Innovative Strategy Based on a Novel Carbon-BlackâÎ²-Cyclodextrin

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The Innovative Strategy for the Simultaneous Determination of Anticancer Drug Flutamide and Environmental Pollutant 4-Nitrophenol Based on Novel Carbon black and #-Cyclodextrin Nanocomposite Subbiramaniyan Kubendhiran, Rajalakshmi Sakthivel, ShenMing Chen, Mutharani Bhuvanenthiran, and Tse-Wei Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00989 • Publication Date (Web): 26 Apr 2018 Downloaded from http://pubs.acs.org on April 26, 2018

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Analytical Chemistry

The Innovative Strategy for the Simultaneous Determination of Anti-cancer Drug Flutamide and Environmental Pollutant 4-Nitrophenol Based on Novel Carbon black and β-Cyclodextrin Nanocomposite Subbiramaniyan Kubendhiran,

a,‡

a,‡

Rajalakshmi Sakthivel, , Shen-Ming Chena,*, Bhuvanenthiran

a

Mutharani , Tse-Wei Chena a

Department of Chemical Engineering and Biotechnology, National Taipei University of

Technology, Taipei 106, Taiwan, ROC. Corresponding Author: *S.M. Chen, Fax: +886227025238, Tel: +886227017147, E mail: [email protected]

1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract In this present work, the noncovalent and eco-friendly approach was proposed to prepare the carbon black/β-cyclodextrin (CB/β-CD) nanocomposite. The CB/β-CD nanocomposite modified screen-printed carbon electrodes were applied for the simultaneous determination of anti-cancer drug flutamide (Flut) and environmental pollutant 4-nitrophenol (4-NP). The electrochemical performance of the proposed sensor was relies the conductivity of the CB, different binding strengths of the guests (Flut & 4-NP) to the host (β-CD) and different reduction potentials of the nitroaromatic compounds. Fascinatingly, the proposed sensor exhibits the excellent electrochemical performance with the high sensitivity, selectivity and reproducibility. The obtained wide linear ranges were 0.05 to 158.3 µM and 0.125 to 225.8 µM for Flut and 4-NP. On the other hand, the low detection limit of 0.016 and 0.040 µM with higher sensitivity of 5.476 and 9.168 µA µM-1 cm-2 was achieved for the determination of Flut and 4-NP. The practical feasibility of the proposed sensor was studied in tap water and human serum samples.

Keywords: carbon black, β-cyclodextrin, Host-guest interaction, Reduction potential, Activation energy.


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Introduction Nitroaromatic compounds are the important class of industrial chemicals, which are used in the variety of diverse products such as explosives, pesticides, synthetic intermediates, dyes and drugs, etc. In addition, the biological functions of the nitro‐heterocyclic drugs are depends on the reduction of the nitro group, reactivity of redox intermediates and stability of radicals.1,2 Nevertheless, the nitro group-containing aromatic compounds has strong electron-withdrawing groups, it become poorly biodegradable in the environment.3 Hence, the researchers are devoted to developing simple, inexpensive and rapid analytical technique to identify and quantify the nitroaromatic compounds. To date, several methods have been employed including luminescence,4 fluorescence,5 chromatographic6 and electrochemistry7 for the determination of nitroaromatic compounds. Among them, electrochemical methods are hitherto known to be a low-cost, reliable to operate and highly sensitive analytical technique.8 On the contrary, the selectivity is the crucial parameter that largely affects the electrochemical performance of the sensor. The main drawback during electrochemical reduction is the interfering effects of similar nitro-aromatic compounds.9 Flutamide (Flut) and 4-nitrophenol (4-NP) belongs to a class of nitroaromatic compounds. Flut is a synthetic non–steroidal antiandrogenic agent used in the treatment of prostate cancer.10 However, the continuous usage of Flut induced health disorders such as rectal bleeding, inflamed prostate, blood in urine, liver malfunction and methemoglobinemia.11,12 On the other hand, the 4-NP was used as a intermediate in chemical industries for the production of pesticides, paints, plasticizers, explosives and medicines.13 Nonetheless, 4-NP considered as an environmental pollutant due to its carcinogenic, mutagenic and teratogenic activity against animals, human beings and plants even at the presence of lowest concentration.14,15 Therefore, in this work we have selected the anti-cancer drug Flut and the environmental pollutant 4-NP as a model analytes for the simultaneous determination. To solve the selectivity problem, fabrication of nanomaterials for electrochemical sensor was often used rather than molecularly imprinted polymers due to its risk factor.16,3 Therefore, we have utilized the nanomaterial fabrication to the electrochemical sensor. Carbon nanomaterials such as graphite, graphene, carbon nanotubes hitherto known to be used for the fabrication of electrochemical sensor.17-19 Owing to the tough synthesis procedure, the usage of graphene, carbon nanotubes and fullerenes were neglected.20 Avoiding those drawbacks, we have chosen 3 ACS Paragon Plus Environment


the super conductive, inexpensive and easily available CB for the simultaneous determination of Flut and 4-NP. CB is a spherically shaped carbon nanomaterial with an average size of 30-100 nm.21 Remarkably, CB has a suitable pore-size distribution, electronic conductivity, high surface area, chemical and electrochemical stability.22 However, the poor solubility nature in water and most common solvents that limits the manipulation, characterization and practical application of CB.23

The traditional acid treatment improves the solubility of CB through the surface

modification.24 However, it causes the problems of corrosion and environmental pollution. Thus, in this work we have utilized the noncovalent, eco-friendly technique using β-cyclodextrin (βCD) as a dispersing agent. Cyclodextrins (CDs) are captivating molecules, classified as ɑ, β and γ-CD which contain six, seven, and eight glucose units. Since the CDs have been used in both research and applied technologies. 25 The CDs are toroidal in shape with a hydrophobic as well as hydrophilic an inner and exterior cavity. Besides, CDs containing two rims of hydroxyl groups, one is primary (tail) and another one is secondary (head) hydroxyl group.26 These amazing molecular structures of the CDs are well known for the forming inclusion complex with different inorganic, organic and biological guest molecules by host-guest interaction.27 On the other hand, the CDs are exhibits the high molecular selectivity and enantioselectivity by the supramolecular assemblies in their hydrophobic cavity.28 In particular, β-CD has a stronger binding constant with target molecules (more than 103) when compared with other two CDs.29 As a result, the β-CD has been utilized in the fields of catalysis, increasing solubility, molecular recognition and environmental protection.25 These interesting properties of the β-CD used in the fabrication of different electrochemical sensor.28,30 Hence, the β-CD for the preparation of CB/β-CD nanocomposite and fabrication of the modified electrodes. This manuscript describes the preparation of the stable aqueous suspension of CB using β-CD for the first time via the simple ultrasonication process. The presence of β-CD in the CB/β-CD composite enhances the supramolecular function and solubility. Especially, the CB/β-CD nanocomposite exhibits the improved electrochemical performance, very high supramolecular recognition and enrichment capability towards the simultaneous determination of Flut and 4-NP. Moreover, the practical applicability of the sensor was demonstrated in tap water and human serum samples to attain the prototype level.


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Experimental section Preparation of CB/β-CD nanocomposite and fabrication of the modified electrodes The CB/β-CD nanocomposite was prepared by simple ultrasonication method. The list of chemicals and reagents used in this work were given in supporting information. The 5 mg of CB was added in 5 mL of water and sonicated well for 30 minutes. Then, the 10 mg of β-CD was mixed into the above solution and ultra-sonicated for 1 hour at the operating frequency of 40 kHz. Finally, well-dispersed black colored homogeneous CB/β-CD solution was obtained. Then, the CB/β-CD dispersed solution about 5 µL was pipetted out on SPCE and dried well. The as prepared CB/β-CD modified SPCE was used for further electrochemical studies. Scheme-1 illustrated the material preparation and application of the CB/β-CD nanocomposite.

Scheme 1. The preparation and applications of CB/ β-CD nanocomposite.

Figure S-1 shows the photographic images of CB and CB/β-CD dispersed solution. It can be seen that the CB particles are not dispersed well due to the poor solubility. In the case of CB/βCD, the obtained homogeneous and well dispersed black color solution ascribed that the interaction between the hydroxyl groups of β-CD and the polar groups of the water molecules.31 Moreover, the formation of the homogeneous CB/β-CD solution and interaction between the CB 5 ACS Paragon Plus Environment


and β-CD can be explained based on the previous report.32,

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The van der Waals forces and

hydrogen-bonding interactions are played major role in the formation of nanocomposite. The van der Waals forces occurred between the β-CD and CB also the hydrogen-bonding interaction occurred between the neighbouring β-CD molecules. These two are the major interactions that involved and elevating the composite formation32, 36. This noncovalent formation was confirmed by FTIR spectrum of CB, β-CD and CB/β-CD (Figure S-2). Results and discussion Investigation of surface morphology and elemental composition The surface morphology of the prepared materials was examined by field emission scanning electron microscopy (FESEM) and the obtained images are displayed in Figure 1 (A-C). The FESEM image of the CB (A) shows the aggregated and small sphere like structures. In the case of β-CD (B), the flake like structure with smooth surface was observed. On the other hand, the βCD was absorbed on the surface of small spherical shaped CB and observed in the CB/β-CD nanocomposite (C). Moreover, the elemental composition of the prepared nanocomposite was estimated quantitatively by energy-dispersive X-ray spectroscopy (EDX). The obtained EDX spectrum of CB/β-CD (Figure 1D) exhibited the signals for carbon and oxygen with the atomic weight percentage of 72.7 % and 27.3 %, respectively. The signal for the oxygen with 27.3% weight percentage asserts the presence of β-CD in the prepared nanocomposite. Furthermore, the elemental mapping was performed to survey the elemental distribution in the prepared CB/β-CD nanocomposite. Figure S3 shows the FESEM image of CB/β-CD (A), corresponding EDS layered image of CB/β-CD (B) and the elemental mapping of carbon (C) and oxygen (D). As shown in Figure S-3C& D, the higher amount of red color particles exemplifies the carbon distribution and lower amount of green color represents the oxygen distribution. These elemental mapping and EDX analysis results clearly indicate the existence of the elements and its quantity (Wt %) in the CB/β-CD nanocomposite.


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Figure 1. FESEM images of CB (A), β-CD (B), CB/β-CD (C) and elemental analysis results of CB/β-CD (D).

XPS and EIS spectra X-ray photoelectron spectroscopy (XPS) is an important tool for the investigation of surface oxidation state, integrity, purity of the nano and micro materials. The binding energy was used to identify the chemical composition and oxidation state of the elements. Figure 2 exhibits the XPS survey spectrum of CB/β-CD (A), and core-level spectrum of C 1s (B) & O 1s (C) states. The survey spectrum of the CB/β-CD shows the intense signal for C 1s and it located at the binding energy of 285.4 eV. In addition, the small peak located at the binding energy of 534.6 eV for O1s which attributed to the existence of oxygen and the presence of β-CD in the nanocomposite. From the observed results, we have confirmed that there are no impurities presented in the CB/βCD composite. 7 ACS Paragon Plus Environment


Figure 2. XPS survey spectrum of (A) CB/β-CD and core-level spectrum of (B) C 1s and (C) O 1s states. (D) EIS spectrum of bare SPCE, CB/SPCE and CB/β-CD/SPCE and inset shows the Randless equivalent circuit model.

The electron transfer resistance property of the different modified electrodes was evaluated by electrochemical impedance spectroscopy (EIS). Figure 2D depicted the Nyquist plots of bare SPCE, CB/SPCE and CB/β-CD/SPCE in 0.1 M KCl solution containing 5 mM K3 [Fe(CN)6] and 5 mM K4 [Fe(CN)6]. The inset shows the Randless equivalent circuit model, where the Rs, Cdl, Ret and Zw represents the solution resistance, double layer capacitance, electron transfer resistance and Warburg diffusion process. The electron transfer resistance of the different modified electrodes was measured directly from the diameter of the semicircle. As can be seen from the Figure 2D, the bare SPCE and CB/SPCE exhibits the higher Ret values about 780 and 339 Ω with the larger semicircles. On the other hand, CB/β-CD/SPCE shows the small circle 8 ACS Paragon Plus Environment

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with the Ret value of 94 Ω. These results suggested that the CB/β-CD quicken the electron transfer from the electrolyte solution to the electrode surface. Hence, the CB/β-CD modified SPCE shows the lower Ret value. This proves the excellent conductivity of our proposed modified electrode. Electrochemistry of the different electrocatalysts The electrocatalytic behavior of the different electrocatalysts were assessed by cyclic voltammetry (CV) using [Fe(CN)6]-3/4 as a redox probe. Figure S-4 displays the cyclic voltammograms of the bare SPCE, CB/SPCE and CB/β-CD/SPCE in 0.1 M KCl containing 5 mM [Fe(CN)6]

-3/4

at the scan rate of 50mV/s . As shown in Figure S-4A, a pair of redox peaks

with higher peak-to-peak separation (∆Ep) value about 487 mV was observed for bare SPCE. After the modification with CB and CB/β-CD, the ∆Ep value was decreased to 242 and 212 mV. On the other hand, the cathodic to anodic peak current ratio (Ipc/Ipa) of bare SPCE, CB and CB/ β-CD about 0.40, 0.65 and 0.76 was obtained. Fascinatingly, the CB/β-CD revealed a good reversible behavior with the higher redox peak currents and then smaller peak separation. In addition, the electroactive surface area of the modified electrodes was calculated using the Randles – Sevcik equation.33 Thereby we have conducted the scan rate studies by differentiate the scan rates from 20 to 200 mV/s and the obtained CV image was displayed in Figure S-4B. The Randles – Sevcik equation as follows ip = 2.69×105 n3/2 A D1/2C υ1/2

(1)

In which, the ip is the anodic or cathodic peak current, n is the number of transferred electrons, A is the electroactive area (cm2), D is the diffusion coefficient of [Fe (CN)6]-3/4 (cm2 s-1), C is the concentration of the [Fe (CN)6]-3/4-(mol cm-3), and υ1/2 is the square root of scan rate (V s-1). For the calculations, we have chosen the slopes of Ipa versus υ1/2 (Figure S-4B, inset). The electrochemical active surface area was calculated about 0.053, 0.093 and 0.150 cm2 for bare SPCE, CB/SPCE and CB/β-CD/SPCE. In the comparison, the CB/β-CD/SPCE exhibited the lowest ∆Ep value, higher Ipc/Ipa ratio with high surface area. These results suggested that the CB/β-CD could be a better electrocatalytic material for the fabrication of electrochemical sensor.



Electrochemical behavior of Flut and 4-NP at different modified electrodes The electrochemical behavior of Flut and 4-NP at different modified electrodes was explored by CV in 0.05 M PB solution at the scan rate 50 mV/s. Figure 3A shows the cyclic voltammograms (CVs) of bare SPCE, β-CD/SPCE, CB/SPCE and CB/β-CD/SPCE for the electrochemical reduction of 100 µM Flut. The bare SPCE, β-CD/SPCE and CB/SPCE shows the reduction peak at ‒0.63, ‒0.62 and ‒0.61 V with the lowest reduction peak current of 9.52, 6.43 and 10.13 µA, respectively. Besides, the CB/β-CD/SPCE exhibited the well defined sharp reduction peak (R1) at –0.57 V with enhanced peak current value of 42.45 µA. In addition, during the reverse scan the CB/β-CD/SPCE exhibits the cathodic peak (R2) at ‒0.05 V and oxidation peak (O1) at 0.03 V. Figure 3B depicts the CVs of bare SPCE, β-CD/SPCE, CB/SPCE and CB/β-CD/SPCE for the electrochemical reduction of 100 µM 4-NP. As can be seen, the bare SPCE, β-CD/SPCE and CB/SPCE demonstrated the negatively shifted cathodic peaks (R1) at ‒0.84, ‒0.81 and ‒0.78 V with lower peak current values about 8.75, 5.97 and 11.40 µA. Interestingly, the CB/β-CD/SPCE display the positively shifted cathodic peak at ‒0.69 V with the improved peak current response about 40.44 µA. Additionally, the cathodic peak (R2) at ‒0.10 V and the anodic peak (O1) at 0.12 V was observed in the reverse scan. Generally, the electrochemical reduction mechanism of nitro aromatic compounds largely depends on the NO2 group, which is reduced to hydroxylamine (R1) and the reversible (O1/R2) conversion of hydroxylamine to nitroso compound formation. Herein, the electro reduction of Flut and 4-NP also exhibited the same mechanism at CB/β-CD modified SPCE. However, the bare SPCE, β-CD/SPCE and CB/SPCE shows small response for R2 and O1 peaks due to the poor catalytic activity, poor electrode surface area and poor conductivity nature of β-CD. 36 On the other hand, the CB/β-CD/SPCE provides more contact area for the adsorption also the host-guest interaction between the β-CD and nitroaromatic compounds enhanced the electrocatalytic activity of the proposed modified electrode.34,35 As well as, the β-CD was developed their supramolecular recognition capability and it forms the inclusion complexes with the analytes.36 Figure 3C&D exhibited the CV curves for the simultaneous detection of Flut and 4-NP under the same experimental conditions.


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Figure 3. (A) and (B) exhibits the CV response of bare SPCE, β-CD/SPCE, CB/SPCE and CB/βCD/SPCE in presence of 100 µM of Flut and 4-NP containing 0.05 M PB solution and scan rate at 50 mV/s. (C) CVs obtained at bare SPCE, β-CD/SPCE, CB/SPCE and CB/β-CD/SPCE for the simultaneous addition of 100 µM of Flut and 4-NP in 0.05 M PB solution. (D) CV response at CB/β-CD/SPCE in the absence of 100 µM Flut and 4-NP, presence of 100 µM Flut, 100 µM 4-NP and simultaneous additions of 100 µM Flut+100 µM 4-NP in 0.05 M PB solution.

As shown in Figure 3C, the bare SPCE, β-CD/SPCE and CB/SPCE exhibited the inferior electro catalytic performance with broad and unresolved cathodic peaks. On the other hand, the CB/βCD/SPCE exhibits good peak to peak separation with well defined peak shape. Moreover, the peak current response of the Flut and 4-NP was not changed in the simultaneous detection for the 100 µM addition of each analytes. Also, there are no changes in the peak potential which confirms the negligible interference between Flut and 4-NP (Figure 3D). These extraordinary 11 ACS Paragon Plus Environment


electrocatalytic performances of the proposed sensor material can be explained by the synergistic effect of CB (high surface area and high conductivity) and β-CD (host-guest inclusion complexes with analyte molecules). In addition, the electron with drawing and donating groups of the nitroaromatic compounds played the vital role in the reduction potential of the analytes. Whereas, the electron donating groups activate the ring and electron with drawing groups deactivate the ring. The Flut has an electron with drawing groups and 4-NP has an electron donating group

37

Due to the presence of electron with drawing groups, the NO2 becomes an

electron deficient and ready to receive the electrons from the electrode surface also it decreases the activation energy of the NO2 group.7 Hence, the cathodic peak for the electro reduction of Flut was aroused in lowest overpotential at 0.57 V. On the other hand, the 4-NP has an electron donating group which activates the ring thus the electron density was increased at NO2 group. Therefore, the electro reduction of 4-NP appeared with higher overpotential at 0.69 V. Furthermore, the different binding energy of the analytes influenced the reduction potential of the nitroaromatic compounds. Hence, the well defined and well resolved peaks were observed for the simultaneous detection of Flut and 4-NP. Effect of scan rate and pH The effect of scan rate on the electrochemical reduction of Flut and 4-NP was investigated by CV. Figure 4A display the CV responses at CB/β-CD/SPCE for different scan rate analysis in 0.05 M PB solution containing 100 µM of Flut and 4-NP. Whereas, the peak current responses of Flut and 4-NP increased gradually when increasing the scan rate from 20 to 300 mV/s. Figure 4B shows the calibration plot between the cathodic peak current (R1) of Flut (pink) and 4-NP (blue) versus different scan rates. As can be seen, the cathodic peak currents of the Flut and 4-NP had a linear relationship with different scan rates. Moreover, the linear regression equation Ipc = -0.266 x ‒13.897 (R2 = 0.994) for Flut and Ipc = -0.279 x ‒19.181 (R2 = 0.995) for 4-NP was obtained. The obtained results are revealed that the reduction of Flut and 4-NP at CB/β-CD/SPCE is an adsorption-controlled process.38,39 In order to evaluate the pH dependence of the proposed sensor, different pH (3 to 11) study was performed using CV and the images were presented in Figure 4C. It can be seen that the acidic pHs (3 & 5) exhibits well resolved peaks. In the case of basic pHs (9 & 11), the imperfect peak shapes with inferior electrochemical response was


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observed. Interestingly, well defined sharp peaks with enhanced peak current response was observed at neutral pH (pH=7).

Figure 4. (A) CVs obtained at different scan rate analysis (20 to 300 mV/s) on CB/β-CD/SPCE for the simultaneous addition of 100 µM Flut + 100 µM 4-NP in 0.05 M PB solution. (B) The linear plot between scan rates versus cathodic peak current of Flut and 4-NP. (C) CVs obtained for the simultaneous addition of 100 µM Flut +100 µM 4-NP in different pH solutions (3 to 11) at CB/β-CD/SPCE. (D) The calibration plot between different pH values versus cathodic peak current of Flut and 4-NP.

In addition, the potential shifts in the peaks (R1, R2 & O1) indicate that the direct involvement of the proton in the electro reduction process. Figure S-5A shows the CV curves of different pH analysis and Figure S-5B shows the calibration plot between different pH and cathodic peak potential of Flut and 4-NP. The obtained slope values from the calibration plots were applied in 13 ACS Paragon Plus Environment


the Nernst equation = −

.

+ to calculate the m/n ratio.40 Whereas, the m is the

number protons of and n is the number of electrons. Using that equation, the m/n ratio was calculated about 0.60 and 0.81 for Flut and 4-NP, respectively. These results exhibited that the equal number of proton and equal number of electrons were involved in the electro reduction of Flut and 4-NP.41,42 Moreover, the electro reduction of Flut and 4-NP at CB/β-CD/SPCE is four electrons and four protons transferred process (Figure S-6A&B).43,44 Simultaneous determination of Flut and 4-NP DPV is the sensitive and suitable voltammetric technique for the simultaneous determination of nitroaromatic compounds. Hence, we have utilized the DPV for the simultaneous determination of Flut and 4-NP in 0.05 M pH-7 PB solution. Figure 5A shows the DPV responses for the different concentration (0.05 to 400 µM) additions of Flut in presence of 50 µM 4-NP at CB/βCD/SPCE. It can be seen that the cathodic peak at ‒0.51 V was increased for the various concentration addition of Flut. In addition, the cathodic peak at ‒0.67 V appeared for the addition of 4-NP and its peak current response was increased little during the addition of different concentration of Flut. The obtained peak current responses were plotted against the Flut concentration and this image was displayed in Figure 5B. As can be seen, the peak current responses had a linear relationship with the different concentrations with the linear regression equation of I = -0.136 µA µM-1 ‒ 7.866 (R2 = 0.998). Moreover, the wider linearity from 0.05 to 158.3 µM and the limit of detection about 0.016 µM with higher sensitivity of 5.476 µA µM-1 cm-2 was observed for the determination of Flut. The electrochemical determination of 4-NP in presence of 50 µM Flut was recorded by DPV and the obtained image was presented in Figure 5C. As shown in Figure 5C, the cathodic peak at ‒0.67 V observed for electro reduction of 4-NP and Flut exhibits the cathodic peak at ‒0.51 V. Furthermore, the peak current responses at ‒0.67 V was increased when adding the different concentrations of 4-NP from 0.125 to 225.8 µM. The linear relationship between the peak current responses and different concentration was displayed in Figure 5D. The linear regression equation I = ‒0.229 µA µM-1 ‒ 4.808 with the correlation coefficient R2 = 0.994 was obtained for the determination of 4-NP. Interestingly, CB/β-CD modified SPCE exhibited the long linear range from 0.125 to 225.8 µM and appreciable LOD about 0.040 µM with higher sensitivity of 9.168 µA µM-1 cm-2.


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Figure 5. (A) DPV responses obtained at CB/β-CD/SPCE for the successive addition of Flut (0.05-400 µM) in presence of 50 µM 4-NP. (B) The calibration plot for the cathodic peak current versus Flut concentrations. (C) DPV responses of CB/β-CD/SPCE for the successive addition of 4-NP (0.125-225.8 µM) in presence of 50 µM Flut. (D) The plot between the cathodic peak current versus 4-NP concentrations.

In addition, the obtained analytical parameter values for the determination of Flut and 4-NP were compared with previous reports and tabulated in Table-S1&S2. On the comparison, our proposed sensor material exhibited the competitive performance than other modified electrodes. Besides, the simultaneous additions of Flut and 4-NP were performed in the same experimental conditions. In this experiment, the equal concentrations of Flut and 4-NP were added from 5 µM to 65 µM and the obtained DPV responses were displayed in Figure 6A. Fascinatingly, well resolved and sharp cathodic peaks were observed and the peak current responses were increased when increases the concentrations of the analytes. Moreover, the obtained peak current responses 15 ACS Paragon Plus Environment


were plotted against the concentrations of Flut and 4-NP (Figure 6B). It can be seen that the peak currents had a linear relationship with the linear regression equation I = ‒1.061 µA µM-1 ‒15.611 (R2 = 0.995) for Flut and I = ‒1.062 µA µM-1 + 2.147 (R2 = 0.997) was obtained. These results reveal that the CB/β-CD is an excellent electrode material for the determination of anti-cancer drug Flut and toxic 4-NP. The host-guest interaction between the CB/β-CD and analytes were enhancing the electrocatalytic performance of the modified electrode.27 Based on the previous reports, the higher interaction between nitroaromatic compounds and β-CD requires more energy for the reduction of nitro group that shifts the reduction potential to negative side.3

Figure 6. (A) DPV response at CB/β-CD/SPCE for the simultaneous additions of Flut and 4-NP from 5 to 65 µM in 0.05 M PB solution. (B) The calibration plot for the cathodic peak current of Flut and 4-NP versus concentrations of Flut and 4-NP.

Selectivity, stability and reproducibility The selectivity of the sensor was evaluated by DPV in presence of the interfering molecules and ions. Figure S-7A displayed the DPV responses of 10 µM Flut and 4-NP (a) in presence of 25 µM uric acid (b), glucose (c), catechol (d), 4-nitroaniline (e) phenol (j), 2,4-dinitrophenol (k), paracetamol (l) hydroquinone (m), and 100 µM addition of common ions Cu2+ (f), K+ (g), Cl‒ (h) and Br‒ (i). Similar experimental conditions which were used for the simultaneous determination of Flut and 4-NP utilized for this experiment. Figure-S-7B shows the peak current responses of the aforementioned molecules and ions. It can be seen that the CB-β-CD/SPCE exhibits the appreciable selectivity in the interference study. Moreover, the reproducibility of the sensor was 16 ACS Paragon Plus Environment

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evaluated in 0.05 M PB solution. For this experiment five parallel determinations were performed using 5 independent CB/β-CD/SPCE. The relative standard deviation (RSD) about 3.5 % was observed for the reproducibility study. On the other hand, the stability of the sensor is an important parameter. Therefore, the stability of the sensor towards the simultaneous determination was evaluated for four weeks. The result shows that the peak current responses of the sensor decreased only 5 % from the initial current value. This selectivity, stability and reproducibility results demonstrated that the CB/β-CD is good electrode material and suitable for the simultaneous determination of Flut and 4-NP. Real sample analysis The practical applications of the proposed sensor were explored in tap water and human serum samples by standard addition method using DPV. Prior to experiment the tap water samples were collected and the pH of the sample was adjusted equal to the PB solution. On the other hand, human serum samples were diluted with the pH-7 buffer solution and this samples are Flut and 4-NP free samples. Therefore, the known concentration of Flut and 4-NP was spiked into the above samples and used for the real sample analysis experiment. Furthermore, the obtained recovery results are tabulated in Table-S-3. As can be seen, the proposed sensor material exhibits the reasonable recovery results. Hence, we suggested that the CB-β-CD/SPCE is feasible for the real time applications. Conclusion In summary, the solubility of the CB was successfully enhanced by the non-covalent approach using β-CD as a dispersing agent. The simple ultrasonication method was used for the preparation of CB/β-CD nanocomposite. Then, the CB/β-CD was used to fabricate the simple, low cost and highly sensitive and selective electrochemical sensor for the simultaneous determination of Flut and 4-NP in aqueous solution. The high conductivity and high surface area of CB and host-guest inclusion complexes with β-CD molecules enhance the electrochemical performance of the proposed sensor. Interestingly, the wide linear ranges about 0.05 to 158.3 µM for Flut and 0.125 to 225.8 µM for 4-NP was obtained. On the other hand, the low detection limit of 0.016 and 0.040 µM with the higher sensitivity 5.476 and 9.168 µA µM-1 cm-2 was achieved for the determination of Flut and 4-NP, respectively. Moreover, the practical feasibility of the proposed sensor was studied in tap water and human serum samples. We believe that this work 17 ACS Paragon Plus Environment


opens a new gate to the preparation of nanocomposites using inexpensive and high conductive CB for determination of various nitroaromatic compounds. Acknowledgments This project was supported by the Ministry of Science and Technology (MOST 106-2113-M- 16 027003), Taiwan, ROC.

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