CN112326749A - Preparation method of novel coronavirus electrochemical immunoassay kit, product and use method of novel coronavirus electrochemical immunoassay kit - Google Patents
Preparation method of novel coronavirus electrochemical immunoassay kit, product and use method of novel coronavirus electrochemical immunoassay kit Download PDFInfo
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Abstract
The invention relates to a preparation method of a novel coronavirus kit for electrochemical immunodetection, a product and a use method thereof, belonging to the technical field of biological product preparation. In the invention, after the specific binding of the novel coronavirus antigen and the antibody, polyaniline (SPAN) and Poly Ethylenedioxythiophene (PEDOT) are respectively used as electric signal substances, and magnetic Fe is utilized3O4The electric signals are amplified by the microspheres and the gold nanoparticles and then detected by an electrochemical immune method, so that the aim of quickly and rapidly preparing the novel coronavirus with high sensitivity and specificity in real time is fulfilled. The two electric signal substances can be matched with different new coronavirus antigens at will to form detection liquid for detecting the corresponding new coronavirus antibodies, and in actual detection, one detection liquid can be used or the two detections can be mixed according to actual needs, so that the detection of one or two new coronavirus antibodies is realized.
Description
Technical Field
The invention belongs to the technical field of biological product preparation, and particularly relates to a preparation method of a novel coronavirus electrochemical immunoassay kit, a product and a using method of the novel coronavirus electrochemical immunoassay kit.
Background
2019 the novel coronavirus (COVID-19) can cause novel human coronavirus pneumonia, mainly manifested by fever, debilitation and dry cough, about half of patients have dyspnea after one week, and severe patients rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis which is difficult to correct and blood coagulation dysfunction. The new coronavirus transmission routes including direct transmission, aerosol transmission and contact transmission have strong infectivity and long latency, so that great challenge is brought to epidemic prevention work.
IgM and IgG antibodies are immune defense proteins generated after a human body is infected with new coronavirus, part of patients can find the IgM antibody to be positive when nucleic acid detection is negative, the IgM antibody is one of effective methods for diagnosing the new coronavirus, IgG is an antibody which appears 14 days after infection, the IgG exists continuously after being generated, the IgM antibody can be used as an index of previous infection, the IgM and IgG antibodies are monitored simultaneously, diagnosis is more accurate, and a doctor is assisted to quickly identify a suspected patient with the new coronavirus, so that the condition of illness can be judged by detecting the existence condition of the two substances.
At present, a plurality of methods for detecting the new coronavirus at home and abroad comprise an enzyme-linked immunosorbent assay, a nucleic acid sequencing method, a PCR (polymerase chain reaction) method and the like. However, most of the methods are complicated to operate or require marking, large-scale equipment is required, the operation is time-consuming, and the sensitivity is not high. Therefore, it is highly desirable to develop a simultaneous detection technique with high sensitivity, high speed and low cost.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a method for preparing a novel coronavirus electrochemical immunoassay kit; the second purpose is to provide a novel coronavirus electrochemical immunoassay kit; the third purpose is to provide a using method of the novel coronavirus electrochemical immunodetection kit.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a preparation method of a novel coronavirus kit for electrochemical immunodetection comprises a working electrode and detection liquid, wherein the detection liquid comprises detection liquid I and detection liquid II, and the method comprises the following steps:
(1) preparation of working electrode
Setting a certain area at one end of the strip-shaped filter paper as a reaction area, and loading gold nanowires on the reaction area;
(2) preparation of detection solution
1) Preparation of AuNPs/SPAN @ Fe3O4Composite material
Mixing polyvinylpyrrolidone and magnetic Fe3O4Adding the microspheres into water, and performing ultrasonic dispersion to obtain a solution I; adding aniline, m-aminobenzene sulfonic acid and HCl into water, and uniformly mixing to obtain a solution II; adding the solution II into the solution I, stirring for reaction, adding an ammonium persulfate solution, stirring for reaction at 0-4 ℃, finally taking a solid phase, washing and drying the solid phase to obtain SPAN @ Fe3O4Core-shell nano-materials;
the SPAN @ Fe3O4Dispersing the core-shell nano material in water, adding polydiallyldimethylammonium chloride aqueous solution, performing ultrasonic treatment, washing with water to remove redundant polydiallyldimethylammonium chloride, and preparing the positive-charged SPAN @ Fe3O4Core-shell nano-materials;
the positively charged SPAN @ Fe3O4Adding the core-shell nano material into the gold colloid solution, stirring for reaction, taking a solid phase, washing and drying the solid phase to obtain AuNPs/SPAN @ Fe3O4A complex;
2) system for makingAuNPs/PEDOT @ Fe preparation3O4Composite material
3, 4-ethylenedioxythiophene, toluene and magnetic Fe3O4After the microspheres are ultrasonically mixed, adding a sodium persulfate solution, stirring for reaction, taking a solid phase, washing the solid phase, and drying to obtain PEDOT @ Fe3O4Core-shell nano-materials;
adding the PEDOT @ Fe3O4Dispersing the core-shell nano material in water, adding polydiallyldimethylammonium chloride aqueous solution, performing ultrasonic treatment, washing with water to remove redundant polydiallyldimethylammonium chloride, and preparing the PEDOT @ Fe with positive electricity3O4Core-shell nano-materials;
(iii) charging said positively charged PEDOT @ Fe3O4Adding the core-shell nano material into the gold colloid solution, stirring for reaction, taking a solid phase, washing and drying the solid phase to obtain AuNPs/PEDOT @ Fe3O4A complex;
3) preparing detection solution I and detection solution II
Mixing the AuNPs/SPAN @ Fe3O4Dispersing the complex in PBS buffer, adding new coronavirus antigen S1Placing the dispersion liquid on a shaking table for reaction to prepare a detection liquid I;
mixing the AuNPs/PEDOT @ Fe3O4Dispersing the complex in PBS buffer, adding new coronavirus antigen S2Placing the dispersion liquid on a shaking table for reaction to prepare a detection liquid II;
said S1And S2Are different new coronavirus antigens.
Preferably, the density of the gold nanowires on the reaction area is 400-2。
Preferably, in the step (1), the certain region ranges from 0.4 to 0.5 × 0.4 to 0.5cm, width × length.
Preferably, the method for loading gold nanowires on the reaction zone specifically comprises the following steps: the gold nanowire solution is slowly pumped and filtered to the reaction area through a pump.
Preferably, in step 1), 0.3g of polyvinylpyrrolidone and 0.06g of magnetic Fe are mixed3O4Adding the microspheres into 66mL of water, and performing ultrasonic treatmentObtaining a solution I after dispersion; adding 28.2 mu L of aniline, 0.05g of m-aminobenzene sulfonic acid and 9mL of 0.2mol/L HCl into 60mL of water, and uniformly mixing to obtain a solution II; adding the solution II into the solution I, stirring for reaction for 30min, adding 15mL of 0.06mol/L ammonium persulfate solution, stirring for reaction for 8h at 0-4 ℃, finally taking a solid phase, washing and drying the solid phase to obtain SPAN @ Fe3O4A core-shell nano material.
Preferably, the time for ultrasonic dispersion is 20-30 min.
Preferably, the solid phase is obtained by magnetic separation.
Preferably, the washing is specifically: washing with water for 3-5 times, and washing with anhydrous ethanol for 3-5 times.
Preferably, the drying specifically comprises: vacuum drying at 50-65 deg.C for 6-7 hr.
Preferably, in step 1), 10mg of SPAN @ Fe is added3O4Dispersing the core-shell nano material in 4mL of water, adding 1mL of polydiallyldimethylammonium chloride aqueous solution with the weight percent of 3, ultrasonically treating, washing with water to remove redundant polydiallyldimethylammonium chloride, and preparing the positive-charged SPAN @ Fe3O4A core-shell nano material.
Preferably, the time of the ultrasonic treatment is 2-3 min.
Preferably, in step 1), 10mg of the positively charged SPAN @ Fe3O4Adding the core-shell nano material into 30mL of 0.5mM gold colloid solution, stirring for reaction for 8h, taking a solid phase, washing and drying the solid phase to obtain AuNPs/SPAN @ Fe3O4And (c) a complex.
Preferably, the solid phase is obtained by magnetic separation.
Preferably, the washing is specifically: washing with water for 3-5 times, and washing with anhydrous ethanol for 3-5 times.
Preferably, the drying specifically comprises: vacuum drying at 50-65 deg.C for 6-7 hr.
Preferably, in step 2), 15. mu.L of 3, 4-ethylenedioxythiophene, 15. mu.L of toluene and 20mg of magnetic Fe3O4After the microspheres were ultrasonically mixed, 300. mu.L of persulfuric acid containing 16mg of sodium persulfate was addedStirring the sodium solution for reaction for 12-15h, taking a solid phase, washing the solid phase, and drying to obtain PEDOT @ Fe3O4A core-shell nano material.
Preferably, the time of ultrasonic dispersion is 10-15 min.
Preferably, the solid phase is obtained by magnetic separation.
Preferably, the washing is specifically: washing with water for 3-5 times, and washing with anhydrous ethanol for 3-5 times.
Preferably, the drying specifically comprises: vacuum drying at 50-65 deg.C for 6-7 hr.
Preferably, in step 2), 10mg of said PEDOT @ Fe3O4Dispersing the core-shell nano material in 4mL of water, adding 1mL of 3 wt% polydiallyldimethylammonium chloride aqueous solution, performing ultrasonic treatment, washing with water to remove redundant polydiallyldimethylammonium chloride, and preparing the positive-charged PEDOT @ Fe3O4A core-shell nano material.
Preferably, the time of the ultrasonic treatment is 2-3 min.
Preferably, in step 2), 10mg of said positively charged PEDOT @ Fe3O4Adding the core-shell nano material into 30mL of 0.5mM gold colloid solution, stirring for reaction for 8h, taking a solid phase, washing and drying the solid phase to obtain AuNPs/PEDOT @ Fe3O4And (c) a complex.
Preferably, the solid phase is obtained by magnetic separation.
Preferably, the washing is specifically: washing with water for 3-5 times, and washing with anhydrous ethanol for 3-5 times.
Preferably, the drying specifically comprises: the drying specifically comprises the following steps: vacuum drying at 50-65 deg.C for 6-7 hr.
Preferably, in step 3), the AuNPs/SPAN @ Fe3O4Complexes and novel coronavirus antigens S1The mass ratio of (A) to (B) is 15-20: 1-2; the AuNPs/PEDOT @ Fe3O4Complexes and novel coronavirus antigens S2The mass ratio of (A) to (B) is 15-20: 1-2.
Preferably, said S1Is a new coronavirus antigen specifically combined with new coronavirus antibody IgM or IgG.
Preferably, said S2Is a new coronavirus antigen specifically combined with new coronavirus antibody IgM or IgG.
Preferably, when the detection solution I and the detection solution II are prepared, the rotation speed of the shaking table is 150-200r/min, and the reaction time is 12-15 h.
2. The novel coronavirus electrochemical immunoassay kit prepared by the method.
3. The use method of the electrochemical immunoassay novel coronavirus kit comprises the following steps:
and dropwise adding a sample to be detected into a reaction area of the working electrode, incubating and washing, then blocking the active sites with BSA, dropwise adding at least one of the detection solution I or the detection solution II according to the detection requirement, and performing electrochemical immunodetection after re-incubation.
Preferably, the incubation is specifically at 37-40 ℃ for 12-15 h.
Preferably, the re-incubation is specifically at 37-40 ℃ for 2-3 h.
The invention has the beneficial effects that: the invention provides a preparation method of a novel coronavirus kit for electrochemical immunodetection, a product and a using method thereof3O4The electric signals are amplified by the microspheres and the gold nanoparticles and then detected by an electrochemical immune method, so that the aim of quickly and rapidly preparing the novel coronavirus with high sensitivity and specificity in real time is fulfilled. The two electric signal substances can be matched with different new coronavirus antigens at will to form detection liquid for detecting the corresponding new coronavirus antibodies, and in actual detection, one detection liquid can be used or the two detections can be mixed according to actual needs, so that the detection of one or two new coronavirus antibodies is realized. In addition, in the process of preparing the kit, the mass ratio of each compound in the solution to the corresponding new coronavirus antigen is detected by optimizing the density of the gold nanowires on the working electrode, so that the high effect on the new coronavirus is better realizedSensitivity, high specificity and real-time rapid detection.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
Drawings
For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:
FIG. 1 shows the magnetic Fe in example 1 in FIG. 13O4Microspheres, SPAN @ Fe3O4Core-shell nano material and AuNPs/SPAN @ Fe3O4TEM and SEM images of the composite; (in FIG. 1, A is magnetic Fe3O4TEM image of microsphere, B in FIG. 1 is SPAN @ Fe3O4TEM image of core-shell nano material, C in FIG. 1 is AuNPs/SPAN @ Fe3O4TEM image of the composite, D in FIG. 1 being magnetic Fe3O4SEM image of microsphere, E in FIG. 1 is SPAN @ Fe3O4SEM picture of core-shell nano material, in figure 1, F is AuNPs/SPAN @ Fe3O4SEM image of Compound
FIG. 2 is a schematic diagram of the detection of the novel coronavirus using the kit prepared in example 1.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example 1
Preparing a novel coronavirus electrochemical immunoassay kit, wherein the kit comprises a working electrode and detection liquid, and the detection liquid comprises detection liquid I and detection liquid II;
(1) preparation of working electrode
Setting a 0.5 multiplied by 0.5cm (width multiplied by length) area at one end of the strip filter paper as a reaction area, slowly pumping and filtering a gold nanowire solution with the concentration of 1mg/mL onto the reaction area through a pump until the density of the gold nanowire loaded on the reaction area is 400 mu g/cm2Then, the method is carried out;
(2) preparation of detection solution
1) Preparation of AuNPs/SPAN @ Fe3O4Composite material
0.3g of polyvinylpyrrolidone and 0.06g of magnetic Fe3O4Adding the microspheres into 66mL of water, and performing ultrasonic dispersion for 30min to obtain a solution I; adding 28.2 mu L of aniline, 0.05g of m-aminobenzene sulfonic acid and 9mL of 0.2mol/L HCl into 60mL of water, and uniformly mixing to obtain a solution II; adding the solution II into the solution I, stirring for reaction for 30min, adding 15mL of 0.06mol/L ammonium persulfate solution, stirring for reaction for 8h at 4 ℃, magnetically separating to obtain a solid phase, washing the solid phase with water for 3 times, washing with anhydrous ethanol for 3 times, and vacuum-drying at 60 ℃ for 6h to obtain SPAN @ Fe3O4Core-shell nano-materials;
10mg of SPAN @ Fe as described above3O4Dispersing the core-shell nano material in 4mL of water, adding 1mL of polydiallyldimethylammonium chloride aqueous solution with the weight percent of 3 percent, carrying out ultrasonic treatment for 3min, washing with water to remove redundant polydiallyldimethylammonium chloride, and preparing the SPAN @ Fe with positive electricity3O4Core-shell nano-materials;
10mg of the above-mentioned positively charged SPAN @ Fe3O4Adding the core-shell nano material into 30mL of 0.5mM gold colloid solution, stirring for reaction for 8h, performing magnetic separation to obtain a solid phase, washing the solid phase with water for 3 times, washing with absolute ethyl alcohol for 3 times, and performing vacuum drying at 50 ℃ for 6h to obtain AuNPs/SPAN @ Fe3O4A complex;
2) preparation of AuNPs/PEDOT @ Fe3O4Composite material
Mixing 15 μ L of 3, 4-ethylenedioxythiophene, 15 μ L of toluene and 20mg of magnetic Fe3O4Mixing the microspheres for 15min, addingStirring 16mg sodium persulfate solution 300 mu L sodium persulfate, reacting for 12h, magnetically separating to obtain solid phase, washing the solid phase with water for 3 times, washing with anhydrous ethanol for 3 times, and vacuum drying at 60 deg.C for 6h to obtain PEDOT @ Fe3O4Core-shell nano-materials;
10mg of the above-mentioned PEDOT @ Fe3O4Dispersing the core-shell nano material in 4mL of water, adding 1mL of 3 wt% polydiallyldimethylammonium chloride aqueous solution, performing ultrasonic treatment for 3min, and washing with water to remove excessive polydiallyldimethylammonium chloride to obtain positive-charged PEDOT @ Fe3O4Core-shell nano-materials;
10mg of the above-mentioned positively charged PEDOT @ Fe3O4Adding the core-shell nano material into 30mL of 0.5mM gold colloid solution, stirring for reaction for 8h, performing magnetic separation to obtain a solid phase, washing the solid phase with water for 3 times, washing with absolute ethyl alcohol for 3 times, and performing vacuum drying at 60 ℃ for 6h to obtain AuNPs/PEDOT @ Fe3O4A complex;
3) preparing detection solution I and detection solution II
Mixing the above AuNPs/SPAN @ Fe3O4Dispersing the complex in PBS buffer solution, adding new coronavirus antigen S specifically combined with new coronavirus antibody IgM1The dispersion is put on a shaking table with the rotating speed of 150r/min for reaction for 12 hours to prepare a detection liquid I, wherein AuNPs/SPAN @ Fe3O4Complexes and novel coronavirus antigens S1In a mass ratio of 20: 1;
mixing the AuNPs/PEDOT @ Fe3O4Dispersing the complex in PBS buffer solution, adding new coronavirus antigen S specifically combined with new coronavirus antibody IgG2The dispersion is placed on a shaking table with the rotating speed of 150r/min for reaction for 12 hours to prepare a detection liquid II, wherein AuNPs/PEDOT @ Fe3O4Complexes and novel coronavirus antigens S2In a mass ratio of 20: 1.
FIG. 1 shows magnetic Fe in example 13O4Microspheres, SPAN @ Fe3O4Core-shell nano material and AuNPs/SPAN @ Fe3O4TEM and SEM images of the composite, wherein A in FIG. 1 is magnetic Fe3O4TEM image of microspheresIn FIG. 1, B is SPAN @ Fe3O4TEM image of core-shell nano material, C in FIG. 1 is AuNPs/SPAN @ Fe3O4TEM image of the composite, D in FIG. 1 being magnetic Fe3O4SEM image of microsphere, E in FIG. 1 is SPAN @ Fe3O4SEM picture of core-shell nano material, in figure 1, F is AuNPs/SPAN @ Fe3O4SEM image of the composite.
The kit prepared in example 1 is used for detecting the novel coronavirus, the detection principle is shown in figure 2, and the specific method is as follows:
dropping a sample to be detected containing new coronavirus antibodies IgM and IgG on a reaction area of a working electrode, connecting the new coronavirus antibodies IgM and IgG with gold nanowires loaded on the reaction area through Au-S bonds, incubating for 15h at 37 ℃, washing, dropping 0.01mg/mL BSA, incubating for 2h at 37 ℃ to seal active sites, dropping a mixed solution of a detection solution I and a detection solution II (the detection solution I and the detection solution II are mixed according to a volume ratio of 1: 1), and then dropping a new coronavirus antigen S in the mixed solution1、S2The activated carbon is respectively combined with IgM and IgG of new coronavirus antibodies on the reaction area in a special way, the processed working electrode is placed into an electrochemical immunodetection system for detection, and SPAN and PEDOT electrochemical response characteristic peaks appear under the potentials of 0.25V and 0.1V respectively.
In the invention, two electric signal substances can be matched with different new coronavirus antigens at will to form detection liquid for detecting corresponding new coronavirus antibodies, and in actual detection, one detection liquid can be used or the two detections can be mixed according to actual needs to realize the detection of one or two new coronavirus antibodies.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Claims (10)
1. The preparation method of the novel coronavirus electrochemical immunodetection kit is characterized by comprising a working electrode and detection liquid, wherein the detection liquid comprises detection liquid I and detection liquid II, and the method comprises the following steps:
(1) preparation of working electrode
Setting a certain area at one end of the strip-shaped filter paper as a reaction area, and loading gold nanowires on the reaction area;
(2) preparation of detection solution
1) Preparation of AuNPs/SPAN @ Fe3O4Composite material
Mixing polyvinylpyrrolidone and magnetic Fe3O4Adding the microspheres into water, and performing ultrasonic dispersion to obtain a solution I; adding aniline, m-aminobenzene sulfonic acid and HCl into water, and uniformly mixing to obtain a solution II; adding the solution II into the solution I, stirring for reaction, adding an ammonium persulfate solution, stirring for reaction at 0-4 ℃, finally taking a solid phase, washing and drying the solid phase to obtain SPAN @ Fe3O4Core-shell nano-materials;
the SPAN @ Fe3O4Dispersing the core-shell nano material in water, adding polydiallyldimethylammonium chloride aqueous solution, performing ultrasonic treatment, washing with water to remove redundant polydiallyldimethylammonium chloride, and preparing the positive-charged SPAN @ Fe3O4Core-shell nano-materials;
the positively charged SPAN @ Fe3O4Adding the core-shell nano material into the gold colloid solution, stirring for reaction, taking a solid phase, washing and drying the solid phase to obtain AuNPs/SPAN @ Fe3O4A complex;
2) preparation of AuNPs/PEDOT @ Fe3O4Composite material
3, 4-ethylenedioxythiophene, toluene and magnetic Fe3O4After the microspheres are ultrasonically mixed, adding a sodium persulfate solution, stirring for reaction, taking a solid phase, washing the solid phase, and drying to obtain PEDOT @ Fe3O4Core-shell nano-materials;
adding the PEDOT @ Fe3O4Dispersing the core-shell nano material in water, and adding polydiallyl dimethyl chlorideDissolving ammonium aqueous solution, performing ultrasonic treatment, washing with water to remove excessive polydiallyldimethylammonium chloride, and preparing the PEDOT @ Fe with positive electricity3O4Core-shell nano-materials;
(iii) charging said positively charged PEDOT @ Fe3O4Adding the core-shell nano material into the gold colloid solution, stirring for reaction, taking a solid phase, washing and drying the solid phase to obtain AuNPs/PEDOT @ Fe3O4A complex;
3) preparing detection solution I and detection solution II
Mixing the AuNPs/SPAN @ Fe3O4Dispersing the complex in PBS buffer, adding new coronavirus antigen S1Placing the dispersion liquid on a shaking table for reaction to prepare a detection liquid I;
mixing the AuNPs/PEDOT @ Fe3O4Dispersing the complex in PBS buffer, adding new coronavirus antigen S2Placing the dispersion liquid on a shaking table for reaction to prepare a detection liquid II;
said S1And S2Are different new coronavirus antigens.
2. The method of claim 1, wherein in step 1), 0.3g of polyvinylpyrrolidone and 0.06g of magnetic Fe are added3O4Adding the microspheres into 66mL of water, and performing ultrasonic dispersion to obtain a solution I; adding 28.2 mu L of aniline, 0.05g of m-aminobenzene sulfonic acid and 9mL of 0.2mol/L HCl into 60mL of water, and uniformly mixing to obtain a solution II; adding the solution II into the solution I, stirring for reaction for 30min, adding 15mL of 0.06mol/L ammonium persulfate solution, stirring for reaction for 8h at 0-4 ℃, finally taking a solid phase, washing and drying the solid phase to obtain SPAN @ Fe3O4A core-shell nano material.
3. The method of claim 1, wherein in step 1), 10mg of the SPAN @ Fe is added3O4Dispersing the core-shell nano material in 4mL of water, adding 1mL of polydiallyldimethylammonium chloride aqueous solution with the weight percent of 3, ultrasonically treating, washing with water to remove redundant polydiallyldimethylammonium chloride, and preparing the positive-charged SPAN @ Fe3O4A core-shell nano material.
4. The method of claim 1, wherein in step 1), 10mg of the positively charged SPAN @ Fe is added3O4Adding the core-shell nano material into 30mL of 0.5mM gold colloid solution, stirring for reaction for 8h, taking a solid phase, washing and drying the solid phase to obtain AuNPs/SPAN @ Fe3O4And (c) a complex.
5. The method of claim 1, wherein in step 2), 15 μ L of 3, 4-ethylenedioxythiophene, 15 μ L of toluene, and 20mg of magnetic Fe are mixed3O4After the microspheres are ultrasonically mixed, 300 mu L of sodium persulfate solution containing 16mg of sodium persulfate is added, the mixture is stirred to react for 12 to 15 hours, then a solid phase is taken, and the solid phase is washed and dried to obtain PEDOT @ Fe3O4A core-shell nano material.
6. The method of claim 1, wherein 10mg of said PEDOT @ Fe is added in step 2)3O4Dispersing the core-shell nano material in 4mL of water, adding 1mL of 3 wt% polydiallyldimethylammonium chloride aqueous solution, performing ultrasonic treatment, washing with water to remove redundant polydiallyldimethylammonium chloride, and preparing the positive-charged PEDOT @ Fe3O4A core-shell nano material.
7. The method of claim 1, wherein 10mg of said positively charged PEDOT @ Fe is added in step 2)3O4Adding the core-shell nano material into 30mL of 0.5mM gold colloid solution, stirring for reaction for 8h, taking a solid phase, washing and drying the solid phase to obtain AuNPs/PEDOT @ Fe3O4And (c) a complex.
8. The method of claim 1, wherein in step 3), the AuNPs/SPAN @ Fe3O4Complexes and novel coronavirus antigens S1The mass ratio of (A) to (B) is 15-20: 1-2; the AuNPs/PEDOT @ Fe3O4Complexes and novel coronavirus antigens S2The mass ratio of (A) to (B) is 15-20: 1-2.
9. A novel kit for electrochemical immunoassay of coronavirus prepared by the method of any one of claims 1-8.
10. The method for using the novel coronavirus electrochemical immunoassay kit of claim 9, wherein the method comprises the following steps:
and dropwise adding a sample to be detected into a reaction area of the working electrode, incubating and washing, then blocking the active sites with BSA, dropwise adding at least one of the detection solution I or the detection solution II according to the detection requirement, and performing electrochemical immunodetection after re-incubation.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103342980A (en) * | 2013-06-26 | 2013-10-09 | 哈尔滨工业大学 | Preparation method for MWCNT/Fe3O4/PANI/Au multilayer wrapped nanotube |
| CN103920537A (en) * | 2014-04-28 | 2014-07-16 | 扬州大学 | Method for preparing magnetic polyaniline noble metal nanometer catalyst |
| CN104971778A (en) * | 2015-06-30 | 2015-10-14 | 天津大学 | Preparation method and applications of ferriferrous oxide-polyaniline-gold nano composite material |
| WO2018030785A1 (en) * | 2016-08-09 | 2018-02-15 | 한양대학교 에리카산학협력단 | Bimetal-conductive polymer janus composite nanostructure having electrical stimulus response, colloid self-assembled structure thereof, preparing method, and bio-sensing, bio-imaging, drug delivery, and industrial application |
| CN108295906A (en) * | 2018-03-06 | 2018-07-20 | 燕山大学 | " ringing a bell " type platinum base magnetism space confinement catalyst and preparation method thereof |
| CN111537720A (en) * | 2020-05-25 | 2020-08-14 | 浙江峻山生物科技有限公司 | Preparation method of novel coronavirus 2019-nCoV IgM/IgG antibody combined detection kit |
-
2020
- 2020-10-26 CN CN202011156552.3A patent/CN112326749B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103342980A (en) * | 2013-06-26 | 2013-10-09 | 哈尔滨工业大学 | Preparation method for MWCNT/Fe3O4/PANI/Au multilayer wrapped nanotube |
| CN103920537A (en) * | 2014-04-28 | 2014-07-16 | 扬州大学 | Method for preparing magnetic polyaniline noble metal nanometer catalyst |
| CN104971778A (en) * | 2015-06-30 | 2015-10-14 | 天津大学 | Preparation method and applications of ferriferrous oxide-polyaniline-gold nano composite material |
| WO2018030785A1 (en) * | 2016-08-09 | 2018-02-15 | 한양대학교 에리카산학협력단 | Bimetal-conductive polymer janus composite nanostructure having electrical stimulus response, colloid self-assembled structure thereof, preparing method, and bio-sensing, bio-imaging, drug delivery, and industrial application |
| CN108295906A (en) * | 2018-03-06 | 2018-07-20 | 燕山大学 | " ringing a bell " type platinum base magnetism space confinement catalyst and preparation method thereof |
| CN111537720A (en) * | 2020-05-25 | 2020-08-14 | 浙江峻山生物科技有限公司 | Preparation method of novel coronavirus 2019-nCoV IgM/IgG antibody combined detection kit |
Non-Patent Citations (3)
| Title |
|---|
| XU GUIYUN 等: "Electrochemical detection of nitrite in food based on poly (3, 4-ethylenedioxythiophene) doped with Fe3O4 Nanoparticles loaded carboxylated nanocrystalline cellulose", 《ACTA CHIMICA SLOVENICA》 * |
| ZHU YUMEI 等: "Ternary Fe3O4@ PANI@ Au nanocomposites as a magnetic catalyst for degradation of organic dyes", 《SCIENCE CHINA TECHNOLOGICAL SCIENCES》 * |
| 李建平等: "磁性纳米金共价固定癌胚抗原单克隆抗体的电流型免疫传感器", 《高等学校化学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114441612A (en) * | 2021-12-30 | 2022-05-06 | 广州市赛特检测有限公司 | Electrochemical biosensor, detection method and application |
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