The Research Of Biosensor Based On DNA Isothermal Signal Amplification For Heavy Metal Ions Detection In Water Environment

With the increase in human activities such as industry,agriculture and life,the environmental enrichment of heavy metal ions have intensified,and human health have been seriously threatened.Therefore,in order to effectively control the harm of heavy metal ions pollution,a series of traditional analysis techniques,such as atomic absorption spectrometry（AAS） and atomic fluorescence spectrometry（AFS）,have been massively applied in actual water detection.However,the instruments required by these methods are relatively precise,the operation processes are cumbersome and need the professional scientific research personnel to operate.It’s impossible to be carried out in actual on-site inspection.Hence,high specificity,low detection limit,and low cost analysis technologies are urgently needed to make up for this deficiency.In this context,the biosensors with excellent performances,such as convenient,efficient,and low cost,have attracted wide attention of researchers.This study have constructed three highly sensitive and high identification biosensors using the specific T-Hg²⁺-T structure for mercury ion(Hg²⁺) and the“8-17”DNAzyme corresponding to lead ion(Pb²⁺) combined with DNA isothermal signal amplification technologies,and been successfully applied in the detection of trace heavy metal ions in the natural water samples.The main research content is as follows:Firstly,the specific T-Hg²⁺-T structure was used as the recognition element of the target mercury ions,and a colorimetric biosensor for mercury ions based on strand displacement reaction and exonuclease Ⅲ has been constructed in a pure homogeneous environment.In this working system,the stable hybrid double strand consisted of a thymine-rich single strand and the other single strand that could bind to the gold nanoparticles-modified short DNA strand.In the presence of mercury ions,another thymine-rich single chain was hybridize with the double strand undergo the strand displacement reaction through a mismatched T-Hg²⁺-T structure,leading the exonuclease Ⅲ-induced dual signal amplification process.As the concentration of mercury ions increased,the short DNA strand on the surface of the gold nanoparticles was gradually reduced,and the distance between the particles was narrowed.Due to the surface plasmon resonance effect of the gold nanoparticles,the color of the sample solution gradually changed from wine red to blue.In the absorption spectrum,the absorption wavelength displayed an obvious red shift and the absorbance was gradually reduced.Compared with the reported analysis method for mercury ions,the designed colorimetric biosensor had a wider detection range（1 nM to 10μM）,a lower limit of detection（0.90 nM）,a faster detection time（30 minutes）,and the relatively simpler operation（one step）,and it could be used in the detection and analysis of trace mercury ions in actual water.Secondly,the interface of the reaction was transferred from the pure homogeneous phase to the electrode surface,and the exonuclease Ⅲ with good digestion activity was continue to employed to construct a labeled electrochemical biosensor for mercury ions.One of the DNA strands that used to form the specific T-Hg²⁺-T recognition structure was subtly designed into a hairpin configuration（the tail contained a large number of thymine）.After the addition of mercury ions,the tail of the hairpin was hybridize with the single strand through the T-Hg²⁺-T structure,inducing the exonuclease Ⅲ-assisted dual signal amplification process.Eventually,a large number of methylene blue were far away from the electrode surface and the electrochemical signal was greatly reduced.By optimizing the experimental parameters,the limit of detection of the electrochemical biosensor for target mercury ions was as low as 0.227nM（ranging from 500 pM to 5μM）,the reproducibility and stability were improved,and the accuracy of detection in the actual water samples was also in good agreement with the atomic fluorescence spectrometry（AFS）.Therefore,the target-induced and enzyme-assisted labeled electrochemical biosensing platform has the potential to detect and analyze actual water samples.Thirdly,following the electrochemical biosensing detection technology of the second work,an enzyme-free and non-labeled electrochemical biosensor for lead ions detection has been constructed based on the catalytic hairpin assembly and the bridging function of gold nanoparticles.The substrate chain of the“8-17”DNAzyme was designed to be a stable hairpin configuration（the cleavage site was located in the loop region of the hairpin）,and the tail end was conjugated to the gold nanoparticles via 10 adenine（A）.The lead ion could cleave the substrate chain into two single DNA strands（one single strand was liberated,and the other single strand was continue to be attached on the surface of the gold nanoparticles）.The liberated strand was alternately hybridize with three hairpin probes that in a metastable state to complete the catalytic hairpin assembly process,forming a branched DNA polymer that could bind to the single DNA strand that modified on the surface of gold nanoparticles,to generate a three-dimensional DNA/gold nanoparticles network system and immobilized on the electrode.These negatively charged DNA strands on the electrode surface were capable of adsorbing positively charged hexaammineruthenium（Ⅲ） chloride by electrostatic interaction,resulting in a strong electrochemical signal.Compared to the previously reported assays for the detection of lead ions,the electrochemical biosensors using the“8-17”DNAzyme-induced triple signal amplification strategy had a limit of detection up to 0.095 nM for the target lead ions ranging from 100 pM to 5μM.Therefore,without the participation of nucleases,the designed lead ion-specific label-free electrochemical biosensor provided a new powerful platform for the analysis and detection of trace lead ions in actual water samples.