A sensitive nanocomposite design via carbon nanotube and silver nanoparticles: Selective probing of Emedastine Difumarate

Journal Pre-proof A sensitive nanocomposite design via carbon nanotube and silver nanoparticles: Selective probing of Emedastine Difumarate Hamideh Imanzadeh (Data curation)WritingOriginal draft preparation) (Visualization) (Investigation)Writing - reviewing and editing), Nurgul K Bakirhan (Data curation)Writingoriginal draft preparation), Biuck Habibi (Software) (Validation), Sibel A Ozkan (Supervision)

PII:

S0731-7085(19)32433-1

DOI:

https://doi.org/10.1016/j.jpba.2020.113096

Reference:

PBA 113096

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

5 October 2019

Revised Date:

31 December 2019

Accepted Date:

2 January 2020

Please cite this article as: Imanzadeh H, Bakirhan NK, Habibi B, Ozkan SA, A sensitive nanocomposite design via carbon nanotube and silver nanoparticles: Selective probing of Emedastine Difumarate, Journal of Pharmaceutical and Biomedical Analysis (2020), doi: https://doi.org/10.1016/j.jpba.2020.113096

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

A sensitive nanocomposite design via carbon nanotube and silver nanoparticles: Selective probing of Emedastine Difumarate

Hamideh Imanzadeha,b, Nurgul K Bakirhanc*, Biuck Habibib, Sibel A Ozkana* a

Ankara University, Faculty of Pharmacy, Department of Analytical Chemistry, 06560, Ankara, Turkey Electroanalytical Chemistry Laboratory, Department of Chemistry, Faculty of Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran c Hitit University, Faculty of Art & Science, Department of Chemistry, Corum, Turkey b

ro

of

*Corresponding Authors: [email protected]; [email protected]

Jo

ur na

lP

re

-p

Graphical abstract

1

Highlights -

In this study, we proposed a possible sensitive sensor for detection of emedastine drug.

-

This is the first time a novel sensor was developed for detecting drug emedastine using carbon and metal nanoparticles. Adsorptive stripping differential pulse voltammetry was applied for trace analysis of

of

-

ro

EDD in an eye drop and human serum with good satisfactory results.

-p

Abstract

In this study, a novel and sensitive nanocomposite of carboxylate-functionalized multiwalled carbon

re

nanotube (COOH-fMWCNT) and silver nanoparticles (AgNPs) was fabricated and used to modify a glassy carbon electrode (GCE) by a simple drop casting method. Modified electrode was then applied for selective

lP

determination of emedastine difumarate (EDD). The COOH-fMWCNT/AgNPs nanocomposite was characterized by electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and cyclic voltammetry (CV). EDD showed two oxidation

ur na

peaks at 0.634 and 1.2 V on the GCE surface. CV results of COOH-fMWCNT/AgNPs/GCE displayed superior electrocatalytic performance in terms of anodic peak current of EDD when compared to AgNPs/GCE, MWCNT/GCE, and COOH-fMWCNT/GCE. The experimental conditions such as effect of pH, supporting electrolyte, effect of accumulation time and potential, scan rate were optimized for getting intense current signals of the target analyte. Under optimized conditions, COOH-fMWCNT/AgNPs/GCE showed a linear current response for oxidation of EDD in the range of 1.0×10-7-1.0×10-4 M, with a limit of

Jo

detection (LOD) and quantification (LOQ) of 5.25 nM, 15.9 nM, respectively, in 0.1M phosphate buffer solution at pH 2.0 using differential pulse adsorptive stripping voltammetry technique. The proposed method was successfully applied for determination of EDD in pharmaceutical dosage form. Satisfactory recovery percentages were achieved from eye drop sample with acceptable RSD values (less than 2%). Furthermore, the reproducibility, stability and repeatability of the modified electrode were studied.

Keywords: Emedastine Difumarate; Electrochemistry; Silver Nanoparticles; Multiwalled Carbon Nanotube; Nanosensor 2

1.

Introduction

Allergic conjunctivitis is an eye inflammation which can cause severe ocular surface disease. Emedastine difumarate (EDD) is 1-(2-ethoxyethyl)-2-(hexahydro-4-methyl-1-H-1, 4-diazepin-1yl), benzimidazole difumarate. EDD is a second-generation H1-receptor antagonist with antiallergic activity, used in eye drops to treat allergic conjunctivitis. The mechanism of action of this drug is that it blocks histamine by binding to H1 receptors [1-3]. It is used in allergic conjunctivitis

of

treatment on patients with 0.05% EDD [4].

ro

Several analytical methods have been used for the detection of EDD in pharmaceutical formulations and biological fluids such as capillary GC method [5], HPLC coupled with

-p

electrospray ionization/mass spectrometry (ESI-MS) [6] and radioimmunoassay (RIA) [7]. In addition, electrochemical studies with carbon paste electrode (CPE) and glassy carbon electrode (GCE) [1] have been reported for the quantification of EDD. Although, electrochemical studies

re

with bare electrodes have been used for detection of EDD, but there is no report of sensitive determination of EDD with the modified electrode. In recent years, electrochemical techniques

lP

utilizing modified electrodes have attracted a lot of attention as a powerful tool for detection of pharmaceutical drugs. Compared to other methods, the electrochemical sensors are simple, low cost, and sensitive and they can be easily adjusted to detect a wide range of analytes with low

ur na

detection limit and high performance.

In the past few decades, nanomaterials have played a vital role in the development of electrochemical sensors. They have created a new area for application in electrochemical sensors technology due to the important features of nanomaterials, such as small-scale, great active surface

Jo

area, good biocompatibility and good stability. Different methods of fabrication nanoparticles and their nanocomposites are proposed to increase the surface area of electrochemical sensors for fabricating and modification of electrodes [8-10]. Carbon-based nanomaterials are widely used for modification electrodes for electrochemical sensing. One of the most important types of carbon nanomaterials are carbon nanotubes, which owing to their unique chemical and physical properties, have found a special place in electrochemical studies [5, 11]. MWCNTs generally exhibit extremely high thermal and electrical conductivity, high surface areas and mechanical 3

strength. MWCNTs-based electrodes have been successfully used for detecting applications. In order to improve water dispersibility and absorption capacity, CNTs can be functionalized with some groups. On the other hand, the use of metallic NPs, especially silver nanoparticles (AgNP) for analytical sensors has been taken into consideration due to their ability to increase the conductivity of the electrode. Another advantage of these materials is their low-cost and simple synthesis steps as well as their ability to facilitate electron transfer. It is well-known that CNT‐ based nanocomposites are capable of delivering very strong and ultra-lightweight materials. In

of

fact, CNT‐based nanocomposites are the most promising electrode materials for improvement of electrochemical sensors. Recent reports displayed that some carbon/silver nanocomposites such as reduced graphene oxide/silver nanocomposite [12], carbon quantum dots/silver nanoparticles [13]

ro

and MWCNT/silver nanocomposite [14] were applied as electrochemical nanosensors.

-p

In this research, we have developed an electrochemical nanosensor of carboxylate-functionalized multiwalled carbon nanotube (COOH-fMWCNT) and silver nanoparticles (AgNPs) as a new

re

electrochemical nanosensor for the trace level detection and determination of EDD. The analytical parameters of the COOH-fMWCNT/AgNPs/GCE were also assessed as well as its reproducibility, selectivity and stability investigations. The results showed that proposed nanosensor has excellent

Experimental section

ur na

2.

lP

electrochemical efficiency with high sensitivity.

2.1. Materials and reagents

The standard reference compound, Emedastine difumarate (EDD) and its commercial form Emadine were kindly supplied by Novartis® Pharmaceuticals (Istanbul/Turkey). COOHfMWCNTs were obtained from DropSens (Llanera, Spain). They have a specific surface BET of

Jo

300 m2/g, an average diameter of 10 nm and an average length of 1.5 μm. AgNPs (10 nm, containing 0.1mM Ag in 20 mL water) were purchased from NanoCS®. For electrochemical measurements different buffer solutions were prepared such as phosphate (pH 2.0; 3.0; 6.0; 7.0; 8.0) and acetate (pH 4.0; 5.0). Other materials such as H2SO4, N, N-dimethylformamide, (DMF), acetonitrile and potassium ferrocyanide were purchased from Merck. All aqueous solutions were prepared with doubly distilled deionized water. 2.2. Instrumentation 4

A PalmSens 5.2 electrochemical analyzer was used for performing voltammetric experiments. A conventional three-electrode system was used with bare GCE (3 mm diameter) and COOHfMWCNT/AgNPs modified GCE as working electrodes; Ag/AgCl (3M KCl solution) as a reference electrode and a Pt wire as the counter electrode. pH measurements were accomplished by a pH meter Model 538 (WTW, Austria) with a merged electrode (reference electrode-glass electrode) for the preparing of the buffer solutions. Scanning electron microscopy (SEM) images and EDX spectra acquired using ZEISS EVO 40 (Merlin, Carl Zeiss). EV018 vacuum oven was

of

used for drying the electrode. For adsorptive stripping differential pulse voltammetry (AdSDPV) studies, optimum stripping conditions were found to be as 0.3 V for accumulation potential and 45

ro

s for accumulation time.

2.3. Preparation and characterization of the modified COOH-fMWCNT/AgNPs /GCE

-p

Prior to use, GCE was polished by the alumina powder (0.05 µm) on a polishing pad and rinsed with distilled water. COOH-fMWCNT suspension was prepared in DMF in the concentration of 1

re

mg/mL and sonicated for 2 h. Then, COOH-fMWCNT/AgNPs suspension was prepared by mixing 400 μL fMWCNT and 200 μL AgNPs under ultrasonic stirring during 30 min. Then, an aliquot of

lP

10 μL of COOH-fMWCNT/AgNPs suspension was coated on the surface of the pre-cleaned GCE and dried in a vacuum oven for 10 min. COOH-fMWCNT/AgNPs nanocomposite was characterized by electrochemical methods and scanning electron microscopy (SEM) with energy

ur na

dispersive X-ray (EDX) analysis using ZEISS EVO 40 (Merlin, Carl Zeiss). 2.4. Preparation of standard and pharmaceutical dosage form Stock solution of 1×10-3 M EDD was prepared by dissolving required amount EDD in deionized water. The stock solution was diluted up with 0.1 M PB solution at pH 3.0 for calibration curve.

Jo

Eye drop sample of EDD was used for real sample application. 1 mL of Emadine eye drop contains 0.5 mg EDD. Eye drop and recovery experiments were performed with good accuracy. To assess the reproducibility of the proposed nanosensor, 5×10-5 M of standard solution was measured for five times in the same day and between the days.

5

3. 3.1.

Results and discussion Electrochemical characterization of the modified electrode

Electrochemical Impedance Spectroscopy (EIS) is a very sensitive technique to study electron transfer processes at interfaces between electrode and electrolyte solution [15, 16]. EIS measurements of bare and modified glassy carbon electrodes were carried out in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 solution. Fig.1A shows the Nyquist curves for the bare CGE, AgNPs/GCE, MWCNT/GCE, COOH-fMWCNT/GCE and COOH-fMWCNT/AgNPs/GCE. As shown in Fig. 1,

of

Nyquist plot contains of a semicircular segment representing the electron transfer process in the high frequency region and its diameter is equivalent to the charge-transfer resistance (Rct) and a

ro

linear segment in the low frequency range representing Warburg resistance (Rw). The value of Rct is an important parameter for describing the interfacial attributes of the modified electrode. The

-p

Rct value of bare GCE (2930 Ω) significantly decreased after modification with AgNPs (2320 Ω), MWCNT (520 Ω) and COOH-fMWCNT (184 Ω) due to excellent conductivity and high

re

electrocatalytic activity of AgNPs, MWCNT and COOH-fMWCNT. In addition, The Rct value further decreased to 23 Ω for the COOH-fMWCNT/AgNPs nanocomposite. Therefore, the

lP

nanocomposite demonstrates a lower semicircle value compared to the other modified electrodes, which represents fast electron transfer kinetics at COOH-fMWCNT/AgNPs/GCE. Parameters calculated from the Nyquist plots are listed in Table 1. In addition, CV was used for the bare GCE

ur na

and COOH-fMWCNT/AgNPs/GCE at different scan rates in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 solution as redox probe. The slope acquired from a linear plot of Ipa versus ѵ1/2 was utilized to estimate the active surface area of bare GCE and COOH-fMWCNT/AgNPs/GCE according to the Randles Sevcik equation (Eq. 1).

(1)

Jo

𝐼𝑝 = 2.69 × 105 𝑛2⁄3 𝐴 𝐷1⁄2 𝑉 1⁄2 𝐶

In this equation; ‘n’ denotes the number of transferred electrons, ‘D’ is diffusion coefficient as 7.6×10−6 cm2s-1, ‘C’ is concentration of K3Fe(CN)6/K4Fe(CN)6 in solution, IP is the peak current (μA), A represents active surface area (cm2) of the electrode. The calculated values of the active surface area of the electrodes are given in Table 1. All CV and EIS results showed good agreement. The results clearly show that COOH-fMWCNT/AgNPs nanocomposite has larger surface area than other individually modified electrodes, which indicates that the nanocomposite with great active 6

surface area increases the electrocatalytic activity and electron transfer rate between analyte and electrode. Thus, nanocomposite represents successful construction of the GCE with increasing of active surface area and decreasing of resistance.

-----FIGURE 1 HERE---------TABLE 1 HERE----Investigation of morphology on COOH-fMWCNT/AgNPs/GCE

of

3.2.

The morphological characterization of the COOH-fMWCNT/AgNPs/GCE was performed by

ro

SEM armed with EDX analysis. The surface morphologies of (A) bare GCE (B) AgNPs/GCE, (C) MWCNT/GCE, (D) COOH-fMWCNT/GCE and (E) COOH-fMWCNT/AgNPs/GCE portrayed by SEM images are exposed in Fig. 2. In Fig. 2(B) it can be seen that the silver particles are distributed

-p

homogeneously well on GCE. MWCNT and COOH-fMWCNT show uniform and rough surface of randomly distributed tubes on GCE. The SEM image of the COOH-fMWCNT/AgNPs

re

nanocomposite displays that the spherical AgNPs are placed uniformly on the surface of the COOH-fMWCNT and provides more porosity in surface area. EDX spectrum of COOH-

electrode.

3.3.

ur na

-----FIGURE 2 HERE-----

lP

fMWCNT/AgNPs film in Fig. 2F confirmed the presence of silver, carbon and oxygen on modified

Electrochemical behavior of EDD at COOH-fMWCNT/AgNPs/GCE

Cyclic voltammograms were recorded at bare GCE and COOH-fMWCNT/AgNPs/GCE between

Jo

the potential range 0.0 and 1.8 V versus Ag/AgCl at a scan rate of 0.1V/s in 0.1 M PB solution at pH 2.0 (containing 0.1 mM EDD) to study electrocatalytic activity of COOH-fMWCNT/AgNPs nanocomposite for electrochemical oxidation of EDD (Fig.1A). EDD demonstrates a reversible couple responses at the potential of 0.634 V and 0.60 V and also another peak at the potential 1.2 V (Fig. 1A inset) on the unmodified GCE. The response of main peak (peak 2) was significantly increased after modification. The COOH-fMWCNT/AgNPs/GCE nanocomposite displayed a synergistic electrocatalysis effect of COOH-fMWCNT and Ag nanoparticles towards EDD 7

oxidation with increasing peak current of EDD with a negative shifting in the peak potential (1.173 V). In Fig. 3B, the differential pulse voltammograms are shown with bare GCE, AgNPs/GCE, MWCNT/GCE, COOH-fMWCNT/GCE/GCE in 0.1 M PB (pH 2.0) solution in the presence of 0.1 mM EDD. In all cases, two anodic responses were observed during the anodic potential scanning of the EDD. Comparing with all other electrodes, the COOH-fMWCNT/AgNPs nanocomposites modified electrode showed the highest peak current (peak 2) at 1.099 V. The second oxidation

of

peak at COOH-fMWCNT/AgNPs/GCE was nearly 20 times higher than the bare GCE, thus all next evaluations were based on the measurement of the second anodic peak. The increasing of the

ro

peak current and the decreasing of the peak potential can be ascribed not only to the to the high electroactive surface area and electrocatalytic activity of the COOH-fMWCNT, but also the effect

-p

of AgNPs that enhanced the conductivity of the nanocomposite on the GCE surface [17]. In addition, MWCNT has better conductivity and molecules can penetrate through the conductive

re

porous channels onto the modified electrode more simply, leading to selectivity and more sensitivity [18]. From these results, it can be concluded that the COOH-fMWCNT/AgNPs

3.4.

ur na

-----FIGURE 3 HERE-----

lP

nanocomposite could be used as a nanosensor for determination of EDD.

The effect of pH on voltammetric response

The effect of pH was examined on the oxidation of EDD by DPV at COOHfMWCNT/AgNPs/GCE in the range of 1 to 8 using sulfuric acid solution and acetate and phosphate buffers. Fig. 4 (A, B) exhibits differential pulse and cyclic voltammograms of EDD at COOH-

Jo

fMWCNT/AgNPs/GCE with various pHs of buffer solutions. The results demonstrated that the anodic peak potentials (Epa) are shifted to less positive potentials by increasing pH for EDD that means involvement of protons during the oxidation process. The graphics shown in Fig. 4 represents the plot of Ep versus pH for the main peak of EDD. It can be observed that the variation of peak potential (Ep) as a function of pH was linear with the below equation: Ep = 1.338 - 0.074pH;

(r =0.9913)

(pH: 1.0 - 8.0); CV

(1)

8

Ep = 1.254 - 0.075pH;

(r =0.9970)

(pH: 1.0 - 8.0); DPV

(2)

The highest current and the best peak shape were obtained at 0.1 M PB solution at pH 2.0. Thus, the 0.1 M PB (pH 2.0) solution was selected as the supporting electrolyte for the quantification of EDD. With considering the resulted slope for peak potential, equal numbers proton and electron are involved in electrochemical oxidation of EDD [19].

of

-----FIGURE 4 HERE-----

3.5. The effect of potential sweep rate

ro

The effect of the scan rate on the 0.1mM EDD voltammetric response was investigated by ranging the scan rate of CV from 5 to 250 mV s−1 at pH 2.0 PB solution on COOH-fMWCNT/AgNPs/GCE

-p

nanosensor to understand whether the process on electrode surface was diffusion or adsorption controlled. The results exhibited that the anodic peak current of EDD oxidation is linearly

(r = 0.9988)

(3)

lP

IP (μA) = 0.190 v (mVs-1) + 1.065

re

proportional to the scan rate, according to the following expression:

Also the relation between logarithm of peak current "log (IP)” was plotted versus logarithm of scan

ur na

rate "log (ѵ)” in the range between 5 and 250 mV/s. A linear dependence was obtained with a slope of 0.7809. It can be stated that the electrochemical oxidation of EDD at COOHfMWCNT/AgNPs/GCE nanosensor is the adsorption controlled process [20]. Related equations are noted under:

(r = 0.9929)

(4)

Jo

log IP (μA) = 0.781 logv (mVs-1) - 0.245

Equation ΔEp – Ep/2 = 47.7/αn was used to calculate the number of electrons involved in the oxidation process of EDD. If the coefficient 'α' was assumed to be 0.5, the desired value for 'n' is equal to 1.862, which indicates the interference of two electrons for the oxidation reaction studied [21].

9

CV and DPV techniques were used to study the electrochemical oxidation mechanism of EDD. Two organic compounds, diazepam and benzydamine HCl, which have the chemical structures like EDD, were studied in 0.1 M PB (pH 2.0, 6.0) solution at COOH-fMWCNT/AgNPs/GCE (Table 2). As shown in Fig.5 (A, B) the diazepam showed no response in 0.1 M PB solutions of pH 2.0 and of pH 6.0. Though, benzydamine HCl showed similar response with a well-defined peak in 0.1 M PB solutions of pH 2.0 and of pH 6.0 in the potentials 1.019 V, 0.859 V, respectively. Therefore, we can propose that our possible oxidation reaction may occur from benzydamine

of

moiety at EDD. Diezapane part is not oxidable according to pure diazepam electrooxidation response.

ro

-----TABLE 2 HERE-----

re

-p

The possible oxidation mechanism of EDD can be shown by the following reaction (Scheme 1):

-----FIGURE 5 HERE-----

The effect of accumulation potential and time

ur na

3.6

lP

-----SCHEME 1 HERE-----

To acquire the best parameters for electrochemical oxidation determination of EDD, accumulation time (tacc) and potential (Eacc) parameters were investigated by AdSDPV using COOHfMWCNT/AgNPs/GCE nanosensor (Fig. 6 A, B and C, D.). The effect of Eacc on the anodic

Jo

oxidation peak current (IPa) of 0.1mM EDD in 0.1 M PB (pH 2.0) solution was investigated between -0.2 and 0.4 V as depicted in Fig.7 (A and B). The peak current for EDD firstly increased when the accumulation potential was increased and achieved maximum at 0.3 V. Then, the effect of tacc was studied between 5 and 90 seconds; the optimum value being 45 s. Therefore, 0.3 V and 45 s were selected as the optimum conditions of stripping technique. In addition, Fig.8 shows the comparison of DP and AdSDP voltammograms in 0.1mM of EDD at 0.1M PB solution (pH 2.0). The oxidation peak current of EDD has significantly increased from 25.39 µA to 60.4 µA with AdSDPV in optimal conditions (Eacc 0.3V and tacc 45s). 10

-----FIGURE 6 HERE-----

-----FIGURE 7 HERE-----

3.7. Analytical applications 3.7.1. Calibration curve

of

In order to find linear concentration range and detection limit (LOD) of EDD, AdSDPV technique was used. AdSDP voltammograms of different concentrations of EDD in 0.1M PB with pH 2.0

ro

were recorded using a COOH-fMWCNT/AgNPs/GCE nanosensor under optimized experimental conditions (Eacc= 0.3 V, tacc= 45 s). These results are presented in Fig 8. The results clearly show

-p

that the anodic peak signal increases with increasing concentration. A linear range of 1.0×10−7 to 1.0×10−4 M with a determination coefficient (r) of 0.9994 were obtained for EDD. The linear

(r = 0.9994)

(5)

lP

Ip (μA) = 0.594C (µM) + 0.113;

re

regression equation can be expressed as;

The analytical parameters which calculated from calibration plot are listed in Table 3. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated as 5.25 nM and 15.9 nM,

ur na

respectively. For these calculations; LOD=3.3 s/m and LOQ=10 s/m [22] equations were used. In addition, the literature comparison is presented for electrochemical EDD analysis in Table 4. It can be clearly seen that the LOD value of the proposed COOH-fMWCNT/AgNPs/GCE nanosensor is lower than those reported studies. The reproducibility of the proposed nanosensor in the same day and between the days were calculated for peak current and peak potential in the standard

Jo

solution. Their relative standard deviation (RSD %) values are listed in Table 3. As shown in Table 3, RSD% was obtained for peak current and potential in the standard solution as less than 2 which confirms good reproducibility of the proposed method.

-----FIGURE 8 HERE-----

11

-----TABLE 3 HERE---------TABLE 4 HERE-----

3.8

Analytical application

Analytical utility of the modified electrode was tested with eye drop sample to estimate the accuracy, validity and applicability of the proposed nanosensor. The results of the determination of the drug in eye drop forms of EDD are presented in Table 5 and are fairly near to the labeled

of

values, displaying the reliability of the experiments. Recovery values obtained after addition of known amount of pure drug into eye drop, using the obtained calibration curves. The excellent

ro

recoveries (99.6%) confirm the reliability and accuracy of the proposed method for eye drop formulation.

re

3.9

-p

-----TABLE 5 HERE-----

Interference study

lP

To investigate the selectivity of the COOH-fMWCNT/AgNPs/GCE nanosensor, the effect of some biological interfering agents like ascorbic acid (AA) and dopamine (DA) and of some ions such as Cl− , NO3− , K + , Na+ , Ca2+ on the voltammetric response of EDD were studied at 1:1, 1:10, 1:100,

ur na

1:1000 ratios (analyte: interferent) by AdSDPV technique. Fig. 9 shows the current intensity of 5×10-5 M EDD in the presence of 1, 10, 100 and 1000 times of interfering agents at 0.1 M PB (pH 2.0) solution. From the results, it can be seem that these compounds have had only a little effect on the EDD signals with less than 5% deviations. These results are representing the valuable

Jo

selectivity of the COOH-fMWCNT/AgNPs/GCE nanosensor towards the determination of EDD.

-----FIGURE 9 HERE-----

12

4. Conclusion In this study, the electrochemical behavior of EDD compound was investigated by CV and DPV and AdSDPV methods. A new COOH-fMWCNT/AgNPs nanocomposite was used to modify the GC electrode surface for selective determination of EDD in 0.1 M PB (pH 2.0) solution. The influences of pH value of the buffer solution and scan rate were studied. This method provides a new method for making a modified electrode for the rapid, simple, and cost-effective analysis of EDD in eye drop, successfully. According to the results COOH-fMWCNT/AgNPs/GCE

of

nanosensor displayed excellent sensitivity and good electrocatalytic activity toward EDD electrooxidation. The LOD and LOQ values were found as 5.25 nM, 15.9 nM with modified electrode,

ro

respectively. This value of LOD and LOQ illustrates excellent sensitivity of the proposed method for ultra-trace level detection of EDD. In addition, the high recovery of 99.6% reflect the successful

may be used as a chip in clinical applications.

re

AUTHR STATEMENT

-p

usage of the designed method for real sample analysis. In the future studies, the developed method

ur na

lP

Nurgul K Bakirhan: Conceptualization, Methodology, Software Hamideh Imanzadeh: Data curation, Writing- Original draft preparation. Hamideh Imanzadeh: Visualization, Investigation. Sibel A Ozkan: Supervision. Buick Habibi: Software, Validation.: Hamideh Imanzadeh: Writing- Reviewing and Editing, Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Jo

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

13

of

No financial support.

Acknowledgements

ro

The authors are grateful to the Ministry of Science, Research and Technology of Iran for financial

ur na

References

lP

re

-p

support.

[1] S.A. Abdel-Razeq, M.M. Foaud, N.N. Salama, S. Abdel-Atty, N. El-Kosy, Voltammetric determination of azelastine-HCl and emedastine difumarate in micellar solution at glassy carbon and carbon paste electrodes, Sensing in Electroanalysis 6 (2011) 289-305. [2] A.-R.A. Sawsan, S.N. Nahla, F.M. Manal, A.-A. Shimaa, E.-K. Naglaa, Spectrophotometric

Jo

determination and thermodynamic studies of the charge transfer complexation of emedastine difumarate with some π-acceptors, Arab J Chem 10 (2017) S1855-S1861. [3] H. Murota, I. Katayama, Emedastine difumarate: a review of its potential ameliorating effect for tissue remodeling in allergic diseases, Expert opin on pharmaco 10(11) (2009) 1859-1867. [4] A. Secchi, A. Leonardi, M. Discepola, J. Deschenes, M. Abelson, E.S. Group, An efficacy and tolerance comparison of emedastine difumarate 0.05% and levocabastine hydrochloride 0.05%: 14

reducing chemosis and eyelid swelling in subjects with seasonal allergic conjunctivitis, Acta Ophthalmol. Scand. 78 (2000) 48-51. [5] T. HAMADA, M. KADOWAKI, Y. NAKAMURA, N. AWATA, Determination of the antiallergic agent KB-2413 in plasma by means of capillary gas chromatography with a nitrogensensitive detector, Chem. Pharm. Bull. 34(3) (1986) 1168-1171. [6] Y. Tian, Z. Zhang, J. Liang, W. Li, Y. Chen, High-Performance Liquid ChromatographyElectrospray Ionization-Mass Spectrometric Determination of Emedastine Difumarate in Human

of

Plasma and Its Pharmacokinetics, J. Chromatogr. Sci. 45(3) (2007) 158-164.

ro

[7] B. Jansen, U. Graselli, S. Dallinger, B. Kiss, V. Wacheck, H. Schlagbauer-Wadl, A. Assandri, M. Müller, Pharmacokinetics and pharmacodynamics of the novel H 1-receptor antagonist

-p

emedastine in healthy volunteers, Eur. J. Clin. Pharmacol. 55(11-12) (2000) 837-841. [8] U. Yogeswaran, S.-M. Chen, A review on the electrochemical sensors and biosensors

re

composed of nanowires as sensing material, Sensors 8(1) (2008) 290-313.

& Sons 2008.

lP

[9] R.C. Alkire, Y. Gogotsi, P. Simon, Nanostructured materials in electrochemistry, John Wiley

[10] S. Viswanathan, P. Manisankar, Nanomaterials for electrochemical sensing and

ur na

decontamination of pesticides, J. Nanosci. Nanotechno. 15(9) (2015) 6914-6923. [11] S. Kurbanoglu, S.A. Ozkan, Electrochemical carbon based nanosensors: a promising tool in pharmaceutical and biomedical analysis, J. Pharmaceut. Biomed. 147 (2018) 439-457. [12] Y. Cheng, H. Li, C. Fang, L. Ai, J. Chen, J. Su, Q. Zhang, Q. Fu, Facile synthesis of reduced

Jo

graphene oxide/silver nanoparticles composites and their application for detecting heavy metal ions, J. Alloy. Compd. 787 (2019) 683-693. [13] S. Yousefi, M. Saraji, Developing a fluorometric aptasensor based on carbon quantum dots and silver nanoparticles for the detection of adenosine, Microchem. J. 148 (2019) 169-176.

15

[14] A. Shams, A. Yari, A new sensor consisting of Ag-MWCNT nanocomposite as the sensing element for electrochemical determination of Epirubicin, Sensor. Actuat B: Chem. 286 (2019) 131-138. [15] A. Lasia, Electrochemical impedance spectroscopy and its applications, Modern aspects of electrochemistry, Springer 2002, pp. 143-248. [16] Z. He, F. Mansfeld, Exploring the use of electrochemical impedance spectroscopy (EIS) in

of

microbial fuel cell studies, Energ. Environ. Sci. 2(2) (2009) 215-219. [17] S. Kumar-Krishnan, E. Prokhorov, M. Hernández-Iturriaga, J.D. Mota-Morales, M. Vázquez-

ro

Lepe, Y. Kovalenko, I.C. Sanchez, G. Luna-Bárcenas, Chitosan/silver nanocomposites: Synergistic antibacterial action of silver nanoparticles and silver ions, Eur. Polym. J. 67 (2015)

-p

242-251.

[18] M.H. Esfe, A. Naderi, M. Akbari, M. Afrand, A. Karimipour, Evaluation of thermal

re

conductivity of COOH-functionalized MWCNTs/water via temperature and solid volume fraction by using experimental data and ANN methods, J. Therm. Anal. Calorim. 121(3) (2015) 1273-1278.

lP

[19] R.N. Goyal, N. Bachheti, A. Tyagi, A.K. Pandey, Differential pulse voltammetric determination of methylprednisolone in pharmaceuticals and human biological fluids, Anal. Chim.

ur na

Acta 605(1) (2007) 34-40.

[20] G. Ozcelikay, S. Kurbanoglu, B. Bozal-Palabiyik, B. Uslu, S.A. Ozkan, MWCNT/CdSe quantum dot modified glassy carbon electrode for the determination of clopidogrel bisulfate in tablet dosage form and serum samples, J. Electroanal. Chem. 827 (2018) 51-57. [21] H. Imanzadeh, N.K. Bakirhan, B. Habibi, S.A. Ozkan, Investigation of Electrochemical

Jo

Oxidation Mechanism, Thermodynamic Parameters and Sensor Design for Analgesic and Relaxant Drug: Phenyramidol in Aqueous Medium by NH2fMWCNT, J. Electrochem. Soc. 166(13) (2019) B1209-B1216. [22] H. Imanzadeh, N.K. Bakirhan, B. Habibi, S.A. Ozkan, Magnetic Nanosensor Design and Assay of an Anti-Tuberculosis Drug, J. Electrochem. Soc. 166(12) (2019) B933-B941.

16

of

ro

-p

re

lP

ur na

Jo Figures

17

O

O N

2e

N

H

N

N

N

N

N

N

H O

O

O H

N

N

N

H

N + H2O

N

C5H10O2

N

N

N

N

N

of

N

N

Jo

ur na

lP

re

-p

ro

Scheme 1. Possible oxidation pathway of EDD in aqueous solution.

18

9000

1600

A

1400

8000

1200

-Z''(Ω)

1000

7000

800 600

6000 400

-Z''(Ω)

200

5000 0 0

500

1000Z'(Ω)1500

2000

2500

of

4000

ro

3000

1000

-p

Bare GCE Ag NPs/GCE MWCNT/GCE .COOH-fMWCNT/GCE .COOH-fMWCNT/AgNPs/GCE

2000

0 0

2000

4000

6000 Z'(Ω)

10000

re

350

8000

B

lP

250

ur na

150

I(µA)

50

-50

Jo

-150

-250

COOH-fMWCNT/AgNPs/GCE COOH-fMWCNT/GCE MWCNT/GCE AgNPs/GCE Bare GCE

-350

-0.6

-0.3

0

0.3 E/(V) vs. Ag/Ag Cl

0.6

0.9

Fig.1. (A) Nyquist diagram (Z″ versus Z′) for the EIS measurements and (B) CVs of bare GCE, AgNPs/GCE, MWCNT/GCE, COOH-fMWCNT/GCE and COOH-fMWCNT/AgNPs/GCE in 5 mM K3Fe (CN) 6/K4Fe (CN) 6. 19

of ro -p re lP ur na Jo

Fig. 2. (A) SEM images of bare GCE; (B) AgNPs/GCE; (C) MWCNT/GCE; (D) COOH-fMWCNT/GCE; (E) COOH-fMWCNT/AgNPs/GCE; (F) EDX spectra of COOH- fMWCNT/AgNPs/GCE.

20

800 110

700

A

A

90 70

I(µA)

600 500

50 30 10

400 0

0.3

0.6 0.9 1.2 E/(V) vs. Ag/Ag Cl

300

1.5

1.8

- - COOH-fMWCNT/AgNPs/GCE – Bare GCE

200

of

I(µA)

-10

ro

100 0

-p

-100

-200 0.2

0.4

0.6

B

lP

20

COOH-fMWCNT/AgNPs/GCE COOH-fMWCNT/GCE

17

MWCNT/GCE

1.4

20

5

Jo

2

15 10

ur na

8

1.8

25

5

Bare GCE

11

1.6

30

AgNPs/GCE

14

I(µA)

1.2

Ip(µA)

26 23

0.8 1 E/(V) vs. Ag/Ag Cl

re

0

0

-1 -4

0.3

0.5

0.7

0.9 1.1 E/(V) vs. Ag/Ag Cl

1.3

1.5

1.7

Fig.3. (A) Cyclic voltammograms of bare CGE and COOH-fMWCNT/AgNPs/GCE (B) DP voltammograms at bare CGE, AgNPs/GCE, MWCNT/GCE, COOH-fMWCNT/GCE and COOHfMWCNT/AgNPs/GCE of 0.1mM EDD in 0.1M PB solution pH 2.0; the inset shows plots of Ip for the different electrocatalysts. 21

800

43

1.4

B

1.4

A

1.2

1.2

38

700

33

1

Ep(V)

Ep(V)

1

0.8 0.6

r = 0.9970

0.2

0.2

0

0 1

2

3

4

5

6

7

8

9

pH

pH 1

0

500

I(µA)

5

6

7

8

9

pH 3

pH 4

300

pH 5

pH 5

ro

I(µA)

4

pH 2

400

pH 4

pH 6

13

3

pH 1

pH 3

18

2

pH

pH 2

23

1

of

0

28

r = 0.9913

0.6

0.4

600

0.4

0.8

pH 6

200

pH 7

pH 7

-p

pH 8 100

3

0

-2

pH 8

re

8

-100

0.5

0.7 0.9 E/(V) vs. Ag/Ag Cl

1.1

1.3

lP

0.3

-0.1

0.1

0.3

0.5

0.7 0.9 E/(V) vs. Ag/Ag Cl

1.1

1.3

1.5

Jo

ur na

Fig.4. (A) DPVs & (B) CVs of 0.1mM EDD in different pH values (1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0) at COOH-fMWCNT/AgNPs/GCE; the inset show plots of Ep vs. pH.

22

1.7

1200

1400

A

1200

B

1000

1000

Emedastine Difumarate

800

Emedastine Difumarate

Benzydamine HCl

Benzydamine HCl

Diazepam

I(µA)

I(µA)

800 600

Diazepam

600 400

400 200 0

0

0

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 E/(V) vs. Ag/Ag Cl

0

2

ro

-200

-200

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 E/(V) vs. Ag/Ag Cl

Emedastine Difumarate

23

23

Benzydamine HCl

20

20

Diazepam

17

26

C

17

D

2

Emedastine Difumarate Benzydamine HCl Diazepam

re

14

-p

26

29

14

I(µA)

I(µA)

of

200

11 5 2 -1

0.65

0.85 1.05 1.25 E/(V) vs. Ag/Ag Cl

1.45

ur na

-4 0.45

lP

8

1.65

11

8 5 2

-1 -4

0.3

0.5

0.7 0.9 E/(V) vs. Ag/Ag Cl

1.1

1.3

Jo

Fig.5. CV and DPV curves of 0.1mM EDD at COOH-fMWCNT/AgNPs/GCE (A&C) in 0.1M PB solution of pH 2.0; (B&D) in 0.1M PB solution of pH 6.

23

80

70

B

A

30

50

40

20

30

10

20

0

10 0

-10

0.8

0.9

1

1.1

1.2

1.3

0

1.4

E/(V) vs. Ag/Ag Cl

10 20 30 40 50 60 70 80 90 100 Time(s)

70

-p

59

D

C

60

49

50

-0.1 V 0V

40

0.2 V

29

0.3 V

19

9

-1

0.9

1 1.1 1.2 E/(V) vs. Ag/Ag Cl

30 20 10 0

1.3

1.4

-0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 Deposition Potanitial (V)

Jo

0.8

ur na

0.4 V

lP

0.1 V

Ip(µA)

39

E= 0.3 V

re

-0.2 V

I(µA)

ro

I(µA)

40

60

Ip(µA)

50

Time= 45 s

70

5s 10 s 15 s 30 s 45 s 60 s 75 s 90 s

of

60

Fig.6. Effect of accumulation time (A & B) and accumulation potential (C & D) on peak current of 0.1mM EDD using COOH-fMWCNT/AgNPs/GCE in 0.1 M PB (pH 2.0) solution.

24

70

60

of

50

ro

30

-p

I(µA)

40

re

20

lP

10

ur na

0

-10

Jo

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

E/(V) vs. Ag/Ag Cl

Fig.7. DP (green line) and AdSDP (blue line) voltammograms of COOH-fMWCNT/AgNPs/GCE in 0.1M PB solution of pH 2.0 containing 0.1mM EDD (optimized experimental conditions for AdSDPV were Eacc= 0.3 V, tacc= 45 s).

25

66

56

100 µM

36

0.1 µM

of

I(µA)

46

26

6

-4 0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

re

0.7

-p

ro

16

E/(V) vs. Ag/Ag Cl

Jo

ur na

lP

Fig.8. AdSDPV responses in various concentrations of EDD at COOH-fMWCNT/AgNPs/GCE in 0.1 M PB (pH 2.0) solution under optimized conditions

26

35 30 25

Ip (µA)

EDD

20

(1/1) (1/10) (1/100)

15

of

(1/1000)

5 0 B

C

D

E

F

G

-p

A

ro

10

H

Jo

ur na

lP

re

Fig 9. Bar graphs peak currents of 5×10-5 M EDD at 0.1 M PB solution (pH 2.0) with COOHfMWCNT/AgNPs/GCE in presence at 1:1, 1:10, 1:100, 1:1000 ratios (analyte: interferent); pure drug EDD (A), EDD + dopamine (B), EDD + ascorbic acid (C), EDD + Ca 2+ (D), EDD + K+ (E), EDD + Na+ (F), EDD + Cl− (G) EDD + NO− 3 (H) in optimal conditions.

27

Tables Table 1. Parameters calculated from the Nyquist plots and cyclic voltammograms. Rct/ 103 (Ω)

Active surface area (cm2)

Bare GCE

397

2.93

0.037

AgNPs/GCE

376

2.32

0.051

MWCNT/GCE

313

0.520

0.071

COOH-fMWCNT/GCE

345

0.184

0.090

COOH-fMWCNT/AgNPs/GCE

293

0.023

of

Re /(Ω)

lP

re

-p

ro

0.113

Table 2. Chemical structures of model compounds. Benzydamine

Diazepam

Jo

ur na

Emedastine

28

Table 3. Regression data of the calibration line for voltammetric determination of EDD by AdSDPV. Standard solution 1.099

Linearity Range (M)

1×10−7–1×10−4

Slope (µAM-1)

0.593

Intercept (µA)

0.113

Determination coefficient

0.9994

LOD (M)

5.25×10-9

ro

of

Measured Potential (V)

1.5×10-8

LOQ (M) With-in days of peak current (RSD %)*

-p

0.86

With-in days of peak potential (RSD %)*

Between days of peak potential (RSD %)*

0.97

lP

Obtained from five measurements.

1.85

Jo

ur na

*

re

Between days of peak current (RSD %)*

0.36

29

Table 4. Comparison of proposed sensor with the reported sensor for EDD.

Electrode

Detection Linearity

LOD (M)

LOQ (M)

range (M)

GCE

DPV

8×10−6–2×10−4

1.01×10−7

3.36×10−7

CPE

DPV

1×10−6–8×10−5

1.23×10−7

4.11×10−7

COOH-fMWCNT/AgNPs/GCE

AdSDPV

1×10−7–1×10−4

5.25×10−9

1.59×10−8

[1]

This work

Jo

ur na

lP

re

-p

ro

of

method

Reference

30

Table 5. The results for the determination of EDD in eye drop form.

AdSDPV

5

Amount found (mg)a

4.89

RSD %

0.955

Bias %

2.2

Added (mg)

2.5

Found (mg)a

2.49

Average recovered (%)

99.6

RSD % of recovery

1.68 0.4

Obtained from five measurements.

Jo

ur na

lP

re

a

-p

Bias %

ro

Labeled claim (mg)

of

Eye drop

31