Complementary Cell-Free Protein Synthesis Assay for Quantification of Amino Acids

The concentration of amino acids in blood is often related to different diseases directly or indirectly. 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 fluorescence protein. In this method that harnesses an amino acid-deficient cell-free synthesis system as a conditional signal-generating module, fluorescence signal is generated in response to the presence of specific amino acids in assay samples. 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 to existing methods relying on HPLC, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable bio-fluorescence intensity in a much shorter period, providing a more affordable and flexible option for the quantification of amino acids.