Srinivasan Shruthi, Letran Khoa, Nasir Salim, Sung Kyung, Lam Johnny, Wang Yun, Duncan Timothy V
Department of Biomedical Engineering, San Jose State University, San Jose, California 95192, United States.
Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland 20993, United States.
ACS Omega. 2025 Aug 4;10(32):36466-36474. doi: 10.1021/acsomega.5c04995. eCollection 2025 Aug 19.
Botulinum neurotoxin (BoNT) poses a significant threat to public health as the most potent toxin known to humans. BoNT intoxication can lead to botulism, a life-threatening disease that may result from ingesting food contaminated with biologically active BoNT. The gold standard method for BoNT detection, the mouse bioassay, is time-consuming and raises ethical concerns regarding the use of laboratory animals. Therefore, there is a critical need for alternative methods that are rapid and require minimal training for field use. In this study, with potential in-field use in mind, we developed a microfluidic nanobiosensor that utilizes droplet-based passive pumping for easy operation and quantum dots (QDs) for sensitive detection. With droplet-based passive pumping, sensor preparation reagents and detection samples can be introduced via simple pipetting. Meanwhile, QDs offer a high quantum yield and excellent photostability, providing strong and reliable photoluminescent signals. The QDs were tethered to the surface of microfluidic channels via BoNT serotype-specific peptides, which can be cleaved due to the BoNT endopeptidase activity. In the presence of the active toxin, peptide cleavage released the QDs from the channels and the resulting reduced QD photoluminescence was measured as a detection signal. Using this biosensor, we detected the biologically active catalytic domain (light chain) of BoNT serotype A (LcA) at concentrations ranging from 1 ng/mL to 100 μg/mL, with a limit of detection (LoD) of 1 ng/mL (20 pM) in a small sample volume (1 pg LcA in 1 μL), within 2 h. This low-cost, user-friendly biosensor provides a rapid and effective method for detecting biologically active BoNT based on its endopeptidase activity without the use of laboratory animals, and it offers a scalable solution for potential BoNT screening in field or low-resource settings after further validation.
肉毒杆菌神经毒素（BoNT）作为人类已知的最具毒性的毒素，对公众健康构成重大威胁。BoNT中毒可导致肉毒中毒，这是一种可能因摄入被具有生物活性的BoNT污染的食物而引发的危及生命的疾病。BoNT检测的金标准方法——小鼠生物测定法，耗时且引发了关于使用实验动物的伦理问题。因此，迫切需要快速且在现场使用时所需培训最少的替代方法。在本研究中，考虑到潜在的现场应用，我们开发了一种微流控纳米生物传感器，该传感器利用基于液滴的被动泵送实现易于操作，并利用量子点（QD）进行灵敏检测。通过基于液滴的被动泵送，传感器制备试剂和检测样品可通过简单移液引入。同时，量子点具有高量子产率和出色的光稳定性，可提供强而可靠的光致发光信号。量子点通过BoNT血清型特异性肽连接到微流控通道表面，该肽可因BoNT内肽酶活性而被切割。在活性毒素存在的情况下，肽的切割使量子点从通道中释放出来，然后将由此产生的量子点光致发光降低作为检测信号进行测量。使用这种生物传感器，我们在小样本体积（1 μL中含1 pg LcA）内，在2小时内检测到浓度范围为1 ng/mL至100 μg/mL的BoNT血清型A（LcA）的生物活性催化结构域（轻链），检测限（LoD）为1 ng/mL（20 pM）。这种低成本、用户友好的生物传感器提供了一种基于其内肽酶活性检测生物活性BoNT的快速有效方法，无需使用实验动物，并且在进一步验证后，为现场或资源匮乏环境中的潜在BoNT筛查提供了可扩展的解决方案。
