Enhancing Single-Cell Western Blotting Sensitivity Using Diffusive Analyte Blotting and Antibody Conjugate Amplification.

While there are many techniques to achieve highly sensitive, multiplex detection of RNA and DNA from single cells, detecting protein content often suffers from low limits of detection and throughput. Miniaturized, high-sensitivity Western blots on single cells (scWesterns) are attractive because they do not require advanced instrumentation. By physically separating analytes, scWesterns also uniquely mitigate limitations to target protein multiplexing posed by the affinity reagent performance. However, a fundamental limitation of scWesterns is their limited sensitivity for detecting low-abundance proteins, which arises from transport barriers posed by the separation gel against detection species. Here we address the sensitivity by decoupling the electrophoretic separation medium from the detection medium. We transfer scWestern separations to a nitrocellulose blotting medium with distinct mass transfer advantages over traditional in-gel probing, yielding a 5.9-fold improvement in the limit of detection. We next amplify probing of blotted proteins with enzyme-antibody conjugates, which are incompatible with traditional in-gel probing to achieve further improvement in the limit of detection to 1000 molecules, a 120-fold improvement. This enables us to detect 100% of cells in an EGFP-expressing population using fluorescently tagged and enzyme-conjugated antibodies compared to 84.5% of cells using in-gel detection. These results suggest the compatibility of nitrocellulose-immobilized scWesterns with a variety of affinity reagents─not previously accessible for in-gel use─for further signal amplification and detection of low-abundance targets.