Study Of The Novel Methods For Determination Of Metallothioneins Based On A Thymine-rich Oligonucleotide-Hg And8-hydroxyquinoline-5-sulphonic Acid-Cd (Ⅱ) Complex

Metallothioneins（MTs） have many important biology functions such as scavengeactivated oxygen species （ROS） and toxic metals, resistance to ionizing radiationeffect and inhibiting the apoptosis. MTs have alse been proposed as a potentialbiomarker for metal pollution in the environment. Therefore, it is significant todevelop a simple, rapid, sensitive and practical method for the determination of MTsnot only for monitoring and assessing environmental pollution level, but also forstudying the function of MTs in organism.In the chapter2, a novel colorimentric method to detect MTs has been developedbased on a thymine-rich oligonucleotide（TRO）-Hg complex and gold nanoparticle. Inthe Britton–Robinson buffer solution of pH8.0, Hg²⁺may react with TRO to form theT-Hg²⁺-T complex. After MTs was added to the T-Hg²⁺-T complex solution, theycould interact with Hg from T-Hg²⁺-T complex to release TRO, which can beabsorbed on AuNPs, resulting in a color change of the system. Based on this principle,a colorimetric method for the determination of MTs was developed and used in theanalysis of human urine. In the optimizing condition, a linear correlation existedbetween the (A₆₂₀/A₅₂₀) and the concentration of MTs in the range of2.5×10^-8to3.08×10^-7mol·L1. The equation of linear regression was (A₆₂₀/A₅₂₀)=0.09²⁺0.242c (×10^-7mol·L1) with correlation coefficient （r） of0.9841. The limit ofdetection （LOD） was7.67×10^-9mol·L1. The proposed method is convenient, rapid,inexpensive and sensitive.In the chapter3, based on8-hydroxyquinoline-5-sulphonic acid （HQS）-Cd （II）complex, a new method for the detection of MTs in human urine has been established by fluorescence quenching strategy. In the Britton-Robinson buffer solution of pH9.0,HQS may interact with Cd²⁺to form a binary complex of HQS-Cd²⁺in the present ofcation surfactant CTMAB. Upon addition of MTs to HQS-Cd²⁺-CTMAB system, thefluorescence intensity of the system was decreased. Thereby, a novel fluorescencequenching method to detect MTs in human urine was established. Under optimalcondition, ΔF is directly propotional to the concentration of MTs over the range of3.12×10^-8–1.23×10^-6mol·L1. The equation of linear regression was ΔF=12.79+20.20c (×10^-7mol·L1), r²=0.9919. The limit of detection （LOD） was9.36×10^-9mol·L^-1. The proposed method is simple, rapid, sensitive and selective, and wasapplied for the determination of MTs in human urine with a satisfactory result.In the chapter4, the mechanism of the interaction between HQS-Cd（II） complexand MTs was studied by fluorescence spectra. The results suggested that theinteraction of HQS-Cd（II） complex with MTs was driven mainly by hydrophobicforces. The composition ratio of HQS-Cd（II） and MTs in HQS-Cd（II）-MTs complexwas11.9:1. According to the results of the Stern–Volmer plots and measurement offluorescence lifetime, a static quenching mode was proposed to be primarilyresponsible for the flurescence quenching event.