Point-of-Care Analyte Quantification and Digital Readout via Lysate-Based Cell-Free Biosensors Interfaced with Personal Glucose Monitors

Abstract

Field-deployable diagnostics based on cell-free systems have advanced greatly, but on-site quantification of target analytes remains a challenge. Here we demonstrate that Escherichia coli lysate-based cell-free biosensors coupled to a personal glucose monitor (PGM) can enable on-site analyte quantification, with the potential for straightforward reconfigurability to diverse types of analytes. We show that analyte-responsive regulators of transcription and translation can modulate the production of the reporter enzyme β-galactosidase, which in turn converts lactose into glucose for PGM quantification. Because glycolysis is active in the lysate and would readily deplete converted glucose, we decoupled enzyme production and glucose conversion to increase the end point signal output. However, this lysate metabolism did allow for one-pot removal of glucose present in complex samples (like human serum) without confounding target quantification. Taken together, our results show that integrating lysate-based cell-free biosensors with PGMs enables accessible target detection and quantification at the point of need.

Keywords: analyte quantification; biosensor; cell-free systems; diagnostics; human serum; personal glucose monitor.

Figure 1:

Characterization of glucose production and depletion in CFE reactions. (A) Verification of CFE compatibility with PGM and time-course measurement of glucose depletion in reactions. A slight decrease in PGM output was observed for the same concentration of glucose in the CFE matrix and no incubation time compared to glucose in a water solution (“Standards”). Rapid depletion of glucose signal was observed in all CFE reactions over time. Error bars represent the standard deviation of cell-free reaction triplicates. Dashed gray line represents PGM’s lowest reading threshold, 20 mg/dL. (B) Decoupling enzyme production and glucose conversion in CFE reactions enabled dose-dependent PGM signal output. CFE reactions containing varying concentrations of plasmid constitutively expressing LacZ were incubated for 30 to 60 minutes before each reaction was quenched by naproxen-lactose mix to shift the reaction from enzyme production to glucose conversion. Plasmid concentration-modulated glucose production was detected using the PGM after 15 minutes of incubation. Error bars represent the standard deviation of cell-free reaction triplicates. Dashed gray line represents PGM’s lowest reading threshold, 20 mg/dL.

Figure 2:

Application of PGM-mediated quantification of zinc in CFE reactions. (A) Schematic of zinc-modulated glucose production and PGM-mediated target quantification in CFE reaction. Zinc modulates LacZ production by binding to the constitutively expressed transcription factor ZntR, thereby activating transcription from the ZntR-responsive promoter P_zntA. Following 45 min of LacZ production, a mixture of the naproxen-lactose solution was added to quench the CFE reaction and to start lactose conversion for 15 min. The converted glucose was then read on the PGM for target analyte quantification. (B) Dose-dependent glucose production in CFE reaction with zinc in a water matrix. The same experiment was replicated on different days to verify consistency in glucose output. Replicates (Rep) represent independently assembled reactions and error bars represent the standard deviation of cell-free reaction triplicates in each replicate. Dashed gray line represents PGM’s lowest reading threshold, 20 mg/dL. (C) Dose-dependent glucose production in CFE reaction with zinc in 25% pooled human serum. X-axis zinc concentrations reflect the total zinc in the reaction after accounting for the remaining zinc in chelated serum (Figure S2D). The same experiment was replicated on different days and with an independently assembled reaction to verify consistency in glucose output. Error bars represent the standard deviation of cell-free reaction triplicates in each replicate. Dashed gray line represents PGM’s lowest reading threshold, 20 mg/dL.

Figure 3:

Application of PGM-mediated quantification of nucleic acids in CFE reactions. (A) Schematic of toehold switch-modulated LacZ production and LacZ-catalyzed lactose conversion to glucose output. Following 45 min of LacZ production caused by RNA trigger activating a toehold switch to allow translation of LacZ, a mixture of the naproxen-lactose solution was added to quench the CFE reaction and to start lactose conversion for 15 min. The converted glucose was then read on the PGM for target analyte quantification. (B) Activation of Stx1 toehold switch and glucose output by RNA Stx1 trigger. Linear glucose response was observed with a logarithmic increment of RNA triggers from 20 to 2000 nM. The same experiment was replicated on different days to verify consistency in glucose output. Replicates (Rep) represent independently assembled reactions and error bars represent the standard deviation of cell-free reaction triplicates in each replicate. Dashed gray line represents PGM’s lowest reading threshold, 20 mg/dL. (C) Activation of Stx2 toehold switch and glucose output by linear DNA coding for Stx2 trigger, which can transcribe Stx2 RNA trigger in CFE reaction. Linear glucose response was observed with linear increments of DNA Stx2 trigger from 5 to 40 nM. Replicates (Rep) represent independently assembled reactions and error bars represent the standard deviation of cell-free reaction triplicates in each replicate. Dashed gray line represents PGM’s lowest reading threshold, 20 mg/dL.