Bispecific Coiled-Coil Reporters Enable Rapid, Multiplexed Detection of Mycoplasma via Toehold Switch Sensors

Abstract Cell-free synthetic biology offers a rapid prototyping environment for molecular diagnostics, yet the utility of these systems is often limited by the slow kinetics and restricted multiplexing capabilities of traditional protein reporters. Here, we report the BINOCULAR (BIspecific Novel Orthogonal Coiled-coil-Using LAteral flow Reporter) system based on supramolecular coiled-coil peptide conjugates that dramatically accelerates signal generation and enables robust multiplexing in cell-free transcription-translation reactions. Using BINOCULARs as toehold switch outputs, we achieve visible readout times of less than 5 min in lateral flow assays, achieving a ≥12-fold increase in speed compared to standard cell-free reporters like β-galactosidase or fluorescent proteins. To demonstrate the utility of this platform, we integrated the bispecific reporters with loop-mediated isothermal amplification and lateral flow assays for the detection of mycoplasma, a common adventitious agent in biomanufacturing. The integrated system demonstrates exceptional sensitivity, achieving a detection limit of 50 genomic copies/mL for mycoplasma and a mammalian internal control. Cell-free assay results were available within 5 min following a 20 min amplification step, and we successfully achieved the simultaneous detection of three distinct targets in a single reaction. Crucially, the platform maintains reliable performance in complex matrices and after lyophilization, making it more accessible for point-of-use applications. Our findings suggest that the modular BINOCULAR architecture can overcome current bottlenecks in cell-free sensing systems, providing a versatile and scalable framework for rapid, point-of-use screening across clinical and industrial applications.