Aequorin as a sensitive and selective reporter for detection of dopamine: A photoprotein inhibition assay approach

摘要： Dopamine is a metabolite that plays a key role in the human body and in biomedical and diagnostic applications. Thus, the concentration of this analyte has been considered in various diseases in therapeutic drug monitoring (TDM). In the present study, for the first time, a photoprotein inhibition assay strategy was developed by utilizing aequorin for the direct detection of dopamine as a receptor and reporter simultaneously. The results showed that bioluminescence emission of aequorin was effectively quenched by increasing concentration of dopamine at the range of 1 nM to 100 μM with a detection limit of 53 nM. The viability of this method for the monitoring of dopamine in spiked biological fluids was also established and it was successfully applied for the direct determination of dopamine in a blood serum and urine without preliminary treatment with satisfactory quantitative recovery 90-95% and 82-93%, respectively. The structural investigation using circular dichroism, fluorescence spectroscopy, and docking simulation indicated that, changes in the microenvironment of aromatic residues were significant, while minor conformational alterations of the protein were observed. It seems dopamine inhibits bioluminescence activity with specific binding to the residues involved in the light production.