvinylpyrrolidone-containing ethylene glycol solution having a concentration of 0.001 mM was prepared, and CuCl-containing solution having a concentration of 0.05 mM was added thereto₂2ml of ethylene glycol solution. AgNO with concentration of 0.002M is prepared₃And adding 10ml of ethylene glycol solution into the mixed solution obtained in the previous step for a plurality of times in small amount for synthesis reaction. Controlling the reaction temperature at 160 ℃, controlling the synthesis time at 40min, and centrifuging after the reaction is finished, wherein the centrifugation speed is 5000 r/min, and the centrifugation time is 10 min. Dispersing the obtained silver nano cube in 10ml of methanol (the mass fraction is 0.1 percent), and mixing with 10 mM methanol solution (10 ml) of poly 2-mercaptoaniline for oscillation reaction; controlling the reaction temperature at 10 ℃, controlling the oscillation time at 5h, and centrifuging after the reaction is finished, wherein the centrifugation speed is 6000 r/min, and the centrifugation time is 15 min.

2) 1 ml of 10 mM HAuCl was prepared₄Immersing 0.001g of solution of silver nanocubes protected by sulfhydryl compounds into the mixed solution, reacting for 60min, centrifuging the mixed solution at a speed of 8000r/min for 50min, cleaning with 30ml of ethanol, drying at 60 ℃ for 2h to obtain gold nanoparticlesParticles/mercapto compound protected silver nanocube. Adding carbon ink into the gold nanoparticle/mercapto compound protected silver nano cubic powder, uniformly mixing (the mass ratio is 1: 50), and preparing an electrode on pvc by using the mixed ink through a screen printing technology.

3) Preparing 0.05U/mu L of glutamate oxidase solution, adding 0.2% glutaraldehyde by volume ratio, placing 10 mu L on a working electrode, and drying at 5 ℃ for 10h to obtain the glutamic acid biosensor chip.

Example 3

1) 40ml of a polyvinyl pyrrolidone-containing ethylene glycol solution having a concentration of 0.5mM was prepared, and 0.6ml of a KCl-containing ethylene glycol solution having a concentration of 0.5mM was added thereto. AgNO with concentration of 0.01M₃And adding 8ml of ethylene glycol solution into the mixed solution obtained in the previous step for a plurality of times in small amount for synthesis reaction. Controlling the reaction temperature at 160 ℃, controlling the synthesis time at 40min, and centrifuging after the reaction is finished, wherein the centrifugation speed is 5000 r/min, and the centrifugation time is 10 min. Dispersing the obtained silver nano-cubic in 10ml of dimethyl sulfoxide (the mass fraction is 0.01 percent), and mixing and oscillating the silver nano-cubic with 5mM dimethyl sulfoxide solution (20 ml) of poly-3-mercaptoaniline for reaction; controlling the reaction temperature at 40 ℃, controlling the oscillation time at 3h, and centrifuging after the reaction is finished, wherein the centrifugation speed is 10000 r/min, and the centrifugation time is 20 min.

2) 1 ml of 30 mM HAuCl was prepared₄And immersing 0.005g of solution of silver nanocubes protected by mercapto compounds into the mixed solution, reacting for 40min, centrifuging the mixed solution at the speed of 3000r/min for 50min, washing with 10ml of ethanol, drying at 50 ℃, and drying for 4h to obtain the silver nanocubes protected by gold nanoparticles/mercapto compounds. Adding carbon ink into the gold nanoparticle/mercapto compound protected silver nano cubic powder, uniformly mixing (the mass ratio is 1: 30), and preparing an electrode on alumina by using the mixed ink through a screen printing technology.

4) Preparing 1.5U/mu L glutamate oxidase solution, adding 10% glutaraldehyde by volume, placing 0.5 mu L on a working electrode, and drying at 10 deg.C for 8h to obtain glutamic acid biosensor chip.

Example 4

1) 40ml of a polyvinyl pyrrolidone-containing ethylene glycol solution having a concentration of 0.002 mM was prepared, and 0.4ml of a NaCl-containing ethylene glycol solution having a concentration of 0.01 mM was added thereto. AgNO with concentration of 0.005M₃And adding 10ml of ethylene glycol solution into the mixed solution obtained in the previous step for a plurality of times in small amount for synthesis reaction. Controlling the reaction temperature at 160 ℃, controlling the synthesis time at 40min, and centrifuging after the reaction is finished, wherein the centrifugation speed is 5000 r/min, and the centrifugation time is 10 min. Dispersing the obtained silver nano cube in 10ml ethanol (mass fraction is 0.05%), mixing with 4mM ethanol solution (15 ml) of poly 4-mercaptoaniline, and oscillating for reaction; controlling the reaction temperature at 25 ℃, controlling the oscillation time at 1h, and centrifuging after the reaction is finished, wherein the centrifugation speed is 8000r/min, and the centrifugation time is 10 min.

2) 1 ml of 25 mM HAuCl was prepared₄And immersing 0.008g of solution of silver nanocubes protected by mercapto compounds into the mixed solution, reacting for 20min, centrifuging the mixed solution at a speed of 7000r/min for 20min, washing with 10ml of ethanol, drying at 40 ℃, and drying for 3h to obtain the silver nanocubes protected by gold nanoparticles/mercapto compounds. Adding carbon ink into the gold nanoparticle/mercapto compound protected silver nano cubic powder, uniformly mixing (the mass ratio is 1: 20), and preparing an electrode on PET (polyethylene terephthalate) by using the mixed ink through a screen printing technology.

4) Preparing 1.0U/mu L of glutamate oxidase solution, adding 5% glutaraldehyde by volume, placing 1 mu L on a working electrode, and drying at 15 ℃ for 6h to obtain the glutamic acid biosensor chip.

The specific application method of the glutamic acid biosensor chip prepared in the embodiments 1 to 4 applied to fermentation broth for detecting the concentration of glutamic acid is as follows:

based on the above example, the prepared four glutamic acid biosensor chips are used for testing the concentration of the glutamic acid in the fermentation liquor by an ampere-hour-timing method, and the detection sensitivity is as high as 26.24 muA mM^-1 cm ^-2Linear range of detection 0.01-0.72mM, and Senshem, 2020, 306 and 127587. After the chip is stored in a refrigerator at 4 ℃ for 30 days, the steady-state response current still keeps over 73 percent of the initial sensitivity, which indicates that the prepared glutaminase biosensor chip has good stability.

Specific detection results are shown in table 1:

TABLE 1 results of performance measurements of biosensing chips prepared in examples 1-4

The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.