The Application Of M13 Phage In The Fluorescence Immunoassay For Alpha-Fetoprotein Detection

Phage display technology is a potential,high-throughput technology used for identifying peptide ligands for a given target.In recent years,biopanning for the peptide binding to a certain biomarker from phage display peptide library has attracted increasing attention,which can be utilized to detect a variety of biomarkers.AFP is a critical clinical marker for hepatocellular carcinoma screening.It is of great importance in establishing robust analytical method for AFP,which is also beneficial to humans’ health.In this study,by using phage library biopanning technique,AFP-binding phage was screened and enriched from dodecapeptide phage library through both positive screening and negative screening.A high affinity and specific AFP-binding peptide was obtained by phage capture assay and its peptide sequence was identified by DNA sequencing(sequence:GVLHMYDLPYNG).An AFP detection assay was further performed on the basis of sandwich immunoassay,in which the AFP antigen was captured by the immunomagnetic beads labeled with AFP antibody(the capture antibody).AFP-binding phage(the detection antibody)labeled with gold nanoparticles(AuNPs)was then added.After the immunoassay was finished,the formed sandwich immunocomplex was magnetically separated.The supernatant solution,which contained AuNPs-phage probes,was used to quench the fluorescence of fluorescein isothiocyanate(FITC)by the inner-filter effect(IFE)of AuNPs.The decrease of fluorescence intensity was proportional to the concentration of AuNPs-phage in the supernatant.The concentration of the AuNPs-phage in the supernatant was determined by the concentration of AFP,and the quantitative detection of the AFP was carried out indirectly with the extent of fluorescence quenching.Under the optimal conditions,the obtained fluorescent immunosensor can detect AFP over a linear range of 0.01-0.10 μg mL-1,with a limit of detection of 8 ng mL-1(3σ,n=7)and a RSD of 1.6%(n=7,0.05 μg mL-1).The selectivity test was performed to evaluate the specificity of the immunoassay for AFP compared with other biomarkers which might coexist with AFP in human serum.The experiment was performed under the same conditions,illustrating the high selectivity of the immunosensor to AFP.In addition,the immunosensor was evaluated by analyzing AFP content in human serum,and the recovery for AFP was 92.5-94.3%,indicating the established immunoassay possesses good specificity,repeatability and accuracy.The study showed great potential in the screening of cancer biomarkers and clinical diagnosis.