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。该装置将由一个直径约25微米、长几毫米的微流控通道组成，在该通道中，所需的抗体被结合在一系列局部区段中。当样本液体流过通道时，目标蛋白将与抗体结合。最初，这些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。这种设备架构有几个优点。首先，通过单点捕获方案实现蛋白质的分离。其次，蛋白质可以从毛细管中回收，其生物活性完好无损，可以进行进一步分析。最后，由于蛋白质可以从设备中冲洗出来，芯片可以重复使用，因此既最大限度地减少了昂贵的抗体库的使用，又大大减少了分析一系列样本的成本和工作量。 NIST已经制作了使用硅作为衬底材料的原型器件，其中已经刻蚀了微流控通道，以评估包括阳极键合在内的键合方法和样品引入口的密封剂技术。 推动这一项目的迫切临床需求是对疱疹乳头瘤病毒(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. The device will consist of a microfluidic channel, approximately 25 microns in diameter and a few millimeters in length, in which desired antibodies are bound in a series of localized sections. When the sample fluid flows through the channel, the target proteins will bind to the antibodies. Initially, the proteins will be detected optically, using fluorescent tags. After detection, the captured proteins may be eluted from the channel for further analysis. This device architecture has several advantages. First, the isolation of the proteins is achieved through a single-point capture scheme. Second, the proteins can be recovered from the capillary with their biological activity intact permitting further analysis. Finally, because the proteins can be flushed from the device, the chip can be reused, thus both minimizing the use of expensive antibody libraries and greatly reducing the cost and effort for analysis of a series of samples. Prototype devices have been made at NIST using silicon as the substrate material in which microfluidic channels have been etched to evaluate both bonding methodologies, including anodic bonding, and sealant techniques for sample introduction ports. The immediate clinical need that motivates this project is an epidemiological study of the immune response to Herpes Papilloma Virus (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.