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Specialized cancer treatments have the potential to exploit glutamine dependence to increase patient survival rates. Glutamine diagnostics capable of tracking a patient's response to treatment would enable a personalized treatment dosage to optimize the tradeoff between treatment success and dangerous side effects. Current clinical glutamine testing requires sophisticated and expensive lab-based tests, which are not broadly available on a frequent, individualized basis. To address the need for a low-cost, portable glutamine diagnostic, this work engineers a cell-free glutamine biosensor to overcome assay background and signal-to-noise limitations evident in previously reported studies. The findings from this work culminate in the development of a shelf-stable, paper-based, colorimetric glutamine test with a high signal strength and a high signal-to-background ratio for dramatically improved signal resolution. While the engineered glutamine test is important progress towards improving the management of cancer and other health conditions, this work also expands the assay development field of the promising cell-free biosensing platform, which can facilitate the low-cost detection of a broad variety of target molecules with high clinical value. [ABSTRACT FROM AUTHOR]

Human body fluids contain biomarkers which are used extensively for prognostication, diagnosis, monitoring, and evaluation of different treatments for a variety of diseases and disorders. The application of biosensors based on cell‐free protein synthesis (CFPS) offers numerous advantages including on‐demand and at‐home use for fast, accurate detection of a variety of biomarkers in human fluids at an affordable price. However, current CFPS‐based biosensors use commercial RNase inhibitors to inhibit different RNases present in human fluids and this reagent is approximately 90% of the expense of these biosensors. Here the flexible nature of Escherichia coli‐lysate‐based CFPS was used for the first time to produce murine RNase Inhibitor (m‐RI) and to optimize its soluble and active production by tuning reaction temperature, reaction time, reduced potential, and addition of GroEL/ES folding chaperons. Furthermore, RNase inhibition activity of m‐RI with the highest activity and stability was determined against increasing amounts of three human fluids of serum, saliva, and urine (0%–100% v/v) in lyophilized CFPS reactions. To further demonstrate the utility of the CFPS‐produced m‐RI, a lyophilized saliva‐based glutamine biosensor was demonstrated to effectively work with saliva samples. Overall, the use of CFPS‐produced m‐RI reduces the total reagent costs of CFPS‐based biosensors used in human body fluids approximately 90%. [ABSTRACT FROM AUTHOR]

Highlights • Cell-free Protein Synthesis-based biosensors are quick, portable, and robust. • CFPS systems biosense at transcription, translation, and protein folding levels. • CFPS-based biosensors are effective in sewage, blood, and urine. Abstract Biosensors highjack and reengineer biological systems for highly specific detection of diverse molecules. However, many traditional biosensor devices have slow response times and high operating costs and require specialized training and immobile lab equipment. In an effort to address these limitations, researchers have begun using cell-free protein synthesis (CFPS) systems as biosensors. CFPS-based systems provide the advantages of speed, portability, and robustness at a relatively low cost. Here, we review CFPS-based biosensors according to the three fundamental stages of CFPS: transcription, translation, and protein folding. [ABSTRACT FROM AUTHOR]

Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor β (hERβ). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERβ (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification. [ABSTRACT FROM AUTHOR]