Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol

Abstract

The sensing properties of immunosensors are determined not only by the amount of immobilized antibodies but also by the number of effective antigen-binding sites of the immobilized antibody. Protein A (PA) exhibits a high degree of affinity with the Fc part of IgG antibody to feasibly produce oriented antibody immobilization. This work proposes a simple method to control the PA surface density on gold nanostructure (AuNS)-deposited screen-printed carbon electrodes (SPCEs) by mixing concentration-varied PA and bovine serum albumin (BSA), and to explore the effect of PA density on the affinity attachment of anti-salbutamol (SAL) antibodies by electrochemical impedance spectroscopy. A concentration of 100 μg/mL PA and 100 μg/mL BSA can obtain a saturated coverage on the 3-mercaptoproponic acid (MPA)/AuNS/SPCEs and exhibit a 50% PA density to adsorb the amount of anti-SAL, more than other concentration-varied PA/BSA-modified electrodes. Compared with the randomly immobilized anti-SAL/MPA/AuNS/SPCEs and the anti-SAL/PA(100 μg/mL):BSA(0 μg/mL)/MPA/AuNS/SPCE, the anti-SAL/PA(100 μg/mL): BSA(100 μg/mL)/MPA/AuNS/SPCE-based immunosensors have better sensing properties for SAL detection, with an extremely low detection limit of 0.2 fg/mL and high reproducibility (<2.5% relative standard deviation). The mixture of PA(100 μg/mL):BSA(100 μg/mL) for the modification of AuNS/SPCEs has great promise for forming an optimal protein layer for the oriented adsorption of IgG antibodies to construct ultrasensitive SAL immunosensors.

A Label-Free Electrochemical Impedimetric Immunosensor with Biotinylated-Antibody for SARS-CoV-2 Nucleoprotein Detection in Saliva

“…The AuNS electrodeposition procedure of SPCEs refers to our previous studies [40][41][42]. Initially, the SPCEs were cleaned by a cyclic potential from 0 to 1.3 V for 20 cycles with a 0.1 V/s scanning rate and then oxidized at 2.0 V for 30 s in 0.1 M PBS (pH 7.0) with a three-electrode system using a Pt plate as the counter electrode and a commercial Ag/AgCl electrode as the RE.…”

“…Figure 1a−c show the random Ab immobilization on a short-chain MPA SAM, the oriented Ab immobilization on a MPA SAM, and a long-chain MUA SAM, respectively. The inset of Figure 1a shows the impedance spectra measured at the bare AuNS/SPCEs, which exhibited a dominant linear region, implying an apparent diffusion-controlled behavior, and a small semicircle region of kinetic control attributed to the fast electron transfer rate of the Fe(CN) 6 3−/4− mediator on the high conductive surface of AuNS/SPCEs [40][41][42]. Following MPA modification, the Nyquist plot showed a small linear part and a large semicircle part, implying a decreasing electron transfer rate.…”

“…The corresponding electric elements fitted by the modified Randles equivalent circuit are listed in Table 1. The modified Randles equivalent circuit, consisting of the solution resistance (R s ), the Warburg impedance (Z w ), the constant phase element (CPE), and the electron transfer resistance (R et ), was mentioned in our previous articles to explain the diffusive and kinetic behavior of the solution/electrode interface [40][41][42]. The mean error of all the fitting data was less than 0.3%.…”

“…It is worth noting that the EIS plot only presented a semicircle after the N-protein immunoreaction, implying a slow electron transfer rate to dominate the Faradaic reaction, with little diffusion-controlled behavior. In our previous studies [40][41][42], the 1R//C equivalent circuit, consisting of R s in a series with one parallel circuit comprising a R et and a CPE, was used to analyze the EIS spectrum of only the semicircle part. The corresponding circuit element values are statistically calculated as the mean ± standard deviation from three individual immunosensors in Table 1.…”

Current Advances in Immunoassays for the Detection of β2-Agonists

“…Highly sensitive label-free electrochemical immunosensors have been constructed by immobilizing anti-β 2 -agonist antibody on various types of electrodes which carry a gold nanostructure and other modifiers for detecting β 2 -agonist, such as salbutamol [99], ractopamine [100] and clenbuterol [101].The LOD was at pg/mL or fg/mL level. Cui et al [102] constructed a sandwich-type electrochemical immunosensor for detecting salbutamol.…”