Gomez Martinez, A. E. | Search bioRxiv

bioRxiv2025

Paired Analyses of Nuclear Protein Targets and Genomic DNA by Single-Cell Western Blot and Single-Cell PCR

Single-cell multimodal assays measure multiple layers of molecular information. Existing single-cell tools have limited capability to analyze nuclear proteins and genomic DNA from the same originating single cell. To address this gap, we designed and developed a microfluidic single-cell assay (SplitBlot), that pairs measurements of genomic DNA (PCR-based) and nucleo-cytoplasmic proteins (nuclear histone H3 and cytoplasmic beta-actin). To accomplish this paired multiomic measurement, we utilize microfluidic precision to fractionate protein molecules (both nuclear and cytoplasmic) from genomic DNA (nuclear). We create a fractionation axis that prepends a comet-like encapsulation of genomic DNA in an agarose molded microwell to a downstream single-cell western blot in polyacrylamide gel (PAG). For single-cell genomic DNA analysis, the agarose-encapsulated DNA is physically extracted from the microfluidic device for in-tube PCR, after release of genomic DNA from a molten agarose pallet (86% of pallets resulted in amplification of TurboGFP). For protein analysis, nucleo-cytoplasmic proteins are photocaptured to the PAG (via benzophenone) and probed in-situ (15 kDa histone H3 resolved from 42 kDa beta-actin with a separation resolution Rs = 0.77, CV = 76%). The SplitBlot reported the amplification of TurboGFP DNA and the separation of nuclear histone H3 and cytoplasmic beta-actin from the same single U251 cells engineered to express TurboGFP. Demonstrated here, Split-Blot offers the capacity for precision genomic DNA vs. protein fractionation for subsequent split workflow consisting of in-tube PCR and on-chip single-cell western blotting, thus providing a tool for pairing genotype to nuclear and cytoplasmic protein expression at the single-cell level. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/646125v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@168541corg.highwire.dtl.DTLVardef@69ff17org.highwire.dtl.DTLVardef@ea4fa1org.highwire.dtl.DTLVardef@111daad_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Single-cell Organelle Extraction with Cellular Indexing

Bulk methods to fractionate organelles lack the resolution to capture single-cell heterogeneity. While microfluidic approaches attempt to fractionate organelles at the cellular level, they fail to map each organelle back to its cell of origin--crucial for multiomics applications. To address this, we developed VacTrap, a high-throughput microfluidic device for isolating and spatially indexing single nuclei from mammalian cells. VacTrap consists of three aligned layers: (1) a Bis-gel microwells layer with a trapdoors (BAC-gel) base, fabricated atop a through-hole glass slide; (2) a PDMS microwell layer to receive transferred nuclei; and (3) a vacuum manifold. VacTrap operation begins with cell lysis using DDF to release intact nuclei into the Bis-gel microwells, while cytoplasmic proteins are electrophoresed into the Bis-gel. Upon exposure to DTT and vacuum force, the trapdoors open, allowing nuclei to transfer to the PDMS microwells. VacTrap dissolves the trapdoors within 3-5 minutes and achieve synchronized nuclei transfer with 98% efficiency across 80% of trapdoors in a 256-microwell array, surpassing the <1% efficiency of passive transfer without vacuum. Morphology analysis confirmed preservation of organelle integrity throughout VacTrap operation. By enabling spatial indexing of nuclei back to their original cell, VacTrap provides a robust, high-throughput tool for single-cell multiomics applications.

bioengineering↗