Sun Steven, Ossandon Miguel, Kostov Yordan, Rasooly Avraham
Division of Biology, Office of Science and Engineering Laboratories, FDA, NIH/NCI, Silver Spring, MD 20993, USA.
Lab Chip. 2009 Nov 21;9(22):3275-81. doi: 10.1039/b912097a. Epub 2009 Sep 17.
A Lab-on-a-chip (LOC) was designed, fabricated and tested for the in vitro detection of botulinum neurotoxin serotype A (BoNT-A) activity using an assay that measures cleavage of a fluorophore-tagged peptide substrate specific for BoNT-A (SNAP-25) by the toxin light chain (LcA). LcA cleavage was detected by Förster Resonance Energy Transfer (FRET) fluorescence. FRET fluorescence was measured by a newly developed portable charge-coupled device (CCD) fluorescent detector equipped with multi-wavelength light-emitting diodes (LED) illumination. An eight V-junction microchannel device for BoNTs activity assays was constructed using Laminated Object Manufacturing (LOM) technology. The six-layer device was fabricated with a Poly(methyl methacrylate (PMMA) core and five polycarbonate (PC) layers micromachined by CO2 laser. The LOC is operated by syringe and is equipped with reagents, sample wells, reaction wells, diffusion traps (to avoid cross contamination among channels) and waste reservoirs. The system was detected LcA at concentrations as low as 0.5 nM, which is the reported sensitivity of the SNAP-25 in vitro cleavage assay. Combined with our CCD detector, the simple point of care system enables the detection of BoNTs activity and may be useful for the performance of other complex medical assays without a laboratory. This approach may realize the potential to enhance the quality of health care delivery for underserved populations.
设计、制造并测试了一种芯片实验室（LOC），用于体外检测A型肉毒杆菌神经毒素（BoNT-A）的活性，该检测方法通过测量毒素轻链（LcA）对BoNT-A特异性的荧光团标记肽底物（SNAP-25）的切割来进行。通过Förster共振能量转移（FRET）荧光检测LcA的切割。FRET荧光由新开发的配备多波长发光二极管（LED）照明的便携式电荷耦合器件（CCD）荧光检测器测量。使用分层实体制造（LOM）技术构建了用于BoNTs活性检测的八V型微通道器件。六层器件由聚甲基丙烯酸甲酯（PMMA）芯和通过CO2激光微加工的五层聚碳酸酯（PC）层制成。LOC由注射器操作，配备有试剂、样品孔、反应孔、扩散阱（以避免通道之间的交叉污染）和废液池。该系统能够检测低至0.5 nM浓度的LcA，这是已报道的SNAP-25体外切割检测的灵敏度。结合我们的CCD检测器，这种简单的即时护理系统能够检测BoNTs的活性，可能有助于在无需实验室的情况下进行其他复杂的医学检测。这种方法可能实现提高为服务不足人群提供医疗保健质量的潜力。
