富含胞嘧啶单链DNA-银纳米簇荧光探针用于S1核酸酶的灵敏检测

摘要

以富含胞嘧啶(C)的单链DNA为模板合成银纳米簇, 将其作为功能化探针, 建立了一种无标记荧光检测S1核酸酶的方法.S1核酸酶可以特异性识别单链DNA, 在最适的酶催化反应条件下, 可将其降解为单核苷酸或寡核苷酸片段.当S1核酸酶不存在时, 富含C的单链DNA可以有效地合成荧光银纳米簇;当S1核酸酶存在时, 单链DNA模板被特异性识别并降解, 导致无法形成银纳米簇, 使体系荧光信号降低.实验结果表明, 银纳米簇的荧光强度随着S1核酸酶浓度的增加而降低.在优化的条件下, 体系荧光信号(F/F0)与S1核酸酶的浓度在5.0×10-5~4.0×10-3 U/μL范围内呈线性关系, 检出限为2.0×10-6 U/μL.该荧光探针选择性好, 可用于RPMI 1640细胞培养基中S1核酸酶的检测, 回收率达到91.8%~109.5%.%A label-free fluorescence method for detection of S1 nuclease was developed using oligonucleotide-templated silver nanoclusters(AgNCs).Cytosine-rich single strand DNA(C-rich ssDNA) scaffolded silver nanoclusters were devised and synthesized as a functional probe.S1 nuclease can specifically recognize ssDNA, which is degraded into mononucleotide and oligonucleotide fragment under the optimum enzyme-catalyzed reaction condition.In the absence of S1 nuclease, the silver nanoclusters could be effectively synthesized by C-rich ssDNA, which produced obvious fluorescence intensity.In the presence of S1 nuclease, C-rich ssDNA probes were specifically recognized and effectively digested into small fragments.As a result, the silver nanoclusters synthesis was suspended leading a fluorescence reduction.A good linear correlation was obtained between fluorescence change and the logarithm of the Cu2+ ions concentration over the range from 5.0×10-5 U/μL to 4.0×10-3 U/μL.The detection limit was estimated as 2.0×10-6 U/μL.The proposed method was highly selective and there were no obvious interfering effects on the determination from Bst polymerase, Thrombin, ExoⅢ, E.coli ligase and BSA.The proposed sensor reveals good recovery rates from 91.8% to 109.5% in RPMI 1640 cell medium, indicating that the sensing system was feasible for the detection of S1 nuclease in practical samples.This label-free, simple and cost-effective strategy has a potential application in biomedical research and clinical early diagnosis.