A Cell-Based Electrochemical Biosensor for the Detection of Infectious Hepatitis A Virus

Abstract

Hepatitis A virus (HAV), a major cause of acute liver infections, is transmitted through the fecal-oral route and close contact with infected individuals. Current HAV standardized methods rely on the detection of virus antigen or RNA, which do not differentiate between infectious and non-infectious HAV. The objective of this study was to develop a prototype cell-based electrochemical biosensor for detection of infectious HAV. A cell culture-adapted HAV strain (HM175/18f) and its permissive cells (FRhK-4), along with gold nanoparticle-modified screen-printed electrodes, were used to develop the biosensor. Electrochemical impedance spectroscopy was used to quantify the electrical impedance signal. Nyquist plots showed successful fabrication of the cell-based biosensor. The optimum period of HAV incubation with the biosensor was 6 h. A significant linear relationship (R² = 0.98) was found between the signal and a 6-log range of HAV titers, with a limit of detection of ~5 TCID₅₀/mL (tissue culture infectious dose). The biosensor did not detect non-target viruses such as feline calicivirus and human coronavirus 229E. The biosensor was stable for 3 to 7 days at an abusive temperature (37 °C), retaining ~90 to 60% of the original signal, respectively. In conclusion, this prototype cell-based biosensor is capable of rapidly detecting low levels of infectious HAV.

Keywords: FRhK-4 cells; Hepatitis A virus; cell-based biosensors; foodborne outbreaks; impedance spectroscopy; infectious viruses; infectivity assay; screen-printed electrodes.

Figure 1

Electrode preparation steps, including the drop-cast deposition of the gold nanoparticles (AuNP) on the bare screen-printed electrodes (SPEs), followed by the immobilization of FRhk-4 cells and subsequent testing with hepatitis A virus (HAV).

Figure 2

Nyquist plot of the electrode after each step of the electrode preparation process and the response of the biosensor to incubation with HAV for 6 h.

Figure 3

Optimization of HAV incubation time with a FRhK-4 cell-based biosensor. The % signal change detected by electrochemical impedance spectroscopy (EIS) after various HAV incubation times: 0 (immediate), 0.5, 1, 2, 4, 6, and 12 h. Data are presented as the mean ± standard deviation (SD). Statistical significance was determined using an ANOVA. Asterisks indicate significant differences at * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Sensitivity of the cell-based biosensor for detecting various HAV titers. The graph shows the relationship between the change in charge transfer resistance (ΔR_CT) and HAV titers. The linear regression equation was found to be y = 156.1x − 65.872 with an R² value of 0.9889 (p < 0.0001).

Figure 5

Specificity of the cell-based biosensor for detecting infectious Hepatitis A virus (HAV) compared to non-target viruses: feline calicivirus (FCV) and human coronavirus (HCoV 229E) used at 10² TCID₅₀/mL. Data are presented as the mean ± standard deviation (SD). Statistical significance was determined using an ANOVA. Asterisks indicate significant differences at **** p < 0.0001.

Figure 6

Morphological changes in FRhK-4 cells as observed under the microscope at 10× magnification. The cells were incubated with 10² TCID₅₀/mL infectious hepatitis A virus (HAV), feline calicivirus (FCV), and human coronavirus (HCoV 229E) over 7 days at 37 °C. The control cells were FRhK-4 cells without any added viruses.

Figure 7

Comparison of the ΔR_CT values of various temperature treatments for HAV (6.32 × 10⁴ TCID₅₀/mL): 4 °C for 60 min (control infectious HAV), 60 °C for 60 min (partially infectious HAV), and 100 °C for 10 min (non-infectious HAV). Data are presented as the mean ± standard deviation (SD). Statistical significance was determined using an ANOVA. Asterisks indicate significant differences at * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 8

Stability of the cell-based biosensor for detecting infectious HAV. (a) The cell-based biosensor was stored in DMEM media over a 14 day-period inside a 37 °C incubator, then used for HAV detection on days 0, 3, 7, 10, and 14. (b) The AuNP-modified electrodes were stored at room temperature over a 14 day-period, but was modified with FRhK-4 cells on days 0, 3, 7, 10, and 14, followed by the addition of HAV. Data are presented as the mean ± standard deviation (SD). Statistical significance was determined using an ANOVA. Asterisks indicate significant differences at * p < 0.05, **** p < 0.0001, Non-significant differences (p > 0.05) compared to day 0 are indicated by the letters ns.