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，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或水凝胶器件制作单层模板。虽然分辨率、器件产量和复杂性略低于专用洁净室，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，我们继续改进两种商用干膜抗蚀剂的图案化方案。因此，我们能够在柔性或刚性基板上可靠地图案化横向尺寸小至10微米且高度范围从15微米到几百微米的模板特征。 此外，我们还开发了用于图案化SU-8（一种旋涂抗蚀剂）的几种配方的方案，该方案将我们的能力扩展到几微米的层厚度和5微米的横向分辨率。我们还开发了使用这些模板生成微结构的PDMS，琼脂糖和PEGDA水凝胶的协议。 最后，我们继续开发PDMS和其他聚合物的表面改性协议，包括PDMS与玻璃的不可逆结合，使用我们实验室中的仪器，以及将设备连接到流量控制仪器（如注射泵）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。 在过去的一年里，这些能力已在代表各种利益和机构的一些项目中得到应用。 除了下面讨论的项目外，我们还培训了基本微加工技术的研究人员，包括来自NHLBI，NCI和NIBIB其他实验室的代表。 一个长期运行的项目是与LSB，NIAID合作正在进行的努力，以研究3-D胶原基质中的趋化性，为此我们一直在开发和改进与高分辨率荧光和双光子成像兼容的微流体琼脂糖装置。 今年，我们在一个单独的平台上安装了一个混合三通管，它与可编程注射泵流量控制一起实现了可重现的时变梯度的形成。 此外，我们正在开发一种能够独立控制胶原纤维排列程度和化学梯度的混合多层装置。 第二个项目是与LCE，NHLBI合作，继续开发用于相位衬度X射线成像的光栅，其中涉及使用NIST纳米纤维设施以及我们自己的设备。 在过去的一年中，我们的团队为光栅制造参数的改进以及光栅复制，转移和功能表征的协议的开发做出了贡献。 与LCMB，NCI合作的第三个正在进行的项目是将B-T细胞对限制在微孔中，使得细胞间连接垂直于光轴，以实现突触形成的高速，高分辨率成像;这已经成功地用PDMS实现，我们继续研究使用与培养基更紧密指数匹配的其他材料。 在第四个项目中，我们一直在继续开发，制造和表征薄的混合聚合物薄膜旋涂用于操作员独立，高分辨率，激光捕获显微切割。这项工作是作为NIMH，NCI和NICHD研究人员的研究所间主任挑战项目的一部分开始的，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。 今年开始的第五个项目是开发和实施PDMS微流控梯度发生器，用于在神经细胞培养的基质上沉积硫酸软骨素蛋白聚糖，我们正在与DN，CBPC，NHLBI合作，以更好地了解这些分子在轴突生长和指导中的作用。 最后，我们正在继续开展一个项目，旨在验证LSB，NIDCR开发的荧光素酶免疫沉淀系统（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。我们已经证明了在一组血清样品中以简单的微流体形式用于诊断HSV 2状态的测定的成功功能。 我们正在进行的工作重点是将信号提升到能够使用电池供电的电子设备进行检测的水平，并将被动流量控制纳入设备设计，以提高分析自动化。

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, we have developed an in-house microfabrication capability that enables us to make single-layer templates for PDMS or hydrogel devices using a convenient dry-film resist process. Although the resolution, device yield, and complexity are somewhat 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, we continued to refine protocols for patterning two commercial dry-film resists. As a result, we are able to reliably pattern template features with lateral dimensions as small as 10 microns, and with heights ranging from 15 microns to a few hundred microns, on either flexible or rigid substrates. In addition, we have developed protocols for patterning several formulations of SU-8, a spin-on resist, which extends our capabilities down to layer thicknesses of a few microns and lateral resolution of 5 microns. We have also developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels. Finally, we continue to develop 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 capabilities have found application in a number of projects, representing a broad variety of interests and institutes, over this past year. Aside from the projects discussed below, we have trained researchers in basic microfabrication techniques, including representatives from other laboratories in NHLBI, NCI, and NIBIB. One longer-running project is an ongoing effort in collaboration with LSB, NIAID, to study chemotaxis in 3-D collagen matrices, for which we have been developing and refining a microfluidic agarose device compatible with high-resolution fluorescence and two-photon imaging. This year we implemented a mixing tee on a separate platform, which together with programmable syringe pump flow control has enabled the formation of reproducible time-varying gradients. In addition, we are in the process of developing a hybrid multilayer device capable of independently controlling the degree of collagen fiber alignment and the chemical gradient. A second project is the continuing development, in collaboration with LCE, NHLBI, of gratings for phase-contrast x-ray imaging, which involves the use of the NIST nanofabrication facilities as well as our own equipment. Over this past year, our group has contributed to refinement of the grating fabrication parameters as well as to the development of protocols for the replication, transfer, and functional characterization of gratings. A third ongoing project, in collaboration with LCMB, NCI, is an effort to confine B-T cell pairs in microwells such that the intercell junction is perpendicular to the optical axis, in order to enable high-speed, high-resolution imaging of synapse formation; this has been successfully implemented with PDMS, and we continue to investigate the use of other materials more closely index-matched to the culture media. In a fourth project we have been continuing the development, fabrication, and characterization of thin hybrid polymer films made by spin coating for use in operator-independent, high-resolution, laser capture microdissection. This work, begun as part of an inter-institute Director's Challenge project with researchers in NIMH, NCI, and NICHD, is aimed at developing methods for tissue-based capture of subcellular structures for mass spectrometry-based proteomic analysis. A fifth project, started this year, is the development and implementation of a PDMS microfluidic gradient generator for the deposition of chondroitin sulfate proteoglycans on substrates for neural cell culture, that we are using, in collaboration with DN, CBPC, NHLBI, to gain better understanding of the role these molecules play in axon growth and guidance. Finally, we are continuing work on a project aimed at miniaturizing the luciferase immunoprecipitation system (LIPS) assay developed in LSB, NIDCR, which uses a fusion protein consisting of Renilla luciferase and an antigen of interest to probe for antibodies in human serum. We have demonstrated successful function of the assay in a simple microfluidic format for diagnosis of HSV2 status in a panel of serum samples. Our ongoing work is focused on boosting the signal to a level that would enable the use of battery powered electronics for detection, and on incorporating passive flow controls into the device design in order to increase the assay automation.