An Ultrasensitive Electrochemical Immunosensor for Alpha-Fetoprotein Using an Envision Complex-Antibody Copolymer as a Sensitive Label

Abstract

A novel strategy is presented for sensitive detection of alfa-fetoprotein (AFP), using a horseradish peroxidase (HRP)-functionalized Envision antibody complex (EVC) as the label. The Envision-AFP signal antibody copolymer (EVC-AFP Ab2) was composed of a dextran amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of secondary antibody, and acted as a signal tag in the immunosensor. The sensor was constructed using the following steps: First, gold electrode (GE) was modified with nano-gold (AuNPs) by electro-deposition in HAuCl4 solution. The high affinity of the AuNPs surface facilitates direct formation of a self-assembled thiolated protein G layer. Next, the coated GE was incubated in a solution of AFP capture antibody (AFP Ab1); these antibodies attach to the thiolated protein G layer through their non-antigenic regions, leaving the antigen binding sites for binding of target analyte. Following a sandwich immunoreaction, an EVC-AFP Ab2-AFP-AFP Ab1 immunocomplex was formed on the electrode surface, allowing large amounts of HRP on the complex to produce an amplified electrocatalytic current of hydroquinone (HQ) in the presence of hydrogen peroxide (H2O2). Highly amplified detection was achieved, with a detection limit of 2 pg/mL and a linear range of 0.005–0.2 ng/mL for AFP in 10 μL undiluted serum; this is near or below the normal levels of most cancer biomarker proteins in human serum. Measurements of AFP in the serum of cancer patients correlated strongly with standard enzyme-linked immunosorbent assays. These easily fabricated EVC-modified immunosensors show excellent promise for future fabrication of bioelectronic arrays. By varying the target biomolecules, this technique may be easily extended for use with other immunoassays, and thus represents a versatile design route.

Current Technologies of Electrochemical Immunosensors: Perspective on Signal Amplification

“…One of the methods is to couple several enzymes to a detection antibody using branch-like functionalized structures (e.g., amine-terminated dextran). Xiong et al proposed an antibody-enzyme network structure composed of a dextran-amine skeleton anchoring more than 100 and 15 molecules of HRP and secondary antibody, respectively [ 90 ]. This allowed many enzyme molecules to participate in the binding event, resulting in signal enhancement significant—enough to detect a target analyte that exists in extremely low concentrations.…”

“…This type of enzyme-based signal amplification can be further improved by modulating the redox cycling associated with electron mediators such as ferrocene [ 91 ] and hydroquinone [ 90 ] as shown in Table 2 . Ferrocene transfers one electron to the electrode by a simple redox reaction.…”

Recent Progress in Nanomaterial-Based Electrochemical Biosensors for Cancer Biomarkers: A Review

“…A sandwich immunoassay using anti-AFP conjugated with horseradish peroxidase (HRP) could detect AFP in serum in the range of 5–80 ng mL −1 . A similar protocol provided highly sensitive AFP sensors that showed response to AFP in the range of 0.005–0.2 ng mL −1 [ 32 ]. In another study, impedimetric immunosensors for epidermal growth factor receptor (EGFR) were studied by using an AuNP-modified Au electrode [ 33 ].…”

A novel sandwich-type noncompetitive immunoassay of diethylstilbestrol using β-cyclodextrin modified electrode and polymer–enzyme labels

“…Polymerization-assisted amplification is another strategy that is used for fabricating sensitive labels [31]. EnVision™ polymer reagent (EV) is a polymerase chelate probe containing a poly-dextrin amine skeleton that is made of 100 molecules of HRP and 15 molecules of anti-IgG antibody [32,33]. After EV is incubated with the DES antibody (DES Ab), the resulting copolymers (EV/DES Ab) are considered as the signal tag of the ECI sensor.…”