Robust Bioengineered Apoferritin Nanoprobes for Ultrasensitive Detection of Infectious Pancreatic Necrosis Virus

Infectious pancreatic necrosis virus (IPNV) has been identified as a viral pathogen for many fish diseases that have become a huge hurdle for the growing fishing industry. Thus, in this work, we report a label-free impedance biosensor to quantify IPNV in real fish samples at point-of-care (POC) level. High specificity IPNV sensor with a detection limit of 2.69 TCID₅₀/mL was achieved by conjugating IPNV antibodies to portable Au disk electrode chips using human heavy chain apoferritin (H-AFN) nanoprobes as a binding agent. H-AFN probes were bioengineered through PCR by incorporating pET-28b(+) resulting in 24 subunits of 6 × his-tag and protein-G units on its outer surface to increase the sensitivity of the IPNV detection. The biosensor surface modifications were characterized by differential pulse voltammetry (DPV) and EIS methods for each modification step. The proposed nanoprobe based sensor showed three-fold enhancement in charge transfer resistance toward IPNV detection in comparison with the traditional linker approach when measured in a group of similar virus molecules. The portable sensor exhibited a linear range of 100-10000 TCID₅₀/mL and sensitivity of 5.40 × 10^-4 TCID₅₀/mL in real-fish samples. The performance of the proposed IPNV sensor was fully validated using an enzyme-linked immunosorbent assay (ELISA) technique with a sensitivity of 3.02 × 10^-4 TCID₅₀/mL. Results from H-AFN nanoprobe based IPNV sensor indicated high selectivity, sensitivity, and stability could be a promising platform for the detection of similar fish viruses and other biological molecules of interest.