A novel colorimetric DNA sensor for rapid detection of feline panleukopenia virus using ZnFe2O4 nanoparticles and TMB modulation

A novel colorimetric genosensor was developed for the detection of feline panleukopenia virus (FPV) DNA, based on the peroxidase-like activity of ZnFe₂O₄ (ZFO) nanoparticles. The synthesized ZFO nanoparticles catalytically oxidized 3,3',5,5'-tetramethylbenzidine (TMB), resulting in the generation of a characteristic blue color. Upon the addition of 5% sulfuric acid stop solution, this intermediate was converted into a yellow diimine product, whose absorbance was measured at 450 nm using a microplate reader. ZFO nanoparticles were subsequently coated onto concave glass slides. 5'-amino-modified oligonucleotide probes were immobilized on APTES-modified glass surfaces to serve as capture probes. In the presence of the target sequence and subsequent hybridization with the immobilized probes, the spatial interaction between ZFO nanoparticles and the TMB substrate was inhibited, leading to a reduced intensity of the blue color. The limit of detection was determined to be 300 ng/µL for the amplified gene target in PCR reactions and 10³ virus particles per gram of feces in clinical fecal samples. The results demonstrated that the proposed genosensor is simple, sensitive, and exhibits high selectivity, high reproducibility of fabrication, and stability. This platform may offer a practical tool for the screening of suspected feline panleukopenia cases in resource-limited settings. Moreover, with appropriate oligonucleotide probes, the genosensor can be adapted for the detection of a broad range of microorganisms.