Raman Spectroscopic Analysis of Signaling Molecules-Dopamine Receptors Interactions in Living Cells

The selective interaction of signaling compounds including neurotransmitters and drugs with the dopamine receptors (DARs) is extremely important for the treatment of neurodegenerative diseases. Here, we report a method to probe the selective interactions of signaling compounds with D1 and D2 DARs in living cells using the combined approach of theoretical calculation and surface-enhanced Raman spectroscopy (SERS). When signaling compounds such as DA, amphetamine, methamphetamine, and methylenedioxypyrovalerone interact with D1 dopamine receptors (DRD1), the intracellular cyclic adenosine monophosphate (cAMP) level is increased. However, the intracellular level of cAMP is decreased when D2 dopamine receptors (DRD2) interact with the abovementioned signaling compounds. In our experiments, we have internalized the silica-coated silver nanoparticles (AgNP@SiO₂) in living cells to adsorb biologically generated cAMP which was probed by using SERS. Besides adsorptions of cAMP, AgNP@SiO₂ has a crucial role for the enhancement of Raman cross section of the samples. We observed the characteristic SERS peaks of cAMP when DRD1-overexpressed cells interact with the signaling compounds; these peaks were not observed for other cells including DRD2-overexpressed and DRD1-DRD2-coexpressed cells. Our experimental approach is successful to probe the intracellular cAMP and characterize the selectivity of signaling compounds to different types of DARs. Furthermore, our experimental approach is highly capable for in vivo studies because it can probe intracellular cAMP using a low input power of incident laser without significant cell damage. Our experimental results and density functional theory calculations showed that 780 and 1503 cm^-1 are signature Raman peaks of cAMP. The SERS peak at 780 cm^-1 is associated with C-O, C-C, and C-N stretching and symmetric and asymmetric bending of two O-H bonds of cAMP, whereas the SERS peak at 1503 cm^-1 is contributed by the O₉-H₃ bending mode.