Rosaniline Hydrochloride Encapsulated MCM-48: Fluorescent and Electrochemical Sensor for Dopamine

The dye Rosaniline hydrochloride (RANH) has been successfully incorporated in MCM-48 (designated as RANH@MCM-48) and characterized by various spectroscopic methods including FT-IR, SEM, EDX and N2 adsorption–desorption isotherm. RANH@MCM-48 in aqueous medium acts as fluorescence “on” sensor for neurotransmitter dopamine (DA) in presence of its main biological interfering agent ascorbic acid or vitamin c (AA) along with Glucose, Cholesterol and Uric acid (UA). The limits of detection (LOD) were found to be 65 nM and 51 nM respectively in absence and in presence of AA. The interaction of DA to RANH@MCM-48 is found to be reversible with respect to EDTA2−. The fluorescence intensity vs. pH plot shows a narrow fluorescence window of 7.2 to 8.8. RANH@MCM-48 has been successfully applied for DA detection in artificial cerebrospinal fluid (ACF) and bovine serum albumin (BSA) with LOD values 27 nM and 22.5 nM respectively. Platinum disc electrode has been modified with RANH@MCM-48 which showed distinct oxidation peaks with a separation of 0.188 V in cyclic voltammetry (CV). The LOD for DA in presence of AA determined from oxidation current is 77.5 nM. The voltammetric detection of DA is found to be free from common interfering species Na+, K+, Ca2+, Fe2+, UA, Cholesterol and Glucose. RANH@MCM-48 has been found to be a very effective fluorescence and voltammetric sensor for DA with very low LOD.


Introduction
Dopamine (DA) is a widely studied neurotransmitter belonging to catecholamine family which plays essential roles in the functioning of central nervous system, cardiovascular system, hormonal system, human behavioural responses such as mood, emotion, memory, and addiction [1][2][3]. Abnormally high levels of DA may cause Huntington's disease [4] whereas insufficient secretion may lead to Parkinson's disease [5] and Schizophrenia [6] which ultimately causes muscle rigidity, loss of balance, anxiety, restlessness, depression etc. Regarding the importance of DA detection and to quantify its concentration, numerous techniques such as colorimetry [7], chromatography [8], chemiluminescence [9], electrochemistry [10], capillary electrophoresis [11], UV-Vis spectroscopy [12], flow-injection analysis [13] and fluorescence spectroscopy [14] have been developed. A major concern in the electrochemical detection of DA is its coexistence with ascorbic acid (AA), the level of which is 100-1000 times higher than that of DA in biological media. Owing to their similar redox potential, selective and sensitive detection of DA is interfered by AA [15]. Therefore development of facile, sensitive, selective, cost effective and quantitative methods such as fluorescent sensors and surface modified electrodes are in high demand for the efficient detection of DA in presence of other biomolecules such as ascorbic acid and uric acid.
Nanostructured materials are emerging as efficient tool for analytical applications. Among nanostructured composites, the three dimensional cubic mesoporous silica MCM-48 described by Ia3d space group has notable advantages for use in detection systems [16]. They have a high surface area, high thermal stability, tunable pore size, large pore volume [17,18] and superior to MCM-41 due to its three dimensional pore system which renders MCM-48 resistance to pore blockage and a more efficient diffusion pathway [19]. Due to these outstanding properties, they are capable of incorporating various substances like electrochemically active dyes, organic and inorganic guest molecules, active pharmaceutical ingredients. Mesoporous silica find wide range applications in drug delivery systems [20][21][22], gene delivery [23], catalysis [24,25], adsorption [26,27], sorption and separation [28,29], detection of heavy metals [30,31] and biological molecules [32][33][34].
Dyes have been used as chemo sensors and optical sensors due to their sensitivity towards the environment and strong chromogenic ability [35]. They can be used in designing sensors by introducing them into the pores of mesoporous materials [36]. Haloi et al. reported 2,7-dichlorofluorescein encapsulated Na-MMT clay as UV/Visible, fluorescence and electrochemical sensor for DA and AA [37].
In this paper, we report that Rosaniline Hydrochloride dye encapsulated in MCM-48 (RAN@MCM-48) acts as a fluorescent sensor for the neurotransmitter dopamine in presence of biomolecules which commonly coexists with dopamine in biological fluids. Platinum disc electrode modified with RANH@MCM-48 leads to a significant difference in oxidation potential of dopamine and ascorbic acid in their mixture. The sensor has been successfully applied for dopamine detection in artificial cerebrospinal fluid and bovine serum albumin. The limits of detection for dopamine in different mediums are also reported.

Materials
MCM-48, Rosaniline hydrochloride (RANH), Dopamine (DA), Ascorbic acid (AA), other biomolecules and all other chemicals were purchased either from Sigma Aldrich or LOBA. The chemicals were of analytical grade and used without further purification. The chemical solutions (10 -3 M) were prepared in de-ionised water obtained from quartz double distilled plant. The FT-IR spectra were recorded in a Perkin Elmer RXI spectrometer as KBr pellets. The fluorescence and UV-Visible spectra were recorded in HITACHI 2500 and Shimadzu UV 1800 spectrophotometer respectively using quartz cuvette (1 cm path length). The electrochemical measurements were carried out on a CHI660DCH Instrument electrochemical analyser (USA) consisting of a three-electrode system: Pt electrode as the working electrode, Ag/AgCl (3 M NaCl) as the auxiliary electrode, Pt wire as the reference electrode and 0.1 M NaNO 3 as supporting electrolyte. Scanning Electron Microscopy was done using Zeiss FESEM Sigma 300 and Energy Dispersive X-ray Spectroscopy was studied using Ametek EDAX. Powder X-ray diffraction (PXRD) patterns of the synthesized samples were recorded using a Rigaku Ultima IV X-Ray diffractometer (Cu K α radiation, λ = 1.5418 Å) at 40 kV and 40 mA. The N 2 adsorption-desorption isotherm of the synthesized samples were recorded at 77 K by using Micromeritics Tristar 3000 analyser. The samples were degassed at 110 °C for 4 h prior to the measurement.

Synthesis of the Sensor (RANH@MCM-48)
The sensor RANH@MCM-48 was prepared as follows: 1.0 g of MCM-48 was suspended in 20 mL deionised water and stirred for 30 min. To the solution, 0.25 g RANH dye was added with continuous stirring for 36 h. The solid obtained was centrifuged, washed with deionised water and the maroon solid was dried in an oven at 150 °C for 12 h to yield the dye-MCM-48 composite (RANH@MCM-48).

Synthesis of Artificial Cerebrospinal Fluid (ACF)
The ACF which contains the majority of interferents in the real cerebrospinal fluid was prepared by the reported method [43]. Briefly: 2.1 g NaCl, 0.07 g KCl, 0.08 g CaCl 2 , 0.2 g Glucose, 0.4 g NaHCO 3 and 0.002 g Urea were taken and mixed together in a 250 mL volumetric flask maintaining the pH at 7.41 using phosphate buffer solution (PBS). The solution was immediately used after preparation to avoid hydrolysis of urea.

Preparation of Modified Electrodes (RANH@ MCM-48/Pt)
10 mg of RANH@MCM-48 was dispersed in 1 mL of acetonitrile and 0.3 mL styrene. 1 µL of the solution was placed on the tip of pre cleaned platinum (Pt) electrode with the help of a micropipette and the electrodes were allowed to dry and used for further investigations. The electrode was cleaned as per reported method [44] (Scheme 1).

Characterisation of RANH@MCM-48
The characterizations of RANH@MCM-48 was done by the approaches of FTIR, PXRD, N 2 adsorption-desorption The low angle XRD data (Fig. 2) shows a peak at 2.5° and around 3° corresponding to (211) and (220) plane. Two low intensity peaks appear between 4° and 5° corresponding to (420) and (332) plane. The pattern clearly shows presence of an Ia3d cubic mesoporous framework. The XRD pattern of RANH@MCM-48 shows a similar pattern with a decrease in intensity of the corresponding planes. The decrement in intensity indicates loss of crystallinity and ordering of the mesoporous material after introduction of RANH into it.
The N 2 adsorption-desorption isotherm showed typical type IV isotherm with H3 hysteresis loop which are characteristics of highly ordered mesoporous materials (Fig. 3). A sharp inflection at relative pressure p/p o = 0.2-0.3 was observed, indicating the capillary condensation of N 2 gas within the uniform pores of MCM-48 material. The uniformity of pore size distribution was indicated by the sharpness and height of inflection point. After the introduction of the dye into the channels of cubic mesoporous MCM-48, the surface area, pore volume and pore diameter were found to decrease as evident from Table 1. This suggests the occupation of pores by the dye molecules which results in the decrease of mesoporous ordering.   Figure 4a shows the SEM image of MCM-48 which have spherical morphology. After the introduction of the dye molecule, the morphology showed some distortion and irregularity of spherical shapes with agglomeration (Fig. 4b). This indicates that the dye is deposited onto the surface and into the mesopores of MCM-48. The energy disperse X-ray analysis (EDX) confirms the coating of RANH in the pores of MCM-48 sample (Fig. 4c, d). The energy disperses X-ray (EDX) elemental mapping analysis also demonstrates the uniform distribution of the dye into the MCM-48 material. Figure 5 shows EDX elemental mapping of O, Si, C and N in RANH@MCM-48.

RANH@MCM-48 as Fluorescent Sensor for DA
0.01 g of RANH@MCM-48 composite was dispersed in 5.0 mL of methanol and sonicated for 10 min to obtain a fine dispersion. 10 -3 M DA solution was prepared in deionised water in PBS ( pH 7.41). 10 -3 M solution of other biomolecules such as AA, UA, glucose and cholesterol were similarly prepared. The UV-Vis spectrum of RANH@ MCM-48 in methanol was measured and the excitation wavelength for the fluorescence spectroscopic experiments was fixed at 280 nm. Figure 6 shows the fluorescence spectra of RANH@MCM-48 in the presence of 100 μL of 0.01 M of DA, AA, UA, Glucose and Cholesterol. Fluorescence enhancement was observed only in case of DA while AA, UA, Glucose and Cholesterol had no significant effect on the fluorescence spectra of RANH@MCM-48. Figure 7 shows the fluorescence spectra of RANH@MCM-48 at different added concentration of DA in aqueous solution. Figure 7, inset shows the plot of fluorescence intensity vs. DA concentration which is linear. The linear range was found to be 0.5 × 10 -5 M to 6 × 10 -5 M. Since AA is the main interfering agent of DA detection in biological fluids and therefore we recorded fluorescence spectra of RANH@ MCM-48 in the presence of AA (Fig. 8). From the figure it is clear that DA could enhance the fluorescence intensity UV-Vis spectra of RANH@MCM-48 were recorded in methanol at different added concentration of DA (Fig. 9). It can be seen that the hump at 281 nm of RANH@MCM-48 becomes prominent with the increase in DA concentration. Inset of the figure shows the plot of fluorescence intensity as a function of DA concentration which is linear and the range is similar to that obtained from fluorescence data.
The reversibility of binding of DA to RANH@MCM-48 has been observed with respect to EDTA 2− (Fig. 10). The fluorescence intensity of RANH@MCM-48 in the presence of 100 μL of 0.01 M of DA was found to decrease with increase in added concentration of EDTA 2− . This clearly indicates that the interaction of RANH@MCM-48 with DA is reversible, which is a primary criterion for a good sensor. Figure 11 shows the effect of pH on fluorescence intensity of RANH@MCM-48 and RANH@MCM-48 in presence of

Detection of DA in Artificial Cerebrospinal Fluid (ACF)
The ability of RANH@MCM-48 to determine DA in ACF was carried out. For this purpose, 5 mL solution of RANH@   (Fig. 12) which suggests the successful detection of DA in ACF medium. The LOD in synthetic ACF medium is calculated to be 27 nM.

Determination of DA in Bovine Serum Albumin (BSA)
The sensor RANH@MCM-48 was successfully applied for detection of DA in bovine serum albumin (BSA) in PBS (pH 7.4). Figure 13 shows an enhancement of fluorescence intensity for RANH@MCM-48 in BSA medium by about nineteen times and the inset shows linear enhancement in intensity as a function of DA concentration in the range 0.5 × 10 -5 M to 6.0 × 10 -5 M. The LOD in BSA medium is calculated to be 22.5 nM.

Electrochemical Sensing of DA by RANH@MCM-48
To study the electrochemical behavior of RANH@MCM-48 and its interaction with DA in the presence of AA, the cyclic voltammetry technique was used. A three electrode electrochemical cell consisting of modified platinum (Pt) electrode as working electrode, Ag-AgCl as reference electrode and Pt wire as an auxiliary electrode was used. Figure 14 shows the cyclic voltammogram of RANH@MCM-48/Pt in aqueous medium (PBS, pH = 7.41) in the absence of DA (red line) and in the presence of DA (black line). In absence of DA one irreversible reduction peak at -0.497 V was observed which may be due to the electrochemical reduction of [RAN] + ion into neutral RAN free radical which subsequently breaks down as shown in Scheme 2 [46]. In the presence of DA (black line) the cyclic voltammogram shows a redox couple with reduction peak at + 0.036 V and oxidation peak at + 0.228 V. The separation in peak potential (ΔE) was found to be 0.192 V and E 1/2 was calculated to be + 0.132 V.  Figure 15 shows that RANH@MCM-48/Pt electrode is capable of resolving the overlapping oxidation peaks of DA and AA at bare platinum electrode into two distinct oxidation peaks in the mixture of 0.5 mM DA and 1 mM AA. The peak for AA was observed at + 0.503 V while the peak for DA was observed at + 0.315 V. The separation between the oxidation peaks of AA and DA has been + 0.188 V, hence the simultaneous detection of DA in the presence of AA is feasible at modified electrode. The oxidation currents for DA and AA were found to increase with the increase in their concentration simultaneously (Fig. 16). The plot of oxidation current for DA vs. its concentration in the presence of AA was linear (Inset, Fig. 16).
The Limit of Detection (LOD) of DA in the presence of AA from cyclic voltammogram data was calculated using the formula [47]: 3σ/K, where σ = Standard Deviation, K = slope obtained from the plot of anodic Current vs. concentration (Fig. 16, inset). The LOD was calculated to be 77.5 nM.

Influence of Other Substances in the Voltammetric Detection of DA and AA
To study the influence of biologically important species that generally co-exist with DA in biological fluids, on the voltammetric detection of DA and AA, the cyclic voltammogram was recorded for DA and AA mixture in presence of Na + , K + , Ca 2+ , Fe 2+ , Uric acid, Cholesterol and Glucose. It was found that the presence of these ions/compounds did not interfere with the simultaneous detection of DA and AA by the modified Pt electrode as shown in Fig. 17. The modified electrode has been employed to detect DA in PBS (pH 7.4) spiked with ACF and BSA with LOD and linear range similar to those in PBS (pH 7.4).

Conclusion
The dye Rosaniline hydrochloride has been successfully encapsulated in MCM-48. This encapsulated MCM-48 can detect neurotransmitter dopamine by fluorescence "on" mode in phosphate buffer solution at pH 7.41 as well as in the artificial Cerebrospinalfluid and bovine serum albumin in the presence of ascorbic acid, glucose, cholesterol and uric acid. The linear range of detection is 0.5 × 10 -5 M to 5.0 × 10 -5 M with limits of detection in nano molar range. Platinum disc electrode modified with the dye encapsulated MCM-48 affects 0.188 V separation in oxidation potential of ascorbic acid and dopamine compared to overlapping oxidation potential at a bare platinum electrode. The linear range of detection for dopamine by voltammetry is 5 × 10 -5 M to 50.0 × 10 -5 M with limits of detection in nanomolar range in presence of Na + , K + , Ca 2+ , Fe 2+ , ascorbic acid, uric acid, cholesterol and glucose in phosphate buffer solution at pH 7.41 as well as in artificial Cerebrospinal fluid and bovine serum albumin. When RANH is not encapsulated in MCM-48 it does not act as a sensor for DA. Thus we have established a new dual fluorescent and voltammetric sensor for dopamine in the form of RANH@MCM-48.