(175az) Nano-Immunosensors for Rapid Detection of Foodborne Toxin

Bacterial toxins in food and environmental samples have long been a threat to the public health. Of which, botulinum toxin is regarded the deadliest one that causes thousands of deaths worldwide every year. The lethal dose of botulinum toxin in unvaccinated humans is estimated to be only 1 ng/kg. Conventionally commondetections are high performance liquid chromatography (HPLC), mass spectrometry, and colorimetric Enzyme-Linked ImmunoSorbent Assays (ELISAs). However, these methods may rely on benchtop apparatuses, resulting in tedious and labor-intensive processes. In addition, their sensitivity remains not sufficiently low. Devices and methods for reliable detect several biological toxins samples in parallel and simultaneously, which have proved to be challenging. The abovementioned limitations hence impede their onsite use in industry.

Despite the daunting fact, detection tools for traces of toxic proteins remain scarce. To address the unmet need, we herein proposed a bead-based diffusometric technique to achieve rapid, simple, and quantitative detection of biological toxins. To this end, we fabricated nano-immunosensors by forming sandwiched immunocomplexes, comprising gold nanoparticles (AuNPs), toxic proteins, and antibodies, on fluorescent probe particles. The particle diffusivity tended to decline with increased target proteins. Calibration curves for purified botulinum toxins (0.01–500 ng/mL) in whole milk and bovine serum were conducted. Result suggested that the measurement was independent of background matrices. The activity of the botulinum toxin was also investigated by coating the synaptosomal-associated protein 25 (SNAP-25) on fluorescent probe particles. When the SNAP25 proteins were cleaved by active botulinum, AuNP-conjugated antibody could then attach to the probe particles. Finally, we further used the cell based assay to confirm the performance of this nano-immunosensor of detected the SNAP25 proteins in lysed cells. A neuronal cell (PC12) was treated with different concentrations of botulinum toxin (1 ~ 0.001ng/mL) to exhibit the dose dependent changes of cleaved SNAP25. In this evaluation, nano-immunosensors were mixed with the PC12 cell lysate for a direct measurement of diffusivity. In agreement with the previous results, distinct diffusivity changes with respect to the concentrations of toxin were observed.

In conclusion, trace toxicity was successfully detected by a slight change of diffusivity using our nano-immunosensors. A short turnaround time of 2 min and a limit of detection as low as 10 pg/mL were achieved. The nano-immunosensors demonstrated rapid biosensing capability for the rising demands of onsite screening in food safety, medical instrument hygiene, and cosmetic surgery products.