阅读说明：本技术 一种基于氧化石墨烯蔗糖生物传感器的传感电极制备方法 (Method for preparing sensing electrode based on graphene oxide sucrose biosensor ) 是由 周杰 庄妮莎 董维亮 薛瑞 姜岷 于 2019-08-08 设计创作，主要内容包括：本发明涉及生物传感器的技术领域,更具体地,本发明提供了一种基于氧化石墨烯蔗糖生物传感器的传感电极制备方法。本发明第一方提供了一种基于氧化石墨烯蔗糖生物传感器的传感电极制备方法,包括杂化纳米片的制备、镜面玻碳电极的制备以及镜面玻碳电极的后处理；其中,杂化纳米片的制备原料包括氧化石墨烯与石墨烯量子点；同时采用壳聚糖将蔗糖酶、变旋酶及葡萄糖氧化酶固定在氧化石墨烯修饰后的电极表面。采用本方法制备的蔗糖生物传感电极及含有该电极的传感器线性检测上限较高,弥补了发酵生产中高浓度蔗糖无法直接检测的缺陷,也为后续开发蔗糖在线检测设备奠定了理论与应用基础。(The invention relates to the technical field of biosensors, and particularly provides a method for preparing a sensing electrode based on a graphene oxide sucrose biosensor. The first aspect of the invention provides a method for preparing a sensing electrode based on a graphene oxide sucrose biosensor, which comprises the steps of preparing a hybrid nanosheet, preparing a mirror surface glassy carbon electrode and post-treating the mirror surface glassy carbon electrode; the preparation raw materials of the hybrid nanosheet comprise graphene oxide and graphene quantum dots; meanwhile, the sucrase, the mutarotase and the glucose oxidase are fixed on the surface of the electrode modified by the graphene oxide by adopting chitosan. The sucrose biosensor electrode prepared by the method and the sensor containing the electrode have higher linear detection upper limit, make up the defect that high-concentration sucrose cannot be directly detected in fermentation production, and lay the theoretical and application foundation for the subsequent development of sucrose on-line detection equipment.)

1. A method for preparing a sensing electrode based on a graphene oxide sucrose biosensor is characterized by comprising the steps of preparing a hybrid nanosheet, preparing a mirror surface glassy carbon electrode and post-treating the mirror surface glassy carbon electrode; the preparation raw materials of the hybrid nanosheet comprise graphene oxide and graphene quantum dots.

2. the preparation method of the sensing electrode according to claim 1, wherein the preparation process of the hybrid nanosheet comprises dissolving graphene oxide and graphene quantum dots in water, performing ultrasonic treatment, reacting, centrifuging and drying.

3. the method for preparing the sensing electrode according to claim 1, wherein the post-processing of the mirror glassy carbon electrode comprises the following steps:

A. Dissolving the hybrid nanosheet in water, and then pouring the hybrid nanosheet into a mirror-surface glassy carbon electrode;

B. And B, mixing the mixed enzyme solution of sucrase, mutarotase and glucose oxidase with the chitosan solution, and titrating the mixture on the surface of the electrode obtained in the step A.

4. The method for preparing the sensing electrode according to any one of claims 1 to 3, wherein the particle size of the graphene quantum dots is less than 10nm, and the sheet size of the graphene oxide is less than 500 nm.

5. The preparation method of the sensing electrode according to claim 3, wherein the concentration of the hybrid nanosheets dissolved in water is 0.5-1.5 mg/mL.

6. The method for preparing a sensor electrode according to claim 3, wherein the amount of sucrase is 0.05 to 0.3U and the amount of glucose oxidase is 0 to 10U.

7. The method for preparing a sensor electrode according to claim 3, wherein the mutarotase content is 0.01 to 0.03mg and the chitosan concentration is 0.15 to 0.35 wt%.

8. the method for preparing the sensing electrode according to any one of claims 5 to 7, wherein the method for preparing the sucrase and the mutarotase comprises the following steps:

(1) Constructing a sucrase gene sequence and a mutarotase gene sequence on an expression plasmid to obtain a recombinant plasmid;

(2) transforming the recombinant plasmid into host bacteria to obtain saccharose producing bacteria and mutarotase producing bacteria;

(3) inoculating the sucrase producing strain and the mutarotase producing strain into a culture medium, performing induced expression, and collecting thalli;

(4) Washing, centrifuging and crushing the thalli obtained in the step (3), and collecting supernatant to obtain a crude enzyme solution;

(5) and purifying the crude enzyme solution to obtain a sucrase solution and a mutarotase solution.

9. the method for preparing the sensing electrode according to claim 8, wherein the crude enzyme solution is purified by the following steps: carrying out specific binding on the crude enzyme solution and a Ni column, and eluting by using an imidazole solution to obtain an eluent containing the target protein; and then the eluent containing the target protein is subjected to centrifugal enrichment through an ultrafiltration tube to obtain purified sucrase and mutarotase liquid.

10. the sensing electrode of the sucrose biosensor prepared by the method for preparing the sensing electrode according to any one of claims 1 to 9.

Technical Field

the invention relates to the technical field of biosensors, and particularly provides a method for preparing a sensing electrode based on a graphene oxide sucrose biosensor.

background

sucrose is prepared by condensing and dehydrating a molecule of hemiacetal hydroxyl group of glucose and a molecule of hemiacetal hydroxyl group of fructose. Sucrose is almost ubiquitous in leaves, flowers, stems, seeds and fruits of the plant kingdom, and is particularly abundant in sugarcane, sugar beet and maple sap. Sucrose is a raw material of various products, has wide application range in industry and food, and can be used as a nutrient of yeast, a food sweetener and the like.

at present, most of sucrose researches concentrate on high-efficiency production and later-period utilization of sucrose, and attention on the change of sucrose concentration in the production process is less. Moreover, most sucrose detection methods are chemical methods, and the detection is performed by hydrolyzing hydrochloric acid into glucose. The detection method has various defects of complicated steps, long pretreatment period, high hysteresis and interference, high cost and the like, and the emerging enzyme biosensor takes sucrase, mutarotase and glucose oxidase as immobilized enzyme elements and has the advantages of real-time convenience, high sensitivity, strong specificity and the like. Meanwhile, the sucrose concentration used in industrial production is higher, and the sucrose detection is more difficult. Therefore, it is imperative to develop a sucrose biosensor with a high detection upper limit.

Disclosure of Invention

in order to solve the technical problems, the first aspect of the invention provides a method for preparing a sensing electrode based on a graphene oxide sucrose biosensor, which comprises the steps of preparing a hybrid nanosheet, preparing a mirror surface glassy carbon electrode and post-treating the mirror surface glassy carbon electrode; the preparation raw materials of the hybrid nanosheet comprise graphene oxide and graphene quantum dots.

As a preferred technical scheme of the invention, the preparation process of the hybrid nanosheet comprises the steps of dissolving graphene oxide and graphene quantum dots in water, carrying out ultrasonic treatment, reacting, centrifuging and drying.

As a preferable technical solution of the present invention, the post-processing process of the mirror glassy carbon electrode includes:

A. Dissolving the hybrid nanosheet in water, and then pouring the hybrid nanosheet into a mirror-surface glassy carbon electrode;

B. and B, mixing the mixed enzyme solution of sucrase, mutarotase and glucose oxidase with the chitosan solution, and titrating the mixture on the surface of the electrode obtained in the step A.

in a preferred embodiment of the present invention, the particle size of the graphene quantum dot is less than 10nm, and the sheet size of the graphene oxide is less than 500 nm.

as a preferable technical scheme of the invention, the concentration of the hybrid nanosheet dissolved in water is 0.5-1.5 mg/mL.

as a preferable technical scheme of the invention, the content of sucrase is 0.05-0.3U, and the content of glucose oxidase is 0-10U.

as a preferable technical scheme of the invention, the content of the mutarotase is 0.01-0.03 mg, and the concentration of the chitosan is 0.15-0.35 wt%.

as a preferable technical scheme of the invention, the preparation method of the sucrase and the mutarotase comprises the following steps:

(1) Constructing a sucrase gene sequence and a mutarotase gene sequence on an expression plasmid to obtain a recombinant plasmid;

(2) transforming the recombinant plasmid into host bacteria to obtain saccharose producing bacteria and mutarotase producing bacteria;

(3) Inoculating the sucrase producing strain and the mutarotase producing strain into a culture medium, performing induced expression, and collecting thalli;

(4) Washing, centrifuging and crushing the thalli obtained in the step (3), and collecting supernatant to obtain a crude enzyme solution;

(5) And purifying the crude enzyme solution to obtain a sucrase solution and a mutarotase solution.

as a preferred technical scheme of the invention, the process of purifying the crude enzyme solution comprises the following steps: carrying out specific binding on the crude enzyme solution and a Ni column, and eluting by using an imidazole solution to obtain an eluent containing the target protein; and then the eluent containing the target protein is subjected to centrifugal enrichment through an ultrafiltration tube to obtain purified sucrase and mutarotase liquid.

the second aspect of the invention provides a sensing electrode of the sucrose biosensor prepared by the method for preparing the sensing electrode.

compared with the prior art, the method for the sensing electrode based on the graphene oxide sucrose biosensor, provided by the invention, adopts chitosan to fix sucrase, mutarotase and glucose oxidase on the surface of the electrode modified by graphene oxide. The sucrose biosensor electrode prepared by the method and the sensor containing the electrode have higher linear detection upper limit, make up the defect that high-concentration sucrose cannot be directly detected in fermentation production, and lay the theoretical and application foundation for the subsequent development of sucrose on-line detection equipment.

Drawings

FIG. 1 is a graph showing current-concentration response curves of prepared sensing electrodes to different concentrations of sucrose;

1-sucrose content 1.0 mM; 2-sucrose content 2.0 mM; 3-sucrose content 3.0 mM; 4-sucrose content 4.0 mM;

FIG. 2 is a graph showing current-concentration response curves of prepared sensing electrodes to different concentrations of sucrose;

FIG. 3 is a graph showing the current-concentration response of different materials with the same concentration for a prepared sensing electrode;

1-sucrose; 2-lactose; 3-galactose; 4-fructose; 5-maltose; 6-malic acid; 7-citric acid; 8-sucrose.

Detailed Description

unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.

the technical features of the technical solutions provided by the present invention are further clearly and completely described below with reference to the specific embodiments, and the scope of protection is not limited thereto.

the words "preferred", "preferably", "more preferred", and the like, in the present invention, refer to embodiments of the invention that may provide certain benefits, under certain circumstances. However, other embodiments may be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention. The sources of components not mentioned in the present invention are all commercially available.

The invention provides a method for preparing a sensing electrode based on a graphene oxide sucrose biosensor, which comprises the steps of preparing a hybrid nanosheet, preparing a mirror surface glassy carbon electrode and post-treating the mirror surface glassy carbon electrode; the preparation raw materials of the hybrid nanosheet comprise graphene oxide and graphene quantum dots.

in one embodiment, a method for preparing a sensing electrode based on a graphene oxide sucrose biosensor comprises the following steps:

I. preparing hybrid nanosheets;

II, preparing a mirror glassy carbon electrode;

and III, post-processing the mirror glassy carbon electrode.

in one embodiment, the hybrid nanosheet is prepared by dissolving graphene oxide and graphene quantum dots in water, sonicating, reacting, centrifuging, and drying.

Preferably, the preparation process of the hybrid nanosheet comprises the steps of dissolving graphene oxide and graphene quantum dots in ultrapure water, and carrying out ultrasonic treatment for 1-5 h; then reacting the obtained solution at 150-200 ℃ for 10-20 h; centrifuging the solution obtained by the reaction at 6000-10000 rpm for 3-9 h; and drying the mixture in a vacuum oven at 45-75 ℃ for 10-20 h.

more preferably, the preparation process of the hybrid nanosheet comprises the steps of dissolving graphene oxide and graphene quantum dots in ultrapure water, and carrying out ultrasonic treatment for 2 hours; then reacting the obtained solution at 180 ℃ for 14 h; centrifuging the solution obtained by the reaction at 8000rpm for 6 h; then the mixture is dried in a vacuum oven for 14 hours at the temperature of 60 ℃.

in one embodiment, the concentration of a solution obtained by dissolving graphene oxide and graphene quantum dots in ultrapure water is 0.8-2 mg/mL; preferably, the concentration of a solution obtained by dissolving graphene oxide and graphene quantum dots in ultrapure water is 1-1.5 mg/mL; more preferably, the concentration of the solution obtained by dissolving the graphene oxide and the graphene quantum dots in the ultrapure water is 1.3 mg/mL.

in one embodiment, the particle size of the graphene quantum dots is <10nm, the platelet size of the graphene oxide is <500 nm; preferably, the particle size of the graphene quantum dots is 3-9 nm, and the flake size of the graphene oxide is 100-400 nm; more preferably, the particle size of the graphene quantum dots is 7nm, and the flake size of the graphene oxide is 220 nm.

in one embodiment, the weight ratio of graphene oxide to graphene quantum dots is 1: (0.5 to 1.5); preferably, the weight ratio of the graphene oxide to the graphene quantum dots is 1: 1.

In one embodiment, a method for preparing a mirror-surface glassy carbon electrode comprises the steps of:

(1) Polishing the glassy carbon electrode by using alumina powder;

(2) respectively carrying out ultrasonic treatment on the material obtained in the step (1) for 1-5 h by using a nitric acid solution, absolute ethyl alcohol and ultrapure water;

(3) And (2) drying the material obtained in the step (1) under the condition of nitrogen to obtain the mirror surface glassy carbon electrode.

Preferably, the preparation method of the mirror glassy carbon electrode comprises the following steps:

(1) Polishing the glassy carbon electrode by using alumina powder;

(2) Respectively carrying out ultrasonic treatment on the material obtained in the step (1) for 2h by using 50% (v/v) nitric acid solution, absolute ethyl alcohol and ultrapure water with the same volume;

(3) And (2) drying the material obtained in the step (1) under the condition of nitrogen to obtain the mirror surface glassy carbon electrode.

in one embodiment, the glassy carbon electrode has a diameter of 1 to 3mm, preferably 2 mm.

in one embodiment, the alumina has a diameter of 0.02 to 0.5 μm; preferably, the diameter of the alumina is 0.05 μm and 0.3 μm; further preferably, the weight ratio of 0.05 μm alumina to 0.3 μm alumina is 1: (0.5 to 1.5); more preferably, the weight ratio of 0.05 μm alumina to 0.3 μm alumina is 1: 1.

in one embodiment, the post-treatment process of the mirror-surface glassy carbon electrode comprises:

A. Dissolving the hybrid nanosheet in water, and then pouring the hybrid nanosheet into a mirror-surface glassy carbon electrode;

B. And B, mixing the mixed enzyme solution of sucrase, mutarotase and glucose oxidase with the chitosan solution, and titrating the mixture on the surface of the electrode obtained in the step A.

Preferably, the post-treatment process of the mirror glassy carbon electrode comprises the following steps:

A. Dissolving the hybrid nanosheet in ultrapure water, carrying out ultrasonic treatment for 1-5 h, and pouring 10-30 mu L of the mixed nanosheet into a mirror surface glassy carbon electrode;

B. and B, mixing the mixed enzyme solution of sucrase, mutarotase and glucose oxidase with the chitosan solution, and titrating the mixture on the surface of the electrode obtained in the step A.

more preferably, the post-treatment process of the mirror glassy carbon electrode comprises the following steps:

A. dissolving the hybrid nanosheet in ultrapure water, performing ultrasonic treatment for 2 hours, and pouring 15 mu L of the hybrid nanosheet into a mirror surface glassy carbon electrode;

B. and B, mixing the mixed enzyme solution of sucrase, mutarotase and glucose oxidase with the chitosan solution, and titrating the mixture on the surface of the electrode obtained in the step A.

in one embodiment, the concentration of the hybrid nanosheet dissolved in water is 0.5-1.5 mg/mL; preferably, the concentration of the hybrid nanosheet dissolved in water is 0.8-1.2 mg/mL; more preferably, the concentration of hybrid nanoplatelets dissolved in water is 1.0 mg/mL.

in one embodiment, the solvent in the chitosan solution is a polar solvent, including but not limited to: at least one of glacial acetic acid, anhydrous ethanol, n-butanol, ethyl acetate, diethyl ether and butyl acetate; preferably, the solvent is glacial acetic acid.

in one embodiment, the mass fraction of the chitosan solution is 0.5-5%; preferably, the mass fraction of the chitosan solution is 1-3%; more preferably, the mass fraction of the chitosan solution is 1.25%.

in one embodiment, the amount of sucrase is 0.05 to 0.3U, and the amount of glucose oxidase is 0 to 10U; preferably, the content of sucrase is 0.1-0.2U, and the content of glucose oxidase is 3-7U; more preferably, the sucrase is 0.16U and the glucose oxidase is 5.92U.

In one embodiment, the mutarotase content is 0.01-0.03 mg, and the chitosan concentration is 0.15-0.35 wt%; preferably, the content of mutarotase is 0.01-0.02 mg, and the concentration of chitosan is 0.2-0.3 wt%; more preferably, the mutarotase content is 0.013mg and the chitosan concentration is 0.23 wt%.

In one embodiment, a method of preparing sucrases and mutarotases comprises:

(1) constructing a sucrase gene sequence and a mutarotase gene sequence on an expression plasmid to obtain a recombinant plasmid;

(2) transforming the recombinant plasmid into host bacteria to obtain saccharose producing bacteria and mutarotase producing bacteria;

(3) inoculating the sucrase producing strain and the mutarotase producing strain into a culture medium, performing induced expression, and collecting thalli;

(4) washing, centrifuging and crushing the thalli obtained in the step (3), and collecting supernatant to obtain a crude enzyme solution;

(5) And purifying the crude enzyme solution to obtain a sucrase solution and a mutarotase solution.

Preferably, the preparation method of the sucrase and the mutarotase comprises the following steps:

(1) constructing a sucrase gene sequence and a mutarotase gene sequence on an expression plasmid to obtain a recombinant plasmid;

(2) converting 100-300 ng of recombinant plasmid into 100-300 mu L of host bacteria, standing on ice for 10-50 min, and thermally shocking at 35-55 ℃ for 60-120 s; placing the mixture on ice for 1-5 min, adding 500-1500 mu L of LB culture medium, culturing for 0.5-3 h at 37 ℃ and 200rpm, coating a proper amount of cultured bacterial liquid on an LB flat plate for overnight culture to obtain saccharose producing bacteria and mutarotase producing bacteria;

(3) The saccharose producing strain and the mutarotase producing strain are inoculated in an LB liquid culture medium; adding 0.2-1 mM lactose or isopropyl-beta-D-thiogalactopyranoside for induction expression, and centrifugally collecting thalli;

(4) washing the bacterium obtained in the step (3) with a PBS buffer solution, centrifuging, crushing, and collecting supernatant to obtain a crude enzyme solution;

(5) and purifying the crude enzyme solution to obtain a sucrase solution and a mutarotase solution.

More preferably, the preparation method of sucrase and mutarotase comprises:

(1) synthesizing sucrase and mutarotase gene sequences by Nanjing Kinshire company and constructing the sucrase and mutarotase gene sequences on an expression plasmid to obtain a recombinant plasmid;

(2) 200ng of recombinant plasmid is transformed into 200 mu L of host bacteria, and the host bacteria are stood on ice for 30min and then heat shock is carried out for 90s at the temperature of 45 ℃; placing on ice for 2min, adding 900 μ L LB culture medium, culturing at 37 deg.C and 200rpm for 1h, coating appropriate amount of cultured bacterial liquid on LB plate, and culturing overnight to obtain sucrase producing strain and mutarotase producing strain;

(3) The saccharose producing strain and the mutarotase producing strain are inoculated in an LB liquid culture medium; adding 0.5mM lactose for induction expression, and centrifuging to collect thalli;

(4) washing the bacterium obtained in the step (3) with a PBS buffer solution, centrifuging, crushing, and collecting supernatant to obtain a crude enzyme solution;

(5) and purifying the crude enzyme solution to obtain a sucrase solution and a mutarotase solution.

In one embodiment, the expression plasmid is pET-29a and \ or pET-30 a; preferably, the expression plasmid is pET-29 a.

In one embodiment, the host bacterium is E.coli; preferably, the host bacterium is Escherichia coli BL21(DE 3).

In one embodiment, the crude enzyme solution purification process is: carrying out specific binding on the crude enzyme solution and a Ni column, and eluting by using an imidazole solution to obtain an eluent containing the target protein; and then the eluent containing the target protein is subjected to centrifugal enrichment through an ultrafiltration tube to obtain purified sucrase and mutarotase liquid.

preferably, the crude enzyme solution purification process is as follows: specifically combining the crude enzyme solution with a Ni column, and sequentially eluting with 50mM, 100mM, 200mM and 250mM imidazole solutions to obtain an eluent containing the target protein; and then the eluent containing the target protein is subjected to centrifugal enrichment through an ultrafiltration tube to obtain purified sucrase and mutarotase liquid.

the source of the glucose oxidase is not particularly limited; in one embodiment, the glucose oxidase is of aspergillus niger origin, available from Sigma.

In the experimental preparation process, it can be found that when specific graphene oxide and graphene quantum dots are used, the corresponding degree of oxidation current can be improved, and the graphene oxide and graphene quantum dots are different in particle size and are fully staggered, so that the glassy carbon electrode is finely and uniformly spread on the surface, and the corresponding sensitivity to current is improved; in addition, the applicant also unexpectedly finds that the problem of insensitivity of the sensor can be solved by regulating the dropping sequence of the enzyme solution and the chitosan solution on the surface of the electrode, when the enzyme solution and the chitosan solution are simultaneously dropped, the peak value of oxidation current can be obviously improved, the sensitivity and the detection accuracy are improved, the molecular-level contact between enzyme molecules and chitosan can be better realized under the condition, so that the molecular-level fixation is realized, the sensitivity to oxidation reaction is improved, and the peak value of oxidation current is improved, the specific detection of sucrose detection is realized by utilizing the enzyme solution and the electrode material prepared by the invention, the detection is not influenced by other materials, and meanwhile, the detection upper limit is higher, the compact structure of graphene oxide and graphene quantum dots on the surface of the electrode and the embedding and fixation of the enzyme solution and the chitosan molecule level are favorable for reducing the interference of impurity components and small molecular substances in the detected solution, reduce the adhesion of other proteins, amino acids, etc., thereby improving the specificity and the upper limit of detection.