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

Abstract

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.

Present and Future of Surface-Enhanced Raman Scattering

“…Surface-enhanced Raman scattering spectra from bacterial and eukaryotic cells in cell cultures add significantly to our understanding of the interaction of plasmonic nanostructures with the intracellular and extracellular environments. Therefore, by controlling or defining the interaction of the SERS substrate with the biological system, intrinsic SERS signals can be used to detect specific biomarkers or physiologically relevant biomolecular species inside or near individual cells, such as adenosine monophosphate (AMP), , important metabolites in normal and tumor cells, neurotransmitters, , or trehalose . Surface-enhanced Raman scattering microscopy always indicates the presence of an interaction of the substrate with the samples and enables mapping the position of plasmonic NPs. − An important aspect in SERS experiments of live cells, and when gold or silver NPs are used as plasmonic substrates rather than when cells are attached to solid sensors , or nanoelectrodes, or when substrates are inserted into cells in needle-type approaches, ,− is the control of the plasmonic properties, which are critical for SERS enhancement.…”

“…As examples, spheres, , shells, flowers, nanorods, and nanostars have been used to record intrinsic SERS spectra from cells. Metal NPs for intracellular SERS can be stabilized by silica coating with varying porosity, so that analyte molecules can reach the plasmonic surface, while aggregation in the endosomal system is controlled. , …”

“…Metal NPs for intracellular SERS can be stabilized by silica coating with varying porosity, so that analyte molecules can reach the plasmonic surface, while aggregation in the endosomal system is controlled. 565,571 Experiments quantifying the amount and distribution of NPs in cells by mass spectrometry, 583 together with high-resolution ultrastructural imaging of the cells and the NPs, help us understand the changes in plasmonic properties and SERS enhancement of a particular type of SERS nanoprobe. Such experiments show that the microscopic distribution and SERS spectral signatures are correlated, rather than the number of NPs that are located in a cell, 584 yet the morphology and interparticle distances within nanoaggregates are critical.…”

Surface enhanced Raman scattering for probing cellular biochemistry

“…Although not directly revealing the interaction itself, the intracellular increase or decrease of cAMP can be probed by SERS using silica coated silver nanoparticles and the 780 cm −1 and 1503 cm −1 bands of the molecule as indicators, and therefore whether the different dopamine receptors trigger the same signal cascade in the presence of the signaling molecules. 12 Tricyclic antidepressants inhibit acid sphingomyelinase activity and block lipid degradation pathways, inducing lipid accumulation ( Fig. 1A and B ).…”

“…signal cascade in the presence of the signaling molecules. 12 Tricyclic antidepressants inhibit acid sphingomyelinase activity and block lipid degradation pathways, inducing lipid accumulation (Figure 1A and B). This process can be identified by SERS when cells are incubated with gold nanoparticles and these inhibitors.…”