Department of Microsystems Engineering, School of Mechanical Engineering, Northwestern Polytechnical University, 127 West Youyi Road, Xi'an, 710072, Shaanxi, People's Republic of China.
Institute of Biotechnology, Czech Academy of Science, Průmyslová 595, 252 50, Vestec, Czech Republic.
Sci Rep. 2022 Nov 7;12(1):18911. doi: 10.1038/s41598-022-23426-z.
Microfluidics systems can be fabricated in various ways using original silicon glass systems, with easy Si processing and surface modifications for subsequent applications such as cell seeding and their study. Fluorescent imaging of cells became a standard technique for the investigation of cell behavior. Unfortunately, high sensitivity fluorescent imaging, e.g., using total internal reflection fluorescence (TIRF) microscopy, is problematic in these microfluidic systems because the uneven surfaces of the silicon channels' bottoms affect light penetration through the optical filters. In this work, we study the nature of the phenomenon, finding that the problem can be rectified by using a silicon-on-insulator (SOI) substrate, defining the channel depth by the thickness of the top Si layer, and halting the etching at the buried SiO layer. Then the fluorescent background signal drops by = 5 times, corresponding to the limit of detection drop from = 0.05 mM to = 50 nM of fluorescein. We demonstrate the importance of a flat surface using TIRF-based single-molecule detection, improving the signal to a noise ratio more than 18 times compared to a conventional Si wafer. Overall, using very high-quality SOI substrates pays off, as it improves the fluorescence image quality due to the increase in signal-to-noise ratio. Concerning the cost of microfluidic device fabrication-design, mask fabrication, wafer processing, and device testing-the initial SOI wafer cost is marginal, and using it improves the system performance.
微流控系统可以使用原始的硅玻璃系统以各种方式制造，易于进行 Si 加工和表面改性，以用于后续应用，例如细胞播种及其研究。荧光成像已成为研究细胞行为的标准技术。不幸的是，由于硅通道底部的不平整表面会影响光透过光学滤光片的穿透，因此在这些微流控系统中，高灵敏度荧光成像（例如，使用全内反射荧光（TIRF）显微镜）存在问题。在这项工作中，我们研究了这种现象的本质，发现可以通过使用绝缘体上硅（SOI）衬底来解决该问题，该衬底通过顶层 Si 层的厚度来定义通道深度，并在埋入的 SiO 层处停止刻蚀。然后，荧光背景信号下降了 = 5 倍，相应的荧光素检测下限从 = 0.05 mM 下降到 = 50 nM。我们通过基于 TIRF 的单分子检测证明了平整表面的重要性，与传统的 Si 晶片相比，信号与噪声比提高了 18 倍以上。总体而言，使用高质量的 SOI 衬底是值得的，因为它可以提高信号与噪声比，从而改善荧光图像质量。关于微流控器件制造的成本-掩模制造，晶片加工和器件测试-最初的 SOI 晶片成本微不足道，使用它可以提高系统性能。
