Easy-to-perform, relatively inexpensive blood diagnostics have transformed at-home healthcare for some patients, but they require analytical equipment and are not easily adapted to measuring other biomarkers. The requirement for reliable quantification in complex sample types (such as blood) has been a critical roadblock in developing and deploying inexpensive, minimal-equipment diagnostics. Here, we developed a platform for inexpensive, easy-to-use diagnostics that uses cell-free expression to generate colored readouts that are visible to the naked eye, yet quantitative and robust to the interference effects seen in complex samples. We achieved this via a parallelized calibration scheme that uses the patient sample to generate custom reference curves. We used this approach to quantify a clinically relevant micronutrient and to quantify nucleic acids, demonstrating a generalizable platform for low-cost quantitative diagnostics.

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.

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 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 endpoint signal output. This lysate metabolism did, however, allow for one-pot removal of glucose present in complex samples (like human serum) without confounding target quantification. Taken together, we show that integrating lysate-based cell-free biosensors with PGMs enables accessible target detection and quantification at the point of need.