Colorimetric aptasensor for the detection of H1N1 inactivated virus-based on highly efficient chemical crosslinking
Taking type A H1N1 inactivated influenza virus as an example,a rapid detection method with high sensitivity,specificity,biosafety and easy industrialization was established,referred as colorimetric assay using magnetic crosslinking precipitation coupled with gold nanoparticles(MCP-GNP).Firstly,the efficient capture of the inactivated virus was achieved by performing the chemical coupling between the activated carboxyl group on the magnetic bead and the primary amine group of the protein on the surface of the inactivated virus.Secondly,the aptamer was incubated with magnetic beads containing the captured viruses,followed by the magnetic separation and heat elution of the bead-bound aptamer.The aptamer specifically recognized the H1N1 inactivated virus.Thirdly,the polymerase chain reaction(PCR)was performed to amplify the eluted aptamer.Finally,the colorimetric detection of the H1N1 inactivated virus was realized,which was based on the different binding affinity of single-strand primers and double-strand amplified products on GNP.When H5N1 inactivated virus was used as the blank control,the detection of limit(S/N=3)was 1 ng/L,which was 1/900 of the detection limit of the specific electrochemical impedance sensor previously reported by our research group.The colorimetric detection of clinical H1N1 inactivated virus positive throat swab samples with low viral load was also achieved for the first time.Under non-optimized conditions,the full process from virus capture to detection was completed within 3 h,demonstrating the clinical application value of MCP-GNP.In addition,all the results of colorimetric detection in this study were consistent with those of fluorescence quantitative PCR,which confirmed the reliability of our method.The core innovation of this work is the use of chemical coupling reaction to conveniently achieve the capture of inactivated virus at ultra-low concentration without the need of viral nucleic acid extraction and enrichment.In addition,the DNA aptamer targeting the H1N1 inactivated virus,instead of the active virus,was used for the specific recognition of the virus,enabling the high biosafety.
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