Study On The Construction Of Ratiometric Electrochemical Sensor And Its Application In Detection Of Biomarkers

With the continuous development of life science research,the biological functions of more and more biomolecules have been rapid revealed,such as proteins and nucleic acids.The physiological expression level of these functional biomolecules is treated as an important basis and reference index for large-scale disease screening,clinical diagnosis,dynamic monitoring and prognostic evaluation.However,actual biological samples are usually complex mixtures,disease marker biomolecules often exhibit low abundance.Therefore,the development of new strategies for functional biomolecular analysis with high sensitivity and selectivity,especially for the analysis and detection of disease-related biomarkers at low physiological levels,is of great research value in the fields of life science,biomedical research and clinical diagnosis of diseases.In this thesis,micro RNA（mi RNA）and matrix metalloproteinase（MMP）were used as the research objects,and the signal amplification methods such as enzyme-assisted target recycling,DNA hairpin assembly,host-guest interaction and nanotechnology were skillfully combined with the dual signal output mode to successfully construct a new platform for homogeneous target recognition and interface signal output separation.And realize the highly selective and sensitive detection of disease-related biomarkers.It mainly includes the following two parts:1.A novel ratiometric electrochemical biosensing strategy based on T7exonuclease-assisted homogenous target recycling coupling hairpin assembly triggered double-signal output for multiple amplified detection of mi RNAA novel ratiometric electrochemical biosensing strategy based on T7 exonuclease（T7Exo）-mediated homogenous target recycling coupling hairpin assembly triggered dual-signal output was designed for accurate and sensitive detection of micro RNA-141（mi RNA-141）.Concretely,with the presence of target mi RNA-141,abundant signal transduction probe（STP）were released via the T7 Exo assisted homogenous target recycling amplification,which could be captured by the specially designed ferrocene labeled hairpin probe（Fc-H1）on electrode interface and trigger the nonenzymatic catalytic hairpin assembly（Fc-H1+MB-H2）to realize the cascade signal amplification and dual-signal elements output.Through such a conformational change process,the electrochemical signal of ferrocene（Fc）and methylene blue（MB）is proportionally and substantially decreased and increased.Therefore,the signal ratio of I_MB/I_Fccan be used to accurately reflect the true level of initial mi RNA-141.Benefiting from the efficient integrating T7 Exo-assisted homogenous target recycle and nonenzymatic hairpin assembly,and dual-signal output mode.The proposed sensor could realize effectively amplified detection of mi RNA-141,which have a wide detection range from 1 f M to 100 p M and a detection limit of 200 a M.Furthermore,it exhibits outstanding sequence specificity to discriminate mismatched RNA,acceptable reproducibility and feasibility for real sample.This strategy effectively integrated the advantages of multiple amplification and ratiometric output modes,which could provide an accurate and efficient method in biosensing and clinical diagnosis.2.A novel ratiometric electrochemical biosensing strategy based on homogeneous recognition coupling host and guest interactions for highly sensitive detection of MMP-2A highly sensitive ratiometric metalloproteinase 2（MMP-2）electrochemical biosensor was constructed based on the functionalized magnetic beads mediated homogeneous recognition and liposome signal amplification coupling the dual-signal output triggered by the competitive host-guest interaction between cyclodextrin and electroactive molecule.The presence of target MMP-2 could trigger the phospholipase（PLA2）modified MMP-2substrate peptide functionalized magnetic nanoparticles release of PLA2,destroy the liposome phospholipid bilayer encapsulating the electroactive molecule bisphenol A（BPA）and release a large amount of transduction molecule BPA.As a result,the"one-to-many"amplification from the target MMP-2 to the signal transduction molecule BPA was realized.On the other hand,β-CD is electropolymerized on the electrode interface modified with high-efficiency charge transfer capability Pt Ga NRs@r GO nanocomposites,and the electroactive molecule Rhodamine B（Rh B）can enter into the hydrophobic inner cavity ofβ-CD.The presence of the signal transduction molecule BPA could displace Rh B because the host-guest interaction betweenβ-CD and BPA is stronger than that betweenβ-CD and Rh B,and the oxidation peak current of Rh B(I_{Rh B})decreases and the oxidation peak current of BPA(I_BPA)increases correspondingly,which finally make I_BPA/I_{Rh B}and the target MMP-2’s concentration shows a certain correlation.The proposed strategy showed a wide linear detection range from 1 fg/m L to 0.1 ng/m L with a detection limit of 490 ag/m L.More importantly,by substituting the substrate on the magnetic nanoparticles,the sensing platform could be conveniently expanded into the detection of other MMPs.Therefore,this strategy proposed a universal ratiometric electrochemical sensing mechanism for biomolecule detection,which showing promising potential application in universal molecular diagnosis.