Studies On Signal Amplification Strategy Of Electrochemical Peptide Biosensor

Protease(also called a peptidase or proteinase)is any enzyme that performs proteolysis and protein catabolism byhydrolysis of peptide bonds.It is closely related to a large number of vital processes in the body?s metabolic processes,including cell growth,cell death,tissue remodeling,and immune defense,and also used as biomarkers.The development of reliable,cost-effective,accurate detection methods for biomarkers detection is of great significance clinical diagnosis for early discovery.Electrochemical biosensor has shown potential for biological molecules detection due to its inherent advantages such as simplicity,low cost,high sensitivity and easy miniaturization.They usually take antibody,enzyme,aptamer and peptide as molecular recognition elements.Among them,short binding peptides,which are selected using the phage display technique,have been used as a promising alternative to conventional antibody in bioassays due to their advantages,such as being reliable,cost-effective,stable,resistant to harsh environments,and more amenable to engineering at the molecular level than antibodies.This thesis selects peptide as molecular recognition elements by combining with various signal amplification strategies to construct high sensitive electrochemical peptide biosensors.The details are mainly as follows:The traditionally electrochemical peptide biosensor monotonously relied on target induced cleavage of the redox tag labeling peptide on an electrode,and such signal-off assays may have an intrinsic limitation on sensitivity and easily get false results as the true ones.Currently,metal nanoparticles have been widely used as sensitive labels for analytical signal amplification due to their unique properties,for example,it can overcome some of the problems associated with the inherent instability of biological materials such as enzymes.In chapter one,a sensitive electrochemical peptide biosensor for prostate-specific antigen(PSA)detection was developed based on positively charged gold nanoparticles(Au NPs)as signal enhancer.The positively charged AuNPs was modified on the peptide,which could adsorb the negatively charged redox probe [Fe(CN)6]3-/4-,leading to a low value of electro-transfer resistance.In the presence of target PSA,the peptide with positively charged AuNPs could be specifically cleaved and released from electrode surface,resulting in a significantly increased of electro-transfer resistance.By monitoring the changed electro-transfer resistance,PSA could be detected quantitatively.Under the optimal conditions,the proposed biosensor displayed a wide dynamic working range from 0.2?pg?m L-1 to 45?ng?m L-1 with a low detection limit of 0.06 pg?m L-1.Additionally,this method was based on the target-induced cleavage specific peptide for target detection with good specificity,so it also provided a promising strategy for the determination of other proteins that display similar properties.The transduction of peptide cleavage event into output DNA may be a fascinating approach because it enables further employment of many DNA-based amplification technologies such as hybridization chain reaction(HCR),rolling circle amplification and enzyme-assisted recycling amplification for sensitive detection of target protein.In the second work,we developed a novel electrochemical peptide biosensor for matrix metalloproteinases-2(MMP-2)detection by converting a peptide cleavage event into DNA detection with exonuclease III(Exo III)-assisted cycling for signal amplification.Compared with other protocols,our method has four significant advantages:(1)the ??signal-on?? assay can minimize false positive results which are caused by electrode stripping or fouling;(2)the approach by combining protein analysis with a DNA signal amplification strategy significantly enhances the detection sensitivity;(3)the adoption of the CB[7]/depAu modified electrode may greatly promote the signal acquisition for electrochemical detection,which not only takes advantage of good conductivity and large surface-to-volume ratio of depAu but also profits from the strong and selective supramolecular recognition ability of CB[7] to MB;and(4)this method is based on the target-induced cleavage specific peptide for MMP-2 detection,so it could be extended to the determination of other proteins that display similar propertiesy.The direct transduction of peptide cleavage event into DNA detection always produced output DNA with some amino acid residues,which influenced the DNA amplification efficiency in view of their steric hindrance effect.To solve the problem,in our third work,an ingenious two-stage DNA template was designed to achieve the highly efficient DNA amplification by utilizing DNA exponential amplification reaction(EXPAR)as model.The usage of two-stage DNA template not only accomplished the traditionally inefficient EXPAR triggered by output DNA with some amino acid residues,but also simultaneously produced a newly identical DNA trigger without any amino acid residues to induce an extra efficient EXPAR,which greatly improved the amplification efficiency and generated amouts of product DNA(S1).Then the yielded S1 as catalysts further initiated a catalyzed hairpin assembly(CHA)recycling on electrode surface to output an amplified electrochemical signal for quantitative detection of MMP-7.As a result,this developed assay demonstrated excellent sensitivity with a linear range from 0.1 pg·m L-1 to 50 ng·mL-1 and limit of detection down to 0.02 pg·m L-1.In this work,this system allowed the conversion of input target MMP-7 into output DNA in solution,which diminished complicated electrode fabrication process and improved the cleavage efficiency of target toward peptide substrate.Secondly,peptide cleavage triggered cascade signal amplification reaction including EXPAR and CHA,which not only had rapid amplification kinetics but also performed at constant temperature with great advantages in simplicity and cost effectiveness.Additionally,the method provided in this study can be successfully extended for the determination of other proteins that displayed enzymatic cleavage activity.