Stability insights into synthetic biology enabled biosensors: A case study with SARS-CoV2 toehold-based colorimetric sensor

Improving medical and environmental diagnostics has become a pressing need. Synthetic biologists are steering biomolecular engineering efforts toward this objective, promising novel, cost-effective diagnostic solutions. While conventional antibody-based diagnostics are sensitive, they are slow, costly, and struggle with emerging pathogens or rare diseases. Synthetic biology's rapid design-to-production cycles offer a solution, introducing engineered gene circuits that diversify molecular detection, create dynamic sensors, and enable portable diagnostic tools. Toehold switch-based diagnostics emerge as a promising, inexpensive, rapid, and highly sensitive alternative to RT-qPCR, especially beneficial in resource-limited regions. These devices, adaptable to paper-based platforms, offer potential for widespread use in low-resource settings. Ensuring stability and functionality under varying environmental factors poses a challenge in their practical implementation for diagnostic purposes. To address this, our study focuses on preserving cell fee expression systems under extended temperature stress through lyophilization. Lyophilization emerges as a crucial method, potentially ensuring prolonged stability and convenient transportation of diagnostic components. We emphasize the significance of choosing the appropriate lyoprotectant, underscoring the necessity of exploring various lyoprotectants to ensure scalability and cost-effectiveness in these molecular tools. Our demonstration of dextran's practical utility in enhancing the stability of lyophilized cell-free expression system for colorimetric diagnostics, especially in detecting synthetic triggers for SARS-CoV-2, signifies a promising advancement in molecular diagnostics for resource-limited settings. • Excipients tested for retaining activity of lyophilized synthetic biology-based SARS-CoV-2 colorimetric assay. • Dextran and maltodextrin retained activity at −20 °C for 3 months. • Dextran retained activity at 4 °C for 3 weeks. • Dextran stabilized paper-based assay for 4 weeks at −20 °C.