Microfabrication for Biomedical Research

人们对同时检测单个生物样品中的许多不同蛋白质有强烈的兴趣。对所收集样品的大小的限制要求这些测量在尽可能小的流体体积上进行。这种兴趣一直是生物医学应用的微流体装置的发展背后的驱动力之一。转向这些较小规模的系统有许多优点。首先，它们能够分析较小体积的样本。其次，在毛细管电泳等应用中，微流控系统可以在比大规模系统少得多的时间内实现相同的分离分辨率。最后，分析装置的尺寸减小提高了开发便携式分析设备的可能性。 DBEPS与NIST的科学家合作，正在开发一种用于免疫亲和电泳的微流控设备，其中多种蛋白质将被同时分离和检测。 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体系统。原型装置由具有蛇形图案的长玻璃封装通道（50 mm x 15 mm x 30 cm）组成。侧端口用于将不同的生物素化抗体压力驱动加载到通道的每个区段中;这些抗体与已经共价连接到通道壁的链霉亲和素结合。抗体固定后，待分析的样品流过整个装置。样品流的电气控制允许调节每个区段中的停留时间，以优化结合。最初，捕获的蛋白质将使用荧光标记进行光学检测。在检测之后，捕获的蛋白质可以从通道洗脱以用于进一步分析。与现有的阵列技术相比，通道器件架构具有几个优点：通过单点捕获检测蛋白质，并且可以使用小得多的样品体积。此外，我们希望能够将结合抗体的通道重复用于多个样品。 激发该项目的直接临床需求是对人乳头瘤病毒（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。对取自外周血样本的淋巴细胞的研究表明，与Th-2型应答的患者相比，其细胞对HPV肽应答的患者具有T淋巴细胞辅助细胞1型应答（Th 1型应答）。为了更好地了解分子水平上的这些差异，有必要直接检测和量化宫颈分泌物中的许多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全身探针。 根据这些要求，对诸如正在开发的微流体装置的需求变得清晰。可以收集的宫颈分泌物的体积非常小，通常为25-50微升，并且难以收集足够数量的流体样品用于流行病学研究，这使得必须从单个样品中提取尽可能多的信息。拟议的设备将能够从一微升样品体积中检测十几种蛋白质。 尽管申报器械的功能是针对该特定应用的，但有充分的理由预期，一旦该器械实现，也可以解决许多其他生物和临床应用。

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.