Immobilization of Firefly Bioluminescent System: Development and Application of Reagents

The present study describes the method of preparing reagents containing firefly luciferase (FLuc) and its substrate, D-luciferin, immobilized into gelatin gel separately or together. The addition of stabilizers dithiothreitol (DTT) and bovine serum albumin (BSA) to the reagent is a factor in achieving higher activity of reagents and their stability during storage. The use of immobilized reagents substantially simplifies the procedure of assay for microbial contamination. The mechanism of action of the reagents is based on the relationship between the intensity of the bioluminescent signal and the level of ATP contained in the solution of the lysed bacterial cells. The highest sensitivity to ATP is achieved by using immobilized FLuc or reagents containing separately immobilized FLuc and D-luciferase. The limit of detection of ATP by the developed reagents is 0.3 pM, which corresponds to 20,000 cells·mL−1. The linear response range is between 0.3 pM and 3 nM ATP. The multicomponent reagent, containing co-immobilized FLuc and D-luciferin, shows insignificantly lower sensitivity to ATP—0.6 pM. Moreover, the proposed method of producing an immobilized firefly luciferin-luciferase system holds considerable promise for the development of bioluminescent biosensors intended for the analysis of microbial contamination.