CN113758983B - Construction method of glucose biosensor based on glutathione assembly - Google Patents
Construction method of glucose biosensor based on glutathione assembly Download PDFInfo
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 title claims abstract description 141
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 100
- 239000008103 glucose Substances 0.000 title claims abstract description 100
- 108010024636 Glutathione Proteins 0.000 title claims abstract description 72
- 229960003180 glutathione Drugs 0.000 title claims abstract description 72
- 238000010276 construction Methods 0.000 title abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 97
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 86
- 235000019420 glucose oxidase Nutrition 0.000 claims abstract description 38
- 108010015776 Glucose oxidase Proteins 0.000 claims abstract description 36
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- 229940116332 glucose oxidase Drugs 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 15
- 239000000243 solution Substances 0.000 claims description 64
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 58
- 150000002343 gold Chemical class 0.000 claims description 34
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 33
- 229910052737 gold Inorganic materials 0.000 claims description 33
- 239000010931 gold Substances 0.000 claims description 33
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 29
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 22
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- WBJINCZRORDGAQ-UHFFFAOYSA-N ethyl formate Chemical compound CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 claims description 7
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 19
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- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 description 2
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- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 2
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- 235000012208 gluconic acid Nutrition 0.000 description 2
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
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- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000004433 nitrogen atom Chemical group N* 0.000 description 2
- 150000003057 platinum Chemical class 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 101150003085 Pdcl gene Proteins 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
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- GNURASXBKKXAOM-JGWLITMVSA-N oxido-[(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexylidene]oxidanium Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=[O+][O-] GNURASXBKKXAOM-JGWLITMVSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/49—Systems involving the determination of the current at a single specific value, or small range of values, of applied voltage for producing selective measurement of one or more particular ionic species
Abstract
The invention belongs to the technical field of biosensors, and particularly relates to a construction method of a glucose biosensor based on glutathione assembly. According to the method, a glutathione-modified gold electrode is used as a carrier, carboxylated graphene is activated and then is loaded on the surface of the glutathione-modified gold electrode, so that the carboxylated graphene/glutathione-modified gold electrode is obtained, and then the carboxylated graphene/glutathione-modified gold electrode is soaked in a cross-linking agent and dried, so that the glucose biosensor based on glutathione assembly is obtained. The glutathione-carboxylated graphene self-assembled glucose biosensor is successfully assembled. The glutathione and carboxylated graphene are utilized to fix glucose oxidase, so that the glucose biosensor with high sensitivity, good selectivity, good reproducibility and good stability is constructed.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a construction method of a glucose biosensor based on glutathione assembly.
Background
Graphene (GN) is a novel two-dimensional carbon material called "parent of carbon material", which has excellent conductivity, high carrier mobility (up to 15000cm 2 .v -1 .s -1 ) The electrode material has the characteristics of electrocatalytic performance, high specific surface, good mechanical stability and the like, and is an ideal electrode material for preparing the biosensor. GN effectively improves the sensitivity and response signal of the biosensor in the electrochemical process and promotes the transmission of electrons. GN has a high specific surface area, which can increase the loading amount of biological reagents, so that the sensitivity and other performances of the sensor can be improved. In addition, GN can also maintain the activity of the supported biological reagent, so that the biosensor has good stability.
Glucose analysis and detection are of great importance for diagnosis, treatment and control of human health and diseases, and thus glucose sensor research has always been one of the hot spots in chemical and biological sensor research. Among the many types of glucose biosensors, many studies on glucose electrochemical sensors are underway. The glucose biosensor with enzyme has specificity and high selectivity based on the specific recognition function of enzyme to substrate.
Chinese patent CN103175884a discloses a high sensitivity glucose biosensor and its preparation method. Placing the polished platinum electrode on K 2 PdCl 4 And electrodepositing the mixture with sulfuric acid to obtain a palladium nanoparticle modified platinum electrode, dissolving glucose oxidase and bovine serum albumin in a phosphate buffer solution, then adding glutaraldehyde solution and an acid treated single-wall carbon nanotube solution, mixing to obtain an enzyme solution, dipping the palladium nanoparticle modified platinum electrode in the enzyme solution, and obtaining the glucose biosensor. The patent deposits palladium particles on the surface of a platinum electrode, and dips the platinum electrode modified by the palladium nano particles in an enzyme solution formed by glucose oxidase, bovine serum albumin and a single-wall carbon nanotube solution, thereby obtaining the glucose biosensor.
Chinese patent CN108490055a discloses a high-biocompatibility glucose biosensor based on graphene oxide and a preparation method thereof, comprising the following steps: placing the platinum electrode into absolute ethyl alcohol for ultrasonic cleaning, taking out, placing into a vacuum drying oven, and airing for standby; immersing platinum electrodes in PANI working solution and GO solution alternately, taking out each time, washing with distilled water, repeating for multiple times, and drying to obtain Pt/(PANI/GO) n electrodes; immersing the Pt/(PANI/GO) n electrode into GOx solution, taking out, washing with distilled water, and drying to obtain the Pt/(PANI/GO) n/GOx electrode. The patent uses polyaniline and graphene oxide to modify a platinum electrode.
Chinese patent CN105866226a discloses a method for preparing and using glucose oxidase biosensor, wherein the mixture of glucose oxidase and organic dye is fixed on the surface of the detecting end of carbon felt electrode, and the glucose oxidase biosensor is made. The patent utilizes physical adsorption, uses carbon felt as a matrix electrode, and adopts an organic dye to modify a glucose oxidase biosensor for quantitative analysis of glucose.
There has not been found a report on the production of a biosensor from graphene oxide together with glutathione.
Disclosure of Invention
The invention aims to provide a construction method of a glucose biosensor based on glutathione assembly, which is characterized in that glutathione, carboxylated graphene and glucose oxidase are fixed on the surface of a gold electrode, so that the stability of the sensor is improved, the reproducibility is good, and the service life is long.
According to the method for constructing the glucose biosensor based on glutathione assembly, provided by the invention, a glutathione-modified gold electrode is used as a carrier, carboxylated graphene is activated and then is loaded on the surface of the glutathione-modified gold electrode to obtain carboxylated graphene/glutathione-modified gold electrode, and then the carboxylated graphene/glutathione-modified gold electrode is soaked in a cross-linking agent and dried to obtain the glucose biosensor based on glutathione assembly.
Wherein:
the cross-linking agent is phosphate buffer solution containing glucose oxidase, catalase, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 4-Dimethylaminopyridine (DMAP). EDC in the cross-linking agent is a carbodiimide series compound with higher activity, and can be used as a coupling agent; the lone pair electrons carried by the nitrogen atom on the dimethylamino group in the DMAP molecule and the aromatic ring resonate to increase the nucleophilicity of the nitrogen atom on the pyridine ring, so that the DMAP has excellent catalytic performance.
The concentration of glucose oxidase in the cross-linking agent is 6-8g/L, the concentration of catalase is 0.6-0.8g/L, the mass percentage concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 0.03-0.05 wt%, and the mass percentage concentration of 4-dimethylaminopyridine is 0.004-0.006 wt%.
The pH value of the phosphate buffer solution is 6.8-7.0, and the concentration is 0.2-0.25mol/L.
The invention relates to a construction method of a glucose biosensor based on glutathione assembly, which specifically comprises the following steps:
(1) Preparation of glutathione-modified gold electrode
Placing a gold electrode in a potassium ferricyanide solution, scanning to be stable by using a cyclic voltammetry, and then soaking the gold electrode in a glutathione solution to obtain a glutathione-modified gold electrode;
(2) Preparation of carboxylated graphene
Adding graphite powder, potassium nitrate and potassium permanganate into concentrated sulfuric acid, heating to react, and adding hydrogen peroxide to react after the reaction is finished to obtain graphene oxide; reacting graphene oxide with sodium hydroxide and bromoacetic acid to obtain carboxylated graphene;
(3) Preparation of glutathione Assembly-based glucose biosensor
And (3) loading the carboxylated graphene obtained in the step (2) on the surface of the glutathione-modified gold electrode obtained in the step (1) after activating the carboxylated graphene to obtain the carboxylated graphene/glutathione-modified gold electrode, soaking the carboxylated graphene/glutathione-modified gold electrode in a cross-linking agent, and airing to obtain the glutathione-assembled glucose biosensor.
Wherein:
in the step (1), the gold electrode adopts Al with average granularity of 0.05-0.06 mu m 2 O 3 Polishing the powder, and then placing the powder into a potassium ferricyanide solution; the concentration of the potassium ferricyanide solution is 2.5X10 -3 -3.0×10 -3 The potassium ferricyanide solution contains potassium chloride, and the concentration of the potassium chloride is 5.0-5.5mol/L.
In the step (1), glutathione solution is prepared by dissolving glutathione in phosphate buffer solution with pH value of 6.8-7.0; the concentration of the glutathione solution is 0.04-0.06mol/L; the soaking temperature is 4-6deg.C, and the soaking time is 22-26h.
In the step (2), the dosage ratio of the graphite powder to the potassium nitrate to the potassium permanganate to the concentrated sulfuric acid to the hydrogen peroxide is 1.0-1.2:0.6-0.8:3.0-3.5:23-25:5-7, wherein the mass percentage concentration of the graphite powder to the potassium nitrate to the potassium permanganate to the hydrogen peroxide is 30-35wt.% in terms of g and the concentrated sulfuric acid to ml; the mass ratio of graphene oxide to sodium hydroxide to bromoacetic acid is 0.1-0.3:5-7:4-6.
In the step (2), the temperature-rising reaction is carried out for 2-3 hours at 15-20 ℃ and then the temperature is raised to 35-40 ℃ for 30-35min.
In the step (3), the carboxylated graphene is activated by adopting a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 4-dimethylaminopyridine, wherein the mass percentage concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in the mixed solution is 0.03-0.05 wt%, and the mass percentage concentration of the 4-dimethylaminopyridine is 0.004-0.006 wt%; the activation time is 20-30min; the soaking temperature is 4-6deg.C, and the soaking time is 22-26h.
The invention discloses a glucose biosensor based on glutathione assembly, which is prepared by taking a glutathione-modified gold electrode as a carrier, loading the carboxylated graphene on the surface of the glutathione-modified gold electrode after activation to obtain the carboxylated graphene/glutathione-modified gold electrode, soaking the carboxylated graphene/glutathione-modified gold electrode in a cross-linking agent, and airing the carboxylated graphene/glutathione-modified gold electrode to obtain the glucose biosensor based on glutathione assembly, wherein the preparation process is as follows:
the working principle of the glucose biosensor based on glutathione assembly is as follows: glucose oxidase (Gox) is a homodimeric molecule containing two Flavin Adenine Dinucleotide (FAD) binding sites. Glucose biosensor contacts with glucose oxidase in the process of catalyzing glucose, glucose oxidase can specifically catalyze beta-D-glucose to generate gluconic acid and hydrogen peroxide as hydrogen acceptor under aerobic condition, formula (1) is shown, hydrogen peroxide can continuously generate oxygen and water in the presence of Catalase (Catalase), formula (2) is shown, formula (3) can be obtained by combining formula (1) and formula (2), and 1/2mol of oxygen is consumed to generate gluconic acid per 1mol of glucose oxide. The cyclic voltammetry measurement result of the modified electrode shows that the glucose generates direct electron transfer, the number of electrons transferred by the chemical reaction of the glucose and oxygen can be detected by the current, and the electric signal output by the electrochemical glucose biosensor is directly proportional to the concentration of the glucose. The reaction formula is as follows:
the test method of the glucose biosensor based on glutathione assembly comprises the following steps: the glucose biosensor of the invention adopts a three-electrode system, the saturated calomel electrode is a reference electrode, the platinum electrode is an auxiliary electrode, and the three electrodes are placed in glucose solutions with different concentrations and scanned by cyclic voltammetry under different pH conditions respectively. The cyclic scan is performed at a certain scan rate within the appropriate potential range and the cyclic voltammogram is recorded.
The beneficial effects of the invention are as follows:
according to the invention, a gold sulfide bond is generated by a self-assembly technology of a gold electrode and sulfhydryl in a glutathione structure, and the obtained glutathione modified gold electrode (compound 1); firstly, a mixed solution of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 4-Dimethylaminopyridine (DMAP) is adopted to activate carboxylated graphene, and the oxygen induction of carbonyl in carboxyl of carboxylated graphene is used for absorbing electrons, so that the electronegativity of-OH oxygen in carboxyl is weakened, the constraint capacity of the carboxyl on H atoms is reduced, carboxyl-COOH in carboxylated graphene can react with EDC, and the carboxyl removes H + ，C＝N - Obtaining H + Generating an O-acyl isothiourea intermediate (compound 2), introducing an ester group to activate carboxyl, and realizing the activation of carboxylated graphene; then, after the activation solution of carboxylated graphene is dripped on the surface of a glutathione-modified gold electrode, the carboxylated graphene is catalyzed by DMAPO-acyl isothiourea intermediate (compound 2) can be combined with carboxyl-NH in glutathione modified gold electrode (compound 1) structure 2 And (3) generating amide through reaction to obtain the carboxylated graphene/glutathione modified gold electrode. Finally, immersing the carboxylated graphene/glutathione modified gold electrode in a cross-linking agent containing glucose oxidase, catalase, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 4-Dimethylaminopyridine (DMAP), wherein the 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) can activate two carboxyl groups on the carboxylated graphene/glutathione modified gold electrode to generate the carboxylated graphene/glutathione modified gold electrode with an ester structure, and under the catalysis condition of the DMAP, the ester structure is broken and is respectively connected with the-NH of the glucose oxidase and the catalase 2 And (3) generating amide by reaction, so that glucose oxidase and catalase are modified on the gold electrode modified by carboxylated graphene/glutathione, and the assembled glucose biosensor based on glutathione is obtained.
The glucose biosensor based on glutathione assembly has good symmetry, and because the gold-sulfur bond is positioned in the middle of the molecular formula of the glucose biosensor, the amino group on one side is combined with carboxylated graphene, and glucose oxidase and catalase are respectively modified to the two sides of a gold electrode, so that a macromolecular structure taking the gold-sulfur bond as a symmetry axis is formed, and the stability is good. In addition, the left side and the right side of the gold sulfur bond are tightly connected with an amide bond, and the structural stability of the gold sulfur bond is further promoted by the existence of the amide bond. The unique structure of the glucose biosensor ensures that the stability and reproducibility of the sensor are good, and the service life of the glucose biosensor is prolonged.
The glutathione-carboxylated graphene self-assembled glucose biosensor is successfully assembled. The glutathione and carboxylated graphene are utilized to fix glucose oxidase, so that the glucose biosensor with high sensitivity, good selectivity, good reproducibility and good stability is constructed. Due to the existence of carboxylated graphene, electron transfer of an active center of glucose oxidase can be promoted, and catalytic activity of enzyme is enhanced, so that the active center of the glucose oxidase shows good direct electrochemical behavior, and has good catalytic effect on oxidation of glucose.
Drawings
FIG. 1 is an infrared spectrum of carboxylated graphene in example 1 of the present invention;
FIG. 2 is a cyclic voltammetry scan of a bare gold electrode, a glutathione-modified gold electrode, a glutathione-and carboxylated graphene-modified gold electrode, a glutathione-assembled-based glucose biosensor;
wherein: a: glutathione-assembled-based glucose biosensor, b: glutathione and carboxylated graphene modified gold electrode, c: glutathione-modified gold electrode, d: a bare gold electrode;
FIG. 3 is a cyclic voltammogram of a glutathione-based assembled glucose biosensor in phosphate buffer solutions at different pH values;
FIG. 4 is a cyclic voltammogram of a glucose solution for different scan rates;
wherein: a:0.1V/s, b:0.08V/s, c:0.06V/s, d:0.04V/s, e:0.02V/s;
FIG. 5 is a graph of the square root of the scan rate versus current for a glutathione-based assembled glucose biosensor.
Detailed Description
The invention is further described below with reference to examples.
Example 1
(1) Preparation of glutathione-modified gold electrode
Gold electrode with diameter of 1.0mm was treated with 0.05 μm Al 2 O 3 Polishing the powder to a mirror surface, washing with distilled water for 10min, taking out, naturally airing, and carrying out ultrasonic washing with 75% alcohol for 10min. At 2.5X10 by Cyclic Voltammetry (CV) -3 The solution of potassium ferricyanide (containing 5.0mol/L KCl) was scanned to a steady state with a potential difference within 100 mV.
0.7683g of glutathione is weighed and dissolved in phosphate buffer solution with pH value of 7.00 to prepare 50mL of glutathione solution with concentration of 0.05mol/L, the ground gold electrode is suspended and soaked in the 0.05mol/L glutathione solution, soaked for 24 hours at 4 ℃, and stored in a refrigerator at 4 ℃ for standby.
(2) Preparation of carboxylated graphene
A 250ml reaction bottle is assembled in the ice water bath, and 23ml of concentrated sulfuric acid is poured in lightly; adding a solid mixture of 0.6g of potassium nitrate and 1.0g of graphite powder under stirring, and adding 3.0g of potassium permanganate; controlling the temperature to be less than 20 ℃, and stirring and reacting for 2 hours; then heating to 35 ℃, and continuing stirring for 30min; adding 46ml of deionized water, continuously stirring for 20min, adding 5g of hydrogen peroxide (30 wt.%) to reduce the residual oxidant, and making the solution brown yellow and emitting red smoke; filtering while the solution is hot, and washing the solution with 5wt.% HCl and deionized water until sulfate radical cannot be detected in the filtrate; finally, placing the solid in a drying oven at 60 ℃ for full drying, and preserving the obtained graphene oxide for later use;
and dispersing 200mg of the prepared graphene oxide in 100mL of aqueous solution by ultrasonic, adding 5.0g of bromoacetic acid and 6.0g of sodium hydroxide, performing ultrasonic reaction for 3 hours, centrifuging, washing to be neutral, and drying in a drying oven to obtain carboxylated graphene. And drying a small amount of carboxylated graphene in an infrared dryer. After drying, tabletting with potassium bromide at a ratio of 1:200, and infrared characterization of carboxylated graphene by an infrared spectrophotometer, wherein a spectrogram is shown in figure 1, and 3177.81cm on the spectrogram -1 The absorption peak is strong and the peak width is strong, and is the absorption peak of carboxyl (-COOH) at 1730.43cm -1 Is subjected to stretching vibration of carbonyl (-C=O), 1621.72cm -1 Is provided with a C=C double bond vibration peak without conjugation, 1220.64cm -1 The vibration peak of the ether (C-O) bond is shown, and the generated substance is carboxylated graphene.
(3) Preparation of glutathione Assembly-based glucose biosensor
Glucose oxidase, catalase, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), 4-Dimethylaminopyridine (DMAP) were dissolved in 10mL of phosphate buffer solution (pH 7.00,0.2mol/L, freshly prepared) to give a crosslinker containing 6g/L glucose oxidase, 0.6g/L catalase, 0.03wt.% EDC, 0.004wt.% DMAP, pH7.00 of the buffer solution, and the resulting crosslinker was put in a refrigerator for use.
Weighing 0.0250g of carboxylated graphene, dissolving in 50mL of distilled water, carrying out ultrasonic treatment for 10min to prepare carboxylated graphene solution with the concentration of 0.05mg/mL, adding a mixed solution containing 0.03wt.% EDC and 0.004wt.% DMAP for activation for 30min, transferring 10 mu L of the carboxylated graphene activation solution by a pipette, dripping the carboxylated graphene activation solution on the surface of a gold electrode modified by glutathione, naturally airing to obtain the carboxylated graphene/gold electrode modified by glutathione, and placing the gold electrode in a refrigerator for standby.
And (3) leaching the carboxylated graphene/glutathione-modified gold electrode with distilled water, soaking the gold electrode in the prepared cross-linking agent for 24 hours (4 ℃), and naturally airing to obtain the glutathione-assembled glucose biosensor.
Example 2
(1) Preparation of glutathione-modified gold electrode
Gold electrode with diameter of 1.0mm was treated with 0.06 μm Al 2 O 3 Polishing the powder to a mirror surface, washing with distilled water for 10min, taking out, naturally airing, and carrying out ultrasonic washing with 75% alcohol for 10min. At 3.0X10 by Cyclic Voltammetry (CV) -3 The solution of potassium ferricyanide (containing 5.5mol/L KCl) was scanned to a steady state with a potential difference within 100 mV.
0.9220g of glutathione is weighed and dissolved in phosphate buffer solution with pH value of 7.00 to prepare 50mL of glutathione solution with concentration of 0.06mol/L, the ground gold electrode is suspended and soaked in the 0.06mol/L glutathione solution, soaked for 20 hours at 4 ℃, and stored in a refrigerator at 4 ℃ for standby.
(2) Preparation of carboxylated graphene
A 250ml reaction bottle is assembled in the ice water bath, and 25ml of concentrated sulfuric acid is poured in lightly; adding a solid mixture of 0.8g of potassium nitrate and 1.2g of graphite powder under stirring, and adding 3.5g of potassium permanganate; controlling the temperature to be less than 15 ℃, and stirring and reacting for 2.5h; then heating to 40 ℃, and continuing stirring for 35min; adding 50ml of deionized water, continuously stirring for 20min, adding 6g of hydrogen peroxide (35 wt.%) to reduce the residual oxidant, and making the solution brown yellow and emit red smoke; filtering while the solution is hot, and washing the solution with 5wt.% HCl and deionized water until sulfate radical cannot be detected in the filtrate; finally, placing the solid in a drying oven at 60 ℃ for full drying, and preserving the obtained graphene oxide for later use;
and dispersing 300mg of the prepared graphene oxide in 150mL of aqueous solution by ultrasonic, adding 6.0g of bromoacetic acid and 7.0g of sodium hydroxide, performing ultrasonic reaction for 3 hours, centrifuging, washing to be neutral, and drying in a drying box to obtain carboxylated graphene.
(3) Preparation of glutathione Assembly-based glucose biosensor
Glucose oxidase, catalase, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), 4-Dimethylaminopyridine (DMAP) were dissolved in 10mL of phosphate buffer solution (pH 7.00,0.25mol/L, freshly prepared) to give a cross-linker containing 8g/L glucose oxidase, 0.8g/L catalase, 0.05wt.% EDC, 0.006wt.% DMAP, pH7.00 of the buffer solution, and the resulting cross-linker was put in a refrigerator for use.
Weighing 0.0250g of carboxylated graphene, dissolving in 50mL of distilled water, carrying out ultrasonic treatment for 10min to prepare carboxylated graphene solution with the concentration of 0.05mg/mL, adding a mixed solution containing 0.05wt.% EDC and 0.006wt.% DMAP for activation for 25 min, transferring 10 mu L of the carboxylated graphene activation solution by a pipette, dripping the carboxylated graphene activation solution on the surface of a gold electrode modified by glutathione, naturally airing to obtain the carboxylated graphene/gold electrode modified by glutathione, and placing the gold electrode in a refrigerator for standby.
And (3) leaching the carboxylated graphene/glutathione-modified gold electrode with distilled water, soaking the gold electrode in the prepared cross-linking agent for 22h (6 ℃), and naturally airing to obtain the glutathione-assembled glucose biosensor.
Example 3
(1) Preparation of glutathione-modified gold electrode
Gold electrode with diameter of 1.0mm was treated with 0.05 μm Al 2 O 3 Polishing the powder to a mirror surface, washing with distilled water for 10min, taking out, naturally airing, and carrying out ultrasonic washing with 75% alcohol for 10min. At 2.8X10 by Cyclic Voltammetry (CV) -3 The solution of potassium ferricyanide (containing 5.2mol/L KCl) was scanned to a steady state with a potential difference within 100 mV.
0.6146g of glutathione is weighed and dissolved in phosphate buffer solution with pH value of 7.00 to prepare 50mL of glutathione solution with concentration of 0.04mol/L, the ground gold electrode is suspended and soaked in the 0.04mol/L glutathione solution, the solution is soaked for 24 hours at 5 ℃, and the solution is preserved in a refrigerator at 4 ℃ for standby.
(2) Preparation of carboxylated graphene
A 250ml reaction bottle is assembled in the ice water bath, and 24ml of concentrated sulfuric acid is poured in lightly; adding a solid mixture of 0.7g of potassium nitrate and 1.1g of graphite powder under stirring, and adding 3.2g of potassium permanganate; controlling the temperature to be less than 15 ℃, and stirring and reacting for 2 hours; then heating to 38 ℃, and continuing stirring for 40min; adding 50ml of deionized water, continuously stirring for 20min, adding 5g of hydrogen peroxide (35 wt.%) to reduce the residual oxidant, and making the solution brown yellow and emit red smoke; filtering while the solution is hot, and washing the solution with 5wt.% HCl and deionized water until sulfate radical cannot be detected in the filtrate; finally, placing the solid in a drying oven at 60 ℃ for full drying, and preserving the obtained graphene oxide for later use;
and dispersing 200mg of the prepared graphene oxide in 150mL of aqueous solution by ultrasonic, adding 5.0g of bromoacetic acid and 6.0g of sodium hydroxide, performing ultrasonic reaction for 3 hours, centrifuging, washing to be neutral, and drying in a drying oven to obtain carboxylated graphene.
(3) Preparation of glutathione Assembly-based glucose biosensor
Glucose oxidase, catalase, 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), 4-Dimethylaminopyridine (DMAP) were dissolved in 10mL of phosphate buffer solution (pH 7.00,0.20mol/L, freshly prepared) to give a cross-linker containing 7g/L glucose oxidase, 0.7g/L catalase, 0.04wt.% EDC, 0.005wt.% DMAP, pH7.00 of the buffer solution, and the resulting cross-linker was put in a refrigerator for use.
Weighing 0.0250g of carboxylated graphene, dissolving in 50mL of distilled water, carrying out ultrasonic treatment for 10min to prepare carboxylated graphene solution with the concentration of 0.05mg/mL, adding a mixed solution containing 0.04wt.% EDC and 0.005wt.% DMAP for activation for 20min, transferring 10 mu L of the carboxylated graphene activation solution by a pipette, dripping the carboxylated graphene activation solution on the surface of a gold electrode modified by glutathione, naturally airing to obtain the carboxylated graphene/gold electrode modified by glutathione, and placing the gold electrode in a refrigerator for standby.
And (3) leaching the carboxylated graphene/glutathione-modified gold electrode with distilled water, soaking the gold electrode in the prepared cross-linking agent for 26 hours (4 ℃), and naturally airing to obtain the glutathione-assembled glucose biosensor.
The invention respectively takes a bare gold electrode, a glutathione modified gold electrode, a glutathione and carboxylated graphene modified gold electrode and a glucose biosensor assembled based on glutathione as working electrodes, a platinum electrode as an auxiliary electrode and a saturated calomel electrode as a reference electrode in the embodiment 1, and the ratio of the electrode to the electrode is 2.5X10 -3 In mol/L potassium ferricyanide (containing 10 mmol/LKCl), cyclic voltammetric scan experiments were performed, and the results are shown in FIG. 2. The graph shows that the peak current c is smaller than the peak current d, which indicates that the bare gold electrode is modified by glutathione, the potential difference change is large, the current response is smaller, and the graph indicates that the glutathione is successfully modified to the surface of the gold electrode, and the glutathione obstructs the transfer of electrons. And compared with the curves b and c, the potential difference of the electrode is increased, the reversibility of the electrode is increased, the current response of an oxidation peak is obviously increased, and the modification of carboxylated graphene is proved to promote the transfer of electrons. From the trend of the curve a, the current response of the redox peak of the enzyme electrode is obviously increased, which indicates that the modification of glutathione-carboxylated graphene-enzyme promotes the transfer of electrons and improves the sensitivity of the electrode.
The present invention has made the following studies on the conditions of use of the glutathione-based assembled glucose biosensor in example 1:
(1) Influence of the pH value of the buffer solution on the glucose biosensor
The electrochemical process of glucose oxidase has protons involved, so the effect of pH of 0.2mol/L phosphate buffer solution (PBS solution) on the electrochemical behavior of glucose oxidase was examined. The cyclic voltammogram of the glucose biosensor of the present invention in PBS solutions at different pH values is shown in FIG. 3. As can be seen from the graph, the glucose oxidase can obtain better electrochemical response in the pH value range of 5.0-8.0Should be. At a scanning rate of 100mv/s, the pH of the PBS solution is in the range of 5.00-8.00, the redox peak current gradually decreases with increasing pH, and the peak potential gradually moves negatively due to H with increasing pH + The concentration is reduced, which is unfavorable for the electrochemical reaction. Glucose oxidase is typically detected under neutral conditions, so the assay measures glucose in PBS medium at ph=7.00.
(2) Relationship between current and scan rate of glucose on modified electrode
The cyclic voltammogram of a glucose solution of 1.0mmol/L was tested in a phosphate buffer solution (PBS solution) of 0.20mol/L, pH =7.00 with the glucose biosensor of the present invention as a working electrode at a scanning rate of 0.1V/s in a potential window of-0.20 to 1.0V, and the resulting cyclic voltammogram is shown in FIG. 4. It can be seen that the enzyme-modified electrode has an oxidation peak within this potential window, ep=0.505V. CV test is carried out within the range of 0.02-0.1V/s, oxidation peak potential Ep of the CV test is positively shifted along with the increase of the scanning speed, oxidation peak current Ip is in an increasing trend along with the increase of the scanning speed, and oxidation peak current Ip and square root of the scanning speed are within the range of 0.02-0.1V/sA good linear relationship is shown in FIG. 5, and the linear fitting equation is Ip (μA) =0.0954+1.6749V 1/2 R= 0.9991. The results show that the voltammetric behavior of the electrode modified by the enzyme in the glucose solution is an electrode process controlled by diffusion, which indicates that glucose is not adsorbed on the electrode, and the cyclic voltammogram of the electrode is unchanged after long-time scanning, which indicates that the electrode is not polluted and can be used for detecting in vitro glucose.
The present invention also conducted the following studies on the methodology of the glutathione-based assembled glucose biosensor in example 1:
(1) Drawing of correction curves
The glucose biosensors of the present invention were placed at a concentration of 1.0X10, respectively -3 mol/L、3.0×10 -3 mol/L、5.0×10 -3 mol/L、8.0×10 -3 mol/L、1.0×10 -2 In a mol/L glucose solution, the current obtained by scanning with a cyclic voltammetry at a scanning rate of 0.1V/s in a potential window of-0.20-1.0V is reduced with the increase of the glucose concentration, the linear equation is I= 2.3611-0.1544c, the correlation coefficient R is 0.9995, and the concentration of the glucose is 1.0x10 -3 mol/L～1×10 -2 The linear relation in the mol/L range is good.
(2) Determination of precision
The glucose biosensor of the present invention was continuously measured at a concentration of 1.0X10 -3 The results of the mol/L glucose solutions were 0.3970. Mu.A, 0.3998. Mu.A, 0.3950. Mu.A, 0.3990. Mu.A and 0.3951. Mu.A, respectively, and the RSD was 0.22% as calculated from the above data, indicating that the glucose biosensor was excellent in precision.
(3) Determination of stability
The glucose biosensor of the invention is respectively placed at the concentration of 1.0X10 at 1h, 4h, 8h, 12h and 24h - 3 As a result of measurement in a glucose solution of mol/L, the peak currents were not substantially changed as 2.098. Mu.A, 2.016. Mu.A, 1.901. Mu.A, 1.949. Mu.A and 1.978. Mu.A, and the RSD was 7.43% as calculated from the above data, indicating that the glucose biosensor was excellent in stability.
The glutathione-carboxylated graphene self-assembled glucose biosensor is successfully assembled. The glutathione and carboxylated graphene are utilized to fix glucose oxidase, so that the glucose biosensor with high sensitivity, good selectivity, good reproducibility and good stability is constructed. Due to the existence of carboxylated graphene, the active center of glucose oxidase shows good direct electrochemical behavior, and has good catalytic effect on oxidation of glucose. Experimental results show that the assembly can well fix the glucose oxidase, and the carboxylated graphene can promote electron transfer of the active center of the glucose oxidase, so that the catalytic activity of the enzyme is enhanced. The electrochemical catalytic behavior of glucose on the self-assembled film modified electrode is studied, and the reversibility of glucose on the enzyme modified gold electrode is obviously improved. Preliminary determination of the glucose biosensor Linear response Range of 1.0X10 by methodological investigation -3 mol/L～1×10 -2 mol/L, phaseThe off coefficient R is 0.9995, and the stability is good. The glucose biosensor has a certain application prospect and can provide reference for clinical examination.
The glucose biosensor enzyme obtained by the invention has higher modification stability, repetition stability and storage stability and prolonged service life, so that the glucose biosensor enzyme can continuously monitor the glucose concentration in vitro, timely provide effective information of optimal insulin treatment and metabolism control for glucose patients, and is an effective method for reducing long-term complications of the diabetes patients. The glucose biosensor plays an important role in food analysis, fermentation control, clinical examination and the like.
Claims (9)
1. A method for constructing a glucose biosensor based on glutathione assembly is characterized by comprising the following steps: taking a glutathione-modified gold electrode as a carrier, activating carboxylated graphene, loading the carboxylated graphene on the surface of the glutathione-modified gold electrode to obtain carboxylated graphene/glutathione-modified gold electrode, soaking the carboxylated graphene/glutathione-modified gold electrode in a cross-linking agent, and airing to obtain a glutathione-assembled-based glucose biosensor;
the cross-linking agent is phosphate buffer solution containing glucose oxidase, catalase, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 4-dimethylaminopyridine;
the carboxylated graphene is activated by adopting a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 4-dimethylaminopyridine.
2. The method for constructing a glutathione assembled glucose biosensor according to claim 1, wherein: the concentration of glucose oxidase in the cross-linking agent is 6-8g/L, the concentration of catalase is 0.6-0.8g/L, the mass percentage concentration of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide is 0.03-0.05 wt%, and the mass percentage concentration of 4-dimethylaminopyridine is 0.004-0.006 wt%.
3. The method for constructing a glutathione assembled glucose biosensor according to claim 1, wherein: the pH value of the phosphate buffer solution is 6.8-7.0, and the concentration is 0.2-0.25mol/L.
4. A method of constructing a glutathione assembled glucose biosensor according to any of claims 1-3, characterized by comprising the steps of:
(1) Preparation of glutathione-modified gold electrode
Placing a gold electrode in a potassium ferricyanide solution, scanning to be stable by using a cyclic voltammetry, and then soaking the gold electrode in a glutathione solution to obtain a glutathione-modified gold electrode;
(2) Preparation of carboxylated graphene
Adding graphite powder, potassium nitrate and potassium permanganate into concentrated sulfuric acid, heating to react, and adding hydrogen peroxide to react after the reaction is finished to obtain graphene oxide; reacting graphene oxide with sodium hydroxide and bromoacetic acid to obtain carboxylated graphene;
(3) Preparation of glutathione Assembly-based glucose biosensor
And (3) loading the carboxylated graphene obtained in the step (2) on the surface of the glutathione-modified gold electrode obtained in the step (1) after activating the carboxylated graphene to obtain the carboxylated graphene/glutathione-modified gold electrode, soaking the carboxylated graphene/glutathione-modified gold electrode in a cross-linking agent, and airing to obtain the glutathione-assembled glucose biosensor.
5. The method for constructing a glutathione assembled glucose biosensor according to claim 4, wherein: in the step (1), the gold electrode adopts Al with average granularity of 0.05-0.06 mu m 2 O 3 Polishing the powder, and then placing the powder into a potassium ferricyanide solution; the concentration of the potassium ferricyanide solution is 2.5X10 -3 -3.0×10 -3 The potassium ferricyanide solution contains potassium chloride, and the concentration of the potassium chloride is 5.0-5.5mol/L.
6. The method for constructing a glutathione assembled glucose biosensor according to claim 4, wherein: in the step (1), glutathione solution is prepared by dissolving glutathione in phosphate buffer solution with pH value of 6.8-7.0; the concentration of the glutathione solution is 0.04-0.06mol/L; the soaking temperature is 4-6deg.C, and the soaking time is 22-26h.
7. The method for constructing a glutathione assembled glucose biosensor according to claim 4, wherein: in the step (2), the dosage ratio of the graphite powder to the potassium nitrate to the potassium permanganate to the concentrated sulfuric acid to the hydrogen peroxide is 1.0-1.2:0.6-0.8:3.0-3.5:23-25:5-7, wherein the mass percentage concentration of the graphite powder to the potassium nitrate to the potassium permanganate to the hydrogen peroxide is 30-35wt.% in terms of g and the concentrated sulfuric acid to ml; the mass ratio of graphene oxide to sodium hydroxide to bromoacetic acid is 0.1-0.3:5-7:4-6.
8. The method for constructing a glutathione assembled glucose biosensor according to claim 4, wherein: in the step (2), the temperature-rising reaction is carried out for 2-3 hours at 15-20 ℃ and then the temperature is raised to 35-40 ℃ for 30-35min.
9. The method for constructing a glutathione assembled glucose biosensor according to claim 4, wherein: in the step (3), the carboxylated graphene is activated by adopting a mixed solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 4-dimethylaminopyridine, wherein the mass percentage concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in the mixed solution is 0.03-0.05 wt%, and the mass percentage concentration of the 4-dimethylaminopyridine is 0.004-0.006 wt%; the activation time is 20-30min; the soaking temperature is 4-6deg.C, and the soaking time is 22-26h.
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| CN108181360A (en) * | 2017-12-22 | 2018-06-19 | 齐鲁医药学院 | The construction method of acid phosphoric acid ester enzyme biologic sensor based on mercapto-functionalized graphene self assembly |
| CN108802124A (en) * | 2018-08-28 | 2018-11-13 | 长沙理工大学 | A kind of detection method and sensor of the l-cysteine based on glutathione composite membrane grid gold electrode |
| CN111879836A (en) * | 2020-07-10 | 2020-11-03 | 宁波大学 | Electrochemical method for detecting uric acid and urate oxidase and application thereof |
| CN115112738A (en) * | 2022-07-21 | 2022-09-27 | 闽江学院 | Preparation of laser direct-writing graphene/enzyme electrode and glucose sensing application |
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