Microfabrication for Biomedical Research

人们对同时和快速检测单个生物样品中的多种生物标志物非常感兴趣。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。 正在开发的一种设备能够同时测量八种不同的电泳运行。另一个正在开发的设备是荧光素酶免疫沉淀（LIPS）测定的小型化，该测定在血清样品中寻找免疫反应。我们小组的其他兴趣包括设计和开发能够分析单细胞分泌和生理的设备。我们的设施现在已经发展到一个地步，我们能够建议和合作，与内外部的研究人员就如何生产微流体设备，以满足他们的特定需求。 通过与NIST的科学家合作，并使用NIST的微制造设施，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，以及将热塑性塑料或弹性体模压成微流体设备的模板。 今年，我们进一步开发了校园内的技术，用于从这些模板中形成，密封和涂覆热塑性塑料和弹性体中的微流体通道。 我们还在实验室建立一个基本的光刻系统，用于快速制造单层结构和模板，尺寸低至约25微米。 此外，我们继续致力于一个项目，旨在验证LIPS检测，该检测使用由海肾荧光素酶和感兴趣的抗原组成的融合蛋白来探测人血清中的抗体。在其目前的形式中，该测定在96孔滤板中进行，并且已被证明在检测许多自身免疫性疾病和感染性疾病中有效。将测定移动到微流体格式可以显著加速分析，允许多路复用，并且还促进该测定应用于护理点诊断。使用含有融合蛋白的细胞提取物和商业抗CFLAG抗体代替血清的实验表明，即使在小型化形式中，阳性信号水平也是可接受的高。 今年，我们的工作主要集中在提高测量重现性上，其中包括重新设计发光测量设置、调整通道表面化学、缓冲液优化和微通道内流量控制的实质性改进，以及对测量协议的其他改进。 因此，我们对血清样品的初步测量现在显示在微通道中使用10分钟孵育时间的运行之间的变化小于10%。 在不久的将来，我们计划对一组样品进行测量，以验证微流体测定和原始孔格式之间的对应关系。

There is a strong interest in the simultaneous and rapid detection of a multiple biomarkers in a single biological sample. 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. One device under development is capable of measuring eight different electrophoretic runs simultaneously. Another device under development is the miniaturization of a luciferase immunoprecipitation (LIPS) assay that looks for immune response in serum samples. Other interests of our group include the design and development of devices capable of analyzing the secretions and physiology of single cells. Our facilities have now developed to a point where we are able to advise and collaborate with both intramural and extramural investigators on how to produce microfluidic devices that address their specific needs. In collaboration with scientists at NIST, and using the microfabrication facilities at NIST, we are able to make micrometer-scale glass-encapsulated microfluidic systems with any desired two-dimensional configuration, as well as templates to stamp thermoplastics or mold elastomers into microfluidic devices. This year, we have further developed technology on-campus for forming, sealing, and coating microfluidic channels in thermoplastics and elastomers from these templates. We are also in the process of setting up a basic lithography system in our laboratory, intended for rapid fabrication of single-layer structures and templates with dimensions down to approximately 25 micrometers. In addition, we continue work on a project aimed at miniaturizing the LIPS assay, which uses a fusion protein consisting of Renilla luciferase and an antigen of interest to probe for antibodies in human serum. In its current format, the assay is performed in a 96-well filter plate, and has been shown effective in detecting a number of autoimmune conditions and infectious diseases. Moving the assay to a microfluidic format could significantly speed the analysis, permit multiplexing, and also facilitate the application of this assay to point-of-care diagnostics. Experiments using cell extracts containing the fusion protein and commercial anti-CFLAG antibodies in lieu of serum had shown that the positive signal levels are acceptably high even in the miniaturized format. This year, our efforts focused primarily on increasing measurement reproducibility, which involved redesign of the luminescence measurement setup, adjustment of the channel surface chemistry, buffer optimization, and substantial refinement of flow control within the microchannel, as well as other improvements to the measurement protocol. As a result, our preliminary measurements on serum samples now show less than 10% variation between runs using ten minute incubation times in a microchannel. In the near future, we plan to undertake measurements on a panel of samples to verify correspondence between the microfluidic assay and the original well format.