| Mercury is a widespread heavy metal pollutant with high toxicity and severe adverse effects on human health and ecological environment even at low concentrations, which has been the world's one of the most notable environmental pollutants. Metallothioneins(MTs) are rich of cysteine, low–molecular weight, thermal stability and metal–binding proteins with many important biological functions, such as heavy metal detoxification, free radical scavengers, resistance to radiation and tumor and so on. In addition, the expression of MTs has been used as a key biomarker for metal pollution. Therefore, to develop simple, rapid, sensitive and practical methods for the determinations of mercury and MTs are significant, which are not only for monitoring environmental pollution level, evaluating occupational health risks, but also for studying the biological function of MTs.In the chapter 1, the significance and detection methods of mercury and metallothioneins are introduced, and the application of G–quadruplex DNAzyme and exonuclease III in biological sensing field are outlined.In the chapter 2, a novel strategy for dual–channel detection of metallothioneins and mercury based on the conformational switching of functional chimera aptamer has been proposed. Without Hg2+, the functional chimera aptamer(FCA) designed can form an intact G–quadruplex with flexibility, which was proved to have peroxidase–like activities upon binding to hemin to catalyze the oxidation of ABTS by H2O2. Upon the addition of Hg2+, the production of T–Hg2+–T complexes facilitates the conformational switching of the FCA, inhibiting the formation of G–quadruplex. Thus a declined peroxidase–like activity along with the decrease of absorbance is observed. While upon addition of MTs, they could strongly interact with Hg2+ through thiol to form a MTs–Hg2+ complex, accompanied by the formation of a G–quadruplex DNAzyme. The color and absorbance of the sensing system were also changed accordingly. In the optimizing condition, ?A was directly proportional to the concentrations of Hg2+ or MTs, ranging from 8.84×10-9 mol·L-1 to 1.0×10-6 mol·L-1 with a linear regression equation of ?A = 0.0428 + 0.178c(×10-7 mol·L-1)(r = 0.9989) for Hg2+, and from 7.22×10-9 mol·L-1 to 4.62×10-7 mol·L-1 with a linear regression equation of ?A = 0.0333 + 0.185c(×10-7 mol·L-1)(r = 0.9995) for MTs. The detection limits were 2.65×10-9 mol·L-1 for Hg2+ and 2.34×10–9 mol·L-1 for MTs, respectively. The proposed method is simple, sensitive, selective, inexpensive and has been successfully applied for the determination of MTs in human urine.In the chapter 3, based on exonuclease III–assisted fluorescence signal amplification technology, we established a novel and simple method for mercury ion detection. The molecular beacons with a 6–FAM fluorophore at 5' end and three extra continuous guanine bases at 3' end were designed. The formation of hairpin structure brings deoxyguanosines at 3'–end close to 6–FAM at 5'–end, resulting in a fluorescence quenching of the fluorophore through photoinduced electron transfer from 6–FAM to deoxyguanosines. In the presence of mercury ions, molecular beacons hybridize with aptamer via the T–Hg2+–T coordination, thus opening the molecular beacons to form a partially double stranded DNA with a blunt end at the 3' terminus. This one can be recognized and cleaved by exonuclease III, releasing the fluorophore, mercury ions and aptamer. The released mercury ions and aptamer are able to be recycled to repeate the process of “hybridization–cleavage –release”, leading to a significantly enhanced fluorescence signal. In the optimizing conditions, ?F was directly proportional to the concentration of mercury ions in the range of 1.0×10-9 to 4.68×10-8 mol·L-1. The equation of linear regression was ?F = – 82.1 + 30.4c(nmol·L-1) with a correlation coefficient(r) of 0.9989. The limit of detection(LOD) was 0.3 nmol·L-1. The proposed method has been successfully applied for the determination of mercury ions in water samples with the recoveries of 95.8% to 106.9%. |
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