一种基于微芯片电泳平台的级联化学发光信号放大策略用于HIV-DNA的高灵敏检测

High Sensitive Detection of HIV-DNA by Cascade Chemiluminescence Signal Amplification Strategy Based on Microchip Electrophoresis Platform

该文基于微芯片电泳-化学发光检测(MCE-CL)平台,以辣根过氧化酶标记的DNA(HRP-DNA)作为信号探针,利用HRP催化鲁米诺和双氧水化学发光反应及目标分子与DNA的杂交反应,结合T7Exo酶辅助信号放大,建立了一种MCE分离辅助双循环化学发光信号放大的新方法。结果显示:优化实验条件下,在1.0×10^(-14)~5.0×10^(-9) mol/L范围内,HIV-DNA的浓度对数值与HIV-DNA的化学发光强度呈良好的线性关系,检出限(S/N=3)为1.6×10^(-15) mol/L,在0.10、0.25、1.0、10(×10^(-12) mol/L)4个加标水平下的回收率为93.0%~103%,相对标准偏差(RSD)为0.50%~3.7%,方法具有较好的准确度,可应用于人血清中HIV-DNA的高灵敏检测。

Based on microchip electrophoresis-chemiluminescence detection(MCE-CL)platform,a new method of MCE separation assisted double cycle chemiluminescence signal amplification was established for the detection of HIV gene(HIV-DNA),with horseradish peroxidase-labeled DNA(HRP-DNA)as signal probe,utilizing HRP to catalyze the CL reaction of luminol and hydrogen peroxide and the hybridization of target molecules with DNA,and combined with T7Exo enzyme-assisted signal amplification.In this method,two DNA single strands P1 and P2 firstly formed P1/P2 double-stranded complexes.They then formed a ternary complex when the target HIV-DNA existed,which could be degraded specifically by T7Exo to release the HIV-DNA and P2 single strands.The released HIV-DNA was rehybridized with the P1/P2 double-stranded complex,and the T7Exo continued to degrade the complex.While the released P2 single strand was hybridized with HRP-DNA portion to form P2/HRP-DNA double strand,which was degraded by T7Exo,simultaneously releasing HRP-DNA fragment and P2 single strand.The released P2 sequence was rehybridized with HRP-DNA,which was degraded by T7Exo to produce a large number of HRP-DNA fragments.High sensitive detection of target molecules could be realized by double cycle signal amplification.Under optimized experimental conditions,the logarithmic values of HIV-DNA concentration in the range of 1.0×10^(-14)-5.0×10^(-9) mol/L showed a good linear relationship with the chemiluminescence intensity of the system.The detection limit(S/N=3)was 1.6×10^(-15) mol/L.At four spiked levels of 0.10,0.25,1.0,10(×10^(-12) mol/L),the recoveries range from 93.0%-103%,and relative standard deviations(RSDs)was 0.50%-3.7%.The proposed method was applied to the analysis of HIV-DNA in human serum with satisfactory results.