0 mL of 10 mM MOPS (3-(N-morpholino)propanesulfonic acid) buffer containing 0.1 M NaNO3 (pH 7.50). 5 ��L of 25 �� SG selleck bio was then added to the solution. After incubation for two minutes, different amounts of Cys were added to the solution. The mixture was then immediately used for the fluorescence measurements. For selectivity analysis, various kinds of amino acids with final concentration of 140 nM were used instead of Cys.3.?Results and Discussion3.1. Sensor Operation PrincipleMSD, including seven T-T mismatches, could form a stable hairpin structure upon combination with Hg2+, which could be specifically stained by SG and produce a high fluorescence signal. However, in the presence of Cys, Hg2+ was extracted from the T-Hg2+-T structure because Cys can bind to Hg2+ with higher affinity (the formation constant for Cys to Hg2+ is ca.
1042) [31]. As a result, the hairpin structure dehybridizes and the fluorescence intensity of SG decreases. Scheme 1 depicts the designed fluorescence method for Cys detection. The detection can be completed in less than 5 min.Scheme 1.Schematic illustration of the fluorescent Cys detection.As shown in Figure 1, the fluorescence intensity of MSD/Hg2+/SG is 116.7 a.u., while MSD/Hg2+/SG/Cys is about 15 a.u. when the concentration of Cys is 140 nM.Figure 1.Fluorescence spectra of SG-Hg2+-MSD in the absence (black line) and in the presence of Cys (red line). [MSD] = 10 nM, [Hg2+] = 70 nM, [SG] = 1.225 �� 10?7 M, [Cys] = 140 nM.3.2. Optimization of the AssayIn previous study, Liu et al. [30] found that MSD kinetically accomplishes the hairpin structure at [Hg2+]/7[MSD] = 1.
One equivalent of MSD thus requires seven equivalents of Hg2+ to completely form the hairpin structure due to the T-Hg2+-T chemistry. So we fixed the concentration of MSD to be 10 nM and Hg2+ 70 nM, and then the concentration of SG was optimized. Figure 2 shows the fluorescence spectra of MSD and MSD-Hg2+ upon addition of different concentration of SG.Figure 2.Fluorescence spectra of MSD (a) and MSD-Hg2+ (b) upon addition of different concentration of SG. [MSD] = 10 nM, [Hg2+] = 70 nM, [SG](from bottom to up) = 2.45, 12.25, 24.5, 36.75, 61.25 �� 10?8 M, respectively. (c) Relationship between …We can see from the results that the fluorescence intensity of SG increases both in the absence of Hg2+ [Figure 2(a)] and in the presence of Hg2+ [Figure 2(b)].
The highest ratio of the fluorescence intensity of SG-Hg2+-MSD Brefeldin_A to that of SG-MSD is 21.8 [Figure 2(c)], at which the concentration of SG is 1.225 �� 10?7 M, so we selected http://www.selleckchem.com/products/Cisplatin.html the concentration of SG to be 1.225 �� 10?7 M when the concentration of MSD is 10 nM and Hg2+ 70 nM.3.3. Cys Sensor SensitivityTo evaluate the sensitivity of the assay, different concentrations of Cys were mixed with MSD/Hg2+/SG, and then the fluorescence intensity was immediately measured.