A colorimetric biosensor based on aptamer-gold nanoparticles for rapid detection of Lp-PLA2
Objective The DNA aptamers of lipoprotein-associated phospholipase A2(Lp-PLA2),a marker of vasculitis,were screened and a visual detection method using unlabeled nucleic acid aptamer-gold nanoparticle(AuNP)probe was established.Method Lp-PLA2 aptamers were screened through 8 cycles of incubation binding,ssDNA isolation,PCR amplification and single strand recovery by the magnetic bead fixation SELEX technique.The affinity and specificity of the aptamers were validated using surface plasmon resonance technology and flow cytometry,and the secondary structure of the aptamer and its three-dimensional molecular docking with the target protein were simulated by computer software.Subsequently,aptamer-AuNP complex was prepared,and the color change was caused by salt-induced condensation of the AuNP solution by target competitive binding.Then,the target concentration was detected by measuring the absorbance of the solution with a spectrophotometer.The linear relationship between the sample absorbance and concentration of Lp-PLA2 were established under the optimal determine conditions.Results Three Lp-PLA2 aptamers B76-2,B76-4 and B76-5 with high affinity and strong specificity were obtained,and the dissociation constants were 1.07,1.26 and 1.75 nmol/L,respectively.Then AuNP colorimetric sensing method based on B76-2 aptamer was successfully constructed.The linear range and detection limit of Lp-PLA2 were 20-500 ng/ml and 78 ng/ml,respectively,and the reaction time was 30 min,which could specifically distinguish the target from other thrombotic markers such as thrombin and myeloperoxidase.Conclusion A simple,rapid and specific visual detection method for visually detecting Lp-PLA2 was established by using aptamer-AuNP colorimetric assay.
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维普
