| Mercury is highly toxic heavy metal which has received much attention due to its severe adverse effects to human health as well as ecological environment. Metallothioneins(MTs), as ion-binding protein with low molecular weight and themostability, have many important biology functions such as scavenge activated oxygen species and toxic metals, resistance to ionizing radiation effect and inhibiting the apoptosis. Besides, MTs have also been proposed as a potential biomarker which is widely applied for metal pollution in the environment. Therefore, it is significant to develop a simple, rapid, sensitive and practical method for the determination of mercury and MTs not only for monitoring and assessing environmental pollution level, but also for studying the function of MTs in organism.In the chapter 2, a novel dual-channel strategy for the detection of Hg2+ and metallothioneins(MTs) has been developed based on a mercury-mediated aptamer beacon(MAB) using thymidine-mercury-thymidine complex as a quencher for the first time. A T-rich oligonucleotide with a 6-carboxyfluorescein at 5?-end(TRO-FAM) has been designed. In the Tris-HCl buffer solution of p H 8.5, the T-rich oligonucleotide with a 6-carboxyfluorescein(TRO-FAM) can form an aptamer beacon via the formation of T-Hg2+-T base pairs, which results in a fluorescence quenching of the sensing system owing to the fluorescence resonance energy transfer(FRET) from the fluorophore of FAM to the terminated T-Hg2+-T base pair. MTs have a stronger binding ability to mercury ion. It is found in the experiment that the fluorescence of the system is recovered upon addition of MTs, suggesting that the binding ability between MTs and Hg2+ is much more stronger than that of Hg2+ and TRO to lead to a disruption of T-Hg2+-T base pair. Based on the formation and disruption of the T-Hg2+-T structure, a dual-channel detection system for MTs and Hg2+ could be established. Under optimal experimental condition, a linear correlation existed between ?F and the concentration of Hg2+ in the range of 1.42×10-8–3.0×10-7 mol·L-1. The equation of linear regression was ?F= –18.79 + 301.2c(×10-7 mol·L-1), r=0.9991. The limit of detection(LOD) was 4.28×10-9 mol·L-1. For MTs assay, ΔF is also directly proportional to the concentration of MTs ranging from 5.63×10-9 to 2.75×10-7 mol·L-1. The equation of linear regression was ΔF= –42.27+365.3c(×10-7 mol·L-1) with a correlation coefficient(r) of 0.9987. The limit of detection(LOD) was 1.69×10-9 mol·L-1. The mercury-mediated aptamer beacon designed by us only needs to be marked using single fluorophore, which is inexpensive. The discovery that the formation of T-Hg2+-T base pairs terminated can quench the fluorescence of 6-FAM provides a novel idea and experimental basis for constructing novel molecular beacon. The application for the detection of real samples further demonstrated its reliability.In the chapter 3, based on the suppression of the surface energy transfer that occurs between acridine orange(AO) and gold nanoparticles(Au NPs), a new strategy was proposed. The addition of gold nanoparticles(Au NPs) in the solution of AO resulted in the decrease of fluorescence intensity of the sensing system due to the surface energy transfer. Upon addition of MTs, it could interact with gold nanoparticles to form a MTs-Au NPs comples, releasing the AO from the surface of gold nanoparticles along with a recovery of the fluorescence in the assay system. Thereby, a novel method to detect MTs has been established. Under optimal experimental condition, maximum fluorescence peak lies on 524 nm. ΔF is directly proportional to the concentration of MTs over the range of 9.3×10-9–1.5×10-7 mol·L-1. The equation of linear regression was ΔF= 3.798+337.3c(×10-7 mol·L-1), r=0.9965, The limit of detection(LOD) was 2.79×10-9 mol·L-1. The proposed method is sensitive and selective, and was applied for the determination of MTs in human urine with a satisfactory result. |
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