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

Figures

• 

  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 (ΔRCT) 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 ΔRCT 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.