title: Electrochemical enzyme-linked immunosorbent assay (ELISA) for α-fetoprotein based on glucose detection with multienzyme-nanoparticle amplification.
abstract: Since glucose biosensors are one of the most popular and widely used point-of-care testing devices, a novel electrochemical enzyme-linked immunosorbent assay (ELISA) for protein biomarkers has been developed based on a glucose detection strategy. In this study, α-fetoprotein (AFP) was used as the target protein. An electrochemical ELISA system was constructed using anti-AFP antibodies immobilized on microwell plates as the capture antibody (Ab1) and multi-label bioconjugates as signal tracer. The bioconjugates were synthesized by attaching glucoamylase and the secondary anti-AFP antibodies (Ab2) to gold nanoparticles (AuNPs). After formation of the sandwich complex, the Ab2-glucoamylase-AuNPs conjugates converted starch into glucose in the presence of AFP. The concentration of AFP can be calculated based on the linear relation between AFP and glucose, the concentration of which can be detected by the glucose biosensor. When the AFP concentration ranged from 0.05 to 100 ng/mL, a linear calibration plot (i (µA) = 13.62033 - 2.86252 logCAFP (ng/mL), r = 0.99886) with a detection limit of 0.02 ng/mL was obtained under optimal conditions. The electrochemical ELISA developed in this work shows acceptable stability and reproducibility, and the assay for AFP spiked in human serum also shows good recovery (97.0%-104%). This new method could be applied for detecting any protein biomarker with the corresponding antibodies.
authors.affiliation: Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China. ganning@nbu.edu.cn.
authors.forename: Qin-Lan
authors.initials: QL
authors.lastname: Liu
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Yan
authors.forename: Xiao-Mao
authors.initials: XM
authors.lastname: Yin
authors.forename: Bo
authors.initials: B
authors.lastname: Situ
authors.forename: Han-Kun
authors.initials: HK
authors.lastname: Zhou
authors.forename: Li
authors.initials: L
authors.lastname: Lin
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.forename: Ning
authors.initials: N
authors.lastname: Gan
authors.forename: Lei
authors.initials: L
authors.lastname: Zheng
mesh_terms: D015415:Biomarkers / Q000737:chemistry; D015374:Biosensing Techniques; D001774:Blood Chemical Analysis; D001786:Blood Glucose; D002272:Carcinoembryonic Antigen / Q000097:blood; D003545:Cysteine / Q000097:blood; D055664:Electrochemical Techniques; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry*; D006514:Hepatitis B Surface Antigens / Q000097:blood; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053768:Metal Nanoparticles / Q000737:chemistry*; D012015:Reference Standards; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000737:chemistry; D000509:alpha-Fetoproteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D001786:Blood Glucose; D002272:Carcinoembryonic Antigen; D004800:Enzymes, Immobilized; D006514:Hepatitis B Surface Antigens; D000509:alpha-Fetoproteins; D012710:Serum Albumin, Bovine; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose; D003545:Cysteine