AbstractSimultaneous detection of multiple analytes from a single sample (multiplexing), particularly when at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test. Despite recent advances, multiplexing still typically faces the critical limitation of measuring only one type of molecule per assay platform – for example, only small molecules or only nucleic acids. In this work, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aqueous two-phase system (ATPS) to produce membrane-less “protocells” containing transcription and translation machinery used for analyte detection. Multiple protocells are arrayed in microwells where each protocell droplet performs distinct reactions to detect chemically diverse targets including small molecules, minerals, and nucleic acid sequences, all from the same sample. We demonstrate that these protocell arrays can measure analytes in a human biofluid matrix, maintain function after lyophilization and rehydration, and produce visually interpretable readouts, illustrating its potential for application as a minimal-equipment, field-deployable, multi-analyte detection tool.
Simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays
