Electrochemical Immunoassay Platform for Human Monkeypox Virus Detection

Abstract

In this study, we reported a selective impedimetric biosensor for the detection of A29 which is the target protein of the monkeypox virus (MPXV). The working principle of the biosensor relies on the interaction mechanism between A29, which is an internal membrane protein of MPXV, and the heparan sulfate receptor. For this purpose, after immobilizing heparan sulfate onto the gold screen-printed electrode surface, its interaction with A29 protein was monitored using electrochemical impedance spectroscopy. After the optimization of experimental parameters, the analytical characteristics of the developed MPVX immunosensor were examined. The developed immunosensor exhibited a linear detection range between 2.0 and 50 ng mL^-1, with a detection limit of 2.08 ng mL^-1 and a quantification limit of 6.28 ng mL^-1. Furthermore, a relative standard deviation value of 2.82% was determined for 25 ng mL^-1. Apart from that, sample application studies were also performed with the standard addition of A29 protein to 1:10 diluted real serum samples that were taken from healthy individuals, and very good recovery values were obtained.

Scheme 1. Schematic Representation of the Fabrication of the Developed MPXV Immunosensor

Figure 1

SEM (a–c) and AFM (d–f) images of bare AuSPE, HS@AuSPE, and A29@HS@AuSPE, respectively.

Figure 2

Nyquist diagrams of resistance measurements and their equivalent circuit (A) and CV voltammogram (B) of each preparation stage of the developed electrochemical MPXV biosensor. The frequency was from 0.1 Hz to 100 kHz at 0.1 V potential for EIS, and the working potential range for CV was between −0.8 and 1.0 V with 0.1 mV s^–1 scan rate. The measurements were taken in the presence of 70 μL of a 5 mM [Fe(CN)₆]^3–/4– redox probe in 0.1 M phosphate buffer solution (pH: 7.4). R_S is the resistance of the electrolyte solution. For the equivalent circuit, R_CT is the electron-transfer resistance of the electrode/electrolyte interface, CPE is the constant phase element, and W is the Warburg impedance for semi-infinite diffusion.

Figure 3

Nyquist diagrams (A) and impedance difference excel plot (B) of HS amount (1.0, 2.0, 5.0, and 10.0 ng mL^–1) optimization in terms of resistance difference. Closer demonstrations of impedance differences for different HS amounts (A1–A4). Representation of the equivalent circuit for the optimum condition of 2 ng/mL HS. For the equivalent circuit, R_S is the resistance of the electrolyte solution, R_CT is the electron-transfer resistance of the electrode/electrolyte interface, CPE is the constant phase element, and W is the Warburg impedance for semi-infinite diffusion (C). ΔR: Resistance difference. All other experimental conditions as in Figure 2

Figure 4

Nyquist diagrams (A) and impedance difference excel plot (B) of HS and A29 protein incubation time optimization studies (10, 20, 30, and 60 min). Closer demonstrations of differences in EIS diagrams for HS and A29 protein interaction (A1–A4). Representation of the equivalent circuit for the optimum condition of 30 min incubation time. For the equivalent circuit, RS is the resistance of the electrolyte solution, RCT is the electron-transfer resistance of the electrode/electrolyte interface, CPE is the constant phase element, and W is the Warburg impedance for semi-infinite diffusion (C). ΔR: Resistance difference. All other experimental conditions as in Figure 2

Figure 5

Nyquist diagrams (A) and LOD value included calibration curve (B) of the developed electrochemical MPXV biosensor with increasing A29 protein concentrations (2.0, 5.0, 7.5, 12.5, 25.0, and 50.0 ng mL^–1). Closer demonstration of impedimetric responses for various A29 concentrations (2.0–50 ng mL^–1) (A1–A6). ΔR: Resistance difference. All other experimental conditions are listed in Figure 2

Figure 6

Nyquist diagrams of 1:1 (A) and 1:2 (B) ratios of interferent cocktails for interference studies. All other experimental conditions as in Figure 2

Figure 7

Sample application studies of the developed monkeypox electrochemical immunosensor. Nyquist diagrams (a) and excel graph (b) for standard addition of 7.5, 12.5, 25, and 50 ng mL^–1 A29 protein to 1:10 diluted human serum samples. ΔR: Resistance difference. Working conditions are as in Figure 2