Studies On Electrochemical Biosensor Based On Nanomaterials And DNA Signal Amplification Technology

Due to the deterioration of the environment,the number of cancer patients in China is increasing every year,which is the main cause of deaths in China.It is reported that most early tumors have a high cure rate and probability of survival in a decade,and the cost of treatment is much smaller than that of advanced tumors.However,the early detection of cancer is difficult.On the one hand,early cancer patients usually have no abnormal feelings,which is difficult to diagnose and treat on time.On the other hand,because of the low expression factor of early tumor release,early examination requires high sensitivity.Electrochemical biosensors have significant advantages such as fast response,high specificity,high sensitivity,simple operation and low cost.They are widely used in environmental pollutant detection,drug screening and disease detection.In order to improve the sensitivity of the biosensor,a variety of signal amplification strategies and nanomaterials have been introduced to improve detection sensitivity.The specific work is as follows:1.An electrochemical biosensor combining target-triggered formation of DNAzyme with cascaded signal amplification.Although the enzyme-assisted signal amplification method can improve the sensitivity of the biosensor,they are highly dependent on the protein enzyme,which has inherent disadvantages such as the temperature and pH sensitivity,storage difficulties and reactiontime-dependent enzyme activity.In this work,a new enzyme-free isothermal amplification strategy for microRNA-21 detection based on target-triggered formation of DNAzyme and cascade signal amplification technology was built,using DNAzyme subunits as sensing elements for signal amplification.The enzyme-free amplification strategy eliminates the need for proteinases,making the system simpler,more stable,and less expensive.First,the helper DNA?Helper 1 and Helper 2?collectively recognizes the target to form a stable active DNAzyme 1,which continued to cleave multiple hairpin H1 substrates with concomitantly production of a large number of DNA fragments.The resulting fragment DNAzyme 2 can cleave the hairpin H2 substrate on the electrode,causing the labeled Fc leaving the electrode surface and the current signal is reduced.The cascaded signal amplification strategy significantly improves the sensitivity of the biosensors.The detection process is completed in one step,providing a new idea for constructing a simple,enzyme-free electrochemical sensor.2.An electrochemical biosensor based on 3W-CHA and dendritic DNA NanostructuresMedical research has shown that many human diseases are directly or indirectly related to the abnormal expression of specific genes.At present,about 1000 genetic diseases can be diagnosed by genetic testing,especially some malignant tumors with high incidence.However,due to the large number of gene mutations,the difficulty in detecting specific gene mutations is greatly increased.In this study,we proposed a biosensor based on 3W-CHA signal amplification strategy and dendritic DNA structure for gene detection.On the basis of catalytic hairpin assembly?CHA?,the direct detection of the target T is replaced by indirect detection of the T-TP hybridization chain.The detection selectivity can be double assured by two specific reactions,T-TP recognition and T-TP-CHA reaction,which greatly improves the resolution of alleles.Subsequently,an ordered dendritic DNA nanostructure is formed on the electrode.The dendritic DNA nanostructure on the electrode greatly hinders electron transfer,causing impedance change on the electrode surface.The biosensor was detected by electrochemical impedance spectroscopy?EIS?method,avoiding the complicated marking process and effectively simplifying the experimental steps.This method provides a new way to construct a signal amplifying impedance sensor.3.An enzyme-free electrochemical biosensor combining target recycling with Fe₃O₄/CeO₂@Au nanocatalysts for microRNA-21 detectionIn this study,an electrochemical biosensor was proposed for microRNA-21detection based on Fe₃O₄/CeO₂@Au magnetite nanoparticles?Fe₃O₄/CeO₂@Au MNPs?as nanocatalyst and catalytic hairpin assembly?CHA?for signal application.Firstly,target microRNA-21 hybridized with hairpin H₂ to form H₂-T duplex stranded DNA dsDNA.Simultaneously,the Fe₃O₄/CeO₂@Au-S₁ not only hybridized with single stranded fragment of H₁-H₂ dsDNA with producing long dsDNA to absorb a large amount of electroactive substances of methylene blue?MB?,but also acted as nanocatalyst to directly catalyze the reduction of MB for amplifying the electrochemical signal.Herein,compared with pure Fe₃O₄ nanoparticles,Fe₃O₄/CeO₂@Au MNPs exhibited excellent catalytic performance since the cerium oxide?CeO₂?nanoparticles and Au nanoparticles can greatly improve the catalytic activity of Fe₃O₄ nanoparticles and effectively prevent the agglomeration of Fe₃O₄ nanoparticles.The proposed biosensor exhibited excellent specificity and sensitivity.This strategy provided a novel avenue for the detection of other biomarkers in electrochemical biosensors..