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.

Scheme 1

Schematic representation of (A) the preparation of bioconjugates, and (B) ELISA and measurement procedure.

Figure 1

Transmission electron micrographs images of AuNPs (A) and Ab₂/glucoamylase/AuNPs (B).

Figure 2

(A) Absorbance readings taken at 595 nm, following Coomassie Brilliant Blue G-250 staining, to observe the amount of Ab₂ in pre-labeled and post-labeled Ab₂ solutions. (B) Absorbance readings taken in the UV-Vis spectra (274 nm), for the detection of binding of glucoamylase to AuNPs.

Figure 3

(A) Optimal pH for the conjugation of glucoamylase and Ab₂ to AuNPs. (B) Effect of glucoamylase/Ab₂ ratio on response current. (C) Effect of incubation time on the DPV peak current.

Figure 4

(A) DPV curves acquired at different concentration of glucose produced by Ab₁/AFP/Ab₂-AuNPs-glucoamylase, incubated with (a) 0, (b) 0.05, (c) 0.1, (d) 1, (e) 2, (f) 5, (g) 10, (h) 20, (i) 50, (j) 100 ng/mL AFP antigen. (B) Calibration curves for detecting AFP antigen were obtained using Ab₂-glucoamylase (a) and Ab₂-AuNPs-glucoamylase (b) as the signal tag, respectively. Inset: linear relationship between the amperometric oxidation current response and the logarithm of AFP concentrations.

Figure 5

Selectivity of the electrochemical ELISA system to AFP (5 ng/mL) by comparing it to the interfering protein: glucose (50 ng/mL), carcinoembryonic antigen (CEA, 50 ng/mL), hepatitis B virus surface antigen (HBsAg, 50 ng/mL), L-cysteine (50 ng/mL), bovine serum albumin (BSA, 50 ng/mL), and the mixed sample containing 5 ng/mL AFP, 50 ng/mL glucose, 50 ng/mL CEA, 50 ng/mL HBsAg, 50 ng/mL L-cysteine and 50 ng/mL BSA.