Abstract
Widely used in research and clinical medicine, immunoblots (e.g., western blot) provide target specificity by combining an electrophoretic separation of target species with subsequent immunoassaying steps. While miniaturization has benefited immunoblots in both separation efficiency and applicability to single cells, the widely used ‘microchannel’ and capillary devices make access to resolved targets cumbersome. To design a single-cell immunoblot with the most favorable mass transport conditions at each assay stage, we introduce a microfluidic format that toggles from a planar array of single-cell electrophoresis separations to a suspension of microparticles, with each rectangular microparticle housing one separation. For single-cell electrophoresis, a thin-layer hydrogel on a glass slide is stippled with microwells for cell isolation, each abutting a region for polyacrylamide gel electrophoresis (PAGE) and photocapture of separated proteins to the gel matrix. Array elements are defined by perforations around each rectangular single-cell PAGE region which, upon mechanical release, toggles into a separations-encoded microparticle. The microparticles measure estrogen receptor and 5 protein targets in hundreds of single breast cancer cells and kidney cells. For immunoassays, holding separations-encoded microparticles in suspension enhances diffusion into the gel making single-cell immunoblotting steps (i.e., immunoprobe introduction and wash out) more efficient by 4x. Dehydrating the microparticles from suspension results in isotropic shrinkage of each separations-encoded particle (10% in length), yielding a 1.6x increase in immunoblot fluorescence signal with no penalty on separation resolution. Designed for optimal mass transport and scaling at each assay stage, separations-encoded microparticles provide an adaptable new form factor for precision single-cell analysis.
Statement of significance Direct measurement of proteins from single cells has been realized at the microscale using microfluidic channels, capillaries, and semi-enclosed microwell arrays. Although powerful, these formats are constrained, with the enclosed geometries proving cumbersome for multistage assays, including electrophoresis followed by immunoprobing. We introduce a hybrid microfluidic format that toggles between a planar microwell array and a suspension of microparticles. The planar array is stippled in a thin sheet of polyacrylamide gel, for efficient single-cell isolation and protein electrophoresis of hundreds-to-thousands of cells. Upon mechanical release, array elements become a suspension of separations-encoded microparticles for more efficient immunoprobing due to enhanced mass transfer. Dehydrating microparticles offer improved analytical sensitivity owing to in-gel concentration of fluorescence signal for high-throughput single-cell targeted proteomics.