基于磁性层状双氢氧化物和核酸外切酶Ⅰ的新型大肠杆菌荧光传感器构建

A novel fluorescent sensor for Escherichia coli detection based on magnetic layered double hydroxide and exonucleaseⅠ

目的基于磁性层状双氢氧化物(magnetic layered double hydroxide,MLDH)对修饰在单链和双链DNA上的羧基荧光素(6-car-boxyfluorescein,FAM)的高效淬灭,以及核酸外切酶Ⅰ(exonucleaseⅠ,ExoⅠ)对单链DNA的选择性降解作用,建立测定大肠杆菌的荧光传感器。方法将不同浓度的大肠杆菌特征DNA与FAM标记的互补DNA探针杂交,形成FAM-双链DNA,加入Exo I选择性降解剩余探针,再加入MLDH吸附并淬灭FAM-双链DNA。通过磁铁分离上清液与MLDH,测量上清液的荧光强度。结果在最优实验条件下,本法检测大肠杆菌特征DNA的线性范围为0.05~30nmol/L,相关系数(R^(2))为0.997,检测限(3σ)为0.025nmol/L,对江水、自来水和茶饮料中大肠杆菌DNA的回收率为85.4%~118%,相对标准偏差小于5%。结论利用MLDH的荧光淬灭能力和Exo I对单链DNA的选择性水解能力成功构建了测定大肠杆菌的新型荧光传感器,与传统大肠杆菌检测法相比,所建新型传感器灵敏度高、选择性好、准确度高,能实现对大肠杆菌的快速检测。

Objective A fluorescent sensor for the determination of Escherichia coli was established,based on the efficient quenching of 6-carboxyfluorescein(FAM)modified on both single-stranded DNA and double-stranded DNA by magnetic layered double hydroxide(MLDH)quenchers,and the selective degradation of single-stranded DNA by exonuclease I(Exo I).Methods Dif-ferent concentrations of Escherichia coli characteristic DNA were hybridized with FAM-labeled DNA probes.The remaining single-stranded DNA probes were selectively hydrolyzed by Exo I.Then,MLDH quenchers were added to adsorb and quench the formed FAM-labeled double-stranded DNA.Finally,the supernatant was separated from MLDH quenchers with the help of an external magnet and its fluorescence intensity was measured.Results Under optimal experimental conditions,the linear range of Escherichia coli characteris-tic DNA was 0.05-30 nmol/L,the correlation coefficient(R^(2))was 0.997,and the detection limit(3σ)was 0.025 nmol/L.The recoveries for Escherichia coli characteristic DNA in river water,tap water and tea ranged from 85.4%to 118%with the relative standard devia-tions less than 5%.Conclusion A novel fluorescent sensor for Escherichia coli detection was successfully established.Compared to tra-ditional Escherichia coli detection methods,the proposed sensor exhibited high sensitivity,good selectivity,and high accuracy,which re-alized rapid detection of Escherichia coli.