Complementary Cell-Free Translational Assay for Quantification of Amino Acids

In this study, we present a simple and economical method that enables rapid quantification of amino acids based on their polymerization into a signal-generating protein. This method harnesses amino acid-deficient cell-free protein synthesis systems that generate fluorescence signals in response to exogenous amino acids. When premixed with assay samples containing the amino acids in question, incubation of the cell-free synthesis reaction mixture rapidly resulted in the production of sfGFP, the fluorescence intensity of which was linearly proportional to the concentration of the amino acids. The assay method achieved a limit of detection as low as ∼100 nM and was successfully applied to the quantification of disease-related amino acids in biological samples. Compared with standard methods in current use that require chemical derivatization of amino acids and chromatographic equipment, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable biofluorescence intensity in a much shorter period, providing a more affordable and flexible option for the quantification of amino acids.