CN113203783A - Method for detecting 1, 5-anhydroglucitol based on nanocomposite - Google Patents
Method for detecting 1, 5-anhydroglucitol based on nanocomposite Download PDFInfo
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- CN113203783A CN113203783A CN202110521986.7A CN202110521986A CN113203783A CN 113203783 A CN113203783 A CN 113203783A CN 202110521986 A CN202110521986 A CN 202110521986A CN 113203783 A CN113203783 A CN 113203783A
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- MPCAJMNYNOGXPB-SLPGGIOYSA-N 1,5-anhydro-D-glucitol Chemical compound OC[C@H]1OC[C@H](O)[C@@H](O)[C@@H]1O MPCAJMNYNOGXPB-SLPGGIOYSA-N 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000002114 nanocomposite Substances 0.000 title claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 17
- 229940025294 hemin Drugs 0.000 claims abstract description 17
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- 238000002360 preparation method Methods 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 6
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 claims description 3
- 238000007650 screen-printing Methods 0.000 claims description 3
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 2
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 52
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- OCLOLUFOLJIQDC-HSUXUTPPSA-N 1,5-anhydro-D-fructose Chemical compound OC[C@H]1OCC(=O)[C@@H](O)[C@@H]1O OCLOLUFOLJIQDC-HSUXUTPPSA-N 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 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 description 1
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Images
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
-
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
Abstract
A method for detecting 1,5-AG based on a nano composite material is characterized in that pyranose oxidase (PROD) is used as a recognition molecule, and the PROD is specifically combined with 1,5-AG to form the RGO-CMCS-Hemin/Pt NPs nano composite material. And then, based on the good electron transfer effect and excellent catalytic performance of the material, an electrochemical biosensor capable of specifically identifying and quantitatively analyzing 1,5-AG is constructed. The method has lower detection limit, and can reach 0.0384 mg/mL.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a method for detecting 1, 5-anhydroglucitol based on a nano composite material.
Background
The existing methods for detecting 1, 5-anhydroglucitol (1, 5-AG) include holoenzyme method, reversed phase chromatography, liquid chromatography-mass spectrometry (LC/MS), enzyme-linked immunosorbent assay (ELISA), and the like. The invention patent with publication number CN 108918447a relates to a sensor and a detection method for detecting 1,5-AG based on QCM, but the method has a complicated processing procedure for quartz crystal wafers. The invention patent with publication number CN 110702676A relates to a 1,5-AG detection kit and a method, a reagent R1 and a reagent R2 are kept stable under the interference of high glucose concentration by selecting a proper method and a proper stabilizer, and the 1,5-AG content in a human serum sample is measured by a pyranose oxidase method; however, the method is complex to operate and high in cost. Therefore, it is necessary to develop a method for detecting 1,5-AG easily and rapidly.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for realizing 1,5-AG detection by constructing an electrochemical sensor based on a nano composite material of reduced graphene oxide-carboxymethyl chitosan-heme/nano platinum (RGO-CMCS-Hemin/Pt NPs).
In order to solve the technical problem, an RGO-CMCS-Hemin/Pt NPs composite material is prepared by a one-step reduction method, and the RGO-CMCS-Hemin/Pt NPs composite material and 1,5-AG are fixed on the surface of a screen printing electrode of modified nano-gold in pyranose oxidase (PROD) in a layer-by-layer self-assembly mode, so that the electrochemical biosensor based on the RGO-CMCS-Hemin/Pt NPs is constructed. 1,5-AG is catalyzed by PROD to generate 1, 5-anhydrofructose and hydrogen peroxide (H)2O2)，H2O2Is catalyzed and decomposed into H by RGO-CMCS-Hemin/Pt NPs composite material again2O and O2Generating an obvious current response signal, recording the peak current by adopting Differential Pulse Voltammetry (DPV) of an electrochemical workstation, and then drawing a working curve according to the relation between the concentration of 1,5-AG and the response current of the sensor to realize the electrochemical detection of 1, 5-AG.
The invention is carried out according to the following steps:
step 1: preparation of RGO-CMCS-Hemin/Pt NPs composite material
(1) Preparation of Reduced Graphene Oxide (RGO)
Weighing single-layer Graphene Oxide (GO), putting into distilled water, uniformly mixing, adding Ascorbic Acid (AA) for reduction, and obtaining Reducing Graphene Oxide (RGO).
(2) Preparation of RGO-CMCS
And adding a carboxymethyl chitosan solution into the RGO solution, and ultrasonically mixing uniformly to obtain an RGO-CMCS dispersion liquid.
(3) Preparation of RGO-CMCS-Hemin
Adding Hemin into the RGO-CMCS dispersion liquid, and uniformly stirring to obtain a reducing graphene oxide-carboxymethyl chitosan-heme (RGO-CMCS-Hemin) solution.
(4) Preparation of RGO-CMCS-Hemin/Pt NPs composite material
Adding sodium chloroplatinate and ascorbic acid into the RGO-CMCS-Hemin dispersion, stirring, centrifuging and washing to obtain the RGO-CMCS-Hemin/Pt NPs composite material.
Step 2: construction of electrochemical sensing interface
(1) The screen-printed electrode (SPCE) was activated in a dilute sulfuric acid solution.
(2) And (3) placing the activated SPCE into a chloroauric acid solution, and performing constant potential deposition to obtain Au NPs/SPCE.
(3) And dropwise adding RGO-CMCS-Hemin/Pt NPs composite material suspension on the Au NPs/SPCE electrode for incubation, washing and airing to obtain RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE.
(4) And (3) dropwise adding the PROD on the RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE, incubating and washing to obtain a PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE sensing interface, and airing for later use.
And step 3: drawing of 1,5-AG working curve
(1) And (3) dropwise adding the standard 1,5-AG solution to the PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE sensing interface obtained in the step (2), incubating and washing to obtain a working electrode, and airing for later use.
(2) The working electrode was placed in PBS solution, scanned using Differential Pulse Voltammetry (DPV), and the response current value of the sensor was recorded.
(3) Respectively detecting 1,5-AG with different concentrations, recording peak current, drawing a working curve according to the relation between the peak current and the 1,5-AG concentration, and calculating the lowest detection limit of the method.
And 4, step 4: detection of 1,5-AG in actual serum samples
(1) And (3) fully mixing the normal human serum sample with the 1,5-AG standard solution according to the ratio of 1:1 to prepare a mixed solution, dripping the mixed solution to be detected on the sensing interface prepared in the step (2), incubating and washing to obtain a working electrode, and airing for later use.
(2) The working electrode was placed in PBS solution, scanned using Differential Pulse Voltammetry (DPV), and the response current value of the sensor was recorded.
(3) And (4) calculating the concentration of the 1,5-AG in the actual serum sample to be detected according to the working curve obtained in the step (3).
Wherein, step 1 provides a high-conductivity nanocomposite material for step 2. The construction of the biosensing interface in the step 2 is an essential key step in the electrochemical detection of 1,5-AG in the steps 3 and 4. The working curve of 1,5-AG in step 3 provides a calculation basis for the determination of the concentration of 1,5-AG in the actual serum sample in step 4. As can be seen, the steps 1-4 support each other and act together, so that the detection of 1,5-AG can be realized by using the RGO-CMCS-Hemin/Pt NPs composite material.
Compared with the prior art, the invention has the following advantages:
1. the RGO-CMCS-Hemin/Pt NPs nano composite material prepared by the invention has a unique membrane structure, a larger specific surface area, stronger catalytic activity and higher conductivity, wherein the large specific surface area of the RGO-CMCS-Hemin nano composite material provides effective binding sites for Pt NPs and PROD, the fixation of PROD enzyme on an electrode is increased, the catalytic efficiency of 1,5-AG is enhanced, and the detection sensitivity is improved. In addition, the RGO-CMCS-Hemin/Pt NPs composite material with high-efficiency peroxidase-like performance is formed by utilizing the good biocompatibility and film forming capability of CMCS, the high specific surface area and the high electron transfer efficiency of RGO and the excellent catalysis of nano platinum (Pt NPs) through synergistic action, and H can be efficiently catalytically decomposed2O2A large number of electrons are generated. Meanwhile, the RGO-CMCS-Hemin/Pt NPs composite material has good conductivity, thereby increasing the electron transfer efficiency and effectively amplifyingA sensed current signal.
2. The invention adopts PROD to carry out specific recognition and catalytic decomposition on 1,5-AG to construct the nano electrochemical sensor based on the RGO-CMCS-Hemin/Pt NPs composite material; the sensor can reach the detection limit of 0.0384 mg/mL.
Drawings
FIG. 1 is a schematic diagram of the detection of 1,5-AG based on RGO-CMCS-Hemin/Pt NPs nanocomposite;
FIG. 2 Transmission Electron Microscopy (TEM) images of RGO-CMCS-Hemin (A) and RGO-CMCS-Hemin/Pt NPs (B);
FIG. 3 is a Scanning Electron Microscope (SEM) representation of the electrode surface modification process;
FIG. 4 DPV curves for different concentrations of 1, 5-AG.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.
A detection principle for detecting 1,5-AG based on RGO-CMCS-Hemin/Pt NPs nanocomposite is shown in figure 1. Firstly, an RGO-CMCS-Hemin/Pt NPs composite material with high-efficiency peroxidase-like performance is formed by utilizing the good biocompatibility and film forming capability of CMCS, the high specific surface area and the high electron transfer efficiency of RGO, the peroxidase performance of Hemin and the excellent catalysis of nano platinum (Pt NPs) through synergistic action; the RGO-CMCS-Hemin/Pt NPs composite material and 1,5-AG are fixed on the surface of a nano-gold modified screen printing electrode by adopting a layer-by-layer self-assembly mode, and the electrochemical biosensor based on the RGO-CMCS-Hemin/Pt NPs is constructed. PROD and 1,5-AG are specifically combined to catalyze and generate hydrogen peroxide (H)2O2)，H2O2Is catalyzed and decomposed into H by RGO-CMCS-Hemin/Pt NPs composite material again2O and O2And the generated electrons are transferred to the surface of the electrode through the RGO-CMCS-Hemin/Pt NPs composite nano-film. And recording the peak current response signal by adopting a Differential Pulse Voltammetry (DPV) of an electrochemical workstation, and then drawing a working curve according to the relation between the 1,5-AG concentration and the response current of the sensor to realize the electrochemical detection of the 1, 5-AG.
The specific implementation steps are as follows:
1. preparation of RGO-CMCS-Hemin/Pt NPs composite material
Firstly, weighing 6mg of Graphene Oxide (GO), putting the Graphene Oxide (GO) into distilled water to a constant volume of 60 mL, and carrying out ultrasonic treatment for 1 hour by using an ultrasonic cell disruptor to fully and uniformly dissolve the Graphene Oxide (GO) to prepare a GO aqueous solution of 0.1 mg/mL. Then 10mg Ascorbic Acid (AA) is added and stirred for reduction for 20h, thus obtaining RGO solution.
Secondly, 20mg of CMCS is added into the RGO solution, and ultrasonic crushing is carried out for 30min to obtain the RGO-CMCS dispersion liquid which is uniformly mixed.
Thirdly, 10mL of 1mg/mL Hemin solution is added into the RGO-CMCS solution, and the mixture is crushed by ultrasound for 1h to be mixed evenly, so as to obtain RGO-CMCS-Hemin dispersion liquid.
Fourthly, 4mL of 0.01mg/mL sodium chloroplatinate was added to the RGO-CMCS-Hemin dispersion, 10mg ascorbic acid was added with stirring, and the mixture was stirred for 20 hours to obtain an RGO-CMCS-Hemin/Pt NPs suspension.
Finally, the mixture is centrifuged and washed at 70oAnd C, drying to obtain the RGO-CMCS-Hemin/Pt NPs composite material.
The RGO-CMCS-Hemin/Pt NPs composite was characterized using a Transmission Electron Microscope (TEM) with Tecnai G2F 30S-TWIN, manufactured by FEI, USA, as shown in FIG. 2. FIG. 2A is a TEM image of RGO-CMCS-Hemin, which shows that RGO-CMCS-Hemin is a relatively flat membrane-like pleated structure. FIG. 2B is a TEM image of RGO-CMCS-Hemin/Pt NPs with small dark colored particles appearing in the membrane-like pleated structure and being more clearly flat, indicating the successful preparation of the RGO-CMCS-Hemin/Pt NPs composite.
Construction of an electrochemical biosensing interface
The screen-printed electrode (SPCE) was first soaked at 0.5 mol/L H before use2SO4Cyclic Voltammetry (CV) scanning is carried out in the solution, and 20 circles of scanning are carried out in a voltage range of-0.4V-1.0V; after completion of the scanning, washing with water and drying, activated SPCE was obtained. And (3) putting the activated SPCE electrode into 10mL of 0.01% chloroauric acid solution, depositing for 120s at a constant potential of-0.4V, washing and drying to obtain Au NPs/SPCE. Soaking Au NPs/SPCE electrode in 2.5% glutaraldehyde for 15min, washing with PBS (pH 7.4), blow-drying, adding 6 μ L RGO-CMCS-Hemin/Pt NPs suspension, incubatingWashing with PBS for 60 min, and air drying to obtain RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE. And dripping 3 mu L of PROD onto an RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE interface, incubating for 3h, washing the PROD which cannot be fixed on the interface, and naturally airing to obtain the PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE sensing interface.
The modification process of the electrode surface is characterized by adopting an SU8020 scanning electron microscope produced by Hitachi of Japan. A typical Scanning Electron Micrograph (SEM) is shown in FIG. 3, where FIG. 3A is SPCE, which is relatively flat. FIG. 3B is Au NPs/SPCE, where many shiny small particles of material are observed on the surface, illustrating the deposition of nanogold onto the screen printed electrode. FIG. 3C is RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE, and compared with FIG. 3B, the shiny small particle substances are reduced and the inclusion is appeared, which is caused by the RGO-CMCS-Hemin/Pt NPs attachment; FIG. 3D shows a clear white plate-like structure, illustrating the successful attachment of PROD to RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE, indicating that a 1,5-AG sensor has been successfully fabricated.
Drawing of working curves
Dropping 3 mu L1, 5-AG solution on a PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE sensing interface, incubating for 30min at 37 ℃, alternately washing with PBS solution with pH7.4 and distilled water and drying to obtain 1,5-AG/PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE. The SEM image is shown in FIG. 3E. In comparison with FIG. 3D, there are clearly visible small dots indicating that 1,5-AG has successfully immobilized to the electrode surface by specifically binding to PROD.
1,5-AG/PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE was placed in PBS buffer (0.2 mol/L, pH 7.4) and the peak current was recorded using DPV scanning at the electrochemical workstation. The DPV profile of the different 1,5-AG concentrations is shown in FIG. 4. Within the concentration of 1,5-AG from 0.1 mg/mL to 2 mg/mL, the current response value of the sensor is in a linear relation with the concentration of 1,5-AG, and the linear equation Y =4.01372+2.18401X (wherein Y is the current peak potential response intensity and X is the concentration of 1, 5-AG), and R = 0.98229. By the formula CLOD=3SbThe detection limit of the sensor is 0.0384mg/mL (S) through calculation of/bbStandard deviation calculated for 6 replicates of blank samples, b is the slope of the working curve).
Detection of 1,5-AG in actual serum samples
A normal human serum sample is fully mixed with 1,5-AG standard solutions of 0.5 mg/mL, 1.5 mg/mL and 2.0 mg/mL respectively according to the proportion of 1:1 to prepare a mixed solution. Respectively dropping 3 μ L of the above mixed solution on the surface of PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE to form a working electrode. The working electrode was placed in PBS buffer as described in step 3, scanned with DPV and its current value recorded. According to the standard curve Y =4.01372+2.18401X in step 3, the concentration of 1,5-AG in the corresponding actual serum sample can be calculated, and the detection result is shown in Table 1. The recovery rate is in the range of 99.25-107.60%, and the RSD value is 1.80-6.14%. The results show that the developed 1,5-AG electrochemical sensor has good application prospect.
TABLE 1 results of 1,5-AG detection in actual serum samples
(Note: serum samples were provided by the ninth second and fourth hospitals of the United nations 'society of people's liberation force).
Claims (2)
1. A preparation method of an RGO-CMCS-Hemin/Pt NPs nano composite material is characterized by comprising the following steps:
(1) weighing 6mg of graphene oxide, putting the graphene oxide into distilled water to a constant volume of 60 mL, and carrying out ultrasonic treatment for 1 hour by using an ultrasonic cell disruptor to fully and uniformly dissolve the graphene oxide to prepare a 0.1 mg/mL GO aqueous solution; adding 10mg ascorbic acid, stirring and reducing for 20h to obtain RGO solution;
(2) adding 20mg CMCS into RGO solution, and ultrasonically crushing for 30min to obtain uniformly mixed RGO-CMCS dispersion;
(3) adding 10mL of 1mg/mL Hemin solution into the RGO-CMCS solution, and carrying out ultrasonic crushing for 1h to fully and uniformly mix the solution to obtain RGO-CMCS-Hemin dispersion;
(4) adding 4mL of 0.01mg/mL sodium chloroplatinate into the RGO-CMCS-Hemin dispersion, adding 10mg ascorbic acid while stirring, and stirring for 20h to obtain an RGO-CMCS-Hemin/Pt NPs suspension;
(5) centrifugally washing and drying at 70 ℃ to obtain the RGO-CMCS-Hemin/Pt NPs nano composite material.
2. A method for detecting 1,5-AG based on the nanocomposite material as claimed in claim 1, which is characterized by comprising the following steps:
the method comprises the following steps: construction of an electrochemical biosensing interface
(1) The screen printing electrode is firstly soaked in 0.5 mol/L H before use2SO4Cyclic Voltammetry (CV) scanning is carried out in the solution, and 20 circles of scanning are carried out in a voltage range of-0.4V-1.0V; after scanning, washing with water, and drying to obtain activated SPCE;
(2) placing the activated SPCE electrode into 10mL of 0.01% chloroauric acid solution, depositing for 120s at-0.4V constant potential, washing and blow-drying to obtain Au NPs/SPCE;
(3) soaking the Au NPs/SPCE electrode in 2.5% glutaraldehyde for 15min, washing with PBS (pH 7.4) for drying, then dropwise adding 6 mu L RGO-CMCS-Hemin/Pt NPs suspension, incubating for 60 min, washing with PBS, and drying to obtain RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE;
(4) dripping 3 mu L of PROD onto an RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE interface, incubating for 3h, washing the PROD which cannot be fixed on the interface, and naturally airing to obtain a PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE sensing interface;
step two: drawing of 1,5-AG working curve
(1) Dripping 3 mu L1, 5-AG solution on a PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE sensing interface, incubating for 30min at 37 ℃, alternately washing with PBS solution with pH value of 7.4 and distilled water and drying to obtain 1,5-AG/PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE;
(2) placing 1,5-AG/PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE in PBS buffer solution with the concentration of 0.2 mol/L and the pH value of 7.4, adopting DPV scanning of an electrochemical workstation, and recording the peak current; the concentration of 1,5-AG is from 0.1 mg/mL to 2 mg/mL, the current response value of the sensor and the concentration of 1,5-AG are in a linear relation, Y is the current peak potential response intensity, X is the concentration of 1,5-AG, and the linear equation Y =4.01372+ 2.18401X;
step three: detection of 1,5-AG in actual serum samples
(1) Fully mixing a normal human serum sample with 0.5 mg/mL, 1.5 mg/mL and 2.0 mg/mL of 1,5-AG standard solutions respectively according to the proportion of 1:1 to prepare a mixed solution;
(2) respectively dripping 3 mu L of the mixed solution on the surface of PROD/RGO-CMCS-Hemin/Pt NPs/Au NPs/SPCE to form a working electrode; placing the working electrode in a PBS buffer solution according to the step 3, scanning by adopting DPV, and recording the current value;
(3) and (4) calculating to obtain the corresponding concentration of the 1,5-AG in the actual serum sample according to the standard curve in the step 3.
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