Accurate and sensitive detection of protein biomarkers is critical for advancing in vitro diagnostics (IVD), yet conventional enzyme-linked immunosorbent assays (ELISA) often fall short in terms of sensitivity compared to nucleic acid-based tests. Bridging this sensitivity gap is essential for improving diagnostic accuracy, particularly in diseases where protein levels better reflect disease progression than nucleic acid biomarkers. In this review, we present strategies developed to enhance the sensitivity of ELISA, structured according to the sequential steps of the assay workflow. Beginning with surface modifications, we then discuss the methodologies to improve mixing and washing efficiency, followed by a summary of recent advances in signal generation and amplification techniques. In particular, we highlight the emerging role of cell-free synthetic biology in augmenting ELISA sensitivity. Recent developments such as expression immunoassays, CRISPR-linked immunoassays (CLISA), and T7 RNA polymerase–linked immunosensing assays (TLISA) demonstrate how programmable nucleic acid and protein synthesis systems can be integrated into ELISA workflows to surpass the present sensitivity, affordability, and accessibility. By combining synthetic biology-driven amplification and signal generation mechanisms with traditional immunoassay formats, ELISA is poised to evolve into a highly modular and adaptable diagnostic platform, representing a significant step toward the next generation of highly sensitive and programmable immunoassays.

In this paper, we have developed a novel cell-free biosensor based on a multiplexed pushbutton-activated microfluidic device (mPAMD) that enables simultaneous detection of multiple 16S rRNAs of pathogens in a single device. The multi-step target-responsive cell-free protein synthesis process was seamlessly integrated into a single microfluidic device with an intuitive finger-pumping mechanism, allowing simultaneous mixing, aliquoting, and detection of 16S rRNAs through the production of reporter proteins. The mPAMD incorporates multiplexed detection zones with pathogen-specific probes to enable the identification of multiple 16S rRNAs that allow a simple and intuitive diagnostic platform for cell-free biosensors. Microchannels were designed and optimized to achieve efficient sample mixing and even distribution of common reagents, ensuring uniform reaction conditions across all reaction channels. The developed system achieved a detection limit for 16S rRNA ranging from 1.69 to 7.39 pM, corresponding to approximately 10⁴ to 10⁵ CFU/mL of pathogens. These results address the growing demand for an accessible multiplexed diagnostic system while ensuring high sensitivity and specificity.