Mixed metal oxide based electrochemical sensing of inhibitory neurotransmitter serotonin in the presence of dopamine

A mixed metal oxide modified electrode has been employed for the voltammetric determination of neurotransmitter serotonin in the presence of dopamine. The electrode was morphologically and electrochemically characterized using scanning electron microscope and cyclic voltammetry respectively. Differential pulse voltammetry were performed to measure the serotonin concentration at the electrode surface. A dynamic linear range of 0.2-52 μm was obtained. Viability of the sensor was tested in blood sample.


INTRODUCTION
5-hydroxytryptamine, widely recognized as serotonin is an essential biochemical in the human body that has an ability to act both as a neurotransmitter in the central nervous system as well as a hormone precursor in the pineal gland [1]. It has inhibitory effects on neuron, making it identified as inhibitory neurotransmitter like gamma-aminobutyric acid and glycine. It is synthesised through a dual step pathway in which an amino acid L-tryptophan is converted to L-5OH-tryptophan by an enzyme called tryptophan hydroxylase and this intermediate formed gets converted to serotonin by an aromatic Lamino acid decarboxylase [2]. It plays a vital role in regulating physiological functions such as mood, sleep, appetite and sexuality. Normal serotonin levels in the whole blood are in the range of 0.5-1.2 µM, 0.3-0.7 µM in urine, and less than 0.0591 nM in cerebrospinal fluid [3]. The reduced levels of serotonin may cause mood related disorders, depression, migraine, insomnia etc. whereas the increased levels of serotonin can range from mild shivering to muscle rigidity and also visible in carcinoid syndrome, Sudden Infant Death Syndrome (SIDS) etc. Serotonin cannot be directly taken from food but tryptophan rich foods can be taken through diet and gets converted to serotonin in the brain. Therefore the selective detection and measurement of 5-HT level is of great importance and can help in understanding the serotonin levels inside the body. Serotonin being electroactive can be used in electrochemical systems which possess high sensitivity, low cost, fewer reagents etc. compare to other analytical techniques [4][5][6].
Herein we report an electrochemical sensor for the determination of serotonin in the presence of another common neurotransmitter dopamine. The electrode modification employed here is a mixed metal oxide thin film comprising manganese and molybdenum as the metals used.

Chemicals
Serotonin hydrochloride and Dopamine hydrochloride was obtained from Sigma Aldrich. Pencil lead with a diameter of 0.7 mm (Cello Fine Leads Pvt. Ltd.) was purchased from a stationary store. All other chemicals are of analytical grade and used without further purification. Millipore water (18.2 MΩ cm) was used to prepare all solutions. Phosphate buffer solution (PBS) used in this study was prepared from Na 2 HPO 4 and NaH 2 PO 4 . All experiments were carried out at room temperature.

Instrumentation
Electrochemical studies such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed with electrochemical analyzer (CHI 610E, CH Instruments, USA). Typical three electrode cell setup was used. Mixed metal oxide modified pencil lead was chosen as the working electrode with platinum wire as counter and silver/silver chloride (Ag/AgCl (1 M KCl)) as the reference electrode. All the potentials referenced here are with respect to Ag/AgCl (1M KCl). Morphology analysis was performed with scanning electron microscope JEOL JSM-7610F Plus.

Fabrication of the electrode
The electrode for serotonin measurement was fabricated using electrodeposition procedure. The procedure for the electrodeposition was taken from a previously published article by Nakayama etal. [7]. The deposition bath contains aqueous solutions of 2mM MnSO 4 .7H 2 O with 10 mM (NH 4 ) 2 MoO 4 and 120mM Na 2 SO 4 . CV was used to electrodeposit mixed metal oxide thin film with potential cycling between 0 and +1.0 V vs Ag/AgCl at a scan rate of 20 mV/s. Electrodeposition was carried out on a pencil lead electrode (PLE) with 0.5 cm length exposed and the rest covered up with a teflon tape. The resultant electrode was designated as Mn-Momixed oxide thin films modified pencil lead electrode (Mn-Mo mixed oxide/PLE) and used for further studies. Figure 1 describes the SEM image of PLE and Mn-Mo mixed oxide/PLE. Here the PLE surface was found to be smooth and as the mixed metal oxide was deposited the surface morphology was turned to be rough with a dense morphology.   Figure 2 A shows the cyclic voltammogram obtained for the Mn-Mo mixed oxide/PLE in the presence and absence of serotonin in 0.1 M PBS 8. CV was performed by scanning between -0.3 -0.6 V with a scan rate of 120 mV/s. While anodic scan, no distinct peaks were observed for the Mn-Mo mixed oxide/PLE under the studied potential window. Upon addition of 2 µM serotonin into the PBS containing electrochemical cell, three new anodic peaks were found to arise at potentials -0.191 V (I a1 ), -0.027 V (I a2 ), +0.287 V (I a3 ). These three anodic peaks were found to undergo a reduction reaction upon the reverse scan on the Mn-Mo mixed oxide/PLE surface. Thus serotonin was found to exhibit a redox response over the modified electrode surface in 0.1 M PBS 8. Even though we observed three anodic peaks, the well-defined peak at +0.287 V (I a3 ) was chosen for further studies.

Selection of buffer pH
After selecting PBS as the suitable electrolyte for serotonin electroxidation at Mn-Mo mixed oxide/PLE surface, the influence of different pH of PBS was studied. Results are tabulated in table 2.
Optimum results in terms of both anodic current response and over potential were observed with PBS pH 8. As the pH was increased from 6-8, the current was found to be increasing and then decreased after 8. Hence we have chosen 0.1 M PBS pH 8 as the optimal buffer pH

Optimization of Mn:Mo ratio
Further the ratios of Mn to Mo and Mo to Mn was optimized. Mn:Mo ratios were varied as 1:5, 1:4 and 1:6. As the response current for 1:5 Mn:Mo was higher that was opted. Then a reverse study of the ratios was also performed. Mo:Mn ratios of 5:1, 5:3 and 1:1. The current for 5:1 Mo:Mn was obtained. So for the ratio of final mixed metal deposition bath, we fixed the Mo:Mn ratio as 5:1. Table  3 shows the results obtained for the optimization parameters of metal concentrations in deposition bath.  Figure 3A represents CV of Mn-Mo mixed oxide/PLE in 0.1 M PBS 8 containing 2 µM serotonin at different scan rates ranging from 20-120 mV/s. As the scan rates were increased from 20-120 mV/s, the peak current associated to the serotonin electroxidation also increased. A linear plot between current and square root of scan rate gives a linear fit depicting a diffusion-controlled process ( figure  3B). The corresponding linear equation is I pa = -0.00139 + 0.0162 * v 1/2 with R 2 = 0.9927.  Figure

Selectivity analysis of the sensor
Selectivity study of the sensor was carried out using DPV. The electrochemical analysis of serotonin was conducted in the presence of dopamine, a coexisting molecule in the blood bearing a similar structure. DPV response of the same is shown as figure 5 A and

Real sample analysis
Real sample analysis of serotonin was carried out in real blood matrix with spiked serotonin into it. DPV analysis were performed and obtained a recovery of 111.9 %. Table 4 shows the results observed for the real sample analysis of the fabricated sensor for the determination of serotonin using standard addition method.

CONCLUSION
We have employed a mixed metal oxide modified pencil lead electrode for the facile determination of neurotransmitter serotonin in phosphate buffer medium. The employed electrode possesses good linearity (0.2-52 µM) which satisfies the blood range. Also the sensor can be used to detect serotonin in the presence of another important neurotransmitter dopamine. Functionality of the sensor was ensured by testing in real blood sample matrix and obtained a recovery of 111.9 %.