Microfabrication for Biomedical Research

在单个生物样品中同时检测多个不同的蛋白质引起了人们的浓厚兴趣。对采集样本大小的限制要求这些测量必须在尽可能小的液体体积上进行。这种兴趣一直是生物医学应用微流控设备发展的驱动力之一。转向这些规模较小的系统有很多好处。首先，他们能够分析较小体积的样品。其次，在毛细管电泳法等应用中，微流控系统可以在更短的时间内达到相同的分离分辨率。最后，分析装置的尺寸减小提高了开发便携式分析装置的可能性。 与NIST的科学家合作，DBEPS正在开发一种用于免疫亲和电泳的微流控设备，其中将同时分离和检测多个蛋白质。 使用NIST的微加工设施，我们能够制造具有任何所需二维配置的微米级玻璃封装微流控系统。原型器件由一个50 mm x 15 mm x 30 cm的长玻璃封装通道组成，带有蛇形图案。侧孔用于压力驱动将不同的生物素化抗体加载到通道的每一段；这些抗体与链霉亲和素结合，链霉亲和素已共价连接到通道壁。抗体被固定后，被分析的样本流经整个设备。样品流量的电子控制允许调整每一段中的停留时间，以优化结合。最初，捕获的蛋白质将使用荧光标签进行光学检测。检测后，捕获的蛋白质可以从通道中洗脱出来进行进一步分析。与现有的阵列技术相比，该通道设备架构具有几个优势：通过单点捕获检测蛋白质，并且可以使用小得多的样本量。此外，我们希望能够将结合抗体的通道重复用于多个样本。 推动这一项目的迫切临床需要是对人乳头瘤病毒(HPV)感染的免疫反应以及这种反应与宫颈癌发展之间的关系进行流行病学研究。对从外周血样本中提取的淋巴细胞的研究表明，其细胞对HPV多肽的反应与T淋巴细胞辅助细胞1型反应(Th1型反应)的患者与Th-2型反应的患者之间存在差异。为了在分子水平上更好地了解这些差异，有必要直接在宫颈分泌物中检测和量化一些选定的免疫调节分子，作为局部探针，而不是血液样本作为免疫反应的系统探针。 从这些要求中，对微流控设备的需求变得明显，例如正在开发的设备。可收集的宫颈分泌物的量非常小，通常为25-50微升，而且为流行病学研究收集足够数量的液体样本的困难使得必须从单个样本中提取尽可能多的信息。传统诊断技术，如酶联免疫吸附试验，只允许从这种大小的样本中分析一到两种分析物。拟议中的设备将能够从一微升的样本中检测到十几种蛋白质。 虽然提议的设备的功能是针对这一特定应用的，但有充分的理由预计，一旦该设备实现，许多其他生物和临床应用也将得到解决。

There is a strong interest in the simultaneous detection of a number of different proteins in a single biological sample. Limitations on the size of the collected sample require that these measurements be done on as small a volume of fluid as possible. This interest has been one of the driving forces behind the development of microfluidic devices for biomedical applications. The move to these smaller-scale systems has a number of advantages. First, they are capable of analyzing smaller volumes of sample. Second, in applications such as capillary electrophoresis, the microfluidic system can achieve the same separation resolution in much less time than a larger-scale system. Finally, the reduced size of the analysis setup raises the possibility of developing portable analytical devices. In collaboration with scientists at NIST, DBEPS is developing a microfluidic device for immunoaffinity electrophoresis, in which multiple proteins will be simultaneously isolated and detected. Using the microfabrication facilities at NIST, we are able to make micrometer-scale glass-encapsulated microfluidic systems with any desired two-dimensional configuration. The prototype devices consist of a long glass-encapsulated channel, 50mm x 15 mm x 30cm, with a serpentine pattern. Side ports are used for pressure-driven loading of different biotinylated antibodies into each segment of the channel; these antibodies bind to streptavidin that has been covalently linked to the channel walls. After the antibodies have been immobilized, the sample under analysis flows through the entire device. Electrical control of the sample flow permits adjustment of the residence time in each segment in order to optimize binding. Initially, the captured proteins will be detected optically, using fluorescent tags. After detection, the captured proteins may be eluted from the channel for further analysis. The channel device architecture has several advantages over existing array technology: the proteins are detected by single-point capture, and much smaller sample volumes can be used. In addition, we hope to be able to reuse the channels with the bound antibodies for multiple samples. The immediate clinical need that motivates this project is an epidemiological study of the immune response to Human Papillomavirus (HPV) infection, and the relationship between this response and the development of cervical cancer. Studies of lymphocytes taken from peripheral blood samples have suggested a difference between patients whose cells respond to HPV peptides with a T lymphocyte helper cell type 1 response (Th1-type response) compared to those with a Th-2 type response. In order to better understand these differences at a molecular level, it is necessary to detect and quantify a number of selected immune regulatory molecules directly in cervical secretions, as a local probe, rather than blood samples as a systemic probe of the immune response. From these requirements, the need for a microfluidic device such as the one being developed becomes clear. The volume of cervical secretions that can be collected is quite small, typically 25-50 microliters, and the difficulty in collecting a sufficient number of fluid samples for an epidemiological study makes it imperative to extract as much information as possible from a single sample Conventional diagnostic techniques, such as ELISA, only allow for the analysis of one or two analytes from a sample of this size. The proposed device will be able to detect over a dozen proteins from a one-microliter sample volume. Although the functionality of the proposed device is being targeted for this particular application, there is every reason to expect that, once this device is realized, many other biological and clinical applications could also be addressed.