Biopanning Of Vascular Endothelial Growth Factor VEGF165-specific Peptide And Its Electrochemical Sensing Application

As a vascular growth regulator,vascular endothelial growth factor(VEGF)plays an important role by specifically binding to receptors on vascular endothelial cells.Among the VEGF family,VEGF165 is widely recognized as a potential target for many malignancies.Since the mid-1980 s,phage display has been applied to drug development or disease diagnosis as a breakthrough technology.An exogenous gene encoding a specific protein or polypeptide is cloned into the appropriate location in the phage genome by genetic engineering techniques and co-expressed with the phage’s own genes,and the resulting recombinant protein is displayed on the phage surface.The peptides or antibodies screened via biopanning against different targets(antigens,viruses,pathogens,cancer cells)have been applied to antigen bioassay,pathogenic microorganism detection,drug delivery and tumor therapy.In this study,8 VEGF165-binding phage clones displaying different peptide sequences were obtained using a negative pre-screening strategy.Enzyme linked immunosorbent assay showed that phage W1(displaying peptide SPFLLRM)has affinity and specificity with a dissociation constant of 10.83±1.42 n M with VEGF165 antigen.Molecular docking simulations show that phage peptide interacts with VEGF165 by hydrogen bond.Using specific phage W1,an electrochemical impedance spectroscopy(EIS)immunosensor with phage-modified electrodes was constructed.By optimizing the experimental conditions,the linear range of the developed EIS immunosensor was 0.5～2000 pg/m L,and the detection limit was 0.15 pg/m L VEGF165.The immunosensor showed low response to high concentrations of unrelated antigens,indicating its good specificity.The immunosensor was placed in a 4 °C environment and measured every two days,which still exhibited good stability after 7 days.In addition,the developed phage-based EIS immunosensor was used for the detection of VEGF165 in human serum samples with satisfactory results.Due to its ultra-sensitivity,selectivity,batch reproducibility and stability,the screening-based selective phage EIS sensor has potential applications in diagnosis and therapy.