用基于荧光的波导生物传感器进行无扩增核酸检测

Philip A. Kocheril, Kiersten D. Lenz, Daniel E. Jacobsen, Jessica Z. Kubicek-Sutherland
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引用次数: 0

摘要

使用临床样本中的核酸早期检测病原体通常需要单拷贝水平的灵敏度,这目前需要耗时且昂贵的核酸扩增。在这里,我们描述了1)一个重新设计的梯形减去几何体育场形状的流动池,以及2)改进的实验程序,该程序允许在基于荧光的波导生物传感器上测量微升量的亚attomolar分析物。我们用一个200μL的荧光链霉亲和素缀合物样品在100 zM(100 zeptomol,或100·10−21 mol L−1)下验证了我们的仪器灵敏度,并使用Langmuir吸附模型从理论上探讨了这种改进的传感平台在三明治免疫测定形式中的适用性。我们提出的检测方法证明,使用荧光分子信标在单拷贝水平(200μL,10zm)上特异性检测合成甲型流感DNA(缓冲液中)和RNA(唾液中)寡核苷酸。最后,我们展示了在临床相关浓度下分离的甲型流感基因组RNA的检测。与我们之前的核酸检测工作相比,这项工作的灵敏度提高了12个数量级以上,说明了优化实验设计可以获得的显著增强。
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Amplification-free nucleic acid detection with a fluorescence-based waveguide biosensor
Early detection of pathogens using nucleic acids in clinical samples often requires sensitivity at the single-copy level, which currently necessitates time-consuming and expensive nucleic acid amplification. Here, we describe 1) a redesigned flow cell in the shape of a trapezoid-subtracted geometric stadium, and 2) modified experimental procedures that allow for the measurement of sub-attomolar analytes in microliter quantities on a fluorescence-based waveguide biosensor. We verified our instrumental sensitivity with a 200-μL sample of a fluorescent streptavidin conjugate at 100 zM (100 zeptomolar, or 100·10−21 mol L−1) and theoretically explored the applicability of this modified sensing platform in a sandwich immunoassay format using a Langmuir adsorption model. We present assays that demonstrate specific detection of synthetic influenza A DNA (in buffer) and RNA (in saliva) oligonucleotides at the single-copy level (200 μL at 10 zM) using a fluorescent molecular beacon. Lastly, we demonstrate detection of isolated genomic influenza A RNA at a clinically relevant concentration. This work constitutes a sensitivity improvement of over twelve orders of magnitude compared to our previous nucleic acid detection work, illustrating the significant enhancements that can be gained with optimized experimental design.
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