Bi-enzyme competition based on ZIF-67 co-immobilization for real-time monitoring of exocellular ATP.

ABSTRACT

Monitoring exocellular adenosine-5'-triphosphate (ATP) is a demanding task but the biosensor development is limited by the low concentration and rapid degradation of ATP. Herein, we developed a simple yet effective biosensor based on ZIF-67 loaded with bi-enzymes of glucose (GOx) and hexokinase (HEX) for effective detection of ATP. In the confined space of the porous matrix, the bi-enzymes competed for the glucose substrate in the presence of ATP, facilitating the biosensor to detect low ATP concentrations down to the micromole level (3.75 µM) at working potential of 0.55 V (vs. Ag/AgCl). Furthermore, ZIF-67 with cobalt served as a porous matrix to specifically adsorb ATP molecules, allowing it to differentiate isomers with sensitivity of 0.53 nA/µM, RSD of 5.4%, and recovery rate of 93.3%. We successfully applied the fabricated biosensor to measure ATP secreted from rat PC12 cells in the pericellular space thus realizing time-resolving measurement. This work paved the path for real-time monitoring of ATP released by cells, which will aid in understanding tumor cell glycolysis and immune responses.