Abstract
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.
Highlights
Simultaneous detection of multiple analytes from a single sample, when done at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test
We envision that an array of membrane-less protocell sensors formed by selective compartmentalization of cell-free expression (CFE) reactions in aqueous two-phase system (ATPS) can facilitate simultaneous detection of multiple analytes beyond just proteins, leading to a new class of protocell array-based diagnostics that reports on diverse types of analytes, has a high degree of sensor customizability, and can be used at the point of need
Protocell arrays meet key criteria for fielddeployable sensors and diagnostics: we demonstrate that GFP reporters can be replaced with color-producing enzymatic reporters to enable equipment-free test interpretation, and we demonstrate parallel, simultaneous detection of multiple analytes using protocell arrays that have been lyophilized for storage at ambient temperature
Summary
Simultaneous detection of multiple analytes from a single sample (multiplexing), 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 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. Since many soluble macromolecules, such as the proteins and nucleic acids that enable CFE-based sensing, selectively partition from a relatively more hydrophobic polyethylene glycol (PEG)-rich phase to a hydrophilic dextran- or Ficoll-rich phase[16,20,21], the core of the distinct biosensing machinery stays compartmentalized in each ATPS-formed protocell, driven by thermodynamic forces Because these protocells are membrane-less, this approach resolves challenges in transporting macromolecular analytes from the bulk phase to inside the protocells, a limitation present in most membranebased approaches[22]. We envision that an array of membrane-less protocell sensors formed by selective compartmentalization of CFE reactions in ATPS can facilitate simultaneous detection of multiple analytes beyond just proteins, leading to a new class of protocell array-based diagnostics that reports on diverse types of analytes, has a high degree of sensor customizability, and can be used at the point of need
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