Redox Cycling-based Electrochemical Reporter Gene Assay for Single Cells Using a Scanning Electrochemical Microscope-microwell System

Abstract

Single-cell analyses are important for providing new insights into cellular biology. Here we report an electrochemical reporter gene assay for single cells using a scanning electrochemical microscope (SECM)-microwell system. Each microwell trapped a single cell that synthesized a reporter protein, secreted alkaline phosphatase (SEAP). The SEAP catalyzed the hydrolysis of p-aminophenyl phosphate to p-aminophenol (PAP). A disk electrode in the SECM was positioned above the microwell and monitored the oxidation currents of PAP derived from SEAP. In addition, ring electrodes were prepared on the microwell device to induce redox cycling between the ring and disk electrodes, thus amplifying the electrochemical signals from the reporter protein. The redox cycling-based electrochemical reporter gene assay is useful for single-cell analyses.

Scanning Probe Microscopy for Nanoscale Electrochemical Imaging

“…The respiratory activity of live HeLa microtissues has been assessed by monitoring the oxygen reduction current in constant height mode SECM. We reported SECM investigation of the respiration activity of mouse embryonic stem cells, , rat primary hepatocyte spheroids, muscle cells embedded in a hydrogel-metallic glass wire complex, the β-galactosidase activity of cellular aggregates as a biomarker for senescence, the alkaline phosphatase activity of single cells entrapped in microwells with ring-electrode arrays, and intracellular enzymatic activities . Membrane protein expression levels have also been evaluated using SECM. , …”