A core-shell molybdenum nanoparticles entrapped f-MWCNTs hybrid nanostructured material based non-enzymatic biosensor for electrochemical detection of dopamine neurotransmitter in biological samples

Dopamine (DA) is a critical neurotransmitter and has been known to be liable for several neurological diseases. Hence, its sensitive and selective detection is essential for the early diagnosis of diseases related to abnormal levels of DA. In this study, we reported novel molybdenum nanoparticles self-supported functionalized multiwalled carbon nanotubes (Mo NPs@f-MWCNTs) based core-shell hybrid nanomaterial with an average diameter of 40–45 nm was found to be the best for electrochemical DA detection. The Mo NPs@f-MWCNTs hybrid material possesses tremendous superiority in the DA sensing is mainly due to the large surface area and numerous electroactive sites. The morphological and structural characteristics of the as-synthesized hybrid nanomaterial were examined by XRD, Raman, FE-SEM, HR-TEM, EDX. The electrochemical characteristics and catalytic behavior of the as-prepared Mo NPs@f-MWCNTs modified screen-printed carbon electrode for the determination of DA were systematically investigated via electrochemical impedance spectroscopy, cyclic voltammetry, and amperometry. The results demonstrate that the developed DA biosensor exhibit a low detection limit of 1.26 nM, excellent linear response of 0.01 µM to 1609 µM with good sensitivity of 4.925 µA µM−1 cm−2. We proposed outstanding appreciable stability sensor was expressed to the real-time detection of DA in the real sample analysis of rat brain, human blood serum, and DA hydrochloride injection.

such as high sensitivity and excellent selectivity. Recently several analytical methods have been proposed for the DA detection such as high-performance liquid chromatography (HPLC) 12 , chemiluminescence 13 , fluorescence 14 , capillary electrophoresis 15 , mass spectrometry, calorimetry 16 , and surface-enhanced Raman scattering spectroscopy 17 . Although, these methods associated with some drawbacks such as require complicated pretreatment, time-consuming, expensive instruments, and consume large quantities of chemical, solvents, and reagents which might possibly cause environmental pollution 18,19 . Expectedly, the characteristics of the electrochemical behavior of DA, make it easily detectable by facile electrochemical method. To date, electrochemical techniques have been extensively explored for the detection of DA owing to their remarkable properties such as fast detection, facile operation mode, accessibility, cost-effectiveness, simplicity, and eco-friendly 4,20,21 . However, the electrochemical detection of DA in a biological fluid using bare electrode is often unsuccessful due to interfering molecules such as an Ascorbic acid (AA) and Uric acid (UA) coexisting with DA detection because of its similar oxidation potential 22 . As a result, the accuracy of its determination is remarkably low, so it is impossible to avoid their presence in real samples. The basal concentration of DA is very low (0.01-1 µM), while the concentration of AA and UA is much higher than that of DA (from 100 to 1000 times higher) 23,24 . Nowadays, the variously modified electrodes have been developed for the sensitive and selective DA detection in the routine analysis without the problem of some potential interferents. Though, these modified electrodes are unsuitable for the specific detection of DA due to its poor selectivity, sensing in higher oxidation potential and fouling to the signal response. To overcome this issue, the modified electrode with those requirements that can be employed by exploiting the hybrid nanomaterials.
So far, numerous hybrid nanomaterials have been fabricated for the electrochemical biosensor application. Especially MWCNTs, the unique carbon-based material consists of curled cylindrical tubes, have been widely used in fabricating electrochemical sensors due to their overwhelming advantages such as the high intrinsic carrier mobility, large surface/volume ratio, good flexibility, and conductivity [25][26][27][28][29][30] . Nevertheless, pristine MWCNT alone is unable to produce hydrophilicity. Functionalization of MWCNTs by acid condensation method using concentrated acids such as sulfuric acid (H 2 SO 4 ) and nitric acid (HNO 3 ) imparts higher solubility and allowing them as a catalytic active material for the electrochemical applications 31 . The chemical oxidation of MWCNTs introduces more oxygen functional groups on their surface, which improve their hydrophilicity, chemical reactivity and intended to enhance dispersity of MWCNT in an aqueous solution 32 . The acid mixture has the ability to break the tubes of MWCNTs resulting in an increase in their electrical conductivity and corrosion resistance. Furthermore, due to the presence of abundant functional groups and delocalized π bonding, f-MWCNTs are suitable matrix to anchor metal/metal oxide nanoparticle 33 . In recent days, f-MWCNT supported metal nanoparticles nanocomposite widely studied in the field of electrochemistry to improve the electrocatalytic properties 34 . Among these metal nanoparticles, Molybdenum based materials act as a noble metal-free electrocatalyst which played a prominent role in hydrogen production even with lower over potential 35 . Molybdenum-based nanomaterials have increased much research attention in recent times because of their ultrahigh specific surface area and unique optical, electronic, catalytic, and mechanical properties [36][37][38] . To prepare Mo nanoparticles, many advanced synthesis strategies have been utilized, such as a molten salt technique 39 , carbothermal reduction 40 , hydrogen reduction 41 , microwave assisted combustive reduction and electro reduction 42 . However, these synthetic techniques are owing to the complicated preparation method, high reduction temperature and costly equipment ultimately resulted in casing research on facile and low-temperature synthetic method for the production of Mo nanoparticles. In this paper, Solution combustion synthesis (SCS) is adopted to prepare Mo nanoparticle. The SCS method is a simple and low production cost and energy efficient method 43 , which is based on a redox reaction between fuel and oxidant containing metal cations. Usually, metal precursors themselves act as an oxidant and fuel is an organic reagent for examples citric acid, ethylene glycol, and urea, etc. has the ability to form a complex with the metal cations 44 .
Hence, in this study, a core-shell hybrid nanomaterial of Mo NPs@f-MWCNTs was successfully synthesized by acid condensation method and used to detect DA electrochemically for the first time. Where the negatively charged oxygen functional groups on the MWCNT serve as anchor sites for interaction of positively charged Mo NPs on the surface and cover the nanotubes through the robust electrostatic interaction. Definitively, the as-prepared Mo NPs@f-MWCNTs was employed as the excellent electrocatalytic material for the detection of DA in both buffer and biological samples. , glycine and all the required chemicals were purchased from Sigma-Aldrich. Dopamine hydrochloride injection was purchased from the local medical hospital. Rat brain sample was carried out in accordance with local laws and approved by Chang Gung University, IACUC Approval No. CGU105-079. The human serum sample used in this study was obtained with written informed consent from all subjects. Human serum was acquired from a healthier volunteer in Chang Gung Memorial Hospital and this study was approved by the institutional review board of Chang Gung Memorial Hospital, IRB No. 201601498B0. All experiments involving the human samples were performed in accordance with relevant guidelines and regulations of Chang Gung Memorial Hospital. The electrolyte of phosphate buffer (PB, 0.1 M) was prepared by mixing Na 2 HPO 4 and NaH 2 PO 4 . H 2 SO 4 and NaOH were used for optimizing the pH of the solution. The morphological characterization was studied using Hitachi S-3000 H (FE-SEM) and (HR-TEM) H-7600, Hitachi, (Japan). The elemental percentage and composition of the materials were investigated with the help of Energy-dispersive X-ray (EDX) HORIBA EMAX X-ACT (Sensor + 24 V = 16 W, resolution at 5.9 keV). Crystalline structure confirmed via Powder X-ray diffraction (XRD) XPERT-PRO (PAN analytical B.V. The Netherlands) diffractometer with Cu Kα radiation (k = 1.54 Å). Raman spectra were performed using Micro-Raman spectrometer. The electrochemical measurements were performed by cyclic voltammetry (CV) CHI 205 C and Amperometric (i-t) measurements were obtained with an analytical rotator with a working area of 0.21 cm 2 of rotating ring disc electrode www.nature.com/scientificreports www.nature.com/scientificreports/ (RRDE), platinum wire as the counter electrode, and Ag/AgCl (saturated KCl) as the reference electrode. All the experiments were performed triplicate.
Synthesis of Mo nanoparticles. The Mo NPs were prepared according to the earlier reported procedure with minor modification 37 . The SCS method was utilized in the preparation of the Mo precursor. First, 0.01 M (NH 4 ) 6 Mo 7 O 24 ⋅4H 2 O, 0.2 M NH 4 NO 3 , and 0.1 M glycine were dissolved in 100 mL deionized water (DI). The mixer was stirred under magnetic stirrer until the homogeneous solution formed. The above dispersion was heated using a temperature controlled electric heating furnace. After several minutes, a black precipitate of MoO 2 precursor was obtained. Mo NPs were synthesized from MoO 2 by hydrogen reduction process in a tubular furnace. The MoO 2 was heated up to 2 h under programmed heating rate (10 °C/min; 500 °C, 550 °C, 600 °C, 650 °C, and 700 °C). During the reaction, 100 mL/min of H 2 (99.99%) was maintained during hydrogen reduction. The obtained Mo NPs was allowed to cool room temperature naturally.

Synthesis of core-shell Mo nps@f-MWcnts hybrid nanocomposite. The Mo NPs@f-MWCNTs
nanocomposite was prepared using an acid condensation method. In the typical synthesis procedure, 1:5 ratio of Mo nanoparticle and MWCNTs were taken in the round bottom flask and then (3:1) H 2 SO 4 and HNO 3 were added to the above mixer 32 . The solution then condensed at 24 h under magnetic stirring. The obtained Mo NPs@f-MWCNTs nanocomposite was centrifuged and washed several time with DI water and ethanol to remove the unreacted particles in the product. The f-MWCNTs were prepared without adding Mo NPs for control studies.

Fabrication of Mo NPs@f-MWCNTs modified SPCE. The SPCE was modified by simple drop cast
method. First, the working electrode (SPCE) surface was pre-cleaned by potential cycling between −1.0 V and 1.2 V in 0.1 M phosphate buffer (PB) (pH 7.0) for 5 cycles at a scan rate of 25 mV s −1 . Typically, ~6 µL of dispersed Mo NPs@f-MWCNTs nanocomposite (optimized amount) was drop cast onto the SPCE electrode ( Fig. 1). Then SPCE was allowed to dry in air at room temperature, followed by gently dipped with buffer solution for twice to remove the unbound material prior to further electrochemical measurements and a similar procedure was adhered to prepare control electrode (f-MWCNTs).

Results and Discussion
Structural characteristics of Mo nps@f-MWcnts nanocomposite. Figure  Raman spectroscopy was conducted to confirm the degree of disorder in the MWCNTs structure during the composite formation. Figure 2B shows the Raman spectra of the pristine MWCNTs (a), f-MWCNTs (b), and Mo NPs@f-MWCNTs composites (c). The G-band at 1590 cm −1 is associated with the E 2 g plane in Stretching and vibration modes of the basal plane of graphite, which indicates that the existence of crystalline nature of graphitic carbon in the MWCNT samples. The peak at 1304 cm −1 (D-band) is assigned to the disordered carbon atoms of MWCNTs and defects. The intensities ratio between G-band (I G ) and D-band (I D ) is sensitive to chemical modification and its measure of defects in MWCNTs. Here, the value of I D /I G for MWCNTs, f-MWCNTs, and Mo NPs@f-MWCNTs are found to be 0.93, 1.03, and 1.01. Expectedly, the I D /I G value of f-MWCNTs was increased compared with pristine MWCNTs due to the introduction of plenty of defects during the functionalization process. However, there is no significant difference in the I D /I G values of f-MWCNTs and Mo NPs@f-MWCNTs, which indicates no structural changes during the formation of the composite. The strong bands are obtained in the region of 600-1000 cm −1 . The shape of the spectrum below 1000 cm −1 closely resembles that of the amorphous medium of molybdenum oxide or molybdenum hydroxide, as confirmed by previous reports 45,46 . the characteristic band of Molybdenum oxide due to electrostatic interaction of Mo nanoparticles with oxygen functional groups on the f-MWCNTs.

Morphological characteristics of Mo nps@f-MWcnts nanocomposite. The morphology and
nanostructure of the as-prepared Mo NPs, f-MWCNTs and Mo NPs@f-MWCNTs nanocomposite were investigated by FE-SEM. Figure 3A representing Mo nanoparticles, which seems to be a spherical shape. As shown in Fig. 3B, the size distribution of f-MWCNTs is relatively uniform with a smooth, dense, and typical tubular structure interlinking with each other. As a comparison, the Mo NPs@f-MWCNTs exhibits a rough surface and much wider than f-MWCNTs with a significant morphological change (Fig. 3C). The increment of tube size is due to Mo nanoparticles covered with f-MWCNTs through strong electrostatic interaction between negatively charged oxygen functional groups on the f-MWCNTs surface and positively charged Mo nanoparticles. The central cores of nanotubes still retained the tube-like structure. whereas, it can be clearly observed that the nanotubes (cores) are covered with Mo NPs (shells) indicating the successful synthesis of a core-shell hybrid structure of Mo NPs@f-MWCNTs.
The TEM analysis was conducted to compare the morphologies and to confirm the detailed core-shell structure of Mo NPs@f-MWCNTs. Figure 4A shows that Mo NPs has irregular spherical structure, which seems to be agglomerated. For the comparison, TEM image with the outer diameter of the pristine MWCNTs and f-MWCNTs tubes displayed in Fig. 4B,C, the outer diameter of the f-MWCNTs increased to ~32.1 nm which is thicker than that of pristine MWCNTs (~25.6 nm) due to the formation of a chemical layer on its structure. The chemical layer of carboxyl groups attached to the MWCNTs during the acid functionalization. The TEM images of Mo NPs@f-MWCNTs (Fig. 4D,E) (Fig. 5A). The EIS data of bare SPCE, f-MWCNTs/SPCE, and Mo NPs@f-MWCNTs/ SPCE were plotted using the Randles circuit model (Inset: Fig. 5A). Where, R ct , R s , Z w , and C dl were portraying charge transfer resistance, electrolyte solution resistance, Warburg impedance, and double layer capacitance, respectively. From this EIS plot, it can be seen that all the modified electrode exhibited semicircles of different diameters which correspond to different R ct value. The R ct value obtained for bare SPCE, f-MWCNT/SPCE and Mo NPs@f-MWCNTs/SPCE was 236 Ω, 195 Ω, and 69 Ω respectively. Furthermore, the Mo NPs@f-MWCNTs/ SPCE displays the very smaller semicircle (R ct = 69 Ω) compared to f-MWCNT/SPCE and bare SPCE, which reveals that the composite modified electrode has superior electron transfer ability owing to its unique core-shell structure.
The  (Fig. 6A). Notice that, the bare SPCE did not show any obvious response towards DA. while, the f-MWCNTs modified electrode shows a small redox peak current towards DA, which indicates the poor electrocatalytic activity of f-MWCNTs. When compared with the bare SPCE and f-MWCNTs modified SPCE, Mo NPs@f-MWCNTs modified SPCE enhanced the DA oxidation and reduction currents with good reversibility, and the obtained current response was found to be 15 folds higher than that of bare SPCE. It can reveal that the negatively charged carboxyl groups in the Mo NPs@f-MWCNTs electrostatically interact with the amine group of DA, which significant interaction enhanced the specificity of DA. The interaction and electron transportation between Mo NPs@f-MWCNTs and DA further strengthened by π-π stacking forces. Also, the excellent synergistic effects that appeared between the Mo NPs and f-MWCNTs improve the well-conductive area and promote the electron transfer rate between the DA and the electrode surface. The electrochemical mechanism suggests that dopamine is directly oxidized into dopamine-o-Quinone (DOQ) with an equal number of protons and electrons. The effects of different concentration of DA over the Mo NPs@f-MWCNTs electrode was carried out by varying the concentration of DA (50-300 µM) in 0.1 M PB (7.0), at a scan rate of 50 mV s −1 (Fig. 6B). The anodic peak current was enhanced linearly as the concentration of DA increased. The result also reveals that the Mo NPs@f-MWCNTs hybrid material can be a suitable material for the quantification of DA. www.nature.com/scientificreports www.nature.com/scientificreports/ The electrochemical mechanism between the DA and Mo NPs@f-MWCNTs modified electrode investigated from cyclic voltammogram of 100 μM DA with different scan rate (20-240 mVs −1 ) (Fig. 6C). Figure 6D displays the linear plot between the oxidation peak currents (I pa ) of DA and the scan rates. As it showed that, the linearity was observed for the I pa of DA against scan rates. Furthermore, I pa of DA increased with the increasing of the scan rate, while the oxidation peak potential (E pa ) was shifted towards the positive side and the reduction peak potential (E pc ) was shifted towards the negative side with the increase of scan rate. The linear regression equation was expressed as I pa (μA) = 0.0282 υ (Vs −1 ) + 6.4133 (R 2 = 0.997). From this scan rate study, it was understood that the electrochemical reaction of DA at core-shell Mo NPs@f-MWCNTs electrode was a surface controlled process.
The effect of pH (pH 3.0-11.0) on the response of 100 μM DA in the PB (7.0) at Mo NPs@f-MWCNTs/SPCE was investigated by CV (Fig. 6E). The relationship of pH to the I pa and E pa of DA were plotted and displayed in Amperometric determination of DA. The amperometric (i-t) technique is higher sensitivity and better resolution technique as compared to conventional other analytical techniques. Therefore, the amperometric technique was used for the detection of DA. Figure 7A shows the response for core-shell Mo NPs@f-MWCNTs modified electrode towards each continuous addition of DA (0.01-1609 µM). Every addition of DA injected at the time intervals of 50 s into the continuously stirred 0.1 PB (7.0) under the rotation speed at 1200 rpm. The applied constant working potential of the electrode was held at +0.27 V. The Mo NPs@f-MWCNTs modified electrode shows a well-defined and rapid amperometric current responses were obtained in various addition of DA concentration with the response time of 50 s, which is clearly indicating that the fast electron transfer process occurred between electrode and the electrolyte interface during the addition of DA. The peak currents were increased linearly with increasing the concentration of DA from lower to higher. A calibration plot was plotted for the [DA] vs. I pa (Fig. 7B) and the linear regression equation can be expressed as I pa (µA) = 0.0985 [DA (µM)] + 0.0084. The amperometric current responses were increased linearly with increasing the DA concentration in the linear range from 0.01 to 1609 µM. From the calibration plot, the correlation coefficient is calculated to be R 2 = 0.999, and the limit of detection is estimated to be 1.26 nM according to the IUPAC definitions 47 .
where 'q' is the slope value (0.0985 µA µM −1 ) from the calibration plot, and 'σ' is the standard deviation obtained from the 3 measurements of the blank signal (i.e., 0.0000414 µA). the sensitivity was calculated as 4.925 µAµM −1 cm −2 , respectively. The obtained analytical parameters such as linear range and LOD at core-shell Selectivity study. In order to investigate the selectivity of the core-shell Mo NPs@f-MWCNTs modified electrode towards the detection of DA in the presence of various possibly interfering substances by amperometric technique. As shown in Fig. 7C, the Mo NPs@f-MWCNTs shows well-defined amperometric current responses for the 50 μM DA, at the same time, there is no notable peak current was detected for the 50-fold excess   www.nature.com/scientificreports www.nature.com/scientificreports/ suggesting that the proposed core-shell Mo NPs@f-MWCNTs biosensor possessed excellent selectivity. Therefore, it can serve as a selective electrochemical biosensor for the detection of DA even in the existence of more interfering substances.
Stability, reproducibility, and repeatability of the Mo nps@f-MWcnts sensor. Long-term stability of Mo NPs@f-MWCNTs modified electrode was investigated and stored in a PB (7.0) at room temperature. The DA signal monitored for each day by using the same Mo NPs@f-MWCNTs modified electrode. After 15 days, the CV response of the same Mo NPs@f-MWCNTs electrode has retained about 98.27% from the initial current response (Fig. 7D). This results revealed that the Mo NPs@f-MWCNTs modified electrode has good storage stability. The reproducibility of the modified electrode was evaluated in 0.1 M PB (pH 7.0) containing 50 μM DA at 5 independent modified electrodes with the relative standard deviation (RSD) was 2.8%, which result suggesting that acceptable reproducibility of the biosensor. The repeatability of the Mo NPs@f-MWCNTs sensor was studied by 5 consecutive measurements of 50 μM DA with RSD of 3.2%, indicating good repeatability of the sensor.

Application of the non-enzymatic biosensor in real samples. The real-time application of the Mo
NPs@f-MWCNTs biosensor was demonstrated in biological samples (human serum and rat brain) and pharmaceutical sample (Dopamine hydrochloride injection). First, rat brain sample was directly analyzed using Mo NPs@f-MWCNTs electrode and results were free of DA. About 0 μ, 50 μL, 100 μL, 150 μL volumes of rat brain samples were injected into 0.1 PB (7.0). Next, known concentrations of DA were injected into the rat brain sample and amperometry signals were recorded for the spiked DA sample. The Mo NPs@f-MWCNTs modified electrode showed well defined current responses for DA present in spiked rat brain sample. The real sample analysis was also carried out in dopamine hydrochloride injection by injecting dopamine hydrochloride injection sample into PB (pH 7.0). The capacity of the electrochemical cell was kept at 1 mL. The amperometry response recorded for each addition of DA injection sample. The direct DA detection in DA injection sample revealed the great potential of Mo NPs@f-MWCNTs for practical application in pharmaceutical sampling tests. In order to perform real sample analysis in a human serum sample, a known concentration of DA was spiked into serum sample. The total volume of the electrochemical cell is kept at 1 mL. Subsequently, amperometry response recorded using Mo NPs@f-MWCNTs modified electrode in PB containing spiked DA serum samples of different concentrations. The current response is linear with the concentration of DA in the spiked sample and hence the developed Mo NPs@f-MWCNTs modified electrode can be used for DA sensing in the human serum sample. Moreover, found and recovery values were calculated for specific concentrations of the spiked real samples and given as Table 2. It can be seen from the table that the core-shell Mo NPs@f-MWCNTs biosensor able to detects DA with an acceptable range of recoveries.

conclusions
In this work, we have constructed an efficient and sensitive electrochemical biosensor based on the core-shell Mo NPs@f-MWCNT hybrid nanomaterial for the sensing of DA. Functionalized multiwalled carbon nanotubes (MWCNT) were entrapped by molybdenum nanoparticles (Mo NPs) to form the type of core-shell hybrid structure. The designed DA biosensor plattform with combining the advantages of MWCNT and Mo NPs offers the good electrocatalytic properties, excellent conductivity and exceptional electron transferability with fast and selective response to DA, wide linear range, low detection limit (1.26 nM, S/N = 3) and excellent selectivity. Moreover, the developed DA sensor demonstrates good reproducibility and stability. Finally, the real time application of our developed sensor was verified by the determination of DA in rat brain, human blood serum and dopamine hydrochloride injection with good accuracy. All these features validate that the core-shell Mo NPs@f-MWCNT hybrid nanostructure shows vast potential for the applications in electrochemical biosensing platform.