基于酶促信号放大的氧化石墨烯生物传感器快速检测金黄色葡萄球菌的研究

Rapid Detection of Staphylococcus aureus by Using Graphene Oxide-Based Biosensor Based on Enzyme-Aided Signal Amplification

本研究以设计的与16SrRNA互补的单链DNA(ssDNA)探针作为捕捉探针,羧基荧光素(FAM)标记的探针1、探针2作为信号探针,将两者组装形成两个FAM标记的探针(TFP),并以氧化石墨烯(GO)作为猝灭剂形成TFP/GO复合物。当无靶标时,TFP吸附到GO表面,FAM的荧光被猝灭,当加入靶标后,TFP从GO上解离下来,荧光恢复,因此可以通过荧光值定量检测靶标。此外,在体系中加入脱氧核糖核酸酶Ⅰ(DNaseⅠ),TFP被降解从而释放出靶标RNA用于下一轮反应同时产生大量的FAM,可以极大地提高该传感器的灵敏度及特异性。结果表明该方法对靶标RNA的检测限可达0.3nmol/L,且在1～60nmol/L范围内呈良好的线性关系(R2=0.9984);对靶标菌的检测限低至50CFU/mL,且在102～107 CFU/mL范围内呈良好的线性关系(R2=0.9973)。该方法对靶标菌检测的信噪比要远大于非靶标菌,表明其具有很强的特异性。该研究建立的快速检测金黄色葡萄球菌的纳米生物传感器具有良好的定量能力和操作性能,且灵敏度高、特异性强。

In this study,the ssDNA used as capture probe that is complementary to 16 SrRNA and the carboxyfluorescein(FAM)-labelled probe 1and probe 2used as signal DNA probe is designed.The two FAM-labeled probe(TFP)are assembled by the two probe,meanwhile,TFP/graphene oxide(GO)is formed by using GO as the quencher.In the absence of target,TFP is adsorbed onto the surface of GO,resulting in the fluorescence quenching of the dye.When the target is introduced,the fluorescence is restored due to the probe being dissociated from GO.The target quantum is achieved by fluorescence increment.In addition,DNaseⅠis introduced,target recycling that release the target for the next round will occur,leading to accumulation of FAM,and the sensitivity and the specificity are considerably improved.The biosensor for target RNA analysis shows a good linear range from 1to 60nmol/L(R2=0.9984),with a lower limit of detection of 0.3nmol/L.In addition,a bacterial sample harboring the target RNA is also detected and the detection limit(LOD)peaked to 50colony-forming units(CFU/mL)with a good linear range from 102 to 107 CFU/mL(R2=0.9973).Furthermore,the much higher signal-tonoise(S/N)of the target than that of the nontarget displays that the biosensor is highly selective.The nano-biosensor designed for Staphylococcus aureus assay shows good linear quantitation,operational performance,sensitivity and selectivity which can meet the needs of food and clinical testing.