| There are many types of portable rapid antigen tests,and the most common technique is lateral flow detection(LFT).The colloidal gold and latex particles are common markers for LFTs.Colloidal gold detection is valuable for stable,fast and convenient test,and cost less.However,this technique has shortcomings of low sensitivity and false positives.Therefore,this paper explores a new method that can sense antigen-antibody interaction information at the level of visual signal sensing based on the "sandwich"-like structure of immune sandwiches.We immobilize the spike S1 protein of severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)on the surface of the chip by the capture antibody,and then incubates it with platinum nanoprobes(Pt NPs probes)to form an immune "sandwich" structure of capture antibody @ target antigen @ detection antibody.Subsequently,Pt NPs probe catalyzes the decomposition of hydrogen peroxide to produce bubbles,a new signal sensing method to show the results of antigen-antibody interaction.This paper is mainly divided into three parts:(1)Preparation and characterization of Pt NPs and Pt NPs probes;(2)Construction of capture antibody @ target antigen @ detection antibody immune "sandwich" structure;(3)Preparation of antibody chips and construction of a new sandwich Enzyme-Free Bubbling Antigen Detection(EFBAD)platform.In this experiment,small-particle size Pt NPs were synthesized by sodium borohydride reducing chloroplatinic acid.The small-particle size Pt NPs act as crystal nuclei to synthesize large-particle size Pt NPs under condition that chloroplatinic acid was reduced by L-ascorbic acid.The Pt NPs were successfully verified by means of UV/VIS spectrophotometer and transmission electron microscopy,and Pt NPs with diameters of about 4.2 nm and 22.3 nm were successfully prepared.In this experiment,two methods for preparing probes for Pt NPs were then screened.First,the surface of Pt NPs is achydrazide by 3-(pyridin-2-yldisulfide)propionyl hydrazide(PDPH)and bound to the Fc segment of the aldehyde-ylated antibody to complete the preparation of Pt NPs probes.Second,citrate-terminated Pt NPs are bound to amino groups on antibody molecules by using 1-ethyl-(3-dimethylaminopropyl)carbodiimide(EDC)and N-hydroxysuccinimide(NHS),and the preparation of Pt NPs probes can be completed in one step.The Pt NPs probes prepared by the two methods were verified by ELISA.The Pt NPs probes had the ability to identify and bind to the spike S1 protein,which met the requirements of the detection probe in this detection system.In comparison,the linear relationship shows better between Pt NPs probes by using PDPH crosslinker and detection of spike S1 protein.To form a capture antibody@target antigen@ detection antibody immune "sandwich" structure,we looked for an antibody pair by a method similar to EnzymeLinked Immuno Sorbent Assay(ELISA).Both antibodies in this pair can bind to the target antigen,and there is a difference between their corresponding epitopes,and bring about one target antigen binding to both capture and detection antibodies.Subsequently,we used the Pt NPs probes with horseradish peroxidase(HRP)properties to screen antibody pairs by Enzyme-Free Immunosorbent Assay(EFISA).Then we identify the capture antibody and detection antibody used in this paper for portable rapid detection of spike S1 protein.After EFISA screening,we obtained a pair of antibodies that met the requirements of the detection system construction.By compared with the traditional ELISA,the detection system used in this study has the advantages of shortening the overall experimental cycle,improving the sensitivity of the experiment and reducing non-specific adsorption.The core component of the antibody chip prepared in this study is the antibody fixed on the chip surface.The preparation of the antibody chip uses a silane coupling agent to aminate the glass surface,and then use glutaraldehyde aldehyde to modify the glass surface,and finally used the amino group the antibody molecule to chemically bond the aldehyde to make the antibody chip.Next,the antibody chip is tested by HRPconjugated antibody and Alexa Fluor 488-conjugated antibody to verify the biological activity of a general antibody chip and meets the requirements of this detection system for antibody chips.Combining antibody chips and Pt NPs probes,we have successfully built an EFBAD detection platform for spike S1 protein,which provides a novel visual sensing method.This method uses Pt NPs to catalyze the decomposition of hydrogen peroxide to generate bubbles,and determine whether there is a target antigen according to the bubble generation time and formation characteristics.Based on the above results,this paper screened out Pt NPs with suitable particle size,prepared Pt NPs probes and verified that the probes met the requirements.The capture antibody and detection antibody for the detection of spike S1 protein were screened out by EFISA method,and the immune "sandwich" structure of capture antibody @ target antigen @ detection antibody was established.On this basis,the EFBAD platform was successfully built to detect the spike S1 protein by the prepared antibody chip,and a data visualization analysis method was proposed.The platform uses a visual bubble sensing signal to determine the presence or absence of a target antigen in the sample.This technical method has a short period for detecting spike S1 protein,and has low requirements for the detection environment,which provides a new idea for the portable rapid detection technology of target antigen. |
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