Separation-encoded microparticles for single-cell western blotting

Abstract

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 separation-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.

Figure 1.. Design and operation of the separation-encoded microparticles for high specificity protein isoform analysis.

(A) Separation-encoded microparticle array consisting of approximately 3500 releasable units fabricated on a half glass slide. (B) Schematic view of single-cell resolution western blotting workflow in microparticles. After performing the single-cell settling, electrophoresis, protein photocapture to the gel (immobilization), and immunoprobing, microparticles are released from the microscope glass slide by the help of a blade. Bands of separated proteins in each microparticle are visualized using a fluorescence scanner for quantification. (C) The array is comprised of a thin layer of polyacrylamide gel. Each microparticle contains a 30 μm diameter well where single cells are housed. (D) Single-cell resolution western blotting assay is ran on microparticles. Multiple protein markers can be probed in individual microparticles, the image shows false-colored micrographs of microparticles. (E) Microparticles can be released and collected for downstream analysis. (F) False-colored microscopy images of microparticle array attached on and released from a glass slide was probed and imaged in both hydrated and dehydrated states. Microparticles were probed for two housekeeping protein markers β-Tubulin (50 kDa, magenta) and GAPDH (35 kDa, blue) from single U251 cells. Dehydrating microparticles boosts analytical sensitivity thanks to the geometry-enhanced concentration of fluorescence signal, while the immunoprobe signal intensity in the released format is also increased attributable to enhanced surface area of each gel element.

Figure 2.. Characterization of ERα isoform expression in microparticle assay.

(A) Measured ERα expression from the same array after a multistep probing (including one stripping round), and after probing of designated microparticles (p>0.05, n = 40 microparticles). (B) Fluorescence micrographs of microparticles for Actinin, β-Tubulin, GAPDH and ERα isoforms in MCF 7 cells. RFU, relative fluorescence units. The off-target peak (via ERα antibody) does not coincide with the ERα isoform bands. (C) Log-linear plot of species molecular weight against migration distance in 8%T PAG the fluorescently labeled species in panel A. (x-axis error bars within point size (± S.D., n = 3 separations); GAPDH, 35 kDa; ERα46, 46 kDa; β-Tubulin, 50 kDa; ERα66, 66 kDa; Actinin, 100 kDa). (D) Box plot demonstrating the separation resolution between ERα isoforms (n = 34 cells).

Figure 3.. Characterization of microparticles with different morphologies.

(A) Reduction in peak widths of β-tubulin and GAPDH were 10% and 7%, consistent with observed microparticle shrinkage. Mann-Whitney U-test p value was found to be lower than 0.05 (n = 121). (B) The median fluorescence signal intensity increase was 1.6x on the dehydrated microparticles compared to the hydrated microparticles (CV = 1.2, n = 121). (C) Quantified β-Tubulin and GAPDH expression in microparticles from U251 glioblastoma cells shows a median normalized AUC for dehydrated microparticles ~1.3 to 1.7x higher than hydrated microparticles For all combinations, Mann-Whitney U-test p value was found to be lower than 0.05 (n = 121).

Figure 4.. Expression of ERα isoforms change over confluency of cell culture. MCF 7 cells are shown as the model ERα positive organisms, whereas MDA MB 231 and HEK 293 serve as the ERα negative control cell lines.

(A) Color-coded beeswarm graphs show single-cell protein measurements in subsequent cell culturing days for MCF 7, MDA MB 231, and HEK 293 cell lines. The black bars present the median protein expression level for each group. (B) Signal-to-noise ratio (SNR) for ERα isoforms. Red dashed line presents SNR = 3, above which protein quantification was employed for all measurements, n_total = 447. (C) The grouped box plots show fluctuations in ERα isoform expression over 14 days in MCF 7 cells (n = 478 cells). Microparticles reported an gradual increase in the number of cells expressing ERα46 (green), while the expression of ERα66 (blue) dropped gradually over 14 days.