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Cell-free protein synthesis (CFPS) biosensors provide flexible, fast, and on-demand diagnosis of biomarkers in human body fluids. Considering the high concentrations of many different RNases in human body fluids, RNase inhibitor is an important reagent for the human-body-fluid-based biosensors and accounts for more than 90% of the total cost of E. coli -lysate-based CFPS biosensors. Here an E. coli extract with RNase inhibition activity was developed which reduces the total cost of a CFPS biosensor by ~90%. Murine RNase inhibitor (m-RI) was overexpressed in E. coli cells at different growth temperatures, redox buffers, and folding chaperones. Reduced growth temperature accompanied by overexpression of GroEL/ES folding chaperones were found to be necessary to maintain RNase inhibition activity despite reduced m-RI expression capability. Further, to optimize protein synthesis capability of CFPS biosensors, E. coli extract containing m-RI was mixed with regular E. coli extract that did not contain m-RI. The optimized CFPS biosensor reagent mixture was lyophilized and rehydrated with 0–100% (v/v) of three human body fluids of saliva, serum, and urine. Rehydration with saliva showed the least drop in protein expression compared to serum and urine. Finally, the CFPS biosensor reagents were assessed with a saliva-based CFPS biosensor sensitive to glutamine concentrations. [Display omitted] • CFPS biosensors need RNase inhibitor to function in human body fluids containing different RNases. • An E. coli extract with overexpressed and active murine RNase Inhibitor (m-RI) was developed and tested. • Reduced fermentation temperature to 24 °C and presence of GroEL/ES folding chaperons are necessary for m-RI activity. • Lyophilized CFPS reactions containing m-RI extract were tested with 0–100% (v/v) of human body fluids with superior functionality in saliva. • A saliva-based glutamine biosensor was developed utilizing m-RI extract to inhibit RNases present in human saliva. [ABSTRACT FROM AUTHOR]

Abstract: Virus-like particles (VLPs) consist of a virus''s outer shell but without the genome. Similar to the virus, VLPs are monodisperse nano-capsules which have a known morphology, maintain a high degree of symmetry, and can be engineered to encapsidate the desired cargo. VLPs are of great interest for vaccination, drug/gene delivery, imaging, sensing, and material science applications. Here we demonstrate the ability to control the disulfide bond formation in VLPs by directly controlling the redox potential during or after production and assembly of VLPs. The open cell-free protein synthesis environment, which has been reported to produce VLPs at yields comparable or greater than traditional in vivo technologies, was employed. Optimal conditions for disulfide bond formation were found to be VLP dependent, and a cooperative effect in the formation of such bonds was observed. [Copyright &y& Elsevier]

Illustration of the assembly, distribution, and point-of-care use of a rapidly-deployable, cell-free COVID-19 biosensor: 1) Assemble: Assembling CFPS reagents by mixing E. coli lysate, murine RNase Inhibitor (mRI), energy sources, cofactors, and toehold switch riboregulator plasmid. 2) Print: aliquoting CFPS reagents onto paper substrates housed in a plastic test cassette. 3) Dehydrate: lyophilizing CFPS reagents on paper substrates. 4) Distribute. 5) Saliva sample: applying saliva samples onto cassette without pretreatments. 6) Reaction: bioluminescent protein expression in presence of target RNA (+), or ribosome detachment in absence of target RNA (-). 7) Visual result: bioluminescent output in the presence of target RNA and NanoLuc luciferase expression. [Display omitted] • Progress towards a rapid, low-cost, point-of-care Covid-19 biosensor. • Engineered toehold switches respond to saliva enriched with SARS-CoV-2 RNA. • mRI enables robust paper-based cell-free protein synthesis in the presence of saliva. • Plastic cassette houses paper-based diagnostic test for simple distribution and use. The COVID-19 pandemic has illustrated the global demand for rapid, low-cost, widely distributable and point-of-care nucleic acid diagnostic technologies. Such technologies could help disrupt transmission, sustain economies and preserve health and lives during widespread infection. In contrast, conventional nucleic acid diagnostic procedures require trained personnel, complex laboratories, expensive equipment, and protracted processing times. In this work, lyophilized cell-free protein synthesis (CFPS) and toehold switch riboregulators are employed to develop a promising paper-based nucleic acid diagnostic platform activated simply by the addition of saliva. First, to facilitate distribution and deployment, an economical paper support matrix is identified and a mass-producible test cassette designed with integral saliva sample receptacles. Next, CFPS is optimized in the presence of saliva using murine RNase inhibitor. Finally, original toehold switch riboregulators are engineered to express the bioluminescent reporter NanoLuc in response to SARS-CoV-2 RNA sequences present in saliva samples. The biosensor generates a visible signal in as few as seven minutes following administration of 15 μL saliva enriched with high concentrations of SARS-CoV-2 RNA sequences. The estimated cost of this test is less than 0.50 USD, which could make this platform readily accessible to both the developed and developing world. While additional research is needed to decrease the limit of detection, this work represents important progress toward developing a diagnostic technology that is rapid, low-cost, distributable and deployable at the point-of-care by a layperson. [ABSTRACT FROM AUTHOR]

• Rapid sensing of physiologically relevant glutamine concentrations in human serum. • Metabolic engineering of cell-free protein synthesis with small molecule inhibitors. • Glutamine synthetase inhibition enables glutamine biosensing. Bioavailable glutamine (Gln) is critical for metabolism, intestinal health, immune function, and cell signaling. Routine measurement of serum Gln concentrations could facilitate improved diagnosis and treatment of severe infections, anorexia nervosa, chronic kidney disease, diabetes, and cancer. Current methods for quantifying tissue Gln concentrations rely mainly on HPLC, which requires extensive sample preparation and expensive equipment. Consequently, patient Gln levels may be clinically underutilized. Cell-free protein synthesis (CFPS) is an emerging sensing platform with promising clinical applications, including detection of hormones, amino acids, nucleic acids, and other biomarkers. In this work, in vitro E. coli amino acid metabolism is engineered with methionine sulfoximine to inhibit glutamine synthetase and create a CFPS Gln sensor. The sensor features a strong signal-to-noise ratio and a detection range ideally suited to physiological Gln concentrations. Furthermore, it quantifies Gln concentration in the presence of human serum. This work demonstrates that CFPS reactions which harness the metabolic power of E. coli lysate may be engineered to detect clinically relevant analytes in human samples. This approach could lead to transformative point-of-care diagnostics and improved treatment regimens for a variety of diseases including cancer, diabetes, anorexia nervosa, chronic kidney disease, and severe infections. [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]