Study Of Electrochemical Methods For Low-abundance Protein Quantification

Protein directly reflects life phenomena and the changes of protein content is closely related to life activities.Therefore,protein quantification is very important in life science and clinical diagnosis.However,the content of many proteins,especially biomarkers of diseases is extremely low.The existing methods for protein quantification are often powerless.In order to detect the low abundance protein,researchers need to develop sensitive protein analysis methods.Aiming to improve the sensitivity and simplify the operation steps,we combine the self-assembly(including DNA self-assembly and amyloid beta self-assembly)on the surface of electrode with sensitive electrochemical technology to develop ultrasensitive protein quantification methods,more details is as follows:1.A new assay for protein quantification based on DNA self-assemblyIn this work,we present an enzyme-free electrochemical assay based on DNA self-assembly at the surface of electrode.Our assay can detect target protein with ultra-high sensitivity through dual amplification.Firstly,target protein is specifically captured by its aptamer,and then is released due to toehold-mediated click chemical ligation(the first round of recycling)via DNA strand displacement reaction.Secondly,the overhang of aptamer on the electrode surface can hybridize with RP DNA and trigger DNA self-assembly(the second round of recycling).Consequently,large amount of electrochemical species hexaammineruthenium(?)chloride([Ru(NH3)6]3+)can be embedded into double-stranded DNA to produce a remarkable electrochemical signal,thus the target protein can be quantified with ultra-high sensitivity.Taken thrombin as a model analyte,a wide linear dynamic range from 100 fM to 10 nM can be obtained.Meanwhile,since no enzyme is required for the measurement,the assay is relatively simple and inexpensive.Therefore,the protein assay method proposed in this work may have a great potential for clinical diagnosis and biomedical research in the future.2.A new assay for protein quantification based on amyloid beta self-assemblyIn this work,a new strategy of biosensor design is developed based on the assembly of amyloid beta at the surface of electrode and its multiple interactions with other bioactive species.These interactions can enable amyloid beta peptide as a multifunctional sensing element,so the immobilization of sensing probe and the step-by-step modification of the sensing interface have all been dispensed with.Instead,the kinetics of the assembly of a peptide-based catalytic network serves to convert the quantity of analyte into amplified signal readout.Based on amyloid beta self-assembly,tubulin tyrosine ligase-like protein 12(TTLL12),a biomarker of prostate cancer can be determined in a liner range from 3 pM to 30 nM with the proposed method.This study may not only have a great potential for clinical diagnosis and biomedical research,but also point to the prospective of extending peptide self-assembly strategy to broader range of biosensing applications in the future.