RESUMO
Botulinum is a deadly bacterial toxin that causes neuroparalytic disease. However, appropriate tools to detect trace toxic proteins are scarce. This study presents a bead-based diffusometric technique for the rapid, simple, and quantitative detection of biological toxins. Functionalized particles called nano-immunosensors were fabricated by forming sandwiched immunocomplexes comprising Au nanoparticles (AuNPs), toxic proteins, and antibodies on fluorescent probe particles. Particle diffusivity tended to decline with increasing concentration of the target proteins. Calibration curves of purified botulinum toxins (0.01-500 ng/mL) were obtained from whole milk and bovine serum, and results suggested that measurement was independent of the background matrix. The activity of botulinum toxin was evaluated by coating synaptosomal-associated protein 25 (SNAP-25) on fluorescent probe particles. AuNP-conjugated antibodies attached to the probe particles when SNAP-25 proteins were cleaved by active botulinum. Thus, toxicity could be detected from slight changes in diffusivity. A short measurement time of 2 min and a limit of detection of 10 pg/mL were achieved. The nano-immunosensors demonstrated rapid biosensing capability and met the demands of onsite screening for food safety, medical instrument hygiene, and cosmetic surgery products.
Assuntos
Técnicas Biossensoriais/métodos , Toxinas Botulínicas Tipo A/sangue , Imunoensaio/métodos , Nanopartículas Metálicas/química , Animais , Anticorpos Monoclonais Murinos/imunologia , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/imunologia , Contaminação de Alimentos/análise , Ouro/química , Limite de Detecção , Camundongos , Leite/química , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/imunologiaRESUMO
Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase-mass spectrometry (Endopep-MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep-MS assay. Our strategy was based on reported BoNT/B-substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum.