| Objective: In order to achieve accurate quantification of SARS-Co V-2 N protein,The immune binding of antigens and antibodies induced DNA proximity hybridization to convert into nucleic acid detection.A homogeneous,clean-free and easily operable,ultra-sensitive chemiluminescence(CL)imaging strategy was designed.Methods: The amplified homogeneous CL imaging system was performed with a pair of DNA-antibody conjugates to recognize SARS-Co V-2 protein,which formed a proximity-ligated complex,Ab-1 / SARS-Co V-2 / Ab-2,to induce a strand displacement reaction for releasing primer from a designed block / primer complex.The released primer then triggered RCA with a C-rich circular DNA as template to produce duplicate sequence of G4,which formed abundant DNAzyme units in the presence of hemin to produce strong CL signal for the detection of the target protein by catalyzing the oxidation of luminol by hydrogen peroxide.Results:(1)The effect of SARS-Co V-2 N protein on the CL intensity was verified by CL imaging comparison.The presence of the target protein induced the nucleic acid proximity hybridization to release primer nucleic acid,which stimulated RCA to product G4 / hemin DNAzyme unit resulting in the doubling increase CL signal,which verified the feasibility of our scheme.(2)To simplify the experimental operation,we synthesized circular DNA,which was successfully verified by thermal differential spectrum(TDS)and CL imaging.In addition,a pair of murine monoclonal antibodies of SARS-Co V-2 were loading on the thiol-nucleic acid chain,Staining by nucleic acid and protein of PAGE gel,since the molecular of DNA-antibody increased,electrophoresis band kept in the previous position.(3)We optimized the experimental conditions,determining 5 μM as optimal concentration of hemin and 2 h as the best reaction time of RCA to ensure the maximum for experimental SNR.(4)Under the optimal experimental conditions,we analyzed the performance of the proposed strategy.Primer concentrations were 10 fm-500 p M,and the linear relationship between concentrations and CL intensity was obtained.In order to get the most wide linear range and the lowest detection limit,the purpose antibodies concentration were selected.At corresponding antibody concentration,the linearity between the concentration of SARS-Co V-2 in 20 fg/m L-100 pg/m L and the CL intensity was obtained.the selectivity of scheme was verified by using different antigens,the experiment showed the only response to SARS-Co V-2 N protein of this proposed detection method.(5)The practicability of this proposed method was verified by standard addition.The inactivated SARS-Co V-2 vaccine of different concentrations acted as the test sample,and compared between the commercialized immunocolloidal gold test paper and the proposed method in our experiment.Conclusion: The proposed assay showed a detectable concentration range over 5orders of magnitude with the detection limit down to 6.46 fg/m L.Firstly,RCA was used to amplify nucleic acid.As a method of constant temperature amplification of nucleic acid,the detection limit could reach one copy,which improved the sensitivity of the experiment.Secondly,the detection of other proteins could be achieved by replacing the probe used for recognition.Finally,apart from the preparation of reagents,the reaction process had only two parts containing reaction solution and detection solution,and it was performed in homogeneous solution under room temperature,and the operation was simple.The excellent selectivity,simple procedure,acceptable accuracy and intrinsic high throughput of imaging technique for analysis of serum samples demonstrated the potential applicability of the proposed detection method in clinical screening and diagnosis. |
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