基于聚胸腺嘧啶单链DNA模板铜纳米簇的非标记碱性磷酸酶活性检测

Lable-free detection of alkaline phosphatase activity based on poly(thymine)single strand DNA-templated copper nanoclusters

以聚胸腺嘧啶单链DNA模板合成铜纳米簇(Cu^(2+)NCs)为探针,结合碱性磷酸酶(ALP)的去磷酸化性能和λ核酸外切酶(λexo)的特异性切割能力,构建了一种非标记、灵敏度高的ALP活性检测方法。研究了Cu^(2+)浓度、抗坏血酸钠浓度、ALP去磷酸化时间、λexo用量及反应时间等参数对荧光强度的影响,并对方法的灵敏度和特异性进行了评价。结果表明,优化的Cu^(2+)和抗坏血酸钠浓度分为0.75和2.50 mmol/L,ALP去磷酸化时间30 min,最优λexo用量和水解时间分别为23.3 U/m L和30 min。在优化条件下,ALP活性在0.05~0.5 U/L范围内与体系荧光强度呈较好的线性关系,线性方程为F=-2393.15c+1699.97,检出限为0.013 U/L。方法可用于血清中ALP的检测,加标回收率为96.7%~104.4%。

Taking poly(thymine) single strand DNA template to synthesize copper nanoclusters(Cu^(2+)NCs)probe,combining with the dephosphorylation of alkaline phosphatase(ALP)and the specific cleavage ability of lambda exonuclease(λ exo),a label-free fluorescence strategy had been developed for ALP activity detection.Effects of concentrations of Cu^(2+)and sodium ascorbate,the ALP dephosphorylation time,the dosage of λ exo and reaction time on fluorescence intensity were investigated. Meanwhile,the sensitivity and specificity of the method were evaluated. The results showed that the optimal conditions were concentrations of Cu^(2+)and sodium ascorbate of 0.75 and 2.50 mmol/L,respectively,ALP dephosphorylation time of 30 min,λ exo dosage and hydrolysis time of 23.3 U/mL and 30 min,respectively. Under the optimum conditions,a good linear relationship between the fluorescence intensity and ALP activity in the range of 0.05-0.5 U/L was obtained,where the linear equation was F=-2393.15c+1699.97,and the detection limit was 0.013 U/L. The method was successfully used for the detection of ALP in serum with recoveries of 96.7%-104.4%.