Microfabrication for Biomedical Research

生物医学研究的微加工

• 批准号: 7967872
• 负责人: Nicole Y Morgan
• 金额: $ 18.67万
• 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/7967872
• 关键词: Address; Antibodies; Antigens; Autoimmune Process; Basic Science; Biological; Biological Assay; Biological Markers; Biomedical Engineering; Biomedical Research; Buffers; Capillary Electrophoresis; Cell Extracts; Cells; Chemistry; Chimeric Proteins; Clinical; Clinical Research; Collaborations; Communicable Diseases; Detection; Development; Device or Instrument Development; Devices; Dimensions; Elastomers; Encapsulated; Extramural Activities; Future; Glass; Human; Immune response; Immunoprecipitation; Intramural Research Program; Laboratories; Luciferases; Luminescent Measurements; Measurement; Measures; Methodology; Microfabrication; Microfluidic Microchips; Microfluidics; Miniaturization; Mission; Molds; Physiology; Process; Protocols documentation; Renilla Luciferases; Reproducibility; Research Personnel; Resolution; Running; Sampling; Scientist; Serum; Signal Transduction; Silicon; Speed; Structure; Surface; System; Technology; Time; United States National Institutes of Health; Variant; Work; design; driving force; instrument; interest; lithography; miniaturize; physical science; point-of-care diagnostics; rapid detection; research study; seal; two-dimensional

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.

人们对同时快速检测单个生物样品中的多个生物标志物产生了浓厚的兴趣。这种兴趣一直是生物医学应用微流体装置开发的驱动力之一。转向这些较小规模的系统有很多优点。首先，它们能够分析较小的样本量。其次，在毛细管电泳等应用中，微流体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，分析装置尺寸的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。另一种正在开发的设备是小型化的荧光素酶免疫沉淀（LIPS）检测，用于寻找血清样本中的免疫反应。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物和生理学的设备。我们的设施现已发展到能够为校内和校外研究人员提供建议并与他们合作，研究如何生产满足其特定需求的微流体设备。 通过与 NIST 的科学家合作，并使用 NIST 的微加工设施，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，以及用于将热塑性塑料或将弹性体模压成微流体装置的模板。 今年，我们在校园内进一步开发了利用这些模板在热塑性塑料和弹性体中形成、密封和涂覆微流体通道的技术。 我们还在实验室建立一个基本的光刻系统，旨在快速制造尺寸小至约 25 微米的单层结构和模板。 此外，我们还在继续开展旨在小型化 LIPS 检测的项目，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。目前的检测形式是在 96 孔滤板中进行，并且已被证明可以有效检测多种自身免疫性疾病和传染病。将测定转移到微流体格式可以显着加快分析速度，允许多重分析，并且还有助于该测定在现场诊断中的应用。使用含有融合蛋白和商业抗 CFLAG 抗体的细胞提取物代替血清进行的实验表明，即使在小型化形式中，阳性信号水平也具有可接受的高水平。 今年，我们的努力主要集中在提高测量重现性，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大幅改进，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，运行之间的差异小于 10%。 在不久的将来，我们计划对一组样品进行测量，以验证微流体测定与原始孔格式之间的对应关系。