Study On Novel Electrochemical Sensing Methods For Biomarkers

In recent years,cancer and chronic diseases have become the main killers of human health due to factors such as lifestyle,diet,and environment.Therefore,it is of great biomedical significance to develop disease-related biomarker analysis to provide reliable and powerful detection and monitoring strategies for disease diagnosis and prognosis.However,the bio-molecular concentration in the actual biological sample has a wide dynamic range,while the disease-related biomarkers tend to be low-abundance.Therefore,developing new analysis strategies for low-abundance biomolecules is of great scientific significance to achieve rapid,sensitive and accurate analysis,reveal the law of life activities,and guide the diseases diagnosis.Electrochemical biosensors have become one of the most promising analytical techniques in the field of life analysis and broad application prospects in biomarker analysis due to the property of simple equipment,convenient operation,fast analysis and high throughput.In this thesis,based on the structural characteristics,recognition characteristics,enzyme amplification,nucleic acid recognition and assembly principle of biological nano-probe,the identification probe is designed reasonably and a new signal amplification mode is developed.A new platform for specific recognition and signal transduction was constructed,and a series of novel biosensor strategies for biomarkers related to diseases,such as proteins and nucleic acids,were developed.The main content includes the following three chapters:1.A versatile label-free electrochemical biosensor for circulating tumor DNA based on dual enzyme assisted multiple amplification strategyA versatile label-free electrochemical biosensor based on dual enzyme assisted multiple amplification strategy was developed for ultrasensitive detection of circulating tumor DNA?ctDNA?.The biosensor consists of a triplehelix molecular switch as molecular recognition and signal transduction probe,ribonuclease HII?RNase HII?and terminal deoxynucleotidyl transferase?TdT?as dual enzyme assisted multiple amplification accelerator.The presence of target ctDNA could open THMS and trigger RNase HII-assisted homogenous target recycling amplification to produce substantial signal transduction probe?STP?.The released STP hybridized with the capture probe immobilized on a gold electrode,then TdT and assistant probe were further employed to fulfill TdT-mediated cascade extension and generate stable DNA dendritic nanostructures.The electroactive methyl blue?MB?was finally used as the signal reporter to realize the multiple electrochemical amplification ctDNA detection as the amount of MB is positively correlated with the target ctDNA.Combined with the efficient recognition capacity of the designed THMS and the excellent multiple amplification ability of RNase HII and TdT,the constructed sensing platform could detect KRAS G12DM with a wide detection range from 0.01 fM to 1 pM,and the limit of detection as low as 2.4 aM.Besides,the platform is capable of detecting ctDNA in biological fluid such as plasma.More importantly,by substituting the loop of THMS with different sequences,this strategy could be conveniently expanded into the detection of other ctDNA,showing promising potential applications in clinical cancer screening and prognosis.2.Electrochemical detection platform for phosphoprotein based on Pt/UiO-66An electrochemical sensing platform was constructed based on functionalized Pt/UiO-66composites.Pt/UiO-66 composites were synthesized with excellent electrocatalytic activity of Pt nanoparticles and better phosphoprotein enrichment ability of UiO-66.The morphology,microstructure and thermal stability of materials were characterized by TEM,SEM,XRD,TGA and Nitrogen adsorbed-desorbed test,which confirmed that Pt/UiO-66had uniform surface distribution,good thermal stability and chemical stability.UiO-66 in the composites could identify and enrich phosphoprotein specifically.Meanwhile,the enriched phosphoprotein could cover the Pt nanoparticles on the surface of the material and affect the electrocatalytic activity for the reduction of H₂O₂,which realize the quantitative analysis strategy of electrochemical phosphoprotein.The experimental results show that the sensing platform has good selection specificity for phosphoprotein?-Casein and?-Casein.The sensing platform has a linear range of 0.1ng ml^-1-1 mg ml^-11 and 0.01 ng ml^-1-0.1 mg ml^-1,respectively.This strategy provides a good method for quantitative analysis of phosphorylated proteins and is expected to have potential in phosphorylation proteomics and clinical marker analysis.3.A novel homogeneous ratiometric electrochemical biosensor for circulating tumor DNA based on buoyancy separation and T7 Exo assisted amplification strategyBased on the functionalized buoyancy microsphere probe and T7 exozyme?T7 Exo?assisted amplification strategy,a highly sensitive homogeneous ctDNA proportional electrochemical sensing platform was designed.The strategy constructed a buoyant microsphere firstly based on the recognition probe barcode.The presence of the target molecule triggers the release of the microsphere load capture probe?CP-Fc?and achieves target circular amplification under the action of T7 Exo.The subsequently added hairpin probe?HP-MB?will hybridize with the bare auxiliary probe?AP?in the buoyancy microspheres.And finally,a homogeneous proportional signal amplification system will be triggered under the action of T7 Exo,which exhibits a certain correlation between I_Fc/I_MBB and the concentration of the target molecule,enabling ultrasensitive detection of the target ctDNA.The sensing platform enables highly sensitive detection of ctDNA PIK3CA E542KM with detection range of 0.01 fM-1 pM and detection limit as low as 9.23 aM.The advantages of this strategy are summarized as follows:?I?Flotation separation and homogeneous non-interface recognition system avoid the cumbersome steps of electrode interface modification,maximize the spatial freedom of biomolecules,which is conducive to the realization of efficient molecules identifying.?II?Double-signal reporting result mode can effectively avoid false-positive and false-negative signals.?III?The introduction of flotation separation concept is expected to realize the concept of minimalist detection without external force,and to be quickly promoted to Point-of-Care detection field,which provides a new approach to electrochemical sensing.