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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Microfluidic Western blotting
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Microfluidic Western blotting

机译：微流控蛋白质印迹

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摘要

Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10-60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for protein sizing and, after brief UV exposure, a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. We observe protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.
机译：快速，定量的蛋白质印迹是生命科学中一个长期寻求的生物分析目标。为此，我们描述了在纯电子控制下在单个玻璃微通道中进行的蛋白质印迹分析。 μWestern印迹法包括多个步骤：样品富集，蛋白上浆，蛋白固定（印迹）和原位抗体探测。为了验证微流体分析，我们将μWestern印迹应用于人类血清（HIV免疫反应性）和细胞裂解物（NFκB）的分析。分析性能得到了提高，包括：10-60分钟的短时间检测，多重分析物检测，飞克级质量灵敏度，高灵敏度50 pM检测限以及动态范围为3.6 log的定量能力。性能的提高归因于微观尺度上有利的运输和反应条件。多步骤测定设计依赖于可光图案化（蓝光）和光反应性（紫外线）聚丙烯酰胺凝胶。这种亲水性聚合物既构成蛋白质上浆的分离基质，又构成短暂的紫外线照射后的蛋白质固定支架，用于随后对固定的蛋白质条带进行抗体探测。我们观察到在上浆条件下蛋白质捕获效率超过75％。这种紧凑的微流体设计支持在标准显微镜载玻片形状因子中演示48重μWestern印迹。综上所述，μWestern印迹通过将卓越的特异性与多重分析的通量优势相结合，为快速，靶向蛋白质组学奠定了基础，这与广泛的生物学研究相关。

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  • 作者

    Alex J. Hughes; Amy E. Herr;

  • 作者单位

    Department of Bioengineering California at San Francisco Graduate Program in Bioengineering,University of California, Berkeley, CA 94720,University of California at Berkeley-University of California at San Francisco Graduate Program in Bioengineering,University of California, Berkeley, CA 94720;

    Department of Bioengineering California at San Francisco Graduate Program in Bioengineering,University of California, Berkeley, CA 94720,University of California at Berkeley-University of California at San Francisco Graduate Program in Bioengineering,University of California, Berkeley, CA 94720;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    immunoblotting; medical diagnostics; protein microarrays; systems biology; electrophoresis;

    机译：免疫印迹医学诊断;蛋白质微阵列;系统生物学;电泳;

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