期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 109, 期 16, 页码 5972-5977出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1108617109
关键词
isoelectric focusing; nanoporous reactive polymers; lab-on-a-chip; Western blotting; antibody selection
资金
- National Institutes of Health (NIH) Office of the Director [1DP2OD007294]
- NIH [5R01CA121460]
A dearth of protein isoform-based clinical diagnostics currently hinders advances in personalized medicine. A well-organized protein biomarker validation process that includes facile measurement of protein isoforms would accelerate development of effective protein-based diagnostics. Toward scalable protein isoform analysis, we introduce a microfluidic single-channel, multistage immunoblotting strategy. The multistep assay performs all immunoblotting steps: separation, immobilization of resolved proteins, antibody probing of immobilized proteins, and all interim wash steps. Programmable, low-dispersion electrophoretic transport obviates the need for pumps and valves. A three-dimensional bulk photoreactive hydrogel eliminates manual blotting. In addition to simplified operation and interfacing, directed electrophoretic transport through our 3D nanoporous reactive hydrogel yields superior performance over the state-of-the-art in enhanced capture efficiency (on par with membrane electroblotting) and sparing consumption of reagents (ca. 1 ng antibody), as supported by empirical and by scaling analyses. We apply our fully integrated microfluidic assay to protein measurements of endogenous prostate specific antigen isoforms in (i) minimally processed human prostate cancer cell lysate (1.1 pg limit of detection) and (ii) crude sera from metastatic prostate cancer patients. The single-instrument functionality establishes a scalable microfluidic framework for high-throughput targeted proteomics, as is relevant to personalized medicine through robust protein biomarker verification, systematic characterization of new antibody probes for functional proteomics, and, more broadly, to characterization of human biospecimen repositories.
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