There is thus an intense demand for more sensitive, selective, convenient inhibitor MEK162 and low-cost methods to detect Cys.Growing research interest has been focused on the development of sensitive, selective, and cost-effective biosensors based on target-responsive DNA structural switching [26�C29]. The core technology of these sensors is a kind of special DNA molecules which change their conformations upon binding with the targets. For example, aptamers are in vitro selected functional oligonucleotides that can bind specifically to target molecules. In this work, we use a mercury-specific DNA (MSD) which presents a random coil form in the absence of Hg2+, and forms a hairpin structure in the presence of Hg2+ due to the formation of a thymine-Hg2+-thymine (T-Hg2+-T) complex.
A fluorescent dye, Sybr Green I (SG), was applied to recognize the structural change due to the different interaction of SG with MSD and MSD/Hg2+ complex [30].It is well-known that Cys can form a very stable complex with Hg2+ [31]. By using this property, Mirkin et al. [32] developed a highly sensitive and selective colorimetric detection method for Cys. However, their method needs an elevated temperature, which requires a long time. We recently developed a fluorescent turn-on ��molecular beacon�� probe for the detection of Cys, which is also based on the competitive ligation of Hg2+ ions by Cys and thymine-thymine (T-T) mismatches [33]. The method shows high sensitivity and selectivity, but still needs to be improved in terms of cost and convenience due to its requirement of a labelled ��molecular beacon�� and a solution heating process.
Here we develop a simple, rapid, sensitive and selective method for detection of Cys by using a target-responsive DNA structural change. The MSD/Hg2+ complex is a hairpin structure, which dehybridizes when Hg2+ is extracted from the thymine-Hg2+-thymine complex by Cys due to the high formation constant of Hg2+-Cys complexes. The fluorescence intensity of SG then decreases upon addition of Cys due to the dehybridization of the MSD/Hg2+ complex.2.?Experimental Section2.1. Chemicals and ApparatusAll chemicals used for these investigations were of analytical grade purity. L-Cysteine (Cys, minimum 98.5%) was purchased from Sigma Aldrich Chemical Company and used as received. MSD (5��-TTCTTTCTTCCCCTTGTTTGTT-3��) was synthesized and purified by HPLC (Takara Biotech.
Co., Dalian). SG (10,000��) was purchased from Invitrogen Inc. A stock solution of 400�� was prepared with DMSO/water (volume 1:1) before use. Milli-Q water (18.2 M? cm) was used in all procedures. The fluorescence measurements were recorded at room temperature (RT) on a Perkin Elmer LS-55 spectrophotometer equipped with a xenon Cilengitide lamp excitation source. Fluorescence spectra were measured Calcitriol at an excitation wavelength of 490 nm and the emission range from 500 to 650 nm with the excitation and emission slit widths set at 5 nm.2.2.