A Dual-Structured Chromogenic Enzyme Platform for a Rapid, Sensitive, Durable, and Precise Gene Expression Analysis

The dual luciferase reporter (DLR) assay is a well-known tool for gene expression analysis. Its ability to provide batch-to-batch, side-by-side normalization makes it a valuable method through which to explore actual sample signals. DLRs identify a real signal based on the stimulant's efficacy and can reflect the slightest change in downstream signaling with its unique signal adjustment ability. However, DLR substrates (e.g., d-luciferin and coelenterazine) are expensive and not stable enough to deliver a laborless operating environment. In this study, we introduce a dual-structured chromogenic enzyme (DSCE) platform that uses horseradish peroxidase (HRP) as a proof of concept. The HRP was engineered to be either tethered to the cell membrane or secreted into the extracellular compartment. Optimizing this technology with substrates (ABTS and TMB), we found that sHRP with ABTS as an internal control and mHRP and TMB for sample signal detection provided the most optimized output. Furthermore, we compared the signal sensitivity and durability of DSCE with the DLR. The DSCE provided a broader dynamic range and signal durability. Finally, substrates of the DSCE had a monetary cost that was 30-fold lower than the DLR. In summary, the DSCE platform utilizes enzymes with substrates to provide rapid detection and a durable signal for over 8 h. The platform is cost-friendly and does not compromise the normalization ability.