Chang Mu-Shen, Lee Chia-Yi, Chang Yu-Yen, Wu Hsin-Yu, Wang Yeng-Tseng, Chao Hsuan, Liu En-Shuo, Huang Hsin-Kai, Lin Wen-Wei
PhD Program in Life Science, College of Life Science, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Anal Chem. 2025 Feb 25;97(7):3872-3880. doi: 10.1021/acs.analchem.4c04608. Epub 2025 Feb 12.
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.
双荧光素酶报告基因（DLR）检测是一种广为人知的基因表达分析工具。它能够进行批次间、并行归一化，使其成为探索实际样本信号的一种有价值的方法。DLR基于刺激物的功效识别真实信号，并凭借其独特的信号调节能力反映下游信号传导中最细微的变化。然而，DLR底物（如d - 荧光素和腔肠素）价格昂贵且稳定性不足，无法提供轻松的操作环境。在本研究中，我们引入了一种以辣根过氧化物酶（HRP）为概念验证的双结构显色酶（DSCE）平台。HRP被设计为要么锚定在细胞膜上，要么分泌到细胞外区室。通过用底物（ABTS和TMB）优化该技术，我们发现以ABTS作为内部对照的分泌型HRP（sHRP）以及用于样本信号检测的膜结合型HRP（mHRP）和TMB提供了最优化的输出。此外，我们比较了DSCE与DLR的信号灵敏度和耐久性。DSCE提供了更宽的动态范围和信号耐久性。最后，DSCE的底物成本比DLR低30倍。总之，DSCE平台利用酶和底物提供快速检测以及持续超过8小时的持久信号。该平台成本低廉且不影响归一化能力。
