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.

Optimizing protein production in the One-Pot Pure system: insights into reaction composition and expression efficiency

“…The open nature of these expression platforms allows direct manipulation of the reaction environment. Applications of these platforms span high-throughput screening, [1][2][3] novel protein modifications, 4,5 interfacing biomolecules with synthetic materials, [6][7][8][9] on-demand biosensing [10][11][12] and biomanufacturing, 13,14 and understanding the rules of life by building it from scratch. [15][16][17][18] Nearly all in vitro protein expression platforms can be categorized into two classes -crude lysatebased and reconstituted systems.…”

Metabolic perturbations to anE. coli-based cell-free system reveal a trade-off between transcription and translation

“…In recent years, cell-free systems have become an important tool for protein engineering, structural biology, and drug discovery, enabling production of functional proteins such as enzymes, antibodies, and vaccine candidates [5, 11]. More broadly, cell-free systems have been used for applications such as rapid and high-throughput prototyping of engineered biological components and systems [15, 17, 30, 29, 34], biosensing and diagnostics [25, 37, 21], and the development of synthetic life [8, 36].…”

“…The cellfree approach offers many advantages over engineering cellular systems, including greater control over the reaction environment, the ability to produce toxic small molecules or proteins that are difficult to express in living cells, and scalability for biomanufacturing and other applications. In recent years, cell-free systems have been used for protein engineering 1,2 , rapid and high-throughput prototyping of engineered biological components and systems [3][4][5][6][7] , biosensing and diagnostics [8][9][10] , and the development of synthetic life [11][12][13] .…”