利用分子信标和EXO Ⅲ放大荧光信号快速检测Ag^+

Rapid detection of Ag^+ based on molecular beacons and EXO Ⅲ-assisted fluorescence signals amplification

设计新型的DNA探针,其主要功能区域包含适配体区域和分子信标互补区域;当无靶标存在时,羧基荧光素(FAM)的荧光被黑洞猝灭基团(BHQ)猝灭,加入靶标后,Ag^+与适配体区域特异性结合,使得分子信标与其互补区域发生结合,加入EXO III后,分子信标被酶解,从而释放Ag^+/DNA探针复合物用于下一轮反应,同时产生大量的FAM,因此,可以通过荧光的增强来定量检测靶标的含量.评价了该方法的灵敏度和选择性,探究它在实际样品中的表现能力.结果表明,适配体区域与分子信标间隔2个碱基的DNA探针与分子信标结合效果最好,检测的信噪比最大.该方法对Ag^+的检测最低下限(LOD)为0.1nmol/L,在0.5~200nmol/L范围内,荧光值与靶标浓度成线性关系(R^2=0.994).对实际样品的检测在20~200nmol/L范围内呈线性关系(R^2=0.998),且能很好的从混合样品中区分10nmol/L Ag^+.该研究建立的快速检测Ag^+方法具有良好的线性定量能力和操作性能,且灵敏度高、特异性强,可满足现实的需求.

To establish a lower cost,rapid,higher sensitive and specifical detection method for Ag+,we designed a novel probe containing the aptamer region and the complementary region of molecular beacon(MBs).In the absence of target,the fluorescence of FAM was quenched by BHQ.Upon adding the target,it would specifically bind to the aptamer,leading to the complementary region being recognized by MBs.MBs were cleaved when the EXO III was introduced,which can release Ag+/DNA complex for the next round of reaction and free FAM for signal amplification,simultaneously.Hence,the amount of targets were quantified by fluorescence changes.Meanwhile,the performance of this method in real sample was also investigated to evaluate its sensitivity and selectivity.It showed that there was the optimal binding result between DNA probe and MBs when separating the molecular beacon from the complementary region with two bases,and it was achieved the best single-to-noise ratio(S/N)too.It had also a good linear range from 0.5 to 200nmol/L(R2=0.994),and a lower limit of detection of 0.1nmol/L.Importantly,a good linear relationship in the range of 20~200nmol/L(R2=0.998)was also obtained when this sensor was used for real sample analysis,moreover,it can distinguish 10nmol/L Ag+from the mixed sample.The method for rapid detection of Ag+has a good linear quantitative ability and operational performance,and its high sensitivity and specificity can meet the real demands.