Determination of Benserazide Based on Bioluminescence Inhibition of Aequorin

Aequorin as a bioluminescence protein due to ease of use, non-toxic, and high capability of detecting has long been the interest of researchers. The aim of this study was to design a method for accurate and simple detection of important therapeutic agents using a bioluminescence inhibition based assay by using aequorin.

In this study, important drugs in therapeutic monitoring with structural similarity to Coelenterazine, were selected and their interaction with aequorin was investigated. Further, the conditions of the bioluminescence assay were optimized to achieve the lowest detection limit.

Among the drugs whose effects have been tested on aequorin, the only benserazide resulted in inhibition of the bioluminescence activity. This analyte can significantly reduce the bioluminescence of aequorin in a concentration-dependent manner. The best dose-response curve was obtained and IC50 of 0.26µM was calculated. The linear calibration curve was obtained in a range of about 100 to 1500nM with LOD and LOQ of 79 and 260nM, respectively. Furthermore, we demonstrated the application of the approach in human serum samples with a recovery of 97%. Guddem-Schild graph was plotted to determine the mechanism of inhibition which indicated that the IC50 of benserazide changed in the presence of different concentrations of Coelenterazine.

The proposed method can be used for measuring benserazide which can easily be applicable for real samples. Also, the results show that benserazide inhibits the bioluminescence activity of aequorin by competitive inhibition.