Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China.
Talanta. 2012 May 15;93:330-5. doi: 10.1016/j.talanta.2012.02.044. Epub 2012 Mar 1.
In this work, a new platform for effective sensing cysteine (Cys) was developed based on fluorescence resonance energy transfer (FRET) between FAM-tagged single-stranded DNA (FAM-ssDNA) and graphene oxide (GO). Due to the noncovalent assembly between FAM-ssDNA and GO, fluorescence quenching of the FAM took place because of FRET. This method relied on the competitive ligation of Ag(+) by Cys and "cytosine-cytosine" (C-C) mismatches in a FAM-labeled DNA strand of the self-hybridizing strand. At first, enough amount of Ag(+) was introduced to bind "C-C" mismatches and form double-stranded DNA (dsDNA), which had weak affinity to GO and kept FAM away from GO surface. However, the presence of Cys removed Ag(+) away from "cytosine-Ag(+)-cytosine" (C-Ag(+)-C) base pairs, leading to the formation of ssDNA again and FRET, and then fluorescence of the FAM-ssDNA was efficiently quenched. The fluorescence intensity decrease was found to be proportional to the increase of concentration of Cys in both aqueous buffer (2-200 nM) and human serum (5-200 nM), and the sensitivity of the proposed method towards Cys was much higher than that of other reported assays for Cys.
在这项工作中，基于 FAM 标记的单链 DNA（FAM-ssDNA）与氧化石墨烯（GO）之间的荧光共振能量转移（FRET），开发了一种用于有效感测半胱氨酸（Cys）的新平台。由于 FAM-ssDNA 与 GO 之间的非共价组装，由于 FRET，FAM 的荧光发生猝灭。该方法依赖于 Cys 和 FAM 标记的 DNA 链中的“胞嘧啶-胞嘧啶”（C-C）错配之间的银（Ag(+)）的竞争性连接。首先，引入足够量的 Ag(+) 以结合“C-C”错配并形成双链 DNA（dsDNA），dsDNA 与 GO 的亲和力较弱，并使 FAM 远离 GO 表面。然而，Cys 的存在将 Ag(+) 从“胞嘧啶-银（+）-胞嘧啶”（C-Ag(+)-C）碱基对中去除，导致再次形成 ssDNA 并发生 FRET，然后 FAM-ssDNA 的荧光被有效猝灭。发现荧光强度的降低与水溶液缓冲液（2-200 nM）和人血清（5-200 nM）中 Cys 浓度的增加成正比，并且该方法对 Cys 的灵敏度比其他报道的 Cys 测定方法高得多。
