摘要
The factors that influence the colorimetric gene detection of gold label silver stain and improve the detection signals were studied. The influence of amino DNA probes and thiol DNA modified gold nanoparticles was investigated based on a sandwich hybridization system. An increase in amino probe concentration brought about an increase in hybridization signal which reached a threshold corresponding to the saturated concentration of amino probes bounded onto a glass slide surface. Since the steric hindrance effect of nanoparticles was dominant over the influence of a surface area, the bigger gold nanoparticles led to weaker hybridization signals. The hybridization efficiency enhanced significantly with the increase of the thiol DNA modified nanoparticle concentrations. Experimental results showed that 125 μmol/L of the amino DNA probe concentration, 15 nm of the gold nanoparticle diameter, and 4.07 nmol/L of the thiol DNA modified gold nanoparticle concentration were optimal for the detection system. The hybridization signals can be improved remarkably by choosing optimal hybridization conditions.
研究了金标银染基因检测方法中的影响因素以及提高DNA杂交信号的途径.在"三明治"杂交系统中,着重研究了氨基DNA探针和纳米金标记巯基DNA探针对杂交的影响.随着氨基探针浓度的增加,杂交信号增加,探针达到一定浓度后信号不再增加,形成一个平台,说明连接于玻片表面的探针已达到饱和.由于纳米金颗粒空间位阻效应的影响大于纳米颗粒表面积的影响.标记的纳米金颗粒越大,杂交信号越弱.而随着金标巯基DNA探针浓度的增加,杂交效率显著增加,导致杂交信号提高.优化后的杂交条件为:氨基DNA探针浓度为125μmol／L,纳米金粒径为15 nm,DNA标记后的纳米金颗粒浓度为4.07nmol／L.通过上述杂交条件优化,可提高杂交银染的信号.
基金
TheNationalHighTechnologyResearchandDevelopmentProgramofChina(863Programm)(No.2003AA302270)Trans-CenturyTrainingProgrammeFoundationfortheTalentsbytheStateofEducationCommission.
