Studies Of The Electrochemical Biosensors Based On Functional Nucleic Acids For Heavy Metal Detection

Heavy metal pollution refers to the environmental pollution, including industrial pollution, traffic pollution and domestic waste pollution, caused by heavy metals or their compounds. Many organic compounds can be degradated by physical, chemical or biological purification, but heavy metal pollution can not. Once heavy metals were gathered in the environment, it is difficult to degrade them. These heavy metals were accumulated in the animal and plant by the water, air, soil and the food chain. The accumulation of these heavy metals resulted in inactivation of the protein, which further causing acute, sub-acute, and chronic poisonings of human body.Conventional detection methods for heavy metals are mainly atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, fluorescence spectrometry. Although these methods have high sensitivity, large-scale instruments, complicated sample pretreatment and time-consuming limited their applications. Therefore, it is significant to develop the detection methods of the heavy metal ions with high sensitivity, high specificity, and simple operation. At present, the detection of heavy metal ions has been widely studied to realize the rapid and efficient monitoring of heavy metal ions in environmental and food.Functional nucleic acids refer to combine with specific target or catalytic functions, or have the catalytic function of nucleic acids. In this paper, we use the DNAzyme and T-Hg²⁺-T complexes as the specific recognition elements, combined with highly sensitive electrochemical technique of electrocheemical impedance spectroscopy（EIS）, quartz crystal microbalance（QCM） and chronocoulome. Biosensors with highly sensitive for Cu²⁺ and Hg²⁺ was successfully constructed, it provides a rapid, simple and economical analysis methods for the detection of Cu²⁺ and Hg²⁺ in food and environment. The research contents are as follows: 1. A DNAzyme-based electrochemical impedance biosensor for highly sensitive detection of Cu²⁺ ions in aqueous solutionWe developed a simple, label-free, DNAzyme-based electrochemical impedance sensor for highly sensitive detection of Cu²⁺ ions in aqueous solution. A complex of Cu²⁺-dependent DNAzyme and its corresponding substrate was immobilized on the gold electrode surface though Au-S bond. In presence of Cu²⁺ ions, the cleavage of DNAzyme resulted in a significant change in the resistance signal intensity. The resistance signal change correlated with the concentration of Cu²⁺ ions. The proposed sensor showed excellent sensitivity and selectivity. The limit of detection of this method is 5 nM, which is far below the limit of Cu²⁺ ions（²0 μM） mandated by United States Environmental Protection Agency（EPA）. A series of metal ions, such as Ca²⁺, Mg²⁺, Mn²⁺, Cr3+, Pb²⁺, Zn²⁺, Co²⁺, and Ni²⁺, have little interference with the detection of Cu²⁺ ions. 2. Studies of the biosensor based on DNAzyme and quartz crystal microbalance for Cu²⁺ detection in aqueous solutionQuartz crystal microbalance（QCM） assay is a simple, rapid and real-time method that can detect the change of the nanogram level on the surface of quartz crystal. Therefore, it has become one of the effective, non-labeled methods in the electrochemical assays. In this chapter, we design a biosensor based on the Cu²⁺ specific DNAzyme and QCM. The proposed sensor was constructed by self-assembly of dsDNA（DNAzyme and substrate） on the surface of the electrode at room temperature. In the presence of Cu²⁺, the substrates on the gold surface were cleaved by Cu²⁺ at the cleavage site, thereby resulting in the mass change on the wafer surfaces. The changes of the resonance frequency（△F） are proportional to the concentration of Cu²⁺. The limit of detection of the biosensor is 3 nM. Other metal ions, such as Co²⁺, Ni²⁺, Mg²⁺, Zn²⁺, Ca²⁺, Mn²⁺ and Pb²⁺ have no effect on the detection of Cu²⁺. The biosensor showed high sensitivity and selectivity, which is expected to be applied to the determination of Cu²⁺ in food and environment. 3. Electrochemical biosensor based on charge transfer of DNA duplexs for the detection of Hg²⁺In this paper, combined Hg²⁺ specific recognition of thymine with chronocoulometry, we constructed a highly sensitive electrochemical biosensor based on DNA-mediated charge transport for the detection of Hg²⁺ in aqueous solution. DNA duplexs with one T-T base mismatch were assembled onto a gold electrode surface through Au-S bond. Chronocoulometry was used to detect Hg²⁺ in 2mM [Fe（CN）6]3-（0.1 M KCl, pH 7.4） containing 2.0 μM MB. T-T mismatch blocks the internal charge transfer of the DNA duplexs. In the presence of Hg²⁺, the DNA on the electrode surface specifically recognized Hg²⁺ and formed thymine-Hg²⁺-thymine complexs. Thus, internal charge transfer path in dsDNA was formed, significantly improving charge transport onto the gold electrode surface. The results showed the chronocoulometry of DNA-modified gold electrode increased with the increase of Hg²⁺ concentration at the reduction peak of methylene blue（-380 mV）. The change of chronocoulometry was linear with regard to lgc Hg²⁺ over a concentration range from 1.0 nM to 104 nM（R2=0.995） and with a detection limit of 0.5 nM（S/N=3）. A test for a series of interference metal ions showed that this biosensor based on DNA-mediated charge transport is highly specific and selective toward Hg²⁺.