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，它们能够分析较小的样品体积。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。目前的重点是流行病学研究，通常需要从大量微升样品中同时分离和检测多种蛋白质;然而，该设备最终可用于各种临床和研究应用。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20个玻璃封装通道组成，每个通道50微米x15微米x1 cm，以蛇形图案连接。阵列中微通道末端的侧端口允许每个区段中完整抗体或抗体F（ab）片段的独立电渗加载和固定。使用电渗泵将样品加载到装置中，这允许调节样品在每个区段中的停留时间以优化结合。这种微流体装置，包括固定化的抗体，可以重复用于多个样品。在分析样品后，可以使用酸性缓冲液梯度来破坏抗体-抗原相互作用，释放捕获的抗原，而不破坏F（ab）片段的共价连接或整个抗体与通道壁的链霉亲和素-生物素连接。 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。最近，我们一直在研究不同的化学品的共价连接的减少抗体片段的微通道的表面，以及微调整个抗体上的微通道的沉积。迄今为止，我们有两种方法可用于将完整抗体或抗体片段附着到微通道的壁上，这两种方法对于我们使用的模型抗体/抗原对都表现出良好的特异性和灵敏度。此外，我们已经证明了终止于抗甲状腺素（T4）完整抗体和抗体片段的微通道的可重复使用性。 未来的目标是进一步调整低分子量蛋白质的释放策略，优化电泳泵送效率和通道几何形状，并进一步开发检测方法。此外，我们正在研究使用的几种聚合物材料作为基板的一次性，流通微流控免疫测定。

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 sample volumes. 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. The immediate focus is on epidemiological studies, for which the simultaneous isolation and detection of multiple proteins from a large number of microliter samples is typically required; however, the device could ultimately be used for a variety of clinical and research applications. 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 device consists of twenty glass-encapsulated channels, each 50 micrometers x 15 micrometers x 1cm, connected in a serpentine pattern. Side ports at the ends of the microchannels in the array allow for independent electroosmotic loading and immobilization of either whole antibodies or antibody F(ab) fragments in each segment. The sample is loaded into the device using electroosmotic pumping, which permits adjustment of the sample residence time in each segment in order to optimize binding. This microfluidic device, including the immobilized antibodies, can be reused for multiple samples. After analysis of a sample, an acidic buffer gradient can be used to disrupt the antibody-antigen interaction, releasing the captured antigens without breaking the covalent attachment of the F(ab) fragments, or the streptavidin-biotin link of the whole antibodies to the channel walls. 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. Recently, we have been investigating different chemistries for the covalent attachment of the reduced antibody fragments to the surfaces of the microchannels as well as fine-tuning the deposition of whole antibodies on the microchannels. To date, we have two methods available for attaching whole antibodies or antibody fragments to the walls of the microchannels and both methods demonstrate good specificity and sensitivity for the model antibody/antigen pairs we have used. In addition, we have demonstrated the reusability of microchannels terminated in anti-thyroxine (T4) whole antibodies and antibody fragments. Future goals are to further tune the release strategies for low molecular mass proteins, optimize the electrophoretic pumping efficiency and channel geometry, and further develop the detection methodology. In addition, we are investigating the use of several polymeric materials as substrates for disposable, flow-through microfluidic immunoassays.