CN115656507B - Sugar signal amplification mode-based norovirus detection method, material and application - Google Patents
Sugar signal amplification mode-based norovirus detection method, material and application Download PDFInfo
- Publication number
- CN115656507B CN115656507B CN202211237306.XA CN202211237306A CN115656507B CN 115656507 B CN115656507 B CN 115656507B CN 202211237306 A CN202211237306 A CN 202211237306A CN 115656507 B CN115656507 B CN 115656507B
- Authority
- CN
- China
- Prior art keywords
- npc
- elisa plate
- apba
- norovirus
- pep
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The application provides a method, a material and an application for detecting norovirus (HuNoV) based on a sugar signal amplification mode. The method mainly comprises the steps of constructing a HuNoV detection biosensor by using a combination of a polypeptide capable of specifically recognizing HuNoV and a HuNoV antibody. The sensor has the advantages of higher sensitivity, wider detection range, higher detection speed, lower detection limit, special selectivity, convenient operation and the like, can be used for quickly and accurately detecting the HuNoV in an actual sample, and provides a cheap, quick and sensitive method for detecting the HuNoV. The results of the examples show that the detection range of HuNoV detected by the biosensor constructed according to the application is 0.5-10 4 The content of HuNoV in the actual sample can be successfully detected by the copies/mL.
Description
Technical Field
The application belongs to the field of biological sensing, and particularly relates to a method for detecting human norovirus. Specifically, a sandwich immunosensor is constructed by taking human norovirus as a target analyte and adopting a nano enzyme composite material of MBP-pep@MB-CD@Cu-NPC@3-APBA.
Background
Norovirus (NoV) is a pathogen capable of initiating global outbreaks of acute non-bacterial gastroenteritis. NoV is divided into 7 gene groups (GI to GVIII) containing more than 30 genotypes, of which NoV from the GI, GII and GIV gene groups infect humans, while the human norovirus (HuNoV) of the GII.4 genotype is the main genotype responsible for the outbreak of the disease. Investigation showed that in developing countries, huNoV caused 2.67 million people to infect each year, over 20 ten thousand children under 5 years of age die. In China, since the 1 st HuNoV infection case was reported in 1995, the disease was outbreak in a plurality of areas, and serious cases and death cases caused by the disease also threaten the health of human beings seriously. Norovirus is statistically responsible for 2.67 hundred million infections, the most common cause of diarrhea cases in all ages.
The main transmission route of HuNoV is the faecal-oral route, the incubation period is 24-48 h, and the disease course is 12-60 h. Norovirus is highly infectious, requiring only 10 viral particles to cause infection. Norovirus has a high prevalence in communities and high stability in the environment, all of which promote the spread of the virus. It is counted that the situation is similar in various countries, and that the high incidence of norovirus is ubiquitous, although the overall risk of death in developed countries may be much lower. Thus, rapid and sensitive detection of HuNoV is important.
Currently, several methods for detecting HuNoV include reverse transcription polymerase chain reaction, PCR method, ELISA and immune colloidal gold technique. The methods have the defects of low sensitivity, complex operation, expensive instrument, long detection time and the like. Therefore, there is a strong need to develop a rapid, sensitive, and convenient method for detecting HuNoV. In the application, the nano enzyme composite material (MBP-pep@MB-CD@Cu-NPC@3-APBA) is used as a new generation of composite nano material, and has the characteristics of good enzyme activity, water solubility and the like. Meanwhile, the polypeptide (Pep) can specifically recognize HuNoV, and the Cu-NPC nanometer mimic enzyme has good laccase activity and TMB chromogenic activity. The nano enzyme composite material has good signal amplification effect and is used for ultrasensitive detection of HuNoV.
Disclosure of Invention
Aiming at the problems of high detection cost, low sensitivity, poor stability and the like of the current human norovirus, the application provides a norovirus detection method based on a sugar signal amplification mode, which comprises the following steps:
step 1) fully spreading human norovirus antibodies on an ELISA plate, and incubating overnight at 4-6 ℃; specific capture NoV;
step 2) cleaning the ELISA plate by using PBST, fully paving the ELISA plate with bovine serum albumin, and incubating for 20-30min at room temperature for removing the rest active sites of the ELISA plate;
step 3) cleaning the ELISA plate by using PBST, spreading human norovirus solutions with different concentrations on the cleaned ELISA plate, and incubating for 1.5-2h at 4-6 ℃;
step 4) cleaning the ELISA plate by using PBST, spreading MBP-pep@MB-CD solution on the cleaned ELISA plate, and incubating for 1.5-2h at 4-6 ℃; MBP-pep@MB-CD@Cu-NPC@3-APBA specifically recognizes human norovirus and amplifies signals;
step 5) cleaning the ELISA plate by using PBST, and taking the solution A to enable Cu-NPC to have absorption peaks with different intensities at the position of about X nm;
step 6) reacting for 20-30min at 50-60 ℃, then carrying out ultraviolet analysis, scanning the wavelength Y nm, and drawing a working curve; obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
The norovirus detection method provided by the application comprises the following specific steps:
step 1) fully paving human norovirus antibody with the concentration of 0.5-1 mug/mL on an ELISA plate, and incubating overnight at 4-6 ℃; specific capture NoV;
step 2), cleaning the ELISA plate 3 times by using 0.1mol/L of PBST with the pH of 7.4, fully paving 50 mu L of bovine serum albumin with the concentration of 1-2% on the ELISA plate, and incubating for 20-30min at room temperature for removing the rest active sites of the ELISA plate;
step 3) cleaning the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, spreading 50 mu L of human norovirus solution with different concentrations on the cleaned ELISA plate, and incubating for 1.5-2h at 4-6 ℃;
step 4) cleaning the ELISA plate 3 times by 0.1mol/L of PBST with pH of 7.4, spreading 50 mu L of MBP-pep@MB-CD solution with the concentration of 0.5-1mg/mL on the cleaned ELISA plate, and incubating for 1.5-2 hours at the temperature of 4-6 ℃; MBP-pep@MB-CD@Cu-NPC@3-APBA specifically recognizes human norovirus and amplifies signals;
step 5), cleaning the ELISA plate 3 times by using 0.1mol/L pH7.4 PBST, and taking the solution A to enable Cu-NPC to have absorption peaks with different intensities at about X nm;
step 6) reacting for 20-30min at 50-60 ℃, then carrying out ultraviolet analysis, scanning the wavelength Y nm, and drawing a working curve; obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
Step 5) of the application is to wash the ELISA plate 3 times with 0.1mol/L pH7.4 PBST, taking 50 mu L of 2, 4-dichlorophenol (2, 4-DP) with the concentration of 0.5-1mg/mL, 50 mu L of 4-aminoantipyrine (4-AP) with the concentration of 0.5-1mg/mL and 150 mu L of MES buffer solution with the pH of 6.8; the Cu-NPC is enabled to have different intensity absorption peaks at about 510 nm.
The step 6) of the application is that the reaction is carried out for 20 to 30 minutes at 50 to 60 ℃, then ultraviolet analysis is carried out, the scanning wavelength is 400 to 700nm, and a working curve is drawn; obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
In addition, the step 5) of the present application is to wash the ELISA plate 3 times with 0.1mol/L of pH7.4 PBST, and take 50. Mu.L of TMB with a concentration of 10-20mM and 50. Mu.L of H with a concentration of 90-100mM 2 O 2 And 150. Mu.L of acetic acid buffer pH 4.5; the Cu-NPC is enabled to have absorption peaks with different intensities at about 652 nm.
The step 6) of the application is that the reaction is carried out for 15-20min at 30-37 ℃, then ultraviolet analysis is carried out, the scanning wavelength is 500-800nm, and a working curve is drawn; obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
The preparation method of the MBP-pep@MB-CD@Cu-NPC@3-APBA in the step 4) comprises the following steps of:
step 1) synthesis of MBP-Pep@MB-CD, mixing MBP-Pep (5 μg/mL,1 mL) and MB-CD (1 mg/mL,1 mL), standing overnight at 4 ℃, then adding ferrocene (1 mg/mL,0.2 mL), and stirring at 4 ℃ for 2h; the method is used for sealing the cyclodextrin cavity, reducing nonspecific adsorption and obtaining MBP-pep@MB-CD for standby;
step 2) synthesizing MBP-pep@MB-CD@Cu-NPC@3-APBA, directly mixing the synthesized MBP-pep@MB-CD and Cu-NPC@3-APBA (1 mg/mL,1 mL), and stirring at 4 ℃ overnight; then, the mixture was washed three times by centrifugation with PBS (4000-6000 rpm,1-2 min); the resulting precipitate was added to PBS (pH 7.4,1M,1 mL) to fix the volume, to give MBP-pep@MB-CD@Cu-NPC@3-APBA dispersion.
The preparation method of the nano-enzyme (Cu-NPC@3-APBA) in the step (4) comprises the following specific steps:
(1) Synthesizing ZIF-8, mixing zinc nitrate hexahydrate (2.36 g), absolute methanol (10 mL) and deionized water (10 mL) to obtain solution A; 2-methylimidazole (4.0 g) was mixed with anhydrous methanol (10 mL) to obtain solution B. Mixing A and B, stirring for 2h, then centrifuging and washing with methanol for three times (5000-6000 rpm,7-8 min), and drying the obtained precipitate in a vacuum environment at 50-60 ℃ to obtain ZIF-8 for later use;
(2) NPC was synthesized by taking ZIF-8 (1 g), calcining at 920℃for 2h under argon atmosphere, and then adding hydrochloric acid (3M, 10 mL) to the obtained product, and reacting at 50℃for 12h. Then centrifugal washing with deionized water for three times (5000-6000 rpm,5-6 min), and drying the obtained precipitate in a vacuum environment at 50-60 ℃ to obtain NPC for later use;
(3) Synthesizing Cu-NPC, mixing NPC (10 mg), copper nitrate (0.01 g), melamine (0.01 g), sodium dicyandiamide (0.01 g) and deionized water (5 mL), stirring for 2 hours, adding sodium borohydride (10 mM,5 mL), stirring for 30 minutes under nitrogen, centrifuging and washing with deionized water for three times (7000-8000 rpm,5-6 minutes), and drying the obtained precipitate in a vacuum environment at 50-60 ℃ to obtain Cu-NPC for later use;
(4) Synthesizing Cu-NPC@3-APBA, weighing synthesized Cu-NPC (10 mg) and 3-APBA (10 mg) and dispersing in 10mL of water, stirring the mixed solution at room temperature overnight, and then washing and centrifuging for three times (7000-8000 rpm,8-10 min); and drying the obtained precipitate under the vacuum environment of 50-60 ℃ to obtain Cu-NPC@3-APBA.
The application discloses a norovirus detection material based on a sugar signal amplification mode, which is Cu-NPC@3-APBA; the detection material Cu-NPC@3-APBA comprises the following components: 1) Laccase-like activity; 2) Catalyzing the color development of 2, 4-dichlorophenol, 4-aminoantipyrine and MES buffer solution; 3) Catalytic TMB, H 2 O 2 And acetic acid buffer solution.
The application relates to an application of a norovirus detection method based on a sugar signal amplification mode, which is used for detecting HuNoV with a detection range of 0.5-10 4 copies/mL。
The application discloses a norovirus detection method based on a sugar signal amplification mode, which comprises the following specific steps:
(1) After the color of a sandwich type biological immunosensor for detecting human norovirus by using the prepared specific antibody is developed, measuring the absorbance of the biological immunosensor by using an ultraviolet spectrophotometer;
(2) Scanning wavelength is 400-800nm;
(3) Recording absorbance values corresponding to human norovirus at different concentrations;
(4) By using a working curve method, human norovirus with different concentrations is detected, and the result shows that the detection range is 0.5-10 4 The lowest detection limit is 0.17copies/mL.
The application mainly uses a combination of polypeptide (Pep) capable of specifically recognizing HuNoV and a HuNoV antibody to construct a biosensor for detecting HuNoV. The specific principle is as follows: the antibody is bound to the ELISA plate to form a surface of the ELISA plate capable of specifically capturing HuNoV, and the HuNoV is captured and immobilized on the surface. Then through the mode of sugar signal amplification: firstly, modifying maltose-cyclodextrin (MB-CD) to form a polypeptide complex (MBP-pep@MB-CD) by utilizing the effect that maltose binding protein-polypeptide (MBP-Pep) can specifically bind to maltose; then, the combination of 3-aminophenylboronic acid (3-APBA) and a large amount of sugar on Cyclodextrin (CD) is utilized to form five-membered ring ester, and the coordination effect of 3-APBA and Cu-NPC is utilized to obtain nano enzyme composite material (MBP-pep@MB-CD@Cu-NPC@3-APBA) as a probe material. The Cu-NPC in the nano enzyme composite material is utilized to change the color of TMB and the laccase-like activity, and an ultraviolet spectrophotometer is used for measuring the absorbance value to detect the content of HuNoV, so that the biosensor capable of rapidly and effectively detecting the concentration of HuNoV is formed.
The application has the beneficial effects that:
(1) According to the application, the nano enzyme composite material is synthesized and used as a probe material, and the synthesized MBP-pep@MB-CD@Cu-NPC@3-APBA nano enzyme composite material has excellent enzyme activity, and a large amount of sugar on cyclodextrin is utilized to amplify signals, so that the excellent performance of the nano enzyme composite material as the probe material is fully exerted. As the concentration of human norovirus increases, the absorbance detected by ultraviolet analysis increases and is between 0.5 and 10 4 The concentration range of the human norovirus of the copies/mL has good linear relation. The replacement of human norovirus with other substances, such as other viruses, protein molecules, etc., does not allow detection of the corresponding signal. The biosensor constructed by the above method was proved to be successful in detecting human norovirus.
(2) The enzymatic colorimetric signal readings used in the present application have a higher sensitivity.
(3) The present application uses Cu-NPC, has ultra-high enzyme activity, and improves signal generation.
(4) The method has the advantages of higher sensitivity, wider detection range, higher detection speed, simple operation and the like, and can be used for the rapid detection of the actual sample.
(5) The method has the advantages of simple equipment, low cost, easy miniaturization, strong specificity, high sensitivity, good stability and the like, and has important scientific significance and clinical application value for detecting the human norovirus.
Drawings
FIG. 1 is an infrared spectrum of a material;
FIG. 2 is a TEM image of a material;
FIG. 3 is a Mapping graph of Cu-NPC@3-APBA;
FIG. 4 is an XPS diagram of Cu-NPC (A) and Cu-NPC@3-APBA (B);
FIG. 5 is a graph of ultraviolet absorbance spectra (A) and standard curve (B) of human norovirus at various concentrations;
FIG. 6 is a graph of ultraviolet absorbance spectra (A) and standard curve (B) of human norovirus at various concentrations;
FIG. 7 is a graph of actual sample detection (A) and specificity experiments (B) of the sensor.
Detailed Description
The chemicals and solvents used in the examples were all analytically pure; the raw materials can be purchased from chemical reagent companies or biopharmaceutical companies; the stirring adopts a magnetic stirrer stirring mode.
Example 1: the norovirus detection method based on the sugar signal amplification mode comprises the following specific steps:
(1) The ELISA plate is fully paved with human norovirus antibody with the concentration of 1 mug/mL, and incubated overnight at 4 ℃;
(2) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, fully paving the ELISA plate with 50 mu L of 1% bovine serum albumin, and incubating for 30min at room temperature;
(3) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, spreading 50 mu L of human norovirus solution with different concentrations on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(4) Washing the ELISA plate 3 times by 0.1mol/L of PBST (potential of Hydrogen) with pH7.4, taking 50 mu L of MBP-pep@MB-CD@Cu-NPC@3-APBA solution with concentration of 1mg/mL and 50 mu L of PBS with concentration of 0.1mol/mL and pH9.6, fully spreading on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(5) The ELISA plate was washed 3 times with 0.1mol/L pH7.4 PBST, 50. Mu.L of 2,4-DP at a concentration of 1mg/mL, 50. Mu.L of 4-AP at a concentration of 1mg/mL, and 150. Mu.L of MES buffer at pH6.8 were taken;
(6) Reacting at 60 ℃ for 30min, then carrying out ultraviolet analysis, scanning the wavelength to 400-700nm, and drawing a working curve. Obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
The preparation method of the nano enzyme material (Cu-NPC@3-APBA) in the step (4) comprises the following specific steps:
(1) Synthesizing ZIF-8, mixing zinc nitrate hexahydrate (2.36 g), absolute methanol (10 mL) and deionized water (10 mL) to obtain solution A; 2-methylimidazole (4.0 g) was mixed with anhydrous methanol (10 mL) to obtain solution B. A and B are mixed and stirred for 2 hours, then the mixture is centrifugally washed with methanol for three times (6000 rpm,7 min), and the obtained precipitate product is dried under the vacuum environment at 60 ℃ to obtain ZIF-8 for later use.
(2) NPC was synthesized by taking ZIF-8 (1 g), calcining at 920℃for 2h under argon atmosphere, and then adding hydrochloric acid (3M, 10 mL) to the obtained product, and reacting at 50℃for 12h. The precipitate was then dried under vacuum at 50℃with three centrifugal washes (6000 rpm,5 min) of deionized water to give NPC for further use.
(3) Synthesizing Cu-NPC, taking NPC (10 mg), copper nitrate (0.01 g), melamine (0.01 g), sodium dicyandiamide (0.01 g) and deionized water (5 mL), mixing and stirring for 2h, then adding sodium borohydride (10 mM,5 mL), stirring for 30min under nitrogen, then centrifugally washing with deionized water for three times (800 rpm,5 min), and drying the obtained precipitate under a vacuum environment at 50 ℃ to obtain Cu-NPC for standby.
(4) Synthesis of Cu-NPC@3-APBA the synthesized Cu-NPC (10 mg) and 3-APBA (10 mg) were weighed and dispersed in 10mL of water, and the mixture was stirred at room temperature overnight and then centrifuged three times (8000 rpm,8 min) with water. And drying the obtained precipitate under a vacuum environment at 60 ℃ to obtain Cu-NPC@3-APBA.
The preparation method of the nano enzyme complex (MBP-pep@MB-CD@Cu-NPC@3-APBA) dispersion liquid in the step (4) comprises the following specific steps:
(1) MBP-Pep@MB-CD was synthesized, MBP-Pep (5. Mu.g/mL, 1 mL) and MB-CD (1 mg/mL,1 mL) were mixed, left overnight at 4℃and then ferrocene (1 mg/mL,0.2 mL) was added and stirred for 2h at 4 ℃. The method is used for sealing the cyclodextrin cavity, reducing nonspecific adsorption and obtaining MBP-pep@MB-CD for standby.
(2) Synthesis of MBP-pep@MB-CD@Cu-NPC@3-APBA the above synthesized MBP-pep@MB-CD was directly mixed with Cu-NPC@3-APBA (1 mg/mL,1 mL) and stirred overnight at 4 ℃. The PBS was then washed three times by centrifugation (6000 rpm,1 min). The resulting precipitate was added to PBS (pH 7.4,1M,1 mL) to fix the volume, to give MBP-pep@MB-CD@Cu-NPC@3-APBA dispersion.
The application discloses a norovirus detection method based on a sugar signal amplification mode, which comprises the following specific steps:
(1) After the color of a sandwich type biological immunosensor for detecting human norovirus by using the prepared specific antibody is developed, measuring the absorbance of the biological immunosensor by using an ultraviolet spectrophotometer;
(2) Scanning wavelength is 400-700nm;
(3) Recording absorbance values corresponding to human norovirus at different concentrations;
(4) By using a working curve method, human norovirus at different concentrations is detected, and the result shows that the detection range is 0.5-5000copies/mL, and the lowest detection lower limit is 0.17copies/mL.
Example 2: the norovirus detection method based on the sugar signal amplification mode comprises the following specific steps:
(1) The ELISA plate is fully paved with human norovirus antibody with the concentration of 1 mug/mL, and incubated overnight at 4 ℃;
(2) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, fully paving the ELISA plate with 50 mu L of 1% bovine serum albumin, and incubating for 30min at room temperature;
(3) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, spreading 50 mu L of human norovirus solution with different concentrations on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(4) Washing the ELISA plate 3 times by 0.1mol/L of PBST (potential of Hydrogen) with pH7.4, taking 50 mu L of MBP-pep@MB-CD@Cu-NPC@3-APBA solution with concentration of 1mg/mL and 50 mu L of PBS with concentration of 0.1mol/mL and pH9.6, fully spreading on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(5) The ELISA plate was washed 3 times with 0.1mol/L pH7.4 PBST, and 50. Mu.L was takenTMB at a concentration of 10mM, 50. Mu.L H at a concentration of 100mM 2 O 2 And 150. Mu.L of acetic acid buffer pH 4.5;
(6) The reaction is carried out for 15min at 37 ℃, then ultraviolet analysis is carried out, the scanning wavelength is 500-800nm, and a working curve is drawn. Obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
The preparation method of the nano enzyme material (Cu-NPC@3-APBA) in the step (4) comprises the following specific steps: (1) Synthesizing ZIF-8, mixing zinc nitrate hexahydrate (2.36 g), absolute methanol (10 mL) and deionized water (10 mL) to obtain solution A; 2-methylimidazole (4.0 g) was mixed with anhydrous methanol (10 mL) to obtain solution B. A and B are mixed and stirred for 2 hours, then the mixture is centrifugally washed with methanol for three times (6000 rpm,7 min), and the obtained precipitate product is dried under the vacuum environment at 50 ℃ to obtain ZIF-8 for later use.
(2) NPC was synthesized by taking ZIF-8 (1 g), calcining at 920℃for 2h under argon atmosphere, and then adding hydrochloric acid (3M, 10 mL) to the obtained product, and reacting at 50℃for 12h. The precipitate was then dried under vacuum at 60℃with three centrifugal washes (6000 rpm,5 min) of deionized water to give NPC for further use.
(3) Synthesizing Cu-NPC, taking NPC (10 mg), copper nitrate (0.01 g), melamine (0.01 g), sodium dicyandiamide (0.01 g) and deionized water (5 mL), mixing and stirring for 2h, then adding sodium borohydride (10 mM,5 mL), stirring for 30min under nitrogen, then centrifugally washing with deionized water for three times (800 rpm,5 min), and drying the obtained precipitate under a vacuum environment at 50 ℃ to obtain Cu-NPC for standby.
(4) Synthesis of Cu-NPC@3-APBA the synthesized Cu-NPC (10 mg) and 3-APBA (10 mg) were weighed and dispersed in 10mL of water, and the mixture was stirred at room temperature overnight and then centrifuged three times (8000 rpm,8 min) with water. And drying the obtained precipitate under a vacuum environment at 50 ℃ to obtain Cu-NPC@3-APBA.
The preparation method of the nano enzyme complex (MBP-pep@MB-CD@Cu-NPC@3-APBA) dispersion liquid in the step (4) comprises the following specific steps:
(1) MBP-Pep@MB-CD was synthesized, MBP-Pep (5. Mu.g/mL, 1 mL) and MB-CD (1 mg/mL,1 mL) were mixed, left overnight at 4℃and then ferrocene (1 mg/mL,0.2 mL) was added and stirred for 2h at 4 ℃. The method is used for sealing the cyclodextrin cavity, reducing nonspecific adsorption and obtaining MBP-pep@MB-CD for standby.
(2) Synthesis of MBP-pep@MB-CD@Cu-NPC@3-APBA the above synthesized MBP-pep@MB-CD was directly mixed with Cu-NPC@3-APBA (1 mg/mL,1 mL) and stirred overnight at 4 ℃. The PBS was then washed three times by centrifugation (6000 rpm,1 min). The resulting precipitate was added to PBS (pH 7.4,1M,1 mL) to fix the volume, to give MBP-pep@MB-CD@Cu-NPC@3-APBA dispersion.
The application discloses a norovirus detection method based on a sugar signal amplification mode, which comprises the following specific steps:
(1) After the color of a sandwich type biological immunosensor for detecting human norovirus by using the prepared specific antibody is developed, measuring the absorbance of the biological immunosensor by using an ultraviolet spectrophotometer;
(2) Scanning wavelength is 500-800nm;
(3) Recording absorbance values corresponding to human norovirus at different concentrations;
(4) By using a working curve method, human norovirus with different concentrations is detected, and the result shows that the detection range is 1-10 4 The lowest detection limit was 0.33copies/mL.
Example 3: the norovirus detection method based on the sugar signal amplification mode comprises the following specific steps:
(1) The ELISA plate is fully paved with human norovirus antibody with the concentration of 1 mug/mL, and incubated overnight at 4 ℃;
(2) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, fully paving the ELISA plate with 50 mu L of 1% bovine serum albumin, and incubating for 30min at room temperature;
(3) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, spreading 50 mu L of human norovirus solution with different concentrations on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(4) Washing the ELISA plate 3 times by 0.1mol/L of PBST (potential of Hydrogen) with pH7.4, taking 50 mu L of MBP-pep@MB-CD@Cu-NPC@3-APBA solution with concentration of 1mg/mL and 50 mu L of PBS with concentration of 0.1mol/mL and pH9.6, fully spreading on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(5) The ELISA plate was washed 3 times with 0.1mol/L pH7.4 PBST, 50. Mu.L of 2,4-DP at a concentration of 1mg/mL, 50. Mu.L of 4-AP at a concentration of 1mg/mL, and 150. Mu.L of MES buffer at pH6.8 were taken;
(6) Reacting at 60 ℃ for 30min, then carrying out ultraviolet analysis, scanning the wavelength to 400-700nm, and drawing a working curve. Obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
The preparation method of the nano enzyme material (Cu-NPC@3-APBA) in the step (4) comprises the following specific steps:
(1) Synthesizing ZIF-8, mixing zinc nitrate hexahydrate (2.36 g), absolute methanol (10 mL) and deionized water (10 mL) to obtain solution A; 2-methylimidazole (4.0 g) was mixed with anhydrous methanol (10 mL) to obtain solution B. A and B are mixed and stirred for 2 hours, then the mixture is centrifugally washed with methanol for three times (6000 rpm,7 min), and the obtained precipitate product is dried under the vacuum environment at 60 ℃ to obtain ZIF-8 for later use.
(2) NPC was synthesized by taking ZIF-8 (1 g), calcining at 920℃for 2h under argon atmosphere, and then adding hydrochloric acid (3M, 10 mL) to the obtained product, and reacting at 50℃for 12h. The precipitate was then dried under vacuum at 60℃with three centrifugal washes (6000 rpm,5 min) of deionized water to give NPC for further use.
(3) Synthesizing Cu-NPC, taking NPC (10 mg), copper nitrate (0.01 g), melamine (0.01 g), sodium dicyandiamide (0.01 g) and deionized water (5 mL), mixing and stirring for 2h, then adding sodium borohydride (10 mM,5 mL), stirring for 30min under nitrogen, then centrifugally washing with deionized water for three times (800 rpm,5 min), and drying the obtained precipitate under a vacuum environment at 50 ℃ to obtain Cu-NPC for standby.
(4) Synthesis of Cu-NPC@3-APBA the synthesized Cu-NPC (5 mg) and 3-APBA (10 mg) were weighed and dispersed in 10mL of water, and the mixture was stirred overnight at room temperature and then centrifuged with water three times (8000 rpm,8 min). And drying the obtained precipitate under the vacuum environment of 50-60 ℃ to obtain Cu-NPC@3-APBA.
The preparation method of the nano enzyme complex (MBP-pep@MB-CD@Cu-NPC@3-APBA) dispersion liquid in the step (4) comprises the following specific steps:
(1) MBP-Pep@MB-CD was synthesized, MBP-Pep (5. Mu.g/mL, 1 mL) and MB-CD (1 mg/mL,1 mL) were mixed, left overnight at 4℃and then ferrocene (1 mg/mL,0.2 mL) was added and stirred for 2h at 4 ℃. The method is used for sealing the cyclodextrin cavity, reducing nonspecific adsorption and obtaining MBP-pep@MB-CD for standby.
(2) Synthesis of MBP-pep@MB-CD@Cu-NPC@3-APBA the above synthesized MBP-pep@MB-CD was directly mixed with Cu-NPC@3-APBA (1 mg/mL,1 mL) and stirred overnight at 4 ℃. The PBS was then washed three times by centrifugation (6000 rpm,1 min). The resulting precipitate was added to PBS (pH 7.4,1M,1 mL) to fix the volume, to give MBP-pep@MB-CD@Cu-NPC@3-APBA dispersion.
The application discloses a norovirus detection method based on a sugar signal amplification mode, which comprises the following specific steps:
(1) After the color of a sandwich type biological immunosensor for detecting human norovirus by using the prepared specific antibody is developed, measuring the absorbance of the biological immunosensor by using an ultraviolet spectrophotometer;
(2) Scanning wavelength is 400-700nm;
(3) Recording absorbance values corresponding to human norovirus at different concentrations;
(4) By using a working curve method, human norovirus at different concentrations is detected, and the result shows that the detection range is 0.5-5000copies/mL, and the lowest detection lower limit is 0.17copies/mL.
Example 4: the norovirus detection method based on the sugar signal amplification mode comprises the following specific steps:
(1) The ELISA plate is fully paved with human norovirus antibody with the concentration of 1 mug/mL, and incubated overnight at 4 ℃;
(2) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, fully paving the ELISA plate with 50 mu L of 1% bovine serum albumin, and incubating for 30min at room temperature;
(3) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, spreading 50 mu L of human norovirus solution with different concentrations on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(4) Washing the ELISA plate 3 times by 0.1mol/L of PBST (potential of Hydrogen) with pH7.4, taking 50 mu L of MBP-pep@MB-CD@Cu-NPC@3-APBA solution with concentration of 1mg/mL and 50 mu L of PBS with concentration of 0.1mol/mL and pH9.6, fully spreading on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(5) The ELISA plate was washed 3 times with 0.1mol/L pH7.4 PBST, and 50. Mu.L of TMB at a concentration of 10mM and 50. Mu.L of H at a concentration of 100mM were taken 2 O 2 And 150. Mu.L of acetic acid buffer pH 4.5;
(6) The reaction is carried out for 15min at 37 ℃, then ultraviolet analysis is carried out, the scanning wavelength is 500-800nm, and a working curve is drawn. Obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
The preparation method of the nano enzyme material (Cu-NPC@3-APBA) in the step (4) comprises the following specific steps:
(1) Synthesizing ZIF-8, mixing zinc nitrate hexahydrate (2.36 g), absolute methanol (10 mL) and deionized water (10 mL) to obtain solution A; 2-methylimidazole (4.0 g) was mixed with anhydrous methanol (10 mL) to obtain solution B. A and B are mixed and stirred for 2 hours, then the mixture is centrifugally washed with methanol for three times (6000 rpm,7 min), and the obtained precipitate product is dried under the vacuum environment at 60 ℃ to obtain ZIF-8 for later use.
(2) NPC was synthesized by taking ZIF-8 (1 g), calcining at 920℃for 2h under argon atmosphere, and then adding hydrochloric acid (3M, 10 mL) to the obtained product, and reacting at 50℃for 12h. The precipitate was then dried under vacuum at 60℃with three centrifugal washes (6000 rpm,5 min) of deionized water to give NPC for further use.
(3) Synthesizing Cu-NPC, taking NPC (10 mg), copper nitrate (0.01 g), melamine (0.01 g), sodium dicyandiamide (0.01 g) and deionized water (5 mL), mixing and stirring for 2h, then adding sodium borohydride (10 mM,5 mL), stirring for 30min under nitrogen, then centrifugally washing with deionized water for three times (800 rpm,5 min), and drying the obtained precipitate under a vacuum environment at 60 ℃ to obtain Cu-NPC for standby.
(4) Synthesis of Cu-NPC@3-APBA the synthesized Cu-NPC (5 mg) and 3-APBA (10 mg) were weighed and dispersed in 10mL of water, and the mixture was stirred at room temperature overnight and then centrifuged three times (8000 rpm,8 min) with water. And drying the obtained precipitate under a vacuum environment at 50 ℃ to obtain Cu-NPC@3-APBA.
The preparation method of the nano enzyme complex (MBP-pep@MB-CD@Cu-NPC@3-APBA) dispersion liquid in the step (4) comprises the following specific steps:
(1) MBP-Pep@MB-CD was synthesized, MBP-Pep (5. Mu.g/mL, 1 mL) and MB-CD (1 mg/mL,1 mL) were mixed, left overnight at 4℃and then ferrocene (1 mg/mL,0.2 mL) was added and stirred for 2h at 4 ℃. The method is used for sealing the cyclodextrin cavity, reducing nonspecific adsorption and obtaining MBP-pep@MB-CD for standby.
(2) Synthesis of MBP-pep@MB-CD@Cu-NPC@3-APBA the above synthesized MBP-pep@MB-CD was directly mixed with Cu-NPC@3-APBA (1 mg/mL,1 mL) and stirred overnight at 4 ℃. The PBS was then washed three times by centrifugation (6000 rpm,1 min). The resulting precipitate was added to PBS (pH 7.4,1M,1 mL) to fix the volume, to give MBP-pep@MB-CD@Cu-NPC@3-APBA dispersion.
The application discloses a norovirus detection method based on a sugar signal amplification mode, which comprises the following specific steps:
(1) After the color of a sandwich type biological immunosensor for detecting human norovirus by using the prepared specific antibody is developed, measuring the absorbance of the biological immunosensor by using an ultraviolet spectrophotometer;
(2) Scanning wavelength is 500-800nm;
(3) Recording absorbance values corresponding to human norovirus at different concentrations;
(4) By using a working curve method, human norovirus with different concentrations is detected, and the result shows that the detection range is 1-10 4 The lowest detection limit was 0.33copies/mL.
Example 5: the norovirus detection method based on the sugar signal amplification mode comprises the following specific steps:
(1) The ELISA plate is fully paved with human norovirus antibody with the concentration of 1 mug/mL, and incubated overnight at 4 ℃;
(2) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, fully paving the ELISA plate with 50 mu L of 1% bovine serum albumin, and incubating for 30min at room temperature;
(3) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, spreading 50 mu L of human norovirus solution with different concentrations on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(4) Washing the ELISA plate 3 times by 0.1mol/L of PBST (potential of Hydrogen) with pH7.4, taking 50 mu L of MBP-pep@MB-CD@Cu-NPC@3-APBA solution with concentration of 1mg/mL and 50 mu L of PBS with concentration of 0.1mol/mL and pH9.6, fully spreading on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(5) The ELISA plate was washed 3 times with 0.1mol/L pH7.4 PBST, 50. Mu.L of 2,4-DP at a concentration of 1mg/mL, 50. Mu.L of 4-AP at a concentration of 1mg/mL, and 150. Mu.L of MES buffer at pH6.8 were taken;
(6) Reacting at 60 ℃ for 30min, then carrying out ultraviolet analysis, scanning the wavelength to 400-700nm, and drawing a working curve. Obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
The preparation method of the nano enzyme material (Cu-NPC@3-APBA) in the step (4) comprises the following specific steps:
(1) Synthesizing ZIF-8, mixing zinc nitrate hexahydrate (2.36 g), absolute methanol (10 mL) and deionized water (10 mL) to obtain solution A; 2-methylimidazole (4.0 g) was mixed with anhydrous methanol (10 mL) to obtain solution B. A and B are mixed and stirred for 2 hours, then the mixture is centrifugally washed with methanol for three times (6000 rpm,7 min), and the obtained precipitate product is dried under the vacuum environment at 50 ℃ to obtain ZIF-8 for later use.
(2) NPC was synthesized by taking ZIF-8 (1 g), calcining at 920℃for 2h under argon atmosphere, and then adding hydrochloric acid (3M, 10 mL) to the obtained product, and reacting at 50℃for 12h. The precipitate was then dried under vacuum at 50℃with three centrifugal washes (6000 rpm,5 min) of deionized water to give NPC for further use.
(3) Synthesizing Cu-NPC, taking NPC (10 mg), copper nitrate (0.01 g), melamine (0.01 g), sodium dicyandiamide (0.01 g) and deionized water (5 mL), mixing and stirring for 2h, then adding sodium borohydride (10 mM,5 mL), stirring for 30min under nitrogen, then centrifugally washing with deionized water for three times (800 rpm,5 min), and drying the obtained precipitate under a vacuum environment at 60 ℃ to obtain Cu-NPC for standby.
(4) Synthesis of Cu-NPC@3-APBA the synthesized Cu-NPC (10 mg) and 3-APBA (5 mg) were weighed and dispersed in 10mL of water, and the mixture was stirred at room temperature overnight and then centrifuged three times (8000 rpm,8 min) with water. And drying the obtained precipitate under a vacuum environment at 50 ℃ to obtain Cu-NPC@3-APBA.
The preparation method of the nano enzyme complex (MBP-pep@MB-CD@Cu-NPC@3-APBA) dispersion liquid in the step (4) comprises the following specific steps:
(1) MBP-Pep@MB-CD was synthesized, MBP-Pep (5. Mu.g/mL, 1 mL) and MB-CD (1 mg/mL,1 mL) were mixed, left overnight at 4℃and then ferrocene (1 mg/mL,0.2 mL) was added and stirred for 2h at 4 ℃. The method is used for sealing the cyclodextrin cavity, reducing nonspecific adsorption and obtaining MBP-pep@MB-CD for standby.
(2) Synthesis of MBP-pep@MB-CD@Cu-NPC@3-APBA the above synthesized MBP-pep@MB-CD was directly mixed with Cu-NPC@3-APBA (1 mg/mL,1 mL) and stirred overnight at 4 ℃. The PBS was then washed three times by centrifugation (6000 rpm,1 min). The resulting precipitate was added to PBS (pH 7.4,1M,1 mL) to fix the volume, to give MBP-pep@MB-CD@Cu-NPC@3-APBA dispersion.
The application discloses a norovirus detection method based on a sugar signal amplification mode, which comprises the following specific steps:
(1) After the color of a sandwich type biological immunosensor for detecting human norovirus by using the prepared specific antibody is developed, measuring the absorbance of the biological immunosensor by using an ultraviolet spectrophotometer;
(2) Scanning wavelength is 400-700nm;
(3) Recording absorbance values corresponding to human norovirus at different concentrations;
(4) By using a working curve method, human norovirus at different concentrations is detected, and the result shows that the detection range is 0.5-5000copies/mL, and the lowest detection lower limit is 0.17copies/mL.
Example 6: the norovirus detection method based on the sugar signal amplification mode comprises the following specific steps:
(1) The ELISA plate is fully paved with human norovirus antibody with the concentration of 1 mug/mL, and incubated overnight at 4 ℃;
(2) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, fully paving the ELISA plate with 50 mu L of 1% bovine serum albumin, and incubating for 30min at room temperature;
(3) Washing the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, spreading 50 mu L of human norovirus solution with different concentrations on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(4) Washing the ELISA plate 3 times by 0.1mol/L of PBST (potential of Hydrogen) with pH7.4, taking 50 mu L of MBP-pep@MB-CD@Cu-NPC@3-APBA solution with concentration of 1mg/mL and 50 mu L of PBS with concentration of 0.1mol/mL and pH9.6, fully spreading on the washed ELISA plate, and incubating for 2 hours at 4 ℃;
(5) The ELISA plate was washed 3 times with 0.1mol/L pH7.4 PBST, and 50. Mu.L of TMB at a concentration of 10mM and 50. Mu.L of H at a concentration of 100mM were taken 2 O 2 And 150. Mu.L of acetic acid buffer pH 4.5;
(6) The reaction is carried out for 15min at 37 ℃, then ultraviolet analysis is carried out, the scanning wavelength is 500-800nm, and a working curve is drawn. Obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
The preparation method of the nano enzyme material (Cu-NPC@3-APBA) in the step (4) comprises the following specific steps:
(1) Synthesizing ZIF-8, mixing zinc nitrate hexahydrate (2.36 g), absolute methanol (10 mL) and deionized water (10 mL) to obtain solution A; 2-methylimidazole (4.0 g) was mixed with anhydrous methanol (10 mL) to obtain solution B. A and B are mixed and stirred for 2 hours, then the mixture is centrifugally washed with methanol for three times (6000 rpm,7 min), and the obtained precipitate product is dried under the vacuum environment at 50 ℃ to obtain ZIF-8 for later use.
(2) NPC was synthesized by taking ZIF-8 (1 g), calcining at 920℃for 2h under argon atmosphere, and then adding hydrochloric acid (3M, 10 mL) to the obtained product, and reacting at 50℃for 12h. The precipitate was then dried under vacuum at 60℃with three centrifugal washes (6000 rpm,5 min) of deionized water to give NPC for further use.
(3) Synthesizing Cu-NPC, taking NPC (10 mg), copper nitrate (0.01 g), melamine (0.01 g), sodium dicyandiamide (0.01 g) and deionized water (5 mL), mixing and stirring for 2h, then adding sodium borohydride (10 mM,5 mL), stirring for 30min under nitrogen, then centrifugally washing with deionized water for three times (800 rpm,5 min), and drying the obtained precipitate under a vacuum environment at 50 ℃ to obtain Cu-NPC for standby.
(4) Synthesis of Cu-NPC@3-APBA the synthesized Cu-NPC (10 mg) and 3-APBA (5 mg) were weighed and dispersed in 10mL of water, and the mixture was stirred at room temperature overnight and then centrifuged three times (8000 rpm,8 min) with water. And drying the obtained precipitate under a vacuum environment at 50 ℃ to obtain Cu-NPC@3-APBA.
The preparation method of the nano enzyme complex (MBP-pep@MB-CD@Cu-NPC@3-APBA) dispersion liquid in the step (4) comprises the following specific steps:
(1) MBP-Pep@MB-CD was synthesized, MBP-Pep (5. Mu.g/mL, 1 mL) and MB-CD (1 mg/mL,1 mL) were mixed, left overnight at 4℃and then ferrocene (1 mg/mL,0.2 mL) was added and stirred for 2h at 4 ℃. The method is used for sealing the cyclodextrin cavity, reducing nonspecific adsorption and obtaining MBP-pep@MB-CD for standby.
(2) Synthesis of MBP-pep@MB-CD@Cu-NPC@3-APBA the above synthesized MBP-pep@MB-CD was directly mixed with Cu-NPC@3-APBA (1 mg/mL,1 mL) and stirred overnight at 4 ℃. The PBS was then washed three times by centrifugation (6000 rpm,1 min). The resulting precipitate was added to PBS (pH 7.4,1M,1 mL) to fix the volume, to give MBP-pep@MB-CD@Cu-NPC@3-APBA dispersion.
The application discloses a norovirus detection method based on a sugar signal amplification mode, which comprises the following specific steps:
(1) After the color of a sandwich type biological immunosensor for detecting human norovirus by using the prepared specific antibody is developed, measuring the absorbance of the biological immunosensor by using an ultraviolet spectrophotometer;
(2) Scanning wavelength is 500-800nm;
(3) Recording absorbance values corresponding to human norovirus at different concentrations;
(4) By using a working curve method, human norovirus with different concentrations is detected, and the result shows that the detection range is 1-10 4 The lowest detection limit was 0.33copies/mL.
FIG. 1 is the infrared spectra (IR) of ZIF-8, NPC, cu-NPC and Cu-NPC@3-APBA in step (4) of example 1; in the ZIF-8 spectrum: 1421. 1458cm -1 Telescoping vibration attributed to the ring bond in ZIF-8; 421cm -1 The telescoping vibration attributed to the Zn-N bond indicates that ZIF-8 was successfully synthesized. In NPC spectrum, 1585cm -1 The stretching vibration attributed to the carbon-nitrogen double bond is due to the fact that NPC retains the framework of ZIF-8. In the patterns of Cu-NPC and Cu-NPC@3-APBA, after the material is loaded with 3-APBA, the stretching vibration peaks (1618, 1581, 1492 and 1445 cm) of benzene ring framework appear -1 ) The method comprises the steps of carrying out a first treatment on the surface of the C-B bond stretching vibration (994 cm) -1 ) The method comprises the steps of carrying out a first treatment on the surface of the Stretching vibration of B-O bond (1299 cm) -1 ) Indicating successful loading of 3-APBA.
FIG. 2 is a Transmission Electron Microscope (TEM) of ZIF-8, NPC, cu-NPC and Cu-NPC@3-APBA in step (4) of example 1; wherein ZIF-8 is a uniform and regular hexahedron; NPC is formed after calcination, a regular hexahedral framework structure is formed, the whole structure is not collapsed after Cu and 3-APBA are added, and the interior of the material is filled with Cu NPs and 3-APBA (figure A).
FIG. 3 is a Mapping graph of Cu-NPC@3-APBA in step (4) of example 1, and FIG. 2 shows that Cu, B, N, C and O are uniformly distributed in the nanoparticles, indicating successful synthesis of Cu-NPC@3-APBA.
FIG. 4 is an XPS plot of Cu-NPC (A) and Cu-NPC@3-APBA (B) in step (4) of example 1, wherein FIG. A shows a characteristic peak of element Cu, C, O, N in Cu-NPC and FIG. B shows a characteristic peak of element Cu, C, O, N, B in Cu-NPC@3-APBA, indicating that 3-APBA was successfully loaded on Cu-NPC.
FIG. 5 shows UV absorption spectra and standard curves for different concentrations prepared in steps (7) - (8) in example 1. The constructed sensor was used to detect different concentrations of human norovirus using a microplate reader at 400-700nm, with the absorbance value increasing progressively as the concentration of human norovirus increases (fig. 3A). As shown in FIG. 3B, the absorbance has a good linear relationship with the logarithm of the concentration of human norovirus, the correlation coefficient (R 2 ) 0.9970 LOD of 0.17copies mL -1 (S/n=3), showing good linearity and lower LOD values.
FIG. 6 is a graph showing the ultraviolet absorbance spectra and standard curves of human norovirus at different concentrations prepared in steps (7) - (8) in example 2. The constructed sensor was used to detect different concentrations of human norovirus using a microplate reader at 500-800nm, with the absorbance value increasing progressively as the concentration of human norovirus increases (fig. 4A). As shown in FIG. 4B, the absorbance has a good linear relationship with the logarithm of the concentration of human norovirus, the correlation coefficient (R 2 ) 0.9927 and LOD of 0.33copies mL -1 (S/n=3), showing good linearity and lower LOD values.
FIG. 7 shows the actual sample detection (A) and specificity experiments (B) performed with the constructed sensor. As shown in fig. 7A, the absorbance values of the negative samples (263, 269, 277) were not much different from the blank, and the absorbance values of the positive samples (247, 243, 244, 245, 246, 248, 249) were higher, indicating that the constructed sensor was able to detect human norovirus in the actual samples. As shown in FIG. 7B, 1-13 are blank groups, na in this order + 、K + 、Mg 2+ 、Ca 2+ Bovine serum albumin, lysozyme, glucose, EV, RV, E.coli, human norovirusHuman norovirus+mix. Absorbance values do not differ much from the blank when other ions, proteins or viruses are detected with the sensor constructed. When human norovirus and human norovirus+mix are detected, the absorbance values are higher and the two sets of values are substantially the same, indicating that the sensor has good specificity and that other substances do not interfere significantly with the detection of the sensor.
The foregoing description is only a few specific embodiments of the present application (the embodiments are not intended to be exhaustive, and the scope of the application includes the numerical range and other technical gist of the present application), and the details or common sense of the present application are not described (including but not limited to shorthand and abbreviations). It should be noted that the above embodiments do not limit the present application in any way, and it is within the scope of the present application for those skilled in the art to obtain the technical solution by equivalent substitution or equivalent transformation. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (6)
1. A method for preparing a norovirus sandwich type biological immunosensor based on a sugar signal amplification mode, which is characterized by comprising the following steps:
step 1) fully spreading human norovirus antibodies on an ELISA plate, and incubating overnight at 4-6 ℃; specific capture NoV;
step 2) cleaning an ELISA plate by using PBST, fully paving the ELISA plate with bovine serum albumin, and incubating for 20-30min at room temperature;
step 3) cleaning the ELISA plate by using PBST, spreading human norovirus solutions with different concentrations on the cleaned ELISA plate, and incubating for 1.5-2h at 4-6 ℃;
step 4) cleaning the ELISA plate by using PBST, spreading MBP-pep@MB-CD solution on the cleaned ELISA plate, and incubating for 1.5-2h at 4-6 ℃; MBP-pep@MB-CD@Cu-NPC@3-APBA specifically recognizes human norovirus and amplifies signals;
the preparation method of the Cu-NPC@3-APBA comprises the following specific steps:
(1) Synthesis of ZIF-8:
2.36g of zinc nitrate hexahydrate, 10mL of absolute methanol and 10mL of deionized water are mixed to obtain solution A; 4.0g of 2-methylimidazole and 10mL of absolute methanol were mixed to obtain solution B; mixing A and B, stirring for 2h, and then centrifugally washing with methanol for three times at 5000-6000rpm for 7-8min each time; drying the obtained precipitate under a vacuum environment at 50-60 ℃ to obtain ZIF-8 for later use;
(2) Synthesis of NPC:
taking ZIF-8 g, calcining for 2 hours at 920 ℃ under the argon environment, adding 3M 10mL of hydrochloric acid into the obtained product, and reacting for 12 hours at 50 ℃; then centrifugal washing with deionized water for three times at 5000-6000rpm for 5-6min each time; drying the obtained precipitate under the vacuum environment of 50-60 ℃ to obtain NPC for standby;
(3) Synthesizing Cu-NPC, mixing and stirring 10mg of NPC, 0.01g of copper nitrate, 0.01g of melamine, 0.01g of dicyandiamide sodium and 5mL of deionized water for 2 hours, then adding 10mM of 5mL of sodium borohydride, stirring for 30 minutes under nitrogen, and then centrifugally washing with deionized water for three times, 7000-8000rpm,5-6 minutes each time; drying the obtained precipitate under a vacuum environment at 50-60 ℃ to obtain Cu-NPC for standby;
(4) Synthesizing Cu-NPC@3-APBA, weighing 10mg of Cu-NPC and 10mg of 3-APBA synthesized, dispersing in 10mL of water, stirring the mixed solution at room temperature overnight, and then washing and centrifuging for three times at 7000-8000rpm for 8-10min each time; drying the obtained precipitate under a vacuum environment at 50-60 ℃ to obtain Cu-NPC@3-APBA;
step 5) cleaning the ELISA plate by using PBST, and taking the solution A to enable Cu-NPC to have absorption peaks with different intensities at about 510 and nm;
step 6) reacting for 20-30min at 50-60 ℃, then carrying out ultraviolet analysis, scanning the wavelength to 400-700nm, and drawing a working curve; obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
2. The method for preparing the norovirus sandwich type biological immunosensor based on the sugar signal amplification mode according to claim 1, wherein the norovirus detection method comprises the following specific steps:
step 1) fully paving human norovirus antibody with the concentration of 0.5-1 mug/mL on an ELISA plate, and incubating overnight at 4-6 ℃; specific capture NoV;
step 2), cleaning the ELISA plate 3 times by using 0.1mol/L of PBST with the pH of 7.4, fully paving the ELISA plate with 50 mu L of bovine serum albumin with the concentration of 1-2%, and incubating for 20-30min at room temperature;
step 3) cleaning the ELISA plate 3 times by 0.1mol/L pH7.4 PBST, spreading 50 mu L of human norovirus solution with different concentrations on the cleaned ELISA plate, and incubating for 1.5-2h at 4-6 ℃;
step 4) cleaning the ELISA plate 3 times by 0.1mol/L of PBST with pH of 7.4, spreading 50 mu L of MBP-pep@MB-CD solution with the concentration of 0.5-1mg/mL on the cleaned ELISA plate, and incubating for 1.5-2 hours at the temperature of 4-6 ℃; MBP-pep@MB-CD@Cu-NPC@3-APBA specifically recognizes human norovirus and amplifies signals;
step 5) washing the ELISA plate 3 times by using 0.1mol/L pH7.4 PBST, and taking the A solution to enable Cu-NPC to have absorption peaks with different intensities at about 510 and nm;
step 6) reacting for 20-30min at 50-60 ℃, then carrying out ultraviolet analysis, scanning the wavelength to 400-700nm, and drawing a working curve; obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
3. The method for preparing a sandwich type biological immunosensor of norovirus based on a glycosignal amplification mode according to claim 2, wherein the step 5) is to wash the elisa plate 3 times with 0.1mol/L pH7.4 PBST, take 50 μl of 2, 4-dichlorophenol with a concentration of 0.5-1mg/mL, 50 μl of 4-aminoantipyrine with a concentration of 0.5-1mg/mL, and 150 μl of pH6.8 MES buffer solution; the Cu-NPC is enabled to have different intensity absorption peaks at about 510 nm.
4. The method for preparing a sandwich type biological immunosensor of norovirus based on a glycosignal amplification mode according to claim 2, wherein the step 5) is to wash the ELISA plate 3 times with 0.1mol/L pH7.4 PBST, and take 50. Mu.L of TMB with a concentration of 10-20mM and 50. Mu.L of H with a concentration of 90-100mM 2 O 2 And 150. Mu.L of pH4.5 acetate bufferA solution; the Cu-NPC is enabled to have absorption peaks with different intensities at about 652 nm.
5. The method for preparing a sandwich type biological immunosensor of norovirus based on a sugar signal amplification mode according to claim 4, wherein the step 6) is to react for 15-20min at 30-37 ℃, then to perform ultraviolet analysis, scan the wavelength for 500-800nm, and draw a working curve; obtaining the sandwich type biological immunosensor for detecting different concentrations of human norovirus.
6. The preparation method of the norovirus sandwich type biological immunosensor based on the sugar signal amplification mode according to claim 2, wherein the preparation method of the MBP-pep@MB-CD@Cu-NPC@3-APBA in the step 4) comprises the following steps:
step 1) synthesis of MBP-pep@MB-CD, mixing 5 mug/mL, 1mL MBP-Pep and 1mg/mL,1mL MB-CD, standing overnight at 4 ℃, then adding 1mg/mL,0.2mL ferrocene, and stirring for 2h at 4 ℃; the method is used for sealing the cyclodextrin cavity, reducing nonspecific adsorption and obtaining MBP-pep@MB-CD for standby;
step 2) synthesizing MBP-pep@MB-CD@Cu-NPC@3-APBA, directly mixing the synthesized MBP-pep@MB-CD with 1mg/mL and 1mL of Cu-NPC@3-APBA, and stirring at 4 ℃ overnight; then, the mixture is centrifugally washed for three times by PBS (phosphate buffer solution), and the speed is 4000-6000rpm for 2min each time; the resulting precipitate was added to pH7.4,1M,1mL of PBS to volume to give MBP-pep@MB-CD@Cu-NPC@3-APBA dispersion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211237306.XA CN115656507B (en) | 2022-10-09 | 2022-10-09 | Sugar signal amplification mode-based norovirus detection method, material and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211237306.XA CN115656507B (en) | 2022-10-09 | 2022-10-09 | Sugar signal amplification mode-based norovirus detection method, material and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115656507A CN115656507A (en) | 2023-01-31 |
| CN115656507B true CN115656507B (en) | 2023-09-26 |
Family
ID=84988029
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211237306.XA Active CN115656507B (en) | 2022-10-09 | 2022-10-09 | Sugar signal amplification mode-based norovirus detection method, material and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115656507B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107709441A (en) * | 2015-04-20 | 2018-02-16 | 康奈尔大学 | Porous ring dextrin polymeric material and its preparation and application |
| CN109187469A (en) * | 2018-09-10 | 2019-01-11 | 广西师范大学 | A method of with enzymatic oxidation TMB fluorescence spectrometry glucose |
| CN110646392A (en) * | 2019-09-30 | 2020-01-03 | 重庆大学 | Double-emission-ratio fluorescent probe based on carbon dots, preparation method and application in dopamine detection |
| CN110760078A (en) * | 2018-07-26 | 2020-02-07 | 南京理工大学 | Sugar cluster material with cross-linked cyclodextrin polymer as scaffold and preparation method and application thereof |
| CN113155924A (en) * | 2021-03-19 | 2021-07-23 | 云南大学 | Detection method of norovirus |
| CN114289066A (en) * | 2021-12-29 | 2022-04-08 | 云南大学 | Nano mimic enzyme material, preparation method and application thereof, and method for detecting ovomucoid |
| CN115025815A (en) * | 2022-05-31 | 2022-09-09 | 江苏大学 | Bionic nano enzyme Hemin @ BSA @ ZIF-8 and application thereof |
-
2022
- 2022-10-09 CN CN202211237306.XA patent/CN115656507B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107709441A (en) * | 2015-04-20 | 2018-02-16 | 康奈尔大学 | Porous ring dextrin polymeric material and its preparation and application |
| CN110760078A (en) * | 2018-07-26 | 2020-02-07 | 南京理工大学 | Sugar cluster material with cross-linked cyclodextrin polymer as scaffold and preparation method and application thereof |
| CN109187469A (en) * | 2018-09-10 | 2019-01-11 | 广西师范大学 | A method of with enzymatic oxidation TMB fluorescence spectrometry glucose |
| CN110646392A (en) * | 2019-09-30 | 2020-01-03 | 重庆大学 | Double-emission-ratio fluorescent probe based on carbon dots, preparation method and application in dopamine detection |
| CN113155924A (en) * | 2021-03-19 | 2021-07-23 | 云南大学 | Detection method of norovirus |
| CN114289066A (en) * | 2021-12-29 | 2022-04-08 | 云南大学 | Nano mimic enzyme material, preparation method and application thereof, and method for detecting ovomucoid |
| CN115025815A (en) * | 2022-05-31 | 2022-09-09 | 江苏大学 | Bionic nano enzyme Hemin @ BSA @ ZIF-8 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115656507A (en) | 2023-01-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108318477B (en) | Based on TiO2Electrochemiluminescence probe prepared by metal organic framework and competitive immunosensing method of electrochemiluminescence probe for vomitoxin | |
| CN107413313A (en) | A kind of Magnetic solid phases extractant based on covalent organic framework material and its preparation method and application | |
| CN113333024B (en) | Magnetic nano enzyme material with peroxidase catalytic activity, kit for detecting norovirus and application thereof | |
| Liu et al. | Ultrasensitive immunoassay for detection of zearalenone in agro-products using enzyme and antibody co-embedded zeolitic imidazolate framework as labels | |
| CN113155924A (en) | Detection method of norovirus | |
| Ji et al. | Progress in rapid detection techniques using paper-based platforms for food safety | |
| CN112834465B (en) | SPR biological sensing chip, chip modification method, SARS-CoV-2 detection kit and detection method | |
| Viswanath et al. | Recent trends in the development of complementary metal oxide semiconductor image sensors to detect foodborne bacterial pathogens | |
| Liu et al. | Phenylboronic acid polymer brush-enabled oriented and high density antibody immobilization for sensitive microarray immunoassay | |
| CN111346676B (en) | Iron-substituted tungstophosphoric acid polydopamine nano mimic enzyme as well as preparation method and application thereof | |
| CN114113582B (en) | Metal organic framework nanoenzyme biological probe and ELISA kit | |
| CN115656507B (en) | Sugar signal amplification mode-based norovirus detection method, material and application | |
| Roberts et al. | Biological/synthetic receptors (antibody, enzyme, and aptamer) used for biosensors development for virus detection | |
| CN113252631B (en) | Fluorescent colorimetric nucleic acid aptamer sensor for dual detection of profenofos pesticide, and preparation method and application thereof | |
| CN112763561B (en) | Electrochemical sensor for detecting GII 4 norovirus | |
| CN115524489B (en) | Photoelectric dual-signal-based norovirus detection method, material and application | |
| CN110031527B (en) | Double-reading biosensor for human thyroglobulin | |
| Jiang et al. | Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections | |
| CN114152757B (en) | Biological material for detecting beta bungarotoxin and method for detecting beta bungarotoxin in non-diagnosis purpose | |
| CN106124579B (en) | A kind of preparation method of the universal signal amplification system of electrochemical nano-immunosensor | |
| CN112763472B (en) | Detection system for detecting T-2 toxin residue and preparation method and application thereof | |
| Wang et al. | Preparation of a visual protein chip for detection of IgG against Treponema pallidum | |
| CN114381553B (en) | Biological material for African swine fever virus detection, kit and method for detecting African swine fever virus for non-diagnostic purpose | |
| Gong et al. | Portable paper-based molecularly imprinted sensor for visual real-time detection of influenza virus H5N1 | |
| CN115980346A (en) | Method for detecting novel coronavirus by constructing colorimetric immunosensor based on metal organic framework material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |