Study On Novel Methods Based On Target-Induced Ratiometric Electrochemical Detection

Accompanied by the rapid development of science and technology,the detection methods of life related substances such as protein,DNA and metal ions had been widely used in the respect of clinical diagnosis,environmental monitoring and food safety.However,these methods often have low sensitivity.Therefore,it is necessary to develop new methods and technologies for the rapid and sensitive sample analysis.In this dissertation,by combining the specificity of the aptamer,the high affinity of the DNAzyme for metal ions,the exonuclease ?-assisted amplification and the amplification of the bio-bar-coded nanoprobe with the traditional detection methods,a series of novel dual-signal ratiometric electrochemical sensor have been designed for selective detection of nucleic acid,protein and lead ion.The unique "one-to-two" signal output mode could effectively overcame the shortcomings such as "false positive" or "false negative" result,and the proposed electrochemical biosensor provided precise and sensitive measurement.The mean works include three chapters:1.A novel “signal-on/off” sensing platform for selective detection of thrombin based on target-induced ratiometric electrochemical biosensing and bio-bar-coded nanoprobe amplification strategyIn this work,a novel dual-signal ratiometric electrochemical aptasensor for highly sensitive and selective detection of thrombin has been designed on the basis of signal-on and signal-off strategy.Ferrocene labeled hairpin probe?Fc-HP?,thrombin aptamer and methyl blue labeled bio-bar-coded AuNPs?MB-P3-AuNPs?were rationally introduced for the construction of the assay platform,which combined the advantages of the recognition of aptamer,the amplification of bio-bar-coded nanoprobe,and the ratiometric signaling readout.Therefore,the recognition event of the target can be dual-signal ratiometric electrochemical readout in both the “signal-off” of MB and the “signal-on” of Fc.The proposed strategy showed a wide linear detection range from 0.003 to 30 nM with a detection limit of 1.1 pM.Moreover,it exhibits excellent selectivity,good stability,andacceptable reproducibility.By changing the recognition probe of the target molecule,this protocol could be easily expanded into the detection of other targets,showing promising potential applications in disease diagnostics and bioanalysis.2.Quantitative detection of Pb²⁺ based on the “signal-on/off” sensing platform and specific DNAzyme catalytic beacon.In this work,a ratiometric electrochemical sensor based on specific DNAzyme was designed for ultrasensitive detection of Pb²⁺.The sensor was developed on the basis of a“8–17” DNAzyme with high metal-binding affinity and specificity.The sensing platform was designed by self-assembly of methyl blue labeled probe?MB-P1?and ferrocene labeled probe?Fc-P2?.In the presence of Pb²⁺,DNAzyme was activated,and the trans-acting catalytic strand cleaves the sessile phosphodiester of the substrate into two fragments.All of this resulted in the release of MB-P1 chain and the departure of Fc-P2 chain from the electrode.The conformational change of MB-P1 chain caused an increase of MB signal?"signal-on"?and a decrease of Fc signal?"signal-off"?,which was directly related to the concentration of Pb²⁺.Therefore,quantitative detection of Pb²⁺ can be achieved by simultaneous occurrence of "signal-on" and "signal-off ".The linear range of the sensor is0.1 nM to 5 ?M and the detection limit is 45.8 pM?S/N=3?.Compared with other sensors,this sensor showed high selectivity,stability and reproducibility.The sensor is expected to be used in the field of environmental pollution and environmental monitoring.3.Ultrasensitive ratiometric electrochemical sensor for selective detection of DNA based on exonuclease ?-assisted recycling amplification strategy.In this work,A novel ratiometric electrochemical sensor?RES?had been developed to determine target DNA species related to oral cancer over expressed 1 in saliva,which combines the signal amplification of exonuclease ?-assisted target recycling amplification with the dual-signal output mode.The presence of the specific DNA target would hybridize with the specifically designed ferrocene-labeled hairpin probe?Fc-P1?and initiate exonuclease ?-aided target recycling amplification.Then,the remaining Fc-P1 hybridized with methylene blue-labeled hairpin probe?MB-PP1?modified on the electrode,which caused the MB tags away from the electrode surface while the Fc tags close to theelectrode surface,resulting in the oxidation current of Fc increased and that of MB decreased.Therefore,the recognition event can be translate into dual-signal ratiometric electrochemical readout.according to the ratio of the Fc and MB oxidation current,the proposed strategy showed a wide linear detection range from 0.02 pM to 2 nM with a detection limit of 12.8 fM.While the sensor without exonuclease ? showed a linear detection range from 0.02 nM to 160 nM with a detection limit of 17.7 pM.Thus,the nuclease-amplified?coupled with?dual-signal ratiometric readout extends the linear range for 1 order of magnitude,and the LOD value of the proposed strategy was also lower than that of no nuclease amplification.Besides,the assay also exhibits a high distinction ability to single-base mismatch,three-bases mismatch,and noncomplementary DNA sequence.This strategy possessed good capacity to integrate the amplification techniques with ratiometric output modes,and would open new opportunities for sensitive detection of other biorecognition events.