Raman Spectroscopic Signature Markers of DopamineâHuman

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Raman Spectroscopic Signature Markers of DopamineHuman Dopamine Transporter Interaction in Living Cells Achut Prasad Silwal, Rajeev Yadav, Jon E Sprague, and H. Peter Lu ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00048 • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

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ACS Chemical Neuroscience

Raman Spectroscopic Signature Markers of Dopamine-Human Dopamine Transporter Interaction in Living Cells Achut P. Silwal1, Rajeev Yadav1, Jon E. Sprague2, and H. Peter Lu1* 1

2

Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University; The Ohio Attorney General’s Center for the Future of Forensic Science, Bowling Green, Ohio 43403, United States.

ABSTRACT: Dopamine (DA) controls many psychological and behavioral activities in the central nervous system (CNS) through interactions with the human dopamine transporter (hDAT) and dopamine receptors. The roles of DA in the function of the CNS are affected by the targeted binding of drugs to hDAT; thus, hDAT plays a critical role in neurophysiology and neuropathophysiology. An effective experimental method is necessary to study the DAhDAT interaction and effects of variety of drugs like psychostimulants and anti-depressants that are dependent on this interaction. In searching for obtaining and identifying the Raman spectral signatures, we have used surface enhanced Raman scattering (SERS) spectroscopy to record SERS spectrum from DA, Human Embryonic Kidney 293 cells (HEK293), hDAT-HEK293, DA-HEK293, and DA-hDAT-HEK293. We have demonstrated a specific 2D-distribution SERS spectral analytical approach to analyze DA-hDAT interaction. Our study shows that the Raman modes at 807, 839, 1076, 1090, 1538, and 1665 cm-1 are related to DA-hDAT interaction, where Raman shift at 807 and 1076 cm-1 are the signature marker for bound state of DA to probe DA-hDAT interaction. On the basis of density function theory (DFT) calculation, Raman shift of bound state of DA at 807 cm-1 is related to combination of bending modes α(C3-O10-H21), α(C2-O11-H22), α(C7-C8-H18), α(C6-C4-H13), α(C7-C8-H19), α(C7-C8-N9), and Raman shift at 1076 cm-1 is related to combination of bending modes α(H19-N9-C8), γ(N9-H19), γ(C8-H19), γ(N9-H20), γ(C8H18), and α(C7-C8-H18). These findings demonstrate that protein-ligand interactions can be confirmed by probing change in Raman shift of ligand molecules, which could be crucial to understanding molecular interactions between neurotransmitters and their receptors or transporters. KEYWORDS: Dopamine (DA), Human dopamine transporter (hDAT), HEK293 Cell, SERS, DA-hDAT interaction

INTRODUCTION Dopamine (DA) is the catecholamine neurotransmitter, which controls many psychological and behavioral activities in central nervous system (CNS) of Mammalia through biochemical interactions with the dopamine transporter (DAT) and dopamine receptors (DARs).1, 2 Dopaminergic neurons located in the ventral tegmental area, substantia nigra pars compacta, and the arcuate nucleus of the hypothalamus enzymatically convert tyrosine (Tyr) into L-DOPA and finally into DA.3-6 The DA neurotransmitter works in a sequence of several steps. First, the action potential is generated on neuron membrane from environmental stimulation. It causes the opening of voltage gated Ca+2 ions channel followed by the entry of Ca+2 ions into the neuronal cell, which induces the release of DA into the synaptic cleft. 7-9 Then, DA in the synaptic cleft activates G protein-coupled DARs located in pre- or post-synaptic region to generate the dopaminergic response.10-15 High concentration of DA in the synaptic cleft

between neuron cells causes over-stimulation of DARs resulting several neurological and physiological disorders. The excessive neurotransmission due to accumulation of DA as well as other biogenic amino neurotransmitters like serotonin, noradrenaline, and ߛ-aminobutyric acid present in synaptic and perisynaptic space is removed by the reuptake process; DA is preferentially taken up into the dopaminergic nerve terminal by DAT.16-19 DAT belongs to the family of solute carrier 6 (SLC6), 20 which maintains homeostasis of neurotransmitters by Na+ and Cl- ion assisted reuptake and thus it is also known as neurotransmitter sodium symporters (NSS). 21-25 Dysfunction of NSS system is linked with several disorders like schizophrenia, depression26, attention deficit hyperactivity disorder (ADHD)27, orthostatic intolerance28, epilepsy29, Parkinson’s disease and infantile Parkinsonism dystonia.30 The agonists and antagonists of DARs has influence in dopaminergic transmission by enhancing or blocking the actions of DA on receptors.31-36 Inhibition of DA reuptake from synaptic cleft has several side effect, but it is an im-

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portant pharmacological method for the treatment of depression.37, 38 The targeted binding of drugs to the human dopamine transporter (hDAT) has effect in the development and function of the nervous system; thus, study of DA-hDAT interaction has importance in neurophysiology.

Figure 1. (A) Schematic representation of dopamine system in neuron cell showing hDAT, DA, and DA receptor, (B) HEK293 cell, and (C) Silica coated silver nanoparticles.

Different types of experimental techniques and computational modeling have been applied to understand the structure and function of membrane proteins and their interactions with ligands 19, 39-57 Imaging of hDAT has been conventionally studied by fluorescence microscopy, positron emission tomography (PET), and single photon electron tomography (SPET). Booij and coworkers used [123I]CIT single photon emission computed tomography (SPECT) to demonstrate loss of striatal dopamine transporter content in Parkinson's disease in human.45 Trafficking of hDAT has been studied by using Photo switchable fluorescence microscopy and fluorescence resonance energy transfer (FRET) microscopy, and real time trafficking of hDAT in response of the compound like DA and amphetamine has been studied by total internal reflection fluorescence (TIRF) microscopy.39-41 Lukyanov and coworkers reported the position and movement of hDAT protein in living cell using photo switchable cyan fluorescence protein39, similarly, Sorkin and coworkers reported hDAT interaction and oligomerization during trafficking in living cell40 utilizing FRET microscopy. However, solid information about atomic level interaction between DA and hDAT was first time reported by Gouaux and coworkers after their X-ray-crystal based study of dopamine transporter (dDATmfc) 54, 55 They have explained the molecular principle to distinguish binding pattern of chemically distinct ligands to DAT in the vicinity of sodium and chloride ions. In recent years, computational molecular dynamic (MD) simulation has been applied to study atomic level interactions between hDAT and DA.19, 46-53 Bahar and coworker have reported molecular mechanism of DA transport by hDAT using homology modeling and full-atomic microsecond simulation.19 De Felice and coworker used voltage clamp method to understand the

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stimulatory, and inhibitory action of DA and other drugs in neurophysiological system.58 All the conventional techniques applied to study biological sample have intrinsic advantages and limitations. For instance, fluorescence microscopy is one of the powerful tools for biological imaging; however, photobleaching effect, photo toxicity of shorter wavelength light to living cell, and toxicity due to byproduct formed in situ photochemical reaction are its limitation.59-65 In addition, biological property of the sample could be changed after fluorescence labeling of samples. Xu and coworkers found the change in efflux function of different transporters when they were tagged with fluorescent protein.66, 67Although the PET and SPET techniques are strong techniques to study protein trafficking and their interaction in biological samples, the radioactive waste produced in this technique is harmful for in vivo studies. Thus, it is highly desirable and necessary to develop and demonstrate other powerful technique to study biological samples. Here, we found those qualities in SERS technique to study DA-hDAT interaction in live HEK-293 cell. Raman spectroscopy was first used to study bacteriorhodopsin as a biological sample;68 salmon's sperm cell as living cells,69 and eosinophilic granulocytes cells as human living cells.70 Now, it has been established as a powerful analytical technique to study broad range of biological samples and living systems. Initially, Raman study of membrane proteins was very difficult due to their low concentration at the cell membrane, extremely small Raman scattering cross-section and very weak Raman scattering intensity compared to background-noise. However, sensitivity of Raman spectroscopy has been improved tremendously by using different approaches. Surface Enhanced Raman Scattering (SERS) is one of the powerful technique to improve Raman sensitivity, which is done by exploiting electromagnetic field enhancement and chemical enhancement mechanism using metallic substrate especially nanoparticles or rough surface of noble metal.71 Van Duyne and coworkers have quantitatively reported in vivo transcutaneous glucose sensing by using surface enhanced spatially offset Raman spectroscopy (SESORS).72 Henry and coworkers applied dark field microscopy and SERS technique combining nanotags with a microfluidic device for the continuous detection of biomarkers in blood.73 Recently, Haynes and coworkers have reported the sensing of ricin B chain in human blood applying aptamer-conjugated silver film-over-nanosphere substrate, and SERS technique.74 Moskovits and coworkers developed and synthesized SERS-based biotags (SBTs) with highly reproducible optical properties and correctly identified the cancerous cells using this SBTs on the mixture of cancerous and noncancerous prostate cells utilizing deconvolution strategies.75, 76 Nie and coworkers have reported biocompatible and nontoxic pegylated gold nanoparticles and SERS technique for in vivo targeting and detection of tumor.77 Recently, they have developed highly sensitive SERS-based assays which can detect 1 stem cell among 106 cells.78 The cytotoxicity on living cell due to bare metal nanoparticles used as SERS substrate is one of the detriments


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of SERS technique;79 which can be solved by using an ultrathin layer of silica on SERS substrates. We have used thin layer of silica (4-6 nm) over silver nano particles to prevent cytotoxicity without reducing electromagnetic field enhancement. Moreover, the metal nanostructures

Figure 2. DFT Calculated Raman spectrum of (A) unbound DA, and (C) DA in dDATmfc by Gaussian 09 with B3LYP/631G(d) basis functions, scaling factor 0.9614; experimental SERS spectrum from (B) DA, (D) HEK293 cell, (E) hDATHEK293 cell, (F) DA-HEK293 cell, and (G) DA-hDATHEK293 cell using silica coated silver nanoparticle as SERS substrate, with excitation frequency 488.16 nm.

used in SERS technique quenches fluorescence background giving higher signal-to-noise ratio, reduces photobleaching, and water background effect and making SERS as a strong analytical tool for varieties of biological study including protein structure, its interaction to other protein or ligand molecules.80-87 Coronado and co-worker has studied influence of protein kinase D1 on identification, localization, and quantification of neuronal cell membrane receptor using SERS and other plasmonic probes.88 Ben-Amotz and co-worker reported the ultra-filtration

Raman difference (UFRD) method to obtain thermodynamics and structural information after label-free detection and quantitation of protein–ligand binding.87 Some research group have already reported the interactions between neurotransmitters and their binding protein using Raman spetroscopy.88, 89 Peticolas and co-worker applied the time dependent UV Raman spectroscopy to observe conformational change of the acetylcholine analogue during its binding to protein probing the changes in frequency and excited state electronic structure that brings variations in the relative intensity of the Raman bands.89 Here, SERS technique is applied to probe progress of DA-hDAT interaction by analyzing change in Raman mode of DA molecules. Table 1. Prominent Raman wavenumber of the samples -1

Samples

Raman modes (cm )

1.

HEK 293

793, 814, 863, 885, 917, 940, 987, 1041, 1140, 1215, 1241, 1265, 1342, 1414, 1487, 1638

2.

DA-hDATHEK293

733, 745, 763, 791, 807, 839, 855, 885, 911, 936, 958, 1014, 1076, 1090, 1114, 1215, 1234, 1290, 1312, 1331, 1438, 1461, 1538, 1564, 1586, 1665

3.

DA-HEK293

778, 859,898,979, 1029, 1164, 1186, 1236, 1393, 1474, 1526, 1579, 1674

4.

hDATHEK293

775, 791, 863, 885, 911, 967, 1014, 1037, 1110, 1138, 1164, 1190, 1217, 1236, 1311, 1315, 1362, 1438, 1463, 1544, 1564, 1578, 1617, 1654

5.

DA

749, 772, 794, 931, 960, 1012, 1114, 1145,1210, 1287, 1348, 1390, 1451, 1579, 1600, 1617

In this report, the SERS spectra from DA, HEK293, hDAT-HEK293, DA-HEK293, and DA-hDAT-HEK293 has been collected and converted into 2D-distribution Raman spectrum in order to analyze DA-hDAT interaction in live HEK293 cell. Analysis showed that new Raman modes present at 807, 839, 1076, 1090, 1538, and 1665 cm-1 in DAhDAT-HEK293 are marker of DA-hDAT interaction. Similarly, the analysis of DFT calculated Raman spectrum of bound and unbound states of DA shows that Raman modes at 807, 894, 971, 1076, and 1403 cm-1 are signature to bound state of DA (figure 2A and 2C). Our rigorous analysis shows that only two Raman modes present at 807 cm-1 and 1076 cm-1 are signature marker Raman shifts of bound states of DA molecules, which are capable to identify and probe DA-hDAT interactions. Other modes present at 894, 971, and 1403 cm-1 are not used to characterize the DA-hDAT interaction since they are overlapped on Raman shift of hDAT, or other cellular proteins.

RESULTS AND DISCUSSION



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All the SERS spectra for DA, HEK293 cell, DA in HEK293 cell (DA-HEK293), hDAT expressed HEK293 cell (hDATHEK293) and DA with hDAT-HEK293 as represented in the

Figure 3. 2D-distrubution Raman spectrum of (A) DA, (B) HEK293, (C) hDAT-HEK293, (D) DA-HEK293, (E)DA-hDATHEK293 cell generated from 40-50 high quality normalized SERS spectra of corresponding samples where color scale represents occurrence of a Raman modes, higher occurrence showing prominent Raman modes. Red circled Raman modes in 3C and 3E represents characteristic Raman modes of hDAT, and DA-hDAT interaction respectively

Figure 2 were recorded using home modified Raman microscope (Figure S1) to determine the binding interactions between DA and hDAT in live cell. The analysis of SERS spectra of DA, HEK293 cell, DA-HEK293, hDAT-HEK293 and DA-hDAT-HEK293 is remarkably useful to probe DAhDAT interactions. The expression of hDAT protein in HEK293 cell, the interaction of DA to HEK293 cell and

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human dopamine transporter (hDAT) could be noticed by the spectral change in the SERS spectrum of DA, HEK293 cell, and hDAT-HEK293 cell (figure 2B-G). The analysis are easier and precise when 2D-distribution Raman spectrum are generated (Figure 3A-E). At first, the wavenumber of prominent spots in 2D-distribution Raman spectrum are determined on the basis of intensity and their recurrence (table 1). Remarkable spectral difference are found in HEK293 cell after expression of hDAT protein showing five extra peaks located at 1012, 1110, 1311, 1463, and 1578 cm-1 with comparison of HEK293 cell; the new peaks might be contributed by hDAT. New Raman modes appeared in hDAT-HEK293 cell could be assigned on the basis of literature and our DFT calculation (table S2).90 The tentative assignment for the Raman mode of hDAT protein are as following. Raman mode at 1012 cm-1 could be related to (1010 cm-1) deformation frequency of C-O-H bond of alcoholic hydroxyl group present in amino acid residues. The possible contributors of Raman mode at 1110 cm-1 could be summarized as follows: (1113-1115 cm-1) C-asymmetric bending, Cα-Cβ stretch, Nt-Cα, Cβ-Cα-Ct stretch (Ala), and (1108-1113 cm-1) NtH3+ asymmetric rocking of (His). Similarly, Raman mode of hDAT at 1311 cm-1 are given as: (Near 1300 cm-1) amide III band, 40% C-N stretch, 30% N-H bending, and 30% skeleton stretches, (1308-1310 cm-1) Ring stretch of phenyl alanine, (1304-1308 cm-1 ) Hα-Cα-Ct, COO- symmetric stretching, Cβ-Cα-Ct (Ala), and (1308-1310 cm-1) ν ring (Phe). Raman mode at 1463 cm-1 could be originated from synergic contribution of (1464 cm-1) C-asym rocking (Ala), (1464-1470 cm-1) Cδbending (Lys), (1463-1464 cm-1) Cβ asymmetric rocking (Ala), and (1463-1465 cm-1) Cγ1 asym rocking, Cδ asym bending (Ile). The Raman mode at 1578 cm-1 could be related to (1566 cm-1) C=N stretch, and (1582 cm-1) structural stretching of Phe and Trp. Beside these new Raman shifts, original Raman shifts of HEK293 cell are still present in hDAT-HEK293 cell (figure 2E). When DA is added in to hDAT-HEK293 cell and waited for 5-10 minutes, new Raman modes for DA-hDAT interaction are observed. Characteristic Raman modes located at 807, 839, 1076, 1090, 1538, and 1665 cm-1 are landmark to DA-hDAT interaction which are represented by red circles in figure 3E. These new Raman modes are nowhere present in Raman spectrum of DA, hDAT-HEK293, HEK293 cell, and DA-HEK293, and only appeared in DAhDAT-HEK293 system. The new Raman modes observed in DA-hDAT-HEK293 system could be contributed by different sources. The new bond formed between DA and protein residues of hDAT could be one of the chief contributor; crystal structure of hDAT analogue supports those types of bond formation during DA bindings. The change of electronic density in the bonds of DA molecule due to DA-hDAT interaction could be next contributors; the change in electronic density of DA is supported by DFT calculation (Figure 4B). The change of electronic density within structure of protein residue of hDAT could be another contributor of new signature Raman modes. However, contribution of every factor are not equally relevant to analyze. For instance, the dynamics of DA reuptake by hDAT is reported to be 3.3 µM/S-4.0 µM/S


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which means bond formation and breaking phenomenon are faster process90, 91, hence analysis of bond formation between DA and protein residue is not preferred to study DA-hDAT interaction. Similarly, it is harder to study DAhDAT interaction by probing change in Raman modes of many protein residues available in hDAT.

Figure 4 (A) Crystal structure of dDATmfc (PDB ID: 4xp1) showing binding site of DA; (B) Calculated HOMO orbitals for un-bound and bound states of DA, visualized with Avogadro (Avogadro: an open-source molecular builder and visualization tool. Version1.XX.http://Avogadro.openmolecules.net).

Figure 5. Integrated Raman spectrum of DA-hDAT-HEK293 cell (averaged from 32 spectra) showing characteristic Raman peak of bound states of DA molecules related to DA-hDAT interaction, Force vectors (green arrow) in the structure of

DA shows Raman vibrational mode at 807 cm-1 and 1076 cm1 in bound states of DA molecules.

Ultimately, the change in Raman mode of DA due to redistribution of electronic density is preferred to probe DA-hDAT interaction. This approach is even justified since DA is the center of DA-hDAT interaction, which is discussed thoroughly as following. The crystal structure of dDATmfc (PDB ID: 4xp1) mimics structure of hDAT (Figure 4A), which shows location of DA molecule within central binding site sur-rounded by the transmembrane helices (TMs).(59) The amine group of DA interacts with carboxylate group of Asp46 within distance of 3A˚; while its catechol group is enclosed in cavity formed by Ala117, Val120, Asp121, Tyr124, Ser422 and Phe325 and it interacts with carboxylate group of Asp121 through hydrogen bonding. The meta-hydroxyl group of DA molecules interacts with the side chain of Asp121 at a distance of 2.7A˚ where it is oriented towards Ser422 in TM8 at a distance of 3.8A˚. The para-hydroxyl group is interacted with both the carbonyl oxygen of Ala117 and the carboxylate of Asp121 at distances of 2.8 and 3.1A˚ respectively. The coordinates of DA molecule located in binding site of dDATmfc crystal has been used to calculate HOMO orbital and DFT Raman spectrum of bound states of DA. HOMO orbital calculation (Figure4B) of bound and unbound states of DA shows variation of electron density in bound state of DA molecule in O11-H22, O10-H21, C3-C2=C1, N9-H19, and C7-C8 bonds with respect to its unbound state. Raman frequency calculations of unbound states of DA is performed with geometry optimization by density functional theory (DFT) method on B3LYP level with a basis set of 631G (d) using Gaussian 09 package. However, the DFT Raman calculation of bound state of DA is carried out with same basis set without geometry optimization, in order to conserve the coordinates of atoms, as they are present in binding cavity of crystal structure. In biological system, DA molecules are present in soluble form, hence experimental SERS spectrum of DA molecules are required to collect both from solution and dried states to mark the solvent effect. We found SERS spectra of DA are consistent in both states, except there is broadening of some Raman peaks in solution state. The Raman mode assignment of unbound DA molecule on the basis of our DFT calculation and published literature,87 are shown in table S1. The Raman peaks of unbound DA observed in our experiment are consistent to theoretical calculations (Figure2A and 2B). However, bound state of DA has not consistent peak in experimental (Figure2G) and DFT calculated Raman spectra (Figure2C). It might be because experimental SERS spectrum of bound DA is recorded from DA-hDAT-HEK293 sample, and it could be mixed up with Raman peak of hDAT, HEK293, and unbound DA. On the other hand, DFT Raman spectrum of bound DA is obtained by using coordinates of DA molecule located in binding cavity of dDATmfc crystal, and hence should be free from any spectral disturbance. Analysis of DFT Raman spectrum of unbound, and bound states of DA is convenient to determine characteristic



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Raman mode due to DA-hDAT interaction (figure2A and 2C). It shows out of five new Raman modes of bound states of DA, only two Raman mode present at 807, and 1076 cm-1 stand out as a landmark of DA binding to hDAT , because other new Raman modes are indistinguishable from Raman modes of unbound state of DA, HEK293 cell, or hDAT protein. The visualization of vibrational mode of bound states of DA molecule obtained by DFT calculation shows that Raman mode at 807 cm-1 is related to combined contribution of following bending processes: α(C3-O10-H21), α(C2O11-H22), α(C7-C8-H18), α(C6-C4-H13), α(C7-C8-H19), α(C7-C8-N9). Similarly, another Raman mode at 1076 cm-1 is found to be related to combination of bending processes: α(H19-N9-C8), γ(N9-H19), γ(C8-H19), γ(N9-H20), γ(C8-H18), and α(C7-C8-H18). We found this types of study is useful to show the progress of DA-hDAT interaction by probing appearance and disappearance of the intensity of these two peaks. If any drugs, for instance, psychostimulant, anti-depressant or potent are applied to replace DA molecules from its binding sites, relative intensity of these characteristic peaks present at 807 cm-1 and 1076 cm-1 in SERS spectrum could be disappeared or decreased due to interference of DA-hDAT interaction. This statement is supported by the result of our control experiment (Figure S.5).

CONCLUSION We have recorded SERS spectrum of DA, HEK293, hDATHEK293 cell, DA-HEK293, and DA-hDAT HEK293 cell to analyze DA-hDAT interaction. Over expression of hDAT protein in HEK293 cell is remarked by new Raman shift in hDAT-HEK293 cell. Similarly, progress of DA-hDAT interaction are noted by the appearance of new Raman mode after addition of DA into hDAT-HEK293 cell. Our emphasis in this study is finding of marker Raman shift of bound state of DA molecule to examine DA-hDAT interaction. The careful analysis of experimental and DFT Raman spectrum of DA in bound and unbound states shows that Raman shift of bound states of DA at 807 cm-1 and 1076 cm-1 are benchmarks for DA-hDAT interaction. On the basis of our experimental and theoretical study, it is found that the characteristic Raman shifts of bound states of DA observed at 807 cm-1 corresponds to the bending modes of ߙ(C3-O10-H21), ߙ(C2-O11-H22), ߙ(C7-C8-H18), ߙ(C6C4-H13), ߙ(C7-C8-H19), and ߙ(C7-C8-N9)), while Raman shift at 1076 cm-1 corresponds to bending modes of ߙ(H19-N9C8), ߛ(N9-H19), ߛ(C8-H19), ߛ(N9-H20), ߛ(C8-H18), and ߙ(C7C8-H18). Our study concludes that protein-ligand interaction could be confirmed by probing change in Raman shift of ligand molecules, which could be crucial to understand molecular interactions of neurotransmitters with their corresponding receptor or transporters.

METHODS Synthesis of silver nano particles and sample preparation. Silver nitrate (AgNO3), sodium citrate, and sodium silicate were purchased from Sigma Aldrich, and were used as received. Silver nanoparticles (AgNPs) is

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synthesized by a standard sodium citrate reduction method,92 followed by the addition of active sodium silicate to generate ultra-thin silica shells over AgNPs.93 The synthesized nanoparticles are characterized by Transmission electron microscopy (TEM), and UV-Vis spectroscopy. The size of AgNP@SiO2 are found between 50-65 nm with SiO2 shell thickness 4-6 nm. The absorption maximum of AgNP@SiO2 is blue shifted in UV-vis spectrum with respect to bare AgNPs. Prof. Louis J De Felice (Virginia Commonwealth University, Richmond, VA) had generously gifted the hDAT inducible HEK293 cells. The vial of cells was thawed in a 37oC water bath and then decontaminated by spraying with 70% ethanol and transferred into a T-75 flask containing 15 ml of culture medium containing Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich D5796) supplemented with 10% fetal bovine serum (SigmaAldrich, F2442) and 1% penicillin-streptomycin (ATCC, 30-2300). The cells were then maintained in an incubator at a temperature of 37oC with 5% CO2 atmosphere. After 24 hours, the medium is aspirated off and replaced with fresh, complete medium containing 1×10-4 gm/ml zeocin (invivoGen, ant-zn-1), and 1×10-3 gm/ml blasticidin (Millipore/Calbiochem, 203350). When the cells reached 7080% confluence on the surface of T-75 flask, hygromycin B (Sigma-Aldrich, H3274) of final concentration 25 μg/μl was added in to it to select hygromycin resistance cells, which are isolated, and subculture in complete media on a 25 mm circular cover glass in 35 mm petri dish. When cells are attached (approx. 24 hrs) on the cover glass, doxycycline (Tocris Bioscience, 4090) was added at a concentration of 0.5 µg/µl. In 2 to 3 days hDAT expression was completed in the HEK293 cells. Before SERS experiment, samples of HEK293 cells, and hDAT-HEK293 cells were washed with PBS solution to remove culture medium, and wet with 250 µl fresh PBS solution to prevent drying out of cell. DA-hDAT interaction is established adding 50 µl of 50nM DA solution in to cell sample at 15 minute prior to SERS experiment. Surface enhanced Raman measurements. All SERS spectra were collected using home-modified confocal Raman microscope94 (Figure S.1) using 488-nm continuous-wave (CW) argon ion laser of approximately 10-15 µw power with integration time of 30 second. Mercury lamp and cyclohexane were used to calibrate the setup before Raman measurements with spectral resolution 2 cm-1, range of SERS spectrum was set to 700–1700 cm-1, and experimental parameters are set identical for each record to avoid parameter’s effect. Non-reproducibility of Raman spectrum (i.e. fluctuation of peaks position and intensity) are removed by generating 2D-distribution Raman spectrum. Two-dimensional SERS plot of spectral mode frequency vs relative signal peak intensity. The temporal SERS fluctuation observed in individual spectrum makes it complicated to determine exact value of wavenumber for a particular Raman mode. The two-dimensional SERS plot of spectral mode frequency for a sample is obtained by the combination of clearly visible Raman peaks of 5060 normalized SERS spectra. In this picture Raman shift is


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represented by colorful spot instead of peak, where color scale shows the recurrence of the particular Raman mode. The broadening of spot along x-axes shows the trend of wavenumber fluctuation for particular Raman modes over numbers of SERS spectra. The center point of the spot is accredited as the average Raman frequency. The fluctuation in relative intensity of a Raman mode could be noticed from broadening or splitting of spot along y-axes. The colorful spot are promising to select prominent Raman modes and helps to get rid of signal fluctuation problem. Beside this advantage, 2D-distribution Raman spectrum is generated using clearly visible Raman peaks from normalized SERS spectra, hence it is always free from noise and unusual peaks. High degree of consistence is found between individual SERS spectrum and corresponding 2D-distribution Raman spectrum of a sample neglecting some minor discrepancies. Hence, 2D-Raman spectrum are more powerful than relative signal-peak intensity SERS spectrum. Density functional theory calculations. Geometry optimization and Raman frequency calculations were performed using density functional theory (DFT) method on B3LYP level with a basis set of 6-31G (d) and Gaussian 09 package to see the difference between free DA and DA bound to hDAT. According to a comprehensive evaluation of Scott and Radom95, the obtained frequencies were scaled by a factor of 0.9614. Molecular orbitals were calculated with the same basis set and visualized with Avogadro software (Avogadro: an open-source molecular builder and visualization tool). All calculations were carried out on a vector processor (Ohio Supercomputer Center, Columbus, Ohio).

ASSOCIATED CONTENT Supporting information The supporting information is available free of charge on the ACS Publication Website at DOI: Home-modified confocal Raman microscope experimental set up; Raman spectra of DA in its different states; UV-Vis spectrum of bare silver nanoparticles and silica coated silver nanoparticles; SERS spectra of bupropion; effect on characteristic vibrational mode of DA-hDAT interaction due to bupropion addition; table for the Raman peak assignment of dopamine and hDAT-HEK293 cell.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACKNOWLEDGMENTS HPL acknowledges the support from the Ohio Eminent Scholar endowment fund and the Ohio Attorney General’s Center for the Future of Forensic Science Research Fund, Ohio. The hDAT Inducible HEK293 cell line sample was generously gifted by the late Dr. Louis J De Felice

(Virginia Commonwealth University). We acknowledge the stimulating discussion with Dr. Andrew Torreli. We acknowledge the use of Ohio Supercomputer Center, Columbus, for our Density Functional Theory Calculations of Raman spectra.

ABBREVIATIONS DA, dopamine; HEK, human embryonic kidney; hDAT, human dopamine transporter; dDAT, drosophila dopamine transporter; SERS, surface enhanced Raman spectroscopy; Ala, alanine; Ile, isoleucine; Val, valine; Phe, Phenylalanine; Trp, tryptophan; Tyr, Tyrosine; Asp, aspartic acid; His, histidine; Lys, lysine; Ser, serine.

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For Table of Contents Use Only

Raman Spectroscopic Signature Marker of Dopamine-Human Dopamine Transporter Interaction in Living Cells Achut P. Silwal1, Rajeev Yadav1, Jon E. Sprague2, and H. Peter Lu1* 1

Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University; 2The Ohio Attorney General’s Center for the Future of Forensic Science, Bowling Green, Ohio 43403, United States.


1


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Figure 1.


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Figure 2



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Figure 3.


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Figure 4.



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Figure 5.


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Raman Spectroscopic Signature Markers of DopamineâHuman

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