WO2023082544A1 - 一种检测covid-19抗体的血糖生物传感器 - Google Patents

一种检测covid-19抗体的血糖生物传感器 Download PDF

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WO2023082544A1
WO2023082544A1 PCT/CN2022/087002 CN2022087002W WO2023082544A1 WO 2023082544 A1 WO2023082544 A1 WO 2023082544A1 CN 2022087002 W CN2022087002 W CN 2022087002W WO 2023082544 A1 WO2023082544 A1 WO 2023082544A1
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dna
covid
substrate
antibody
concentration
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李调阳
林本慧
林美雅
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福州大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of biological detection, in particular to a blood glucose biosensor for detecting COVID-19 antibodies.
  • the detection technology of new crown antibody is mainly immunoassay method, which uses the preparation of new crown antigen protein to specifically capture and recognize new crown specific antibody (IgM/IgG).
  • the main methods include ELISA, chemiluminescence and immunoassay flow chromatography, etc., among which ELISA ELISA and chemiluminescence not only require relatively expensive optical detection equipment such as microplate readers, but also have high requirements for operators, so ELISA and chemiluminescence are not suitable for large-scale rapid detection.
  • Immunoflow chromatography is the fastest and most convenient antibody detection method among them.
  • the detection signal can be observed with the naked eye, which is very suitable for the rapid detection of the new crown antibody.
  • Subjective error, and quantitative analysis of the new crown antibody cannot be performed.
  • the object of the present invention is to provide a biosensor for detecting COVID-19 antibodies by using blood glucose to address the above problems.
  • the present invention adopts the following technical solutions:
  • a blood glucose biosensor for detecting COVID-19 antibodies comprising: new coronavirus COVID-19 antigen, new coronavirus COVID-19 antibody, new coronavirus COVID-19 secondary antibody modified with hairpin structure DNA fragment H1-DNA , Trigger DNA fragment T-DNA, enzyme-labeled hairpin structure DNA fragment H2-DNA and blood glucose biosensor;
  • the new coronavirus COVID-19 antigen is: the S protein or N protein of the new coronavirus COVID-19; wherein the amino acid sequence of the S protein is as shown in SEQ ID NO.1; wherein the amino acid sequence of the N protein is as shown in SEQ ID NO.2 Show;
  • the new coronavirus COVID-19 antibody is: new coronavirus COVID-19 antibody COVID-19 IgG or COVID-19 IgM;
  • the new coronavirus COVID-19 secondary antibody is: the anti-antibody of the new coronavirus COVID-19 antibody IgG or IgM, that is, COVID-19 Anti-IgG or COVID-19 Anti-IgM;
  • the nucleotide sequence of the modified hairpin structure DNA fragment H1-DNA is shown in SEQ ID NO.3;
  • the nucleotide sequence of the trigger DNA fragment T-DNA is as shown in SEQ ID NO.4;
  • the hairpin structure DNA fragment H2-DNA of described enzyme mark its nucleotide sequence is as shown in SEQ ID NO.5;
  • the enzyme of mark H2-DNA is the enzyme that can convert carbohydrate substrate into glucose, comprises sucrase Any one of (Invertase), maltase (Maltase), and fructose.
  • the above-mentioned blood glucose biosensor for detecting COVID-19 antibody wherein the blood glucose biosensor is a blood glucose biosensor prepared by using screen-printed electrodes;
  • the preparation method of the blood glucose biosensor prepared by using screen-printed electrodes comprises the following steps: first Deposit Prussian blue on the working electrode as an electrical intermediary substance, deposition conditions: hydrochloric acid concentration 0.1-1M, anhydrous ferric chloride concentration 0.25-2.5mM, potassium ferricyanide concentration 0.25-2.5mM, potassium chloride concentration 0.01-0.1M , CV deposition, sweep rate 10-20mV/s, deposition 3-5 cycles, -0.1V-1V; after depositing Prussian blue, rinse with deionized water, dry with nitrogen; then fix glucose oxidase on the surface of Prussian blue (GOx): 0.1-1mg/mL GOx is diluted 10 times with 1wt%-5wt% BSA (solvent is deionized water), take 10uL diluted GOx and add 10
  • a method for using a blood glucose biosensor for detecting COVID-19 antibodies comprising the following steps:
  • Step1 Immobilize the new coronavirus COVID-19 antigen on the substrate, wash and dry the substrate with buffer solution;
  • Step2 Add the sample containing the new coronavirus COVID-19 antibody to the substrate of Step1, the new coronavirus COVID-19 antigen immobilized on the substrate specifically captures the COVID-19 antibody, wash and dry the substrate with a buffer solution;
  • Step3 Add the new coronavirus COVID-19 secondary antibody modified with the hairpin structure DNA fragment H1-DNA to the dried substrate of Step2 to specifically recognize the new coronavirus COVID-19 antibody, wash and dry the substrate with a buffer solution;
  • Step4 Add the trigger DNA fragment T-DNA and the enzyme-labeled hairpin structure DNA fragment H2-DNA to the dried substrate of Step3, T-DNA opens the hairpin structure of H1-DNA to form a T-DNA-H1-DNA complex H2-DNA replaces T-DNA to form a H1-DNA-H2-DNA complex, and the replaced T-DNA circulates again to participate in opening the H1-DNA hairpin structure. After reacting at room temperature for a certain period of time, wash with buffer solution and dry the substrate;
  • Step5 Add the carbohydrate substrate to the dried substrate of Step4, react at room temperature for a certain period of time, and the enzyme labeled on H2-DNA converts the carbohydrate substrate into glucose;
  • Step6 Add the glucose solution of Step5 to the blood glucose biosensor to detect the glucose content of the solution, and then detect the content of the new coronavirus COVID-19 antibody in the sample containing the new coronavirus COVID-19 antibody.
  • the substrate described in Step 1 of the above method of use includes any one of 96-well plate, centrifuge tube, and glass tube; the substrate is pretreated before use, specifically: pretreatment with oxygen plasma, the ratio of argon to oxygen is 2 :1-5:1, power 10W-50W, treatment 5-10min; then add APTES solution to the substrate treated with oxygen plasma, the concentration of APTES solution is 0.1wt%-5wt%, the solvent is absolute ethanol and water The mixed solution, the content of water is 1wt%-10wt%, react at room temperature for 2-6h, after the reaction, clean the substrate with absolute ethanol and deionized water, dry it with nitrogen gas for use.
  • the sample containing the new coronavirus COVID-19 antibody in Step 2 of the above method of use is added to the substrate of Step 1 and incubated at room temperature for 5-15 minutes.
  • the trigger DNA fragment T-DNA and the enzyme-labeled hairpin structure DNA fragment H2-DNA are prepared into a solution with 5 ⁇ SSC buffer solution; the concentration ratio of the two is 1:10; the volume ratio is 1:1; T - The concentration of DNA is 0.01-1 ⁇ mol/L, the concentration of H2-DNA is 0.1-10 ⁇ mol/L; react at room temperature for 10-30 minutes.
  • Step1 immobilizes the new coronavirus COVID-19 antigen (S protein or N protein, Figure 1-12) on the substrate ( Figure 1-11);
  • Step2 will contain the new coronavirus COVID-19 antibody (COVID-19 IgG or COVID-19 IgM,
  • the sample ( Figure 1-13) of Figure 1-14) is added to the substrate of Step1, and the new crown antigen (S protein or N protein) immobilized on the substrate specifically captures the new crown antibody (COVID-19 IgG or COVID-19 IgM);
  • Step3 adds the new coronavirus COVID-19 secondary antibody modified with the hairpin structure DNA fragment H1-DNA (COVID-19 Anti-IgG or COVID-19 Anti-IgM, Figure 1-15) to the dried substrate of Step2 to Specifically recognize COVID-19 IgG or COVID-19 IgM;
  • Step4 will trigger DNA fragment T-DNA ( Figure 1-16) and enzyme-labeled hairpin structure DNA fragment H2-DNA ( Figure 1-18) into
  • the replaced T-DNA (Fig. 1-16) can be circulated again to participate in the reaction of opening the H1-DNA hairpin structure; enzyme-labeled hybrid chain amplification technology (E-HCR)
  • E-HCR enzyme-labeled hybrid chain amplification technology
  • N protein or S protein As a signal probe for COVID-19 IgG/IgM, a new coronavirus antigen (N protein or S protein)-COVID-19 IgG/IgM-E-HCR (H1-DNA-H2-DNA complex) sandwich structure was constructed;
  • the substrate-like substrate is added to the dried substrate of Step4, on the H2-DNA in the sandwich structure of the new coronavirus antigen (N protein or S protein)-COVID-19 IgG/IgM-E-HCR (H1-DNA-H2-DNA complex)
  • the labeled enzyme ( Figure 1-110) converts (sucrose, fructose, maltose, etc.) sugar substrate (Figure 1-111) into glucose ( Figure 1
  • E-HCR enzyme-labeled hybrid chain amplification technology
  • FIG. 1 A schematic diagram of a technical solution for detecting COVID-19 antibodies with a blood glucose biosensor.
  • 11 substrate, 12: new coronavirus COVID-19 antigen, 13: sample, 14: new coronavirus COVID-19 antibody, 15: new coronavirus COVID-19 secondary antibody modified with hairpin structure DNA fragment H1-DNA, 16: trigger DNA fragment T-DNA, 17: T-DNA-H1-DNA complex, 18: enzyme-labeled hairpin DNA fragment H2-DNA, 19: H1-DNA-H2-DNA complex, 110: labeled H2-DNA Enzymes, 111: Carbohydrate Substrates, 112: Glucose, 113: Glucose Biosensors.
  • Figure 2 is the current-time curve of serum solutions with different concentrations of COIVD-19 IgG using the new crown COVID-19N protein as the antigen.
  • Figure 3 is a fitting curve of the new crown COVID-19N protein as the antigen current signal and the concentration of COVID-19 IgG.
  • Figure 4 is the fitting curve of the new crown COVID-19N protein as the antigen current signal and the concentration of COVID-19 IgM.
  • Figure 5 is a fitting curve of the new crown COVID-19S protein as the antigen current signal and the concentration of COVID-19 IgG.
  • Figure 6 is a fitting curve of the new crown COVID-19S protein as the antigen current signal and the concentration of COVID-19 IgM.
  • COVID-19 antibodies COVID-19 IgG, COVID-19 IgM and COVID-19 secondary antibodies COVID-19 Anti-IgG, COVID-19 Anti-IgM were purchased from Inshore Protein Technology Co., Ltd.
  • H1-DNA-COVID-19 Anti-IgG or H1-DNA-COVID-19 Anti-IgM) decorated with hairpin DNA fragment H1-DNA was synthesized by Shanghai Sangon Bioengineering Co., Ltd. .
  • the enzyme-labeled hairpin DNA fragment H2-DNA was synthesized by Shanghai Sangon Bioengineering Co., Ltd.
  • Embodiment 1 A kind of blood glucose biosensor that detects COVID-19 antibody
  • a blood glucose biosensor for detecting COVID-19 antibodies comprising the following components: new coronavirus COVID-19N protein (amino acid sequence shown in SEQ ID NO.2), new coronavirus COVID-19 antibody COVID-19 IgG, modified with hairpin Structural DNA fragment H1-DNA COVID-19 secondary antibody H1-DNA-COVID-19 Anti-IgG, trigger DNA fragment T-DNA, sucrase-labeled hairpin structure DNA fragment H2-DNA and blood glucose biosensor;
  • the nucleotide sequence of the hairpin structure DNA fragment H1-DNA is shown in SEQ ID NO.3:
  • SEQ ID NO.3 H1-DNA: 3'-COOH-TTTTTTTTTTTTGGTGTATGGCGTCTGCCCTCTGTAAGGCAGACGCCATACACCTTTCCAATACC-5';
  • the nucleotide sequence of the trigger DNA fragment T-DNA is as shown in SEQ ID NO.4;
  • SEQ ID NO.4 T-DNA: 3'-GGTATTGGAAAGGTGTATGGCGTCTGCC-5';
  • the hairpin structure DNA fragment H2-DNA has a nucleotide sequence as shown in SEQ ID NO.5:
  • SEQ ID NO.5 H2-DNA: 3'-Enzyme-TTTTTTTTTTTTTT-CTCTGTAAGGTGTATGGCGTCTGCCTTACAGAGGGCAG-5'.
  • the 96-well plate was treated with oxygen plasma, the ratio of argon to oxygen was 4:1, the power was 20W, and the treatment was 5 minutes.
  • H1-DNA-modified COVID-19 Anti-IgG H1-DNA-COVID-19 Anti-IgG
  • T-DNA and H2-DNA solutions Prepare T-DNA and H2-DNA solutions with 5 ⁇ SSC buffer solution, the concentration ratio of the two is 1:10, and modify sucrase on H2-DNA.
  • the concentration of T-DNA was 0.5 ⁇ mol/L, and the concentration of H2-DNA was 5 ⁇ mol/L.
  • Add 50 ⁇ L T-DNA and 50 ⁇ L H2-DNA solution into the 96-well plate at the same time. React at room temperature for 10 min, wash with 1 ⁇ PBS buffer solution of pH 7.4, and dry the 96-well plate with nitrogen gas for later use.
  • the blood glucose biosensor was prepared using screen-printed electrodes. First, Prussian blue was deposited on the working electrode as an electrical intermediary substance. The deposition conditions were: hydrochloric acid concentration 0.2M, anhydrous ferric chloride concentration 0.5mM, potassium ferricyanide concentration 0.5mM, and potassium chloride concentration 0.1M. CV deposition, scan rate 10V/s, deposition 4 cycles, -0.1V-1V. After the Prussian blue was deposited, it was rinsed with deionized water and dried with nitrogen gas. Glucose oxidase (GOx) was then immobilized on the Prussian blue surface.
  • GOx Glucose oxidase
  • step (7) Drop the solution containing glucose in step (7) on the blood glucose biosensor, and test the I-T curve at -0.05V for 60s.
  • Figure 2 is the current-time curve of serum solutions with different concentrations of COIVD-19 IgG using the new crown COVID-19N protein as the antigen. It can be seen from the figure that as the concentration of COVID-19 IgG increases, the current signal of the blood glucose biosensor increases. Can detect COVID-19 IgG as low as 0.1ng/mL.
  • Figure 3 is a fitting curve of the new crown COVID-19N protein as the antigen current signal and the concentration of COVID-19 IgG. It can be seen from the figure that the linear range of detection is 0-2ng/mL.
  • Embodiment 2 A kind of blood glucose biosensor that detects COVID-19 antibody
  • a blood glucose biosensor for detecting COVID-19 antibodies includes the following components: N protein (amino acid sequence shown in SEQ ID NO.2) of new coronavirus COVID-19, new coronavirus COVID-19 antibody COVID-19 IgM, modified hair Clip structure DNA fragment H1-DNA COVID-19 secondary antibody H1-DNA-COVID-19 Anti-IgM, trigger DNA fragment T-DNA, sucrase-labeled hairpin structure DNA fragment H2-DNA and blood glucose biosensor;
  • the nucleotide sequences of the hairpin structure DNA fragment H1-DNA, the trigger DNA fragment T-DNA, and the hairpin structure DNA fragment H2-DNA are the same as those in Example 1.
  • the 96-well plate was treated with oxygen plasma, the ratio of argon to oxygen was 4:1, the power was 20W, and the treatment was 5 minutes.
  • H1-DNA-modified COVID-19 Anti-IgM H1-DNA-COVID-19 Anti-IgM
  • T-DNA and H2-DNA solutions Prepare T-DNA and H2-DNA solutions with 5 ⁇ SSC buffer solution, the concentration ratio of the two is 1:10, and modify sucrase on H2-DNA.
  • the concentration of T-DNA was 0.5 ⁇ mol/L
  • the concentration of H2-DNA was 0.5 ⁇ mol/L.
  • Add 50 ⁇ L T-DNA and 50 ⁇ L H2-DNA solution into the 96-well plate at the same time. React at room temperature for 10 min, wash with 1 ⁇ PBS buffer solution of pH 7.4, and dry the 96-well plate with nitrogen gas for later use.
  • the blood glucose biosensor was prepared using screen-printed electrodes. First, Prussian blue was deposited on the working electrode as an electrical intermediary substance. The deposition conditions were: hydrochloric acid concentration 0.2M, anhydrous ferric chloride concentration 0.5mM, potassium ferricyanide concentration 0.5mM, and potassium chloride concentration 0.1M. CV deposition, scan rate 10V/s, deposition 4 cycles, -0.1V-1V. After the Prussian blue was deposited, it was rinsed with deionized water and dried with nitrogen gas. Glucose oxidase (GOx) was then immobilized on the Prussian blue surface.
  • GOx Glucose oxidase
  • step (7) Drop the solution containing glucose in step (7) on the blood glucose biosensor, and test the I-T curve at -0.05V for 60s.
  • Figure 4 is the fitting curve of the new crown COVID-19N protein as the antigen current signal and the concentration of COVID-19 IgM. It can be seen from the figure that the detection can reach 0.1ng/mL, and the linear range of detection is 0-4ng/mL.
  • Embodiment 3 A kind of blood glucose biosensor that detects COVID-19 antibody
  • a blood glucose biosensor for detecting COVID-19 antibodies includes the following components: new coronavirus COVID-19S protein (amino acid sequence shown in SEQ ID NO.1), new coronavirus COVID-19 antibody COVID-19 IgG, modified with hairpin structure DNA fragment H1-DNA secondary antibody H1-DNA-COVID-19 Anti-IgG of the new coronavirus COVID-19 antibody IgG, trigger DNA fragment T-DNA, maltase-labeled hairpin structure DNA fragment H2-DNA and blood glucose biosensor ;
  • the nucleotide sequences of the hairpin structure DNA fragment H1-DNA, the trigger DNA fragment T-DNA, and the hairpin structure DNA fragment H2-DNA are the same as those in Example 1.
  • the centrifuge tube was treated with oxygen plasma, the ratio of argon to oxygen was 5:1, the power was 50W, and the treatment was 7 minutes.
  • H1-DNA-modified COVID-19 Anti-IgG H1-DNA-COVID-19 Anti-IgG
  • T-DNA and H2-DNA solutions with 5 ⁇ SSC buffer solution, the concentration ratio of the two is 1:10, modify maltase on H2-DNA; the concentration of T-DNA is 1 ⁇ mol/L, the concentration of H2-DNA The concentration is 10 ⁇ mol/L.
  • Add 50 ⁇ L of T-DNA and 50 ⁇ L of H2-DNA solution into the centrifuge tube at the same time; react at room temperature for 10 min, clean the centrifuge tube with 1 ⁇ PBS buffer solution of pH 7.4, blow dry with nitrogen gas and set aside.
  • the blood glucose biosensor was prepared using screen-printed electrodes. First, deposit Prussian blue on the working electrode as an electrical intermediary substance, deposition conditions: concentration of hydrochloric acid 1M, concentration of anhydrous ferric chloride 2.5mM, concentration of potassium ferricyanide 2.5mM, concentration of potassium chloride 0.1M; CV deposition, sweep rate 20V /s, 5 cycles of deposition, -0.1V-1V. After the Prussian blue was deposited, it was rinsed with deionized water and dried with nitrogen gas. Glucose oxidase (GOx) was then immobilized on the Prussian blue surface.
  • deposition conditions concentration of hydrochloric acid 1M, concentration of anhydrous ferric chloride 2.5mM, concentration of potassium ferricyanide 2.5mM, concentration of potassium chloride 0.1M
  • CV deposition sweep rate 20V /s, 5 cycles of deposition, -0.1V-1V.
  • Glucose oxidase GOx
  • step (7) Drop the solution containing glucose in step (7) on the blood glucose biosensor, and test the I-T curve at -0.05V for 60s.
  • Figure 5 is a fitting curve of the current signal and the concentration of COVID-19 IgG using the new crown COVID-19S protein as the antigen. It can be seen from the figure that the detection can reach 0.1ng/mL, and the linear range of detection is 0-2ng/mL.
  • Embodiment 4 A kind of blood glucose biosensor that detects COVID-19 antibody
  • a blood glucose biosensor for detecting COVID-19 antibodies includes the following components: new coronavirus COVID-19S protein (amino acid sequence shown in SEQ ID NO.1), new coronavirus COVID-19 antibody COVID-19 IgM, modified with hairpin structure DNA fragment H1-DNA secondary antibody H1-DNA-COVID-19 Anti-IgM of new coronavirus COVID-19 antibody IgM, trigger DNA fragment T-DNA, fructose-labeled hairpin structure DNA fragment H2-DNA and blood glucose biosensor ;
  • the nucleotide sequences of the hairpin structure DNA fragment H1-DNA, the trigger DNA fragment T-DNA, and the hairpin structure DNA fragment H2-DNA are the same as those in Example 1.
  • the glass tube is treated with oxygen plasma, the ratio of argon to oxygen is 2:1, the power is 10W, and the treatment is 10min.
  • H1-DNA-modified COVID-19 Anti-IgM H1-DNA-COVID-19 Anti-IgM
  • T-DNA and H2-DNA solutions Prepare T-DNA and H2-DNA solutions with 5 ⁇ SSC buffer solution, the concentration ratio of the two is 1:10, and modify fructose on H2-DNA.
  • concentration of T-DNA is 0.01 ⁇ mol/L
  • concentration of H2-DNA is 0.1 ⁇ mol/L
  • the glass tube orifice plate was cleaned with PBS buffer solution, and blown dry with nitrogen gas for later use.
  • the blood glucose biosensor was prepared using screen-printed electrodes. Firstly, Prussian blue is deposited on the working electrode as an electrical intermediary substance. The deposition conditions are: hydrochloric acid concentration 0.1M, anhydrous ferric chloride concentration 0.25mM, potassium ferricyanide concentration 0.25mM, and potassium chloride concentration 0.5M. CV deposition, scan rate 15V/s, deposition 3 cycles, -0.1V-1V. After the Prussian blue was deposited, it was rinsed with deionized water and dried with nitrogen gas. Glucose oxidase (GOx) was then immobilized on the Prussian blue surface.
  • GOx Glucose oxidase
  • GOx-dilute 10 times with 5% BSA solvent is deionized water.
  • solvent is deionized water.
  • 10uL diluted GOx Take 10uL 2% BSA, 10uL 0.8% glutaraldehyde solution, mix well, take 3uL drop on the working electrode, and dry at 25°C to form a film.
  • step (7) Drop the solution containing glucose in step (7) on the blood glucose biosensor, and test the I-T curve at -0.05V for 60s.
  • Figure 6 is the fitting curve of the current signal and the concentration of COVID-19 IgM. It can be seen from the figure that the detection can reach 0.1ng/mL, and the linear range of detection is 0-2ng/mL.

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Abstract

一种检测COVID-19抗体的血糖生物传感器，其包括以下组件：新冠病毒COVID-19抗原（12）、新冠病毒COVID-19抗体（14）、修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗（15）、触发DNA片段T-DNA（16）、酶标记的发夹结构DNA片段H2-DNA（18）和血糖生物传感器（113）。生物传感器构筑新冠病毒抗原-COVID-19 IgG/IgM-(H1-DNA-H2-DNA复合物)三明治结构；三明治结构中H2-DNA上标记的酶（110）将糖类底物（111）转化为葡萄糖（112）；将葡萄糖（112）溶液滴加在血糖生物传感器（113）上检测葡萄糖（112）的量，进而检测出COVID-19 IgG/IgM的含量。

Description

一种检测COVID-19抗体的血糖生物传感器 技术领域
本发明属于生物检测技术领域，具体涉及一种检测COVID-19抗体的血糖生物传感器。
背景技术
目前新冠抗体的检测技术主要为免疫分析法，利用制备新冠抗原蛋白特异性捕获和识别新冠特异性抗体(IgM/IgG)，主要方法有ELISA、化学发光和免疫测流层析法等，其中ELISA和化学发光不仅需要酶标仪等较为昂贵的光学检测设备，而且对操作人员要求较高，因此ELISA和化学发光不适用于大规模的快速检测。
技术问题
免疫测流层析法是其中速度最快、最为简便的抗体检测方法，通过肉眼即可观察到检测信号，非常适用于新冠抗体的快速检测，但由于通过肉眼观察检测结果不仅会造成很大的主观误差，而且不能对新冠抗体进行定量分析。
技术解决方案
本发明的目的在于针对上述问题，提供一种用血糖检测COVID-19抗体的生物传感器。
为实现上述目的，本发明采用如下技术方案：
一种检测COVID-19抗体的血糖生物传感器，所述生物传感器包括：新冠病毒COVID-19抗原、新冠病毒COVID-19抗体、修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗、触发DNA片段T-DNA、酶标记的发夹结构DNA片段H2-DNA和血糖生物传感器；
所述新冠病毒COVID-19抗原为：新冠病毒COVID-19的S蛋白或N蛋白；其中S蛋白的氨基酸序列如SEQ ID NO.1所示；其中N蛋白的氨基酸序列如SEQ ID NO.2所示；
所述新冠病毒COVID-19抗体为：新冠病毒COVID-19抗体COVID-19 IgG或COVID-19 IgM；
所述新冠病毒COVID-19二抗为：新冠病毒COVID-19抗体IgG或IgM的抗抗体，即COVID-19 Anti-IgG或COVID-19 Anti-IgM；
所述修饰有发夹结构DNA片段H1-DNA的核苷酸序列如SEQ ID NO.3所示；
所述触发DNA片段T-DNA的核苷酸序列如SEQ ID NO.4所示；
所述酶标记的发夹结构DNA片段H2-DNA，其核苷酸序列如SEQ ID NO.5所示；标记H2-DNA的酶为能将糖类底物转化为葡萄糖的酶，包括蔗糖酶(Invertase)、麦芽糖酶(Maltase)、果糖酶中的任意一种。
进一步的，上述检测COVID-19抗体的血糖生物传感器，其中所述血糖生物传感器为采用丝网印刷电极制备血糖生物传感器；所述采用丝网印刷电极制备血糖生物传感器其制备方法包括以下步骤：首先在工作电极上沉积普鲁士蓝作为电中介物质，沉积条件：盐酸浓度0.1-1M，无水氯化铁浓度0.25-2.5mM，铁氰化钾浓度0.25-2.5mM，氯化钾浓度0.01-0.1M，CV沉积，扫速10-20mV/s，沉积3-5个循环，-0.1V-1V；沉积完普鲁士蓝后，去离子水清洗干净，氮气吹干；然后在普鲁士蓝表面固定葡萄糖氧化酶(GOx)：0.1-1mg/mL GOx用1wt％-5wt％BSA(溶剂为去离子水)稀释10倍，取10uL稀释后的GOx加入10uL 1wt％-5wt％BSA、10uL 0.5wt％-1wt％戊二醛溶液，混匀，取3uL滴于沉积普鲁士蓝的工作电极上，25℃干燥形成GOx成膜；取3uL浓度为0.1％-0.5％Nafion溶液(以1×PBS，pH＝7.4稀释)，滴加在GOx膜表面，25℃干燥成膜。
检测COVID-19抗体的血糖生物传感器的使用方法，包括以下步骤：
Step1：在基板上固定新冠病毒COVID-19抗原，用缓冲溶液清洗并干燥基板；
Step2：将含有新冠病毒COVID-19抗体的样本加入Step1的基板中，固定在基板上的新冠病毒COVID-19抗原特异性捕获COVID-19抗体，用缓冲溶液清洗并干燥基板；
Step3：将修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗加入Step2干燥后的基板中，以特异性识别新冠病毒COVID-19抗体，用缓冲溶液清洗并干燥基板；
Step4：将触发DNA片段T-DNA和酶标记的发夹结构DNA片段H2-DNA加入Step3干燥后的基板中，T-DNA打开H1-DNA的发夹结构，形成 T-DNA-H1-DNA复合物，H2-DNA将T-DNA置换下来，形成H1-DNA-H2-DNA复合物，置换下来的T-DNA再次循环参与打开H1-DNA发夹结构，室温反应一定时间后，用缓冲溶液清洗并干燥基板；
Step5：将糖类底物加入Step4干燥后的基板中，室温反应一定时间，H2-DNA上标记的酶将糖类底物转化为葡萄糖；
Step6：将Step5的葡萄糖溶液滴加在血糖生物传感器上检测中溶液的葡萄糖含量，进而检测出含有新冠病毒COVID-19抗体的样本中新冠病毒COVID-19抗体的含量。
上述使用方法Step1中所述基板包括96孔板、离心管、玻璃管中的任意一种；使用前对基板进行预处理，具体为：用氧等离子体预处理，氩气与氧气的比例为2:1-5:1，功率10W-50W，处理5-10min；然后将APTES溶液加入氧等离子体处理过的基板中，APTES溶液的浓度为0.1wt％-5wt％，溶剂为无水乙醇和水的混合液，水的含量为1wt％-10wt％；室温下反应2-6h，反应结束后，用无水乙醇、去离子水清洗干净基板，氮气吹干待用。
上述使用方法Step1中新冠病毒COVID-19抗原为用pH＝7.4的1×PBS缓冲溶液配制成浓度为10-20μg/mL的抗原溶液；采用EDC/NHS法将抗原固定在基板上；其中EDC的浓度为1-5mmol/L，NHS的浓度为5-25mmol/L，室温避光反应0.5-1小时；反应结束后，用pH＝7.4的1×PBS缓冲溶液清洗干净基板，氮气吹干，再将1％-5％BSA加入基板中，室温孵育30min。
上述使用方法Step2中含有新冠病毒COVID-19抗体的样本加入Step1的基板中室温孵育5-15min。
上述使用方法Step3中修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗用pH＝7.4的1×PBS缓冲溶液配制成浓度为1-10μg/mL的二抗溶液；加入Step2干燥后的基板中室温孵育5-15min。
上述使用方法中Step1-Step4中所述用缓冲溶液清洗并干燥基板为用pH＝7.4的1×PBS缓冲溶液清洗干净，氮气吹干待用。
上述方法Step4中触发DNA片段T-DNA和酶标记的发夹结构DNA片段H2-DNA用5×SSC缓冲溶液配制配置成溶液；二者浓度比为1:10；体积比为1:1；T-DNA的浓度为0.01-1μmol/L，H2-DNA的浓度为0.1-10μmol/L；室温反应10-30min。
上述方法Step5中用pH＝7.4的1×PBS缓冲溶液配制糖类底物溶液，糖类底物溶液的浓度为0.1-1mmol/L。
上述检测COVID-19抗体的血糖生物传感器在检测COVID-19抗体中的应用。
上述检测COVID-19抗体的血糖生物传感器的工作原理(如图1所示)：
Step1在基板(图1-11)上固定新冠病毒COVID-19抗原(S蛋白或N蛋白，图1-12)；Step2将含有新冠病毒COVID-19抗体(COVID-19 IgG或COVID-19 IgM，图1-14)的样本(图1-13)加入Step1的基板中，固定在基板上的新冠抗原(S蛋白或N蛋白)特异性捕获新冠抗体(COVID-19 IgG或COVID-19 IgM)；Step3将修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗(将COVID-19 Anti-IgG或COVID-19 Anti-IgM，图1-15)加入Step2干燥后的基板中，以特异性识别COVID-19 IgG或COVID-19 IgM；Step4将触发DNA片段T-DNA(图1-16)和酶标记的发夹结构DNA片段H2-DNA(图1-18)加入Step3干燥后的基板中；T-DNA打开H1-DNA的发夹结构，形成T-DNA-H1-DNA复合物(图1-17)，H2-DNA可以将T-DNA置换下来，形成H1-DNA-H2-DNA复合物(图1-19)，置换下来的T-DNA(图1-16)可以再次循环参与打开H1-DNA发夹结构的反应；以酶标记的杂交链式放大技术(E-HCR)作为COVID-19IgG/IgM的信号探针，构筑了新冠病毒抗原(N蛋白或S蛋白)-COVID-19IgG/IgM-E-HCR(H1-DNA-H2-DNA复合物)三明治结构；Step5将糖类底物加入Step4干燥后的基板中，新冠病毒抗原(N蛋白或S蛋白)-COVID-19 IgG/IgM-E-HCR(H1-DNA-H2-DNA复合物)三明治结构中H2-DNA上标记的酶(图1-110)将(蔗糖、果糖、麦芽糖等)糖类底物(图1-111)转化为葡萄糖(图1-112)；Step6将Step5的葡萄糖溶液滴加在血糖生物传感器(图1-113)上用血糖生物传感器检测葡萄糖的量，进而检测出COVID-19 IgG/IgM的含量。
有益效果
本发明的优点在于：
以酶标记的杂交链式放大技术(E-HCR)作为COVID-19 IgG/IgM的信号探针，构筑了新冠病毒抗原(N蛋白或S蛋白)-COVID-19 IgG/IgM-E-HCR(H1-DNA-H2-DNA复合物)三明治结构：E-HCR首先将蔗糖、果糖、麦芽糖等糖类底物转换为葡萄糖，然后用血糖生物传感器检测葡萄糖的量，进而检测出 COVID-19 IgG/IgM，此方法具有检测速度快、灵敏度高的优点，更为重要的是能对COVID-19 IgG/IgM精确定量检测，检测限可达到0.1ng/mL。
附图说明
图1一种用血糖生物传感器检测COVID-19抗体的技术方案示意图。11：基板，12：新冠病毒COVID-19抗原，13：样本，14：新冠病毒COVID-19抗体，15：修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗，16：触发DNA片段T-DNA，17：T-DNA-H1-DNA复合物，18：酶标记的发夹结构DNA片段H2-DNA，19：H1-DNA-H2-DNA复合物，110：标记H2-DNA的酶，111：糖类底物，112：葡萄糖，113：血糖生物传感器。
图2为以新冠COVID-19N蛋白为抗原不同浓度COIVD-19 IgG的血清溶液的电流时间曲线。
图3为以新冠COVID-19N蛋白为抗原电流信号与COVID-19 IgG浓度的拟合曲线。
图4为以新冠COVID-19N蛋白为抗原电流信号与COVID-19 IgM浓度的拟合曲线。
图5为以新冠COVID-19S蛋白为抗原电流信号与COVID-19 IgG浓度的拟合曲线。
图6为以新冠COVID-19S蛋白为抗原电流信号与COVID-19 IgM浓度的拟合曲线。
本发明的实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但下述的实例仅仅是本发明其中的例子而已，并不代表本发明所限定的权利保护范围，本发明的权利保护范围以权利要求书为准。
新冠病毒COVID-19抗体COVID-19 IgG、COVID-19 IgM和新冠病毒COVID-19二抗COVID-19 Anti-IgG、COVID-19 Anti-IgM均购自近岸蛋白质科技有限公司。
饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗(H1-DNA-COVID-19 Anti-IgG或H1-DNA-COVID-19 Anti-IgM)由上海生工生物工程有限公司合成。
酶标记的发夹结构DNA片段H2-DNA由上海生工生物工程有限公司合成。
实施例1一种检测COVID-19抗体的血糖生物传感器
一种检测COVID-19抗体的血糖生物传感器，包括以下组件：新冠病毒COVID-19N蛋白(氨基酸序列如SEQ ID NO.2所示)、新冠病毒COVID-19抗体COVID-19 IgG、修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗H1-DNA-COVID-19 Anti-IgG、触发DNA片段T-DNA、蔗糖酶标记的发夹结构DNA片段H2-DNA和血糖生物传感器；
所述发夹结构DNA片段H1-DNA的核苷酸序列如SEQ ID NO.3所示：
SEQ ID NO.3：H1-DNA：3’-COOH-TTTTTTTTTTGGTGTATGGCGTCTGCCCTCTGTAAGGCAGACGCCATACACCTTTCCAATACC-5’；
所述触发DNA片段T-DNA的核苷酸序列如SEQ ID NO.4所示；
SEQ ID NO.4：T-DNA：3’-GGTATTGGAAAGGTGTATGGCGTCTGCC-5’；
所述发夹结构DNA片段H2-DNA，其核苷酸序列如SEQ ID NO.5所示：
SEQ ID NO.5：H2-DNA：3’-Enzyme-TTTTTTTTTT-CTCTGTAAGGTGTATGGCGTCTGCCTTACAGAGGGCAG-5’。
其使用方法包括，以下步骤：
(1)96孔板用氧等离子体处理，氩气与氧气的比例为4：1，功率20W，处理5min。
(2)将APTES溶液加入氧等离子体处理过的96孔板中，APTES的浓度为2％，溶剂为无水乙醇和水的混合液，水的含量为5％。室温下反应3小时，反应结束后，用无水乙醇、去离子水清洗干净96孔板，氮气吹干待用。
(3)用pH＝7.4的1×PBS缓冲溶液配制15μg/mL的新冠抗原N蛋白，采用EDC/NHS法将其固定在96孔板上。EDC的浓度为2mmol/L，NHS的浓度为10mmol/L。室温避光反应0.5小时。反应结束后，用pH＝7.4的1×PBS缓冲溶液清 洗干净96孔板，氮气吹干待用。将100μL2％BSA加入96孔板中，室温孵育30min，1×PBS缓冲溶液清洗干净，96孔板，氮气吹干待用。
(4)将COVID-19 IgG标准品加入人血清中，配制成含有0、0.1、0.5、1、1.5、2、4ng/mLCOVID-19 IgG的血清溶液。将100μL血清溶液加入96孔板中，室温孵育10min，用pH＝7.4的1×PBS缓冲溶液清洗干净，96孔板，氮气吹干待用。
(5)用pH＝7.4的1×PBS缓冲溶液配制修饰H1-DNA的COVID-19 Anti-IgG(H1-DNA-COVID-19 Anti-IgG)溶液，浓度为5μg/mL，将100μL H1-DNA-COVID-19 Anti-IgG溶液加入96孔板中，室温孵育10min，用pH＝7.4的1×PBS缓冲溶液清洗干净，96孔板，氮气吹干待用。
(6)用5×SSC缓冲溶液配制T-DNA、H2-DNA溶液，二者浓度比为1:10，H2-DNA上修饰蔗糖酶。T-DNA的浓度为0.5μmol/L，H2-DNA的浓度为5μmol/L。将50μL T-DNA和50μL H2-DNA溶液同时加入96孔板中。室温反应10min，用pH＝7.4的1×PBS缓冲溶液清洗干净，96孔板，氮气吹干待用。
(7)用pH＝7.4的1×PBS缓冲溶液配制蔗糖溶液，蔗糖的浓度为0.5mmol/L。100μL蔗糖溶液加入96孔板中，室温反应1min。此时，蔗糖酶将蔗糖水解为葡萄糖。
(8)血糖生物传感器的制备：采用丝网印刷电极制备血糖生物传感器。首先在工作电极上沉积普鲁士蓝作为电中介物质，沉积条件：盐酸浓度0.2M，无水氯化铁浓度0.5mM，铁氰化钾浓度0.5mM，氯化钾浓度0.1M。CV沉积，扫速10V/s，沉积4个循环，-0.1V-1V。沉积完普鲁士蓝后，去离子水清洗干净，氮气吹干。然后在普鲁士蓝表面固定葡萄糖氧化酶(GOx)。0.5mg/mL GOx-用2％BSA(溶剂为去离子水)稀释10倍。取10uL稀释后的GOx加入10uL1％BSA、10uL 0.5％戊二醛溶液，混匀，取3uL滴于工作电极，25℃干燥成膜。取3uL浓度为0.2％Nafion溶液(以1×PBS，pH＝7.4稀释)，滴加在GOx膜表面，25℃干燥成膜。
(9)将第(7)步含有葡萄糖的溶液滴加在血糖生物传感器上，-0.05V测试I-T曲线，60s。
图2为以新冠COVID-19N蛋白为抗原不同浓度COIVD-19 IgG的血清溶液的电流时间曲线。从图中可以看出，随着COVID-19 IgG的浓度增大，血糖生物传感器的电流信号越大。可以检测低至0.1ng/mL的COVID-19 IgG。
图3为以新冠COVID-19N蛋白为抗原电流信号与COVID-19 IgG浓度的拟合曲线。从图中可以看出，检测的线性范围为0-2ng/mL。
实施例2一种检测COVID-19抗体的血糖生物传感器
一种检测COVID-19抗体的血糖生物传感器包括以下组件：新冠病毒COVID-19的N蛋白(氨基酸序列如SEQ ID NO.2所示)、新冠病毒COVID-19抗体COVID-19 IgM、修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗H1-DNA-COVID-19 Anti-IgM、触发DNA片段T-DNA、蔗糖酶标记的发夹结构DNA片段H2-DNA和血糖生物传感器；
所述发夹结构DNA片段H1-DNA、触发DNA片段T-DNA、发夹结构DNA片段H2-DNA的核苷酸序列同实施例1。
其使用方法，包括以下步骤：
(1)96孔板用氧等离子体处理，氩气与氧气的比例为4:1，功率20W，处理5min。
(2)将APTES溶液加入氧等离子体处理过的96孔板中，APTES的浓度为2％，溶剂为无水乙醇和水的混合液，水的含量为5％。室温下反应3小时，反应结束后，用无水乙醇、去离子水清洗干净96孔板，氮气吹干待用。
(3)用pH＝7.4的1×PBS缓冲溶液配制15μg/mL的新冠抗原N蛋白，采用EDC/NHS法将其固定在96孔板上。EDC的浓度为2mmol/L，NHS的浓度为10mmol/L。室温避光反应0.5小时。反应结束后，用pH＝7.4的1×PBS缓冲溶液清洗干净96孔板，氮气吹干待用。将100μL2％BSA加入96孔板中，室温孵育30min，1×PBS缓冲溶液清洗干净，96孔板，氮气吹干待用。
(4)将COVID-19 IgM标准品加入人血清中，配制成含有0、0.1、0.5、1、1.5、2、4ng/mLCOVID-19 IgM的血清溶液。将100μL血清溶液加入96孔板中，室温孵育10min，用pH＝7.4的1×PBS缓冲溶液清洗干净，96孔板，氮气吹干待用。
(5)用pH＝7.4的1×PBS缓冲溶液配制修饰H1-DNA的COVID-19 Anti-IgM(H1-DNA-COVID-19 Anti-IgM)溶液，浓度为5μg/mL，将100μL  H1-DNA-COVID-19 Anti-IgM溶液加入96孔板中，室温孵育10min，用pH＝7.4的1×PBS缓冲溶液清洗干净，96孔板，氮气吹干待用。
(6)用5×SSC缓冲溶液配制T-DNA、H2-DNA溶液，二者浓度比为1:10，H2-DNA上修饰蔗糖酶。T-DNA的浓度为0.5μmol/L，H2-DNA的浓度为0.5μmol/L。将50μL T-DNA和50μL H2-DNA溶液同时加入96孔板中。室温反应10min，用pH＝7.4的1×PBS缓冲溶液清洗干净，96孔板，氮气吹干待用。
(7)用pH＝7.4的1×PBS缓冲溶液配制蔗糖溶液，蔗糖的浓度为0.5mmol/L。100μL蔗糖溶液加入96孔板中，室温反应1min。此时，蔗糖酶将蔗糖水解为葡萄糖。
(8)血糖生物传感器的制备：采用丝网印刷电极制备血糖生物传感器。首先在工作电极上沉积普鲁士蓝作为电中介物质，沉积条件：盐酸浓度0.2M，无水氯化铁浓度0.5mM，铁氰化钾浓度0.5mM，氯化钾浓度0.1M。CV沉积，扫速10V/s，沉积4个循环，-0.1V-1V。沉积完普鲁士蓝后，去离子水清洗干净，氮气吹干。然后在普鲁士蓝表面固定葡萄糖氧化酶(GOx)。0.5mg/mL GOx-用2％BSA(溶剂为去离子水)稀释10倍。取10uL稀释后的GOx加入10uL1％BSA、10uL 0.5％戊二醛溶液，混匀，取3uL滴于工作电极，25℃干燥成膜。取3uL浓度为0.2％Nafion溶液(以1×PBS，pH＝7.4稀释)，滴加在GOx膜表面，25℃干燥成膜。
(9)将第(7)步含有葡萄糖的溶液滴加在血糖生物传感器上，-0.05V测试I-T曲线，60s。
图4为以新冠COVID-19N蛋白为抗原电流信号与COVID-19 IgM浓度的拟合曲线。从图中可以看出，检测可达到0.1ng/mL，检测的线性范围为0-4ng/mL。
实施例3一种检测COVID-19抗体的血糖生物传感器
一种检测COVID-19抗体的血糖生物传感器包括以下组件：新冠病毒COVID-19S蛋白(氨基酸序列如SEQ ID NO.1所示)、新冠病毒COVID-19抗体COVID-19 IgG、修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19抗体IgG的二抗H1-DNA-COVID-19 Anti-IgG、触发DNA片段T-DNA、麦芽糖酶标记的发夹结构DNA片段H2-DNA和血糖生物传感器；
所述发夹结构DNA片段H1-DNA、触发DNA片段T-DNA、发夹结构DNA片段H2-DNA的核苷酸序列同实施例1。
其使用方法，包括以下步骤：
(1)离心管用氧等离子体处理，氩气与氧气的比例为5:1，功率50W，处理7min。
(2)将APTES溶液加入氧等离子体处理过的离心管中，APTES的浓度为5％，溶剂为无水乙醇和水的混合液，水的含量为10％。室温下反应3小时，反应结束后，用无水乙醇、去离子水清洗干净离心管，氮气吹干待用。
(3)用pH＝7.4的1×PBS缓冲溶液配制10μg/mL的新冠抗原S蛋白，采用EDC/NHS法将其固定在离心管上。EDC的浓度为5mmol/L，NHS的浓度为25mmol/L。室温避光反应1小时。反应结束后，用pH＝7.4的1×PBS缓冲溶液清洗干净离心管，氮气吹干待用。将100μL 5％BSA加入96孔板中，室温孵育30min，1×PBS缓冲溶液清洗干净，离心管，氮气吹干待用。
(4)将COVID-19 IgG标准品加入人血清中，配制成含有0、0.1、0.5、1、1.5、2、4ng/mLCOVID-19 IgG的血清溶液。将100μL血清溶液加入离心管中，室温孵育5min，用pH＝7.4的1×PBS缓冲溶液清洗干净离心管，氮气吹干待用。
(5)用pH＝7.4的1×PBS缓冲溶液配制修饰H1-DNA的COVID-19 Anti-IgG(H1-DNA-COVID-19 Anti-IgG)溶液，浓度为10μg/mL，将100μL H1-DNA-COVID-19 Anti-IgG溶液加入离心管中，室温孵育5min，用pH＝7.4的1×PBS缓冲溶液清洗干净，离心管，氮气吹干待用。
(6)用5×SSC缓冲溶液配制T-DNA、H2-DNA溶液，二者浓度比为1:10，H2-DNA上修饰麦芽糖酶；T-DNA的浓度为1μmol/L，H2-DNA的浓度为10μmol/L。将50μL T-DNA和50μL H2-DNA溶液同时加入离心管中；室温反应10min，用pH＝7.4的1×PBS缓冲溶液清洗干净离心管，氮气吹干待用。
(7)用pH＝7.4的1×PBS缓冲溶液配制麦芽糖溶液，麦芽糖的浓度为1mmol/L，100μL麦芽糖溶液加入离心管中，室温反应1min，此时，麦芽糖酶将麦芽糖水解为葡萄糖。
(8)血糖生物传感器的制备：采用丝网印刷电极制备血糖生物传感器。首先在工作电极上沉积普鲁士蓝作为电中介物质，沉积条件：盐酸浓度1M，无水氯化铁浓度2.5mM，铁氰化钾浓度2.5mM，氯化钾浓度0.1M；CV沉积，扫速 20V/s，沉积5个循环，-0.1V-1V。沉积完普鲁士蓝后，去离子水清洗干净，氮气吹干。然后在普鲁士蓝表面固定葡萄糖氧化酶(GOx)。1mg/mL GOx-用1％BSA(溶剂为去离子水)稀释10倍。取10uL稀释后的GOx加入10uL1％BSA、10uL 1％戊二醛溶液，混匀，取3uL滴于工作电极，25℃干燥成膜。取3uL浓度为0.1％Nafion溶液(以1×PBS，pH＝7.4稀释)，滴加在GOx膜表面，25℃干燥成膜。
(9)将第(7)步含有葡萄糖的溶液滴加在血糖生物传感器上，-0.05V测试I-T曲线，60s。
图5为以新冠COVID-19S蛋白为抗原的电流信号与COVID-19 IgG浓度的拟合曲线。从图中可以看出，检测可达到0.1ng/mL，检测的线性范围为0-2ng/mL。
实施例4一种检测COVID-19抗体的血糖生物传感器
一种检测COVID-19抗体的血糖生物传感器包括以下组件：新冠病毒COVID-19S蛋白(氨基酸序列如SEQ ID NO.1所示)、新冠病毒COVID-19抗体COVID-19 IgM、修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19抗体IgM的二抗H1-DNA-COVID-19 Anti-IgM、触发DNA片段T-DNA、果糖酶标记的发夹结构DNA片段H2-DNA和血糖生物传感器；
所述发夹结构DNA片段H1-DNA、触发DNA片段T-DNA、发夹结构DNA片段H2-DNA的核苷酸序列同实施例1。
其使用方法，包括以下步骤：
(1)玻璃管用氧等离子体处理，氩气与氧气的比例为2:1，功率10W，处理10min。
(2)将APTES溶液加入氧等离子体处理过的玻璃管中，APTES的浓度为0.1％，溶剂为无水乙醇和水的混合液，水的含量为1％，室温下反应6小时，反应结束后，用无水乙醇、去离子水清洗干净玻璃管，氮气吹干待用。
(3)用pH＝7.4的1×PBS缓冲溶液配制10μg/mL的新冠抗原S蛋白，采用EDC/NHS法将其固定在玻璃管上。EDC的浓度为1mmol/L，NHS的浓度为5mmol/L。室温避光反应0.75小时。反应结束后，用pH＝7.4的1×PBS缓冲溶液清洗干净玻璃管，氮气吹干待用。将100μL 1％BSA加入玻璃管中，室温孵育30min，1×PBS缓冲溶液清洗干净玻璃管，氮气吹干待用。
(4)将COVID-19 IgM标准品加入人血清中，配制成含有0、0.1、0.5、1、1.5、2、4ng/mLCOVID-19 IgM的血清溶液。将100μL血清溶液加入玻璃管中，室温孵育15min，用pH＝7.4的1×PBS缓冲溶液清洗干净玻璃管，氮气吹干待用。
(5)用pH＝7.4的1×PBS缓冲溶液配制修饰H1-DNA的COVID-19 Anti-IgM(H1-DNA-COVID-19 Anti-IgM)溶液，浓度为1μg/mL，将100μL H1-DNA-COVID-19 Anti-IgM溶液加入玻璃管中，室温孵育15min，用pH＝7.4的1×PBS缓冲溶液清洗干净玻璃管，氮气吹干待用。
(6)用5×SSC缓冲溶液配制T-DNA、H2-DNA溶液，二者浓度比为1:10，H2-DNA上修饰果糖酶。T-DNA的浓度为0.01μmol/L，H2-DNA的浓度为0.1μmol/L；将50μL T-DNA和50μL H2-DNA溶液同时加入玻璃管中，室温反应30min，用pH＝7.4的1×PBS缓冲溶液清洗干净玻璃管孔板，氮气吹干待用。
(7)用pH＝7.4的1×PBS缓冲溶液配制果糖溶液，果糖的浓度为0.1mmol/L，100μL果糖溶液加入玻璃管中，室温反应1min，此时，果糖酶将果糖水解为葡萄糖。
(8)血糖生物传感器的制备：采用丝网印刷电极制备血糖生物传感器。首先在工作电极上沉积普鲁士蓝作为电中介物质，沉积条件：盐酸浓度0.1M，无水氯化铁浓度0.25mM，铁氰化钾浓度0.25mM，氯化钾浓度0.5M。CV沉积，扫速15V/s，沉积3个循环，-0.1V-1V。沉积完普鲁士蓝后，去离子水清洗干净，氮气吹干。然后在普鲁士蓝表面固定葡萄糖氧化酶(GOx)。0.1mg/mL GOx-用5％BSA(溶剂为去离子水)稀释10倍。取10uL稀释后的GOx加入10uL 2％BSA、10uL 0.8％戊二醛溶液，混匀，取3uL滴于工作电极，25℃干燥成膜。取3uL浓度为0.5％Nafion溶液(以1×PBS，pH＝7.4稀释)，滴加在GOx膜表面，25℃干燥成膜。
(9)将第(7)步含有葡萄糖的溶液滴加在血糖生物传感器上，-0.05V测试I-T曲线，60s。
图6为电流信号与COVID-19 IgM浓度的拟合曲线。从图中可以看出，检测可达到0.1ng/mL，检测的线性范围为0-2ng/mL。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。

Claims (10)

  1. 一种检测COVID-19抗体的血糖生物传感器，其特征在于：所述生物传感器包括以下组件：新冠病毒COVID-19抗原、新冠病毒COVID-19抗体、修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗、触发DNA片段T-DNA、酶标记的发夹结构DNA片段H2-DNA和血糖生物传感器；
    所述新冠病毒COVID-19抗原为：新冠病毒COVID-19的S蛋白或N蛋白；其中S蛋白的氨基酸序列如SEQ ID NO.1所示；其中N蛋白的氨基酸序列如SEQ ID NO.2所示；
    所述新冠病毒COVID-19抗体为：新冠病毒COVID-19抗体COVID-19 IgG或COVID-19 IgM；
    所述新冠病毒COVID-19二抗为：新冠病毒COVID-19抗体IgG或IgM的抗抗体，即COVID-19 Anti-IgG或COVID-19 Anti-IgM；
    所述修饰有发夹结构DNA片段H1-DNA的核苷酸序列如SEQ ID NO.3所示；
    所述触发DNA片段T-DNA的核苷酸序列如SEQ ID NO.4所示；
    所述酶标记的发夹结构DNA片段H2-DNA，其核苷酸序列如SEQ ID NO.5所示；标记H2-DNA的酶为能将糖类底物转化为葡萄糖的酶。
  2. 根据权利要求1所述的检测COVID-19抗体的血糖生物传感器，其特征在于：所述标记H2-DNA的酶为能将糖类底物转化为葡萄糖的酶包括蔗糖酶、麦芽糖酶、果糖酶中的任意一种。
  3. 根据权利要求1所述的检测COVID-19抗体的血糖生物传感器，其特征在于：所述血糖生物传感器为采用丝网印刷电极制备血糖生物传感器；所述采用丝网印刷电极制备血糖生物传感器其制备方法包括以下步骤：首先在工作电极上沉积普鲁士蓝作为电中介物质，沉积条件：盐酸浓度0.1-1M，无水氯化铁浓度0.25-2.5mM，铁氰化钾浓度0.25-2.5mM，氯化钾浓度0.01-0.1M，CV沉积，扫速10-20mV/s，沉积3-5个循环，-0.1V-1V；沉积完普鲁士蓝后，去离子水清洗干净，氮气吹干；然后在普鲁士蓝表面固定葡萄糖氧化酶：0.1-1mg/mL葡萄糖氧化酶用1wt％-5wt％BSA稀释10倍，取10uL稀释后的葡萄糖氧化酶加入10uL 1wt％-5wt％BSA、10uL 0.5wt％-1wt％戊二醛溶液，混匀，取3uL滴于沉积普鲁士蓝的工作电极上，25℃干燥形成葡萄糖氧化酶膜；取3uL以1×PBS，pH＝7.4 稀释的浓度为0.1％-0.5％Nafion溶液，滴加在葡萄糖氧化酶膜表面，25℃干燥成膜。
  4. 权利要求1所述检测COVID-19抗体的血糖生物传感器的使用方法，其特征在于，包括以下步骤：
    Step1：在基板上固定新冠病毒COVID-19抗原，用缓冲溶液清洗并干燥基板；
    Step2：将含有新冠病毒COVID-19抗体的样本加入Step1的基板中，固定在基板上的新冠病毒COVID-19抗原特异性捕获COVID-19抗体，用缓冲溶液清洗并干燥基板；
    Step3：将修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗加入Step2干燥后的基板中，以特异性识别新冠病毒COVID-19抗体，用缓冲溶液清洗并干燥基板；
    Step4：将触发DNA片段T-DNA和酶标记的发夹结构DNA片段H2-DNA加入Step3干燥后的基板中，T-DNA打开H1-DNA的发夹结构，形成T-DNA-H1-DNA复合物，H2-DNA将T-DNA置换下来，形成H1-DNA-H2-DNA复合物，置换下来的T-DNA再次循环参与打开H1-DNA发夹结构，室温反应一定时间后，用缓冲溶液清洗并干燥基板；
    Step5：将糖类底物加入Step4干燥后的基板中，室温反应一定时间，H2-DNA上标记的酶将糖类底物转化为葡萄糖；
    Step6：将Step5的葡萄糖溶液滴加在血糖生物传感器上检测中溶液的葡萄糖含量，进而检测出含有新冠病毒COVID-19抗体的样本中新冠病毒COVID-19抗体的含量。
  5. 根据权利要求4所述的使用方法，其特征在于：Step1中所述基板包括96孔板、离心管、玻璃管中的任意一种；使用前对基板进行预处理，具体为：用氧等离子体预处理，氩气与氧气的比例为2:1-5:1，功率10W-50W，处理5-10min；然后将APTES溶液加入氧等离子体处理过的基板中，APTES溶液的浓度为0.1wt％-5wt％，溶剂为无水乙醇和水的混合液，水的含量为1wt％-10wt％；室温下反应2-6h，反应结束后，用无水乙醇、去离子水清洗干净基板，氮气吹干待用。
  6. 根据权利要求4所述的使用方法，其特征在于：Step1中新冠病毒COVID-19抗原为用pH＝7.4的1×PBS缓冲溶液配制成浓度为10-20μg/mL的抗原溶液；采用EDC/NHS法将抗原固定在基板上；其中EDC的浓度为1-5mmol/L，NHS的浓度为5-25mmol/L，室温避光反应0.5-1小时；反应结束后，用pH＝7.4的1×PBS缓冲溶液清洗干净基板，氮气吹干，再将1％-5％BSA加入基板中，室温孵育30min。
  7. 根据权利要求4所述的使用方法，其特征在于：Step3中修饰有发夹结构DNA片段H1-DNA的新冠病毒COVID-19二抗用pH＝7.4的1×PBS缓冲溶液配制成浓度为1-10μg/mL的二抗溶液；加入Step2干燥后的基板中室温孵育5-15min。
  8. 根据权利要求4所述的使用方法，其特征在于：Step4中触发DNA片段T-DNA和酶标记的发夹结构DNA片段H2-DNA用5×SSC缓冲溶液配制配置成溶液；二者浓度比为1:10；体积比为1:1；T-DNA的浓度为0.01-1μmol/L，H2-DNA的浓度为0.1-10μmol/L；室温反应10-30min。
  9. 根据权利要求4所述的使用方法，其特征在于：Step5中用pH＝7.4的1×PBS缓冲溶液配制糖类底物溶液，糖类底物溶液的浓度为0.1-1mmol/L。
  10. 权利要求1所述检测COVID-19抗体的血糖生物传感器在检测COVID-19抗体中的应用。
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