Microfabrication for Biomedical Research

尽管在开发用于生物医学应用的微加工和微流体技术方面已经开展了大量工作，但将该技术广泛应用于生物医学研究实验室仍然存在瓶颈。部分问题在于许多生物医学研究人员对微加工的能力缺乏了解。此外，许多潜在的研究项目都受到限制，需要广泛的定制和多次设计迭代，而这在可用的商业产品数量有限的情况下可能无法实现。 为了降低将微加工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加工能力，可以使用干膜抗蚀剂或 SU-8 制作 PDMS 或水凝胶器件的模板。尽管分辨率、器件良率和复杂性略低于专用洁净室所能达到的水平，但它们仍然足以进行许多细胞实验。此外，仪器复杂性、制造成本和周转时间都大大降低，从而能够根据需要快速循环设计参数。 今年，我们继续完善图案化和器件制造协议，现在能够可靠地图案化单层和双层模板特征，横向尺寸在 5 微米以下，高度范围从几微米到几百微米。 在单个模板上图案化具有不同高度的多个层的能力使得能够生产具有显着更大功能的装置，例如，可用于将细胞捕获在一个隔室中的结构，同时通过更大的通道提供连续的流体输送。 我们还开发了使用这些模板生成微结构 PDMS、琼脂糖和 PEGDA 水凝胶的方案，并且今年一直在改进用于制造薄（<200 微米）PDMS 层的技术，用于多层器件和无底结构。我们继续开发 PDMS 和其他聚合物表面改性的方案，包括使用实验室仪器将 PDMS 与玻璃不可逆结合，以及将设备连接到流量控制仪器（例如注射泵和压力控制器）的技术。通过与 NIST 科学家的持续合作，我们还能够使用 NIST 的纳米加工设施，根据需要制造更复杂、更高耐受性的结构。 今年，我们还开发了使用可编程剃须刀和压敏粘合剂来直接制造高度从25微米到几百微米、横向尺寸为亚毫米的流通池。 这种低成本且方便的方法可用于准备制造具有两个玻璃壁的流通池，以及在已经功能化的表面上提供流体限制。 在过去的一年里，这些功能已在代表广泛利益和机构的许多项目中得到应用。 除了下面讨论的代表性项目和其他仍处于早期阶段的项目外，我们还对基础微加工技术的研究人员进行了培训，包括来自 NHLBI、NCI、NICHD 和 NIBIB 其他实验室的人员。 一个长期运行的项目是与 LSB、NIAID 合作，研究 3-D 胶原蛋白基质的趋化性，为此我们一直在开发和改进一种与高分辨率荧光和双光子成像兼容的微流体琼脂糖装置。单独平台上的混合三通与可编程注射泵流量控制一起，能够形成可重复的时变空间梯度。今年，我们继续探索能够独立控制胶原纤维排列程度和化学梯度的多层设备的不同架构。 此外，我们还开发了原始器件和多层器件的有限元模型，以便为器件设计提供信息并协助表征。 第二个项目是与 LCE、NHLBI 合作，持续开发用于相衬 X 射线成像的光栅，其中涉及使用 NIST 纳米加工设施以及我们自己的设备。今年，我们的小组为光栅制造参数和表征的细化以及金属电沉积到图案化光栅结构的协议的开发做出了贡献。 第三个正在进行的项目是与 MDP、NIDDK 合作，使用微孔限制卵子，以便通过快速、高分辨率显微镜研究受精。 对于此应用，孔需要与现有的荧光成像和培养系统兼容，并且能够以最少的处理捕获卵。 最近的一项改进增加了微通道，以便能够在焦平面上控制精子的输送。 在第四个项目中，我们一直在继续开发、制造和表征通过旋涂制成的混合聚合物薄膜，用于独立于操作员的高分辨率激光捕获显微切割。这项工作是 NIMH、NCI 和 NICHD 研究人员共同发起的研究所间主任挑战项目的一部分，旨在开发基于组织捕获亚细胞结构的方法，用于基于质谱的蛋白质组分析。 第五个项目是开发和实施 PDMS 微流体梯度发生器，用于将硫酸软骨素蛋白聚糖沉积在神经细胞培养基质上，我们与 DN、CBPC、NHLBI 合作使用该发生器，以更好地了解这些分子在轴突生长和引导中所起的作用。 最后，我们正在继续开展一个项目，旨在小型化 LSB、NIDCR 开发的荧光素酶免疫沉淀系统 (LIPS) 测定，该项目使用由海肾荧光素酶和目标抗原组成的融合蛋白来探测人血清中的抗体。今年，我们使用无电源流量控制在玻璃流通池中展示了该测定的成功功能，并通过使用功能化磁珠增加有效捕获表面积，使信号比原始单通道微流体格式增加了三十倍以上。此外，我们开始研究使用大面积光电二极管的电池供电探测器。 我们正在进行的工作重点是进一步增强信号，并将无源流量控制纳入设备设计中，以提高易用性。

Although there has been extensive work developing microfabrication and microfluidic technology for biomedical applications, bottlenecks remain 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 many 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 for making templates for PDMS or hydrogel devices using either a dry-film resist or SU-8. 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 and device fabrication, and are now able to reliably pattern single and double layer template features with lateral dimensions under 5 microns, and with heights ranging from a few microns to a few hundred microns. The ability to pattern multiple layers with different heights on a single template enables the production of devices with substantially greater functionality as an example, structures that can be used for trapping cells in one compartment while providing continuous fluid delivery through larger channels. We have also developed protocols for using these templates to generate microstructured PDMS, agarose, and PEGDA hydrogels, and this year have been refining techniques for making thin (<200 micrometer) PDMS layers for use in multilayer devices and bottomless structures. 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 and pressure controllers. 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. This year, we also developed the use of a programmable razor cutter and pressure-sensitive adhesive to directly make flow cells with heights ranging from 25 to a few hundred microns, and sub-millimeter lateral dimensions. This low-cost and convenient method is useful for ready fabrication of flow cells with two glass walls, as well as for providing fluidic confinement over already-functionalized surfaces. These capabilities have found application in a number of projects, representing a broad variety of interests and institutes, over this past year. In addition to the representative projects discussed below and others still in the early stages, we have also trained researchers in basic microfabrication techniques, including personnel from other laboratories in NHLBI, NCI, NICHD, 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. A mixing tee on a separate platform, together with programmable syringe pump flow control, enables the formation of reproducible time-varying spatial gradients. This year, we have continued to explore different architectures for a multilayer device capable of independently controlling the degree of collagen fiber alignment and the chemical gradient. In addition, we have developed finite element models of the original device as well as the multilayer device in order to inform device design and assist in characterization. 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. This year, our group has contributed to refinement of the grating fabrication parameters and characterization as well as to the development of protocols for electrodeposition of metals into the patterned grating structures. A third ongoing project, in collaboration with MDP, NIDDK, is the use of microwells to confine eggs in order to study fertilization with fast, high-resolution microscopy. For this application, the wells need to be compatible with existing fluorescence imaging and culture systems, and also to enable capture of the eggs with minimal handling. A recent refinement has added microchannels to enable controlled delivery of the sperm at the focal plane. 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 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. This year, we demonstrated successful function of the assay in a glass flow cell using power-free flow control, and achieved more than a thirty-fold increase in signal over the original, single-channel microfluidic format by using functionalized magnetic beads to increase the effective surface area for capture. In addition, we began work on a battery powered detector using a large-area photodiode. Our ongoing work is focused on further boosting the signal, and on incorporating passive flow controls into the device design in order to improve ease of use.