Protocell arrays for simultaneous detection of diverse analytes

Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when done at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test. Despite recent advances, multiplexed analyte sensing still typically faces the critical limitation of measuring only one type of molecule (e.g., small molecules or nucleic acids) per assay platform. Here, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aqueous two-phase system (ATPS), producing membrane-less protocells containing transcription and translation machinery used for detection. We show that multiple protocells, each performing a distinct sensing reaction, can be arrayed in the same microwell to detect chemically diverse targets from the same sample. Furthermore, these protocell arrays are compatible with human biofluids, maintain function after lyophilization and rehydration, and can produce visually interpretable readouts, illustrating this platform’s potential as a minimal-equipment, field-deployable, multi-analyte detection tool.