阳离子铝酞菁红区荧光探针在纳克级RNA定量分析中的应用

Application of Cationic Aluminum Phthalocyanine,a Red-Emitting Fluorescent Probe,for Sensitive Quantitative Analysis of RNA at Nanogram Level

荷正电的四取代三甲基碘化铵铝酞菁（Tetra（trimethylammionio）aluminum phthalocyanine,TTMAAlPc）是新近出现的、很有应用潜力的新型红区荧光化合物。研究表明,弱碱性条件,RNA对TTMAAlPc的荧光具有高效猝灭作用,且猝灭程度与RNA含量呈线性正相关。据此发现建立了可测定纳克级RNA的高灵敏定量分析新方法,并对原理进行了讨论。考察了pH、缓冲体系、反应时间、温度、TTMAAlPc浓度的影响,确定了最佳反应条件（pH 8.0的广泛缓冲液、反应5 min、室温、浓度为2×10-6 mol·L-1的TTMAAlPc）。在最佳条件下,方法的线性回归方程为y=-15.0＋0.51x,相关系数r=0.998 6,线性范围7.71~1 705.57ng·mL-1,检测限为1.55ng·mL-1。本法灵敏度高、线性范围宽、对RNA测定常见干扰物质包括阴、阳离子、表面活性剂和维生素等的抗干扰能力强,且操作简便。该法应用于实际样品RNA含量的测定,取得了满意的结果。还以参比法首次测定了不同pH下TTMAAlPc的荧光量子产率,结果显示TTMAAlPc具有较高的荧光量子产率,且对大范围的酸度稳定,表明TTMAAlPc是极具应用潜力的新型红色荧光探针,值得深入研究,开拓其应用。

Tetrasubstituted trimethyl ammonium iodide aluminum phthalocyanine（TTMAAlPc）,apositively charged phthalocyanine compound,is an emerging and potentially useful red-emitting fluorescence probe.The study showed that the fluorescence of TTMAAlPc could be quenched by RNA with high efficiency in weak alkaline media,and the degree of quenching has a linear relationship with RNA in a wide concentration range.The mechanism of quenching behavior of RNA on TTMAAlPc was discussed.It was attributed by the static interaction between RNA and TTMAAlPc,and the assembly of TTMAAlPc induced by RNA.Based on this new discovery,a novel method for quantitative determination of RNA at nanogram level has been established.The factors,including the pH of medium,buffer system,reaction time,reaction temperature,the usage of TTMAAlPc as well as the interferences,which affected the determination,were investigated and discussed.Under optimum conditions,the linear range of the calibration curve was 7.71~1 705.57ng·mL-1.The detection limit for RNA was 1.55ng·mL-1.This method has been applied to the analysis of practical samples with satisfied results.The constructed method is of high sensitivity and has a wide linear range,it also showed strong ability in the tolerance of foreign substances from anions,cations,surfactants and vitamins,all of which are common interferences encountered in the determination of RNA.Besides,it is the first report that the fluorescence quantum yield of TTMAAlPc has been measured at different pH by reference method in this work.The achieved data indicated that the fluorescence quantum yield of TTMAAlPc is larger than 20% and it keeps constant in a wide range of acidity,implying that TTMAAlPc is a high-quality red-emitting fluorescence probe,it has great potential for practical applications,thus is worthy of further study.This work expands the application of phthalocyanine compound in analytical sciences.