A New DNA Sensor Based On Enzymatic Application In Assay Of Protein And Metal Ion

This research reported new methods based on HCR, RCA and enzyme catalyzed reaction technology et.al, which were used to detect thrombin, PDGF-BB protein and visual Hg2+ in aqueous solution by scanning electrochemical microscope (SECM) and fluorescence spectrophotometer. This thesis mainly consists of the following three parts:1 We developed a new electrochemical aptasensor for highly sensitive detection of thrombin using hybridization chain reaction (HCR) for signal amplification.The sandwich system of aptamer/thrombin/aptamer-primer complex was fabricated as the sensing platform. As the initiator strands, aptamer-primer complex could propagate a chain reaction of hybridization events between the two hairpin probes, and whether long nicked DNA polymers could be formed on the modified electrode. Then the biotin-labeled dsDNA polymers could introduce numerous avidin-labeled horseradish peroxidase (HRP), resulting in significantly amplified electrochemical signal through the electrocatalysis of HRP. On the basis of the enzymatic oxidization of Fe2+ by H2O2 to yield Fe3+, the imaging of thrombin was detected by the reduction current of Fe3+ with the scanning electrochemical microscopic tip. The electrochemical signals had a good linear with logarithm of thrombin concentration in the range from 1.O×10-15 M to 1.0×10-13 M, reaching a detection limit of thrombin as low as 4.0×10-17 M. In addition, the proposed strategy exhibited excellent specificity and was successfully applied in real sample assay which demonstrated the potential application in clinical diagnostics.2 Based on the combination of branched rolling circle amplification chain (BRCA), HCR and enzyme catalyzed reaction for further signal amplification, a novel fluorescent aptamer sensor for the platelet-derived growth factor BB (PDGF-BB) protein detection has been designed. First, the segments of the PDGF-BB aptamers were immobilized on the magnetic beads, another section of the aptamers were added to form a stable sandwich system after rolling circle amplification. After that, The second section of the aptamers hybridized with a seris of DNA sequence that could cause hybridization chain reaction (HCR). Finally, the biotin-labeled dsDNA polymers introduced numerous avidin-labeled horseradish peroxidase (HRP) by HCR and specific binding interaction, resulting in significantly amplified fluorescence signal through the catalysis of HRP, reaching a detection limit of PDGF-BB protein as low as 1.0×10-11 g mL-1. The method provides the basis for diagnosis of early lung cancer with high sensitivity and excellent selectivity.3 The detection of Hg2+ in aqueous solution was achieved by fluorescence spectrophotometer, based on enzyme cutting cycle signal amplification technology and the fluorescence probe technique. To achieve the fluorescence assay, the substrate DNA was modified on the magnetic bead surface, and the rhodamine-labeled singal DNA hybridized with its end. Thymine-thymine (T-T) mismatches in the toehold domains can serve as specific recognition elements for Hg2+ binding with the help of T-Hg2+-T base pairs to initiate toehold-mediated strand displacement reaction. Hg2+ in aqueous solution was fixed to the double stranded DNA by specific binding function of T-Hg2+-T and strand displacement reaction. The double stranded was cut into two segments of single stranded DNA by exonucleaseIII (Exo III), One part is used for circulation to amplify the signal, while the other part is used for hybridization with the substrate DNA, the rhodamine-labeled singal DNA were displaced down for the detection of fluorescence signal in the action of the polymerase. The fluorescence signals had a good linear with Hg2+ concentration in the range from 2.0×10-10 M to 1.0 ×10-9 M, reaching a detection limit of Hg2+ as low as 2.0×10-10 M. Moreover, the proposed strategy exhibited excellent sensitivity and selectivity, provided a new approach for the detection of Hg2+ in the environment.