Microfabrication for Biomedical Research

虽然已经有大量的工作开发微加工和微流体技术的生物医学应用，仍然有有限的进展，在移动该技术广泛进入生物医学研究实验室。 部分问题是生物医学研究人员对微制造的能力缺乏了解。 此外，许多潜在的研究项目都有限制，需要广泛的定制和多次设计迭代，这可能是有限数量的商业产品无法实现的。 为了降低将微制造技术应用于广泛的生物医学问题的障碍，在过去的一年中，我们开发了一种内部微制造能力，使我们能够使用方便的干膜抗蚀剂工艺为PDMS制作单层模板。 虽然分辨率，设备产量和复杂性大大低于专用洁净室，但它们仍然足以用于许多细胞实验。 此外，仪器复杂性、制造成本和周转时间大大降低，使得能够根据需要快速循环设计参数。 今年，相当大的努力花在开发和优化两个商业干膜抗蚀剂和SU-8，旋涂抗蚀剂图案化的协议。 因此，我们能够可靠地图案化横向尺寸小至15微米，高度范围从15微米到几百微米的特征。 此外，我们开发了一种使用数字光投影仪的无掩模光刻能力;使用这种仪器，我们可以在几个小时或更短的时间内从位图图像制作微流体模板。最后，我们还开发了PDMS和其他聚合物的表面改性方案，包括使用我们实验室中的仪器将PDMS不可逆地键合到玻璃上，以及用于将装置连接到诸如注射泵的流动控制仪器的技术。 通过与NIST科学家的持续合作，我们还能够访问NIST的纳米制造设施，根据需要制造更复杂和更高公差的结构。 在过去一年中，这些新的能力已在几个初步项目中得到应用，所有这些项目都在进行中。 第一个是制造琼脂糖微流控装置，在一个平台上兼容高分辨率荧光和双光子成像，在3-D胶原蛋白基质的趋化性的研究。 第二个项目是将B-T细胞对限制在PDMS微孔中，使得细胞间连接垂直于光轴，以便能够对突触形成进行高速高分辨率成像。 第三个项目是开发用于相衬X射线成像的光栅，我们正在使用NIST的纳米纤维设备和我们自己的设备。 此外，我们一直在利用我们的能力，旋涂薄聚合物薄膜，使薄聚（乙烯共-醋酸乙烯酯）（伊娃）薄膜用于操作员独立，高分辨率，激光捕获显微切割。 这项工作是NIMH，NCI和NICHD研究人员的研究所间主任挑战项目的一部分，旨在开发基于组织的亚细胞结构捕获方法，用于基于质谱的蛋白质组学分析。 为此，我们已经开发了用于在柔性和刚性基板上从几种不同等级的伊娃制造厚度范围从0.5微米到几微米的捕获膜的协议。 最后，我们继续致力于一个项目，旨在验证LIPS检测，它使用的融合蛋白组成的海肾荧光素酶和感兴趣的抗原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。先前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已经表明，即使在小型化形式中，阳性信号水平也是可接受的高。今年，我们的工作重点是提高测量的重现性，并使用一组血清样本验证小型化检测的功能。

Although there has been extensive work developing microfabrication and microfluidic technology for biomedical applications, there has still been limited progress in moving the technology broadly into biomedical research laboratories. Part of the issue is a lack of familiarity with the capabilities of microfabrication on the part of biomedical researchers. In addition, many potential research projects have constraints that require extensive customization and multiple design iterations, which may not be achievable with the limited number of commercial products available. In an effort to lower the barriers for applying microfabrication techniques to a wide range of biomedical problems, over the past year we have developed an in-house microfabrication capability which enables us to make single-layer templates for PDMS using a convenient dry-film resist process. Although the resolution, device yield, and complexity are considerably lower than those achievable with a dedicated cleanroom, they are nonetheless sufficient for many experiments on cells. Furthermore, the instrumentation complexity, fabrication cost, and turnaround time are greatly reduced, enabling rapid cycling through design parameters as needed. This year, considerable effort was spent on developing and optimizing protocols for patterning two commercial dry-film resists and SU-8, a spin-on resist. As a result, we are able to reliably pattern features with lateral dimensions as small as 15 microns, and with heights ranging from 15 microns to a few hundred microns. In addition, we developed a capability for maskless photolithography using a digital light projector; using this instrumentation we can make a microfluidic template from a bitmap image in a few hours or less. Finally, we also have developed protocols for surface modification of PDMS and other polymers, including the irreversible bonding of PDMS to glass using instrumentation in our laboratory, as well as techniques for connecting devices to flow-control instruments, such as syringe pumps. Through continued collaboration with scientists at NIST, we are also able to access the nanofabrication facilities at NIST to make more complex and higher-tolerance structures as needed. These new capabilities have found application in several initial projects during this past year, all of which are ongoing. The first is the fabrication of agarose microfluidic devices, on a platform compatible with high-resolution fluorescence and two-photon imaging, for the study of chemotaxis in a 3-D collagen matrix. The second project is an effort to confine B-T cell pairs in PDMS microwells such that the intercell junction is perpendicular to the optical axis, in order to enable high-speed high-resolution imaging of the synapse formation. A third project is the development of gratings for phase-contrast x-ray imaging, for which we are using both the NIST nanofabrication facilities as well as our own equipment. In addition, we have been using our capability to spin-coat thin polymer films to make thin poly(ethylene co-vinyl acetate) (EVA) films for use in operator-independent, high-resolution, laser capture microdissection. This work is part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD, aimed at developing methods for tissue-based capture of subcellular structures for mass spectrometry-based proteomic analysis. To this end, we have developed protocols for making capture films from several different grades of EVA with thicknesses ranging from 0.5 microns to a few microns, on flexible and rigid substrates. Finally, 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. Previous 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 on increasing measurement reproducibility and verifying function of the miniaturized assay with a panel of serum samples.