A Novel Principle And Method For The Detection Of Lead(Ⅱ) Based On Dnazyme And Gold Nanoparticle-Lead Mimic Enzyme

Lead, a kind of toxic heavy metals, can be found in the dust,soil, minerals, water and other medium. Due to its unique physical and chemical properties, lead has been widely applied in industrial production and daily life. Thus it is easy to exposure to lead, and the development of a sensitive and specific method for the detection of Pb2+ ions is of great significance to control environmental pollution and protect human health.In the chapter 1, the research significance and developments in the determination of lead(Ⅱ) are reviewed. Then the concepts and applications of deoxyribozymes, nanomaterials-based enzyme mimetics and rolling circle amplification(RCA) technology are also introduced.Meanwhile, the main topics of this thesis are outlined.In the chapter 2, a novel sensitive fluorescence method for the determination of Pb2+ was established by combining the high sensitive and specific rolling circle amplification technology and Pb2+- specific DNAzyme. N-methylmesoporphyrin Ⅸ(NMM), an asymmetric anion porphyrin characterized by a significant structure selectivity for G-quadruplex, was used as signal output. In the presence of Pb2+, theDNAzyme was activated to cleave the substrate strand into two DNA fragments at the ribonucleotide site. Due to the lack of thermal stability the cleaved substrate strands were released. Then, the resulting free catalytic strand could be hybridized with the two ends of RCA template,which was a cytosine(C)-rich base sequence. Upon addition of ligase,these two ends were ligated to form a circular RCA template. After RCA,a long single-stranded DNA with repeating G-rich sequence was produced. The G-quadruplex formed by the RCA production can specificly bind to NMM. Thereby, a high sensitive method for the detection of Pb2+ ions was established, with a detection limit of 0.21 n M.In addition, the mechanisms of the change of DNAzyme on addition of Pb2+ and the RCA process have been proved by CD, fluorescence spectroscopy and electrophoresis.In the chapter 3, a sensitive method for the detection of Pb2+ has been developed based on an unlabeled DNAzyme-gold nanoparticle(Au NPs) system by using resonance light scattering(RLS). In Tris-HCl buffer solution(p H 7.0, 10 m M), Pb2+-dependent DNAzyme is activated by Pb2+ and the substrate strand is cleaved at the r A site into ss DNA fragments. Consequently, the released ss DNA can adsorb onto the surface of Au NPs through Au–N interaction and greatly increase the density of the negative charges on the Au NPs, preventing them from aggregating in the presence of Na Cl. This decreases RLS intensity. The change of theRLS intensity was propotional to the concentration of Pb2+ over the range of 1.68×10–8~ 12.5×10–7mol/L, and the linear regression equation wasΔIRLS= 22.78+4.46c(×10–8mol/L)(r=0.970). The detection limit was5.04 n M. The proposed method was convenient and rapid, with a higher selectivity and sensitivity.In the chapter 4, an UV-Vis spectrophotometric method for detecting Pb2+ has been developed using gold nanoparticle(Au NPs)-lead mimic enzyme. In the citrate-HCl buffer solution of p H 3.3, Au NPs can interact with Pb2+ to form a Au NPs-Pb peroxidase mimetic, which can catalyze the color reaction of 2,2’-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid(ABTS) by H2O2 to form a green radical product ABTS?+. As a result,the absorbance of the system is improved. We found that there was a good linear relationships in the ranges from 1.62×10–7 mol/L to 7.00×10–6mol/L under optimized conditions. The equations of linear regression areΔA1= 0.005 + 0.072c(×10–6 mol/L) in the ranges of 1.62×10–7 mol/L~1.00×10–6mol/L, and ΔA2 = 0.067 + 0.015c(×10–6 mol/L) ranging from1.00×10–6 mol/L~7.00×10–6mol/L with the correlation coefficients of0.990 and 0.980, respectively. A detection limit of 48.7 n M was obtained.In addition, mechanism on the interaction between Au NPs and Pb2+ was verified by zeta potential and ICP-MS.