A glucose meter interface for point-of-care gene circuit-based diagnostics

Abstract

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

Effective Use of Linear DNA in Cell-Free Expression Systems

“…requires screening multiple (and sometimes many) candidates. Also, triggers can be added directly as linear ssDNA oligos to quickly test for switch functionality for a given target, avoiding the encoding of RNA triggers on DNA templates entirely ( Amalfitano et al, 2021 ).…”

Point-of-care analyte quantification and digital readout via lysate-based cell-free biosensors interfaced with personal glucose monitors

“…Although our current nucleic acid sensors could not detect targets at physiologically relevant concentrations (typically attomolar to femtomolar levels), an upstream amplification step can be implemented to bring initial nucleic acid concentrations up to the detection limit 3, 7, 29 . Previous work has shown robust concentration-dependent toehold switch activation with femto- to pico-molar of triggers amplified via isothermal amplification techniques 29 .…”

“…CFE biosensing reactions have been demonstrated to retain their function after lyophilization 2–4, 27, 33, 34 , so these tests can be stored and shipped to testing sites without cold-chain requirements, significantly enabling their deployment to the point of need. Moreover, the use of lysate-based CFE in this work rather than purified enzymes can reduce the cost of CFE reagents by almost an order of magnitude 2, 7 , making such an approach more feasible for wide-scale deployment and accessible to the developing world as well as to consumers in developed countries. We also show that CFE metabolism can be exploited to remove endogenous glucose initially present in complex samples (like human serum) in a one-pot format, thereby eliminating an upstream processing step to remove endogenous glucose that would otherwise be a requirement of a PGM-based method.…”

“…The first-ever reported use of cell-free systems with a glucose monitor was limited to using reagent complementation strategies 8 , such that it was only used to measure analytes that were required components of the cell-free reaction. A more recent report demonstrated detection of the presence and absence of targets (nucleic acid sequences) but did not aim for analyte quantification, used a purified enzyme strategy with high costs (almost $10 per sample) 7 , and required a separate, overnight enzymatic conversion step necessitating advance knowledge of patient glucose levels and sample-specific variable volumes of reagents to be added to the reaction by the user to clear native glucose from samples, which would not be viable for field-deployable applications. While that report was a significant step forward, the use of a PGM for qualitative, presence/absence diagnosis rather than quantitative measurement meant the strategy did not fully exploit one of the most critical and impactful capabilities offered by PGMs.…”

“…Since the product's commercialization in the 1970s, personal glucose monitors have been through decades of refinement and matured into a robust and easily accessible technology that many patients use daily and almost anywhere 6 . As a result, there has been significant research effort to engineer biosensors that can be read by a glucose monitor [7][8][9][10][11][12][13][14][15] rather than trying to engineer entirely new quantification devices that match the PGM's portability and reliability. Recent efforts have even successfully interfaced glucose monitors with cell-free system-based biosensors, though they were subject to significant limitations.…”