基于切刻内切酶信号放大的电致化学发光DNA生物传感器的研究

Ultrasensitive Electrochemiluminescence Biosensor for DNA Determination Based on Nicking Endonuclease Assisted Signal Amplification

利用切刻内切酶的酶切作用实现信号放大,结合量子点高效的电化学发光性能,构建了一种新型电化学发光DNA生物传感器。将捕获探针DNA(c-DNA)通过自组装的方式固定到金电极表面,后与目标DNA(t-DNA)互补杂交形成双链DNA,利用切刻内切酶Nt.Bst NBI特异性识别双链上的酶切位点(5'-GAGTC-3'),然后在c-DNA相应的切割位点(识别序列3'端后的4个碱基处)对其进行剪切,释放出目标链,参与下一轮的杂交及酶切,通过目标物的循环利用,实现信号放大。利用N-羟基琥珀酰亚胺(NHS)和1-乙基-3-3-二甲基氨丙基碳化二亚胺(EDC)活化羧基化Cd Te量子点表面的羧基,与电极表面残留的c-DNA末端的氨基共价交联,通过测定捕获的量子点的电化学发光信号对目标DNA进行检测。优化后的检测条件为:c-DNA浓度1μmol/L,杂交时间60 min,Nt.Bst NBI浓度0.5 U/μL,酶切反应时间4 h。在优化条件下,目标DNA浓度在2.0×10^(-13)~2.0×10^(-11)mol/L范围内,其对数与电化学发光强度呈线性关系,检出限为7.3×10^(-14) mol/L。人体血样加标回收率为96.4%~108.0%。

A novel electrochemiluminescence( ECL) DNA biosensor based on nicking endonuclease and the efficient ECL property of quantum dots( QDs) assisted signal amplification was developed. The capture probe DNA( c-DNA) was self-assembled on gold electrode via AuS bond,and then hybridized with target DNA( t-DNA) to form double-stranded DNA. Then nicking endonuclease Nt. Bst NBI recognized a cutting site( 5'-GAGTC-3') in the double chain and cleaved c-DNA strand at 4 bases away from the 3' end of its recognition site,thus releasing the t-DNA and achieving a recycle of t-DNA in the next hybridization and signal amplification. Finally,carboxyl groups on the surface of Cd Te QDs were activated with 1-ethyl-3-( 3-dimethylaminopropyl) carbodiimide( EDC) and N-hydroxysuccinimide( NHS),and then reacted with amino groups at the terminal of residual c-DNA on the electrode surface. So the QDs were fabricated and the t-DNA concentration could be determined by measuring the ECL signal of the Cd Te QDs. The experimental conditions were optimized and 1 μmol / L c-DNA,60 min of hybridization time,0. 5 U / μL Nt. Bst NBI and 4 h of endonuclease reaction time were chosen. The experimental results showed that under optimal conditions,t-DNA could be specifically assayed with a linear relationship between the ECL signal intensity and the logarithm of t-DNA concentration in the range of 2. 0 × 10^(-13)- 2. 0 × 10^(-11)mol / L,with a limit of detection of 7. 3 × 10^(-14)mol / L. The biosensor was successfully applied to determine t-DNA concentration in human blood sample with recoveries of 96. 4%-108. 0%.