A Novel Method Based On Rolling Circle Amplification And G4-hemin For Detecting Prostate Specific Antigen

Objective:To develop a novel colorimetric assay for the detection of prostate cancer specific antigen（PSA）based on the integration strategy of rolling loop amplification（RCA）and G4-Hemin DNAzyme catalytic amplification.Methods:Using the shear activity of PSA to specific substrate peptide,the special single strand DNA covalently link to PSA substrate peptide was designed to connect with nanomagnetic beads,which could separate the RCA promoter chain after PSA-targeted shearing of substrate peptides.The promoters next trigger the RCA reaction to obtain a large number of short G4 DNA single strands that bind with Hemin to form DNA mimicking enzyme,which catalyzed TMB chromogenic reaction,and a new PSA colorimetric detection assay was finally constructed.Following,some characterization of key experimental steps such as the electrophoretic,circular dichroism and spectrophotometry were carried out,and the feasibility of entire scheme was analyzed.Then,the key enzyme concentration,reaction buffer and temperature of RCA,and colorimetric detection conditions were optimized.We next evaluated the specificity,repeatability,the detection linear range and LOD,and recovery rate of complex matrix with our developed PSA colorimetric detection assay.Finally,9 clinical blood samples were tested with this assay and clinical standard method.Results:In this study,a new colorimetric detection strategy integrating PSA-targeted shear,nano-magnetic beads enrich,RCA technology and G4-Hemin DNAzyme catalytic amplification was successfully designed and constructed,and a novel PSA detection method with high specificity,high sensitivity,simple and fast based on the mentioned strategy was successfully developed,completing the characterization of key steps and the colorimetric feasibility analysis of the whole scheme were verified.The optimal RCA reaction conditions of the new method were determined with 0.4 UμL^-1Phi29 polymerase,0.3 UμL^-1Nb.Bbv CI nickase in1.5×r Custmart buffer in 37℃,and the optimal concentration of K⁺the G4-Hemin incubated buffer were 100 mmol/L,which optimal p H was 7.5,while the optimum TMB catalytic reaction temperature of G4-Hemin DNAzyme was also 37℃.The results of methodological evaluation showed that this new developed method has high specificity and stability,the good detection linear range from 1 ng/ml to 100 ng/ml and the LOD was 0.732 ng/m L,and the recoveries of different PSA concentrations in the complex matrix of 20%blood were 94.2%implying that the new method has strong anti-interference ability in blood.Finally,this new PSA colorimetric assay realized the detection of actual serum samples from 6 patients with abnormal PSA and3 patients with healthy physical examination,and the results were basically consistent with the current clinical detection results of f PSA after data fitting..Conclusion:Based on RCA and G4-Hemin DNAzyme catalytic amplification strategy,we successfully constructed a novel PSA colorimetric assay with high specificity,high sensitivity,simple and fast,and achieved the detection of prostate cancer patients’blood samples.It provides a new technical tool and research strategy for clinical detection of PSA,which has important potential application value in early detection,periodic screening and curative effect monitoring of prostate cancer.