Microfabrication for Biomedical Research

生物医学研究的微加工

• 批准号: 8340631
• 负责人: Nicole Y Morgan
• 金额: $ 22.2万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8340631
• 关键词: 3-Dimensional; Antibodies; Antigens; Automation; Biological Assay; Biomedical Research; Biomedical Technology; Cell Culture Techniques; Cells; Chemicals; Chemotaxis; Chimeric Proteins; Chondroitin Sulfate Proteoglycan; Collaborations; Collagen; Collagen Fiber; Complex; Culture Media; Deposition; Detection; Development; Device Designs; Devices; Diagnosis; Dimensions; Discipline; Drug Formulations; Electronics; Equipment; Familiarity; Film; Fluorescence; Glass; Height; Housing; Human; Human Herpesvirus 2; Hybrids; Hydrogels; Image; Immunoprecipitation; Institutes; Laboratories; Lateral; Luciferases; Mass Spectrum Analysis; Methods; Microfabrication; Microfluidics; Modification; Molecular Classification of Tumors; National Heart, Lung, and Blood Institute; National Institute of Allergy and Infectious Disease; National Institute of Biomedical Imaging and Bioengineering; National Institute of Child Health and Human Development; National Institute of Dental and Craniofacial Research; National Institute of Mental Health; Neurons; Optics; Pattern; Phase; Play; Polymers; Process; Proteomics; Protocols documentation; Pump; Relative (related person); Renilla Luciferases; Research; Research Personnel; Research Project Grants; Resolution; Role; Running; Sampling; Scientist; Sepharose; Serum; Signal Transduction; Silicon; Speed; Structure; Subcellular structure; Surface; Syringes; System; T-Lymphocyte; Techniques; Technology; Thick; Time; Tissues; Training; Work; axon growth; axon guidance; base; biological systems; cost; design; flexibility; indexing; instrument; instrumentation; interest; laser capture microdissection; miniaturize; nanofabrication; protocol development; research study; synaptogenesis; two-photon

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.

尽管已经开展了广泛的工作来开发用于生物医学应用的微制造和微流控技术，但在将该技术广泛应用于生物医学研究实验室方面仍然进展有限。问题的一部分是生物医学研究人员对微制造能力缺乏熟悉。此外，许多潜在的研究项目具有需要广泛定制和多次设计迭代的限制，这在可用商业产品数量有限的情况下可能无法实现。 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制造用于PDMS或水凝胶设备的单层模板。尽管分辨率、器件成品率和复杂性比专用净化室要低一些，但它们仍然足以在细胞上进行许多实验。此外，极大地降低了仪器的复杂性、制造成本和周转时间，使设计参数能够根据需要快速循环。 今年，我们继续完善了两种商用干膜抗蚀剂的图案化方案。因此，我们能够可靠地在柔性或刚性衬底上绘制横向尺寸小至10微米、高度从15微米到数百微米的模板特征图案。此外，我们还开发了用于对几种SU-8配方进行图案化的协议，SU-8是一种旋转式抗蚀剂，它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PDMS、琼脂糖凝胶和PEGDA水凝胶的方案。最后，我们继续开发PDMS和其他聚合物的表面改性协议，包括使用我们实验室的仪器将PDMS与玻璃进行不可逆粘合，以及将设备连接到流量控制仪器的技术，如注射泵。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，以根据需要制造更复杂和更高公差的结构。 在过去的一年里，这些能力在许多项目中得到了应用，这些项目代表了广泛的利益和机构。除了下面讨论的项目，我们还培训了基础微制造技术的研究人员，包括来自NHLBI、NCI和NIBIB的其他实验室的代表。 一个运行时间更长的项目是与LSB，NIAID合作，研究3D胶原基质中的趋化性，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控琼脂糖设备。今年，我们在一个单独的平台上安装了混合三通，与可编程的注射器泵流量控制一起，形成了可重复的时变梯度。此外，我们正在开发一种混合多层设备，能够独立控制胶原纤维的取向程度和化学梯度。 第二个项目是与LCE、NHLBI合作，继续开发用于相衬X射线成像的光栅，这涉及到使用NIST纳米制造设施以及我们自己的设备。在过去的一年里，我们的团队致力于改进光栅制造参数以及开发用于复制、传输和光栅功能表征的协议。 第三个正在进行的项目是与LC MB，NCI合作，努力限制微孔中的B-T细胞对，使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；这已经通过PDMS成功实现，我们继续研究使用其他与培养基指数更匹配的材料。 在第四个项目中，我们一直在继续开发、制造和表征旋涂法制备的杂化聚合物薄膜，用于独立于操作员的、高分辨率的激光捕获显微解剖。这项工作是与NIMH、NCI和NICHD的研究人员共同开展的机构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，用于基于质谱学的蛋白质组分析。 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底物上沉积硫酸软骨素蛋白多糖，我们正在与DN、CBPC、NHLBI合作使用该设备，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，旨在使NIDCR LSB开发的荧光素酶免疫沉淀系统(LIPS)试验微型化，该系统使用由Renilla荧光素酶和感兴趣的抗原组成的融合蛋白来探测人类血清中的抗体。我们已经以一种简单的微流控形式展示了该检测方法在一组血清样本中诊断HSV2状态的成功功能。我们正在进行的工作集中于将信号提高到能够使用电池供电的电子设备进行检测的水平，并将被动流动控制纳入设备设计，以提高分析自动化。