Electrochemical sensitive determination of isoprenaline at β-cyclodextrin functionalized graphene oxide and electrochemically generated acid yellow 9 polymer modified electrode

Accepted Manuscript Electrochemical sensitive determination of isoprenaline at βcyclodextrin functionalized graphene oxide and electrochemically generated acid yellow 9 polymer modified electrode

Venkata Narayana Palakollu, Tirivashe E. Chiwunze, Atal A.S. Gill, Neeta Thapliyal, Shital M. Maru, Rajshekhar Karpoormath PII: DOI: Reference:

S0167-7322(17)33850-3 doi:10.1016/j.molliq.2017.10.092 MOLLIQ 8054

To appear in:

Journal of Molecular Liquids

Received date: Revised date: Accepted date:

23 August 2017 18 October 2017 19 October 2017

Please cite this article as: Venkata Narayana Palakollu, Tirivashe E. Chiwunze, Atal A.S. Gill, Neeta Thapliyal, Shital M. Maru, Rajshekhar Karpoormath , Electrochemical sensitive determination of isoprenaline at β-cyclodextrin functionalized graphene oxide and electrochemically generated acid yellow 9 polymer modified electrode. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Molliq(2017), doi:10.1016/j.molliq.2017.10.092

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ACCEPTED MANUSCRIPT Electrochemical sensitive determination of isoprenaline at β-cyclodextrin functionalized graphene oxide and electrochemically generated acid yellow 9 polymer modified electrode Venkata Narayana Palakollua, Tirivashe E. Chiwunzea, Atal A. S. Gilla, Neeta Thapliyala,

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Shital M. Marub, Rajshekhar Karpoormatha* Department of Pharmaceutical Chemistry, College of Health Sciences, University of

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KwaZulu-Natal (Westville Campus), Durban-4000, South Africa. Department of Pharmaceutics and Pharmacy Practice, School of Pharmacy, College of

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Health Sciences, University of Nairobi, P.O. Box 19676-00202, Nairobi, Kenya. *Corresponding Author: Rajshekhar Karpoormath, E-mail address: [email protected]

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Fax: +27 31 260 7792; Tel: +27 31 260 7179

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Abstract

A novel electrochemical sensor for isoprenaline (IP) comprising a β-Cyclodextrin (β-

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CD) functionalized graphene oxide (GO) and an electrochemically generated acid yellow 9

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polymer as a composite material modified glassy carbon electrode (GCE) has been developed. The composite material was characterized using infrared spectroscopy and

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scanning electron microscopy. The functionalization of GO with β-CD was scrutinized by varying the content of the β-CD material. Interestingly, the synergistic electrocatalytic

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activity was examined at different β-CD loadings functionalized GO in the sensitivity for the detection of IP. The electrochemical characterization of the proposed sensor (β-CDGO/PAY/GCE) was performed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The developed composite based electrode displayed superior sensitivity towards IP over that of the other modified electrodes. The limit of detection at βCD-GO/PAY/GCE was found to be 3.3×10-8 M. Moreover, the detection potential of IP was notably lower at the proposed composite based sensor. The β-CD-GO/PAY/GCE was also

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ACCEPTED MANUSCRIPT successfully applied for simultaneous resolution of IP in the presence of endogenous interfering biomolecule such as uric acid. The recovery results attained for real samples recommends practical utility of the proposed composite electrode an effective and reliable electrochemical sensor for quantification of IP. The sensor is highly stable, reproducible and

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signifies a feasible platform for the analysis of IP. Keywords: Isoprenaline, Uric acid, Electrochemical impedance spectroscopy, Graphene

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oxide, β-Cyclodextrin, Acid yellow 9.

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ACCEPTED MANUSCRIPT 1 Introduction Isoprenaline or isoproterenol (IP) (IUPAC: 3, 4-dihydroxy-α-[(isopropylamino) methyl] benzyl alcohol) is the synthetic N-isopropyl derivative of norepinephrine. It is one of the catecholamine drugs and was approved as a potent non-selective β-adrenergic agonist and trace-amine associated receptor 1 (TAAR1) agonist by the Food and Drug Administration

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(FDA). An IP is commonly used for the treatment of bradycardia, heart block, styptic and

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Hence, quantification of this compound is very essential.

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bronchial asthma [1,2]. The excess use of the drug may cause arrhythmias and heart failure.

One of the most significant biomolecules that existing in the physiological fluids is

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uric acid (2, 6, 8,-trihydroxypurine, UA), which is a product of purine metabolism in the human body. It has been observed that abnormal levels of UA in the body is the symptom of

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several diseases like gout, Lesch–Nyan syndrome, obesity, cardiovascular and kidney damage [3–5]. Thus, it has become essential to develop a simple, precise and reliable

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analytical tool for the determination of UA.

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Recently, many techniques have been developed for determination of IP and their metabolites in biological fluids such as gas-liquid chromatography (GLC)[6], liquid

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chromatography-mass spectrometry (LC-MS) [7], high performance liquid chromatography (HPLC) [8], selective separation using bismuth silicate ion-exchanger [9] and

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spectrophotometry [10]. These methods usually require complex pre-treatment steps, expensive instrumentations and are time-consuming. In contrast, electrochemical techniques offer an economical and simple analytical tool for the sensitive and selective for rapid quantification of IP. Moreover, the sensors based on electrochemical methods have widely been used for the detection of biomolecules due to their capability of being incorporated into a portable, robust and miniaturized devices for targeted applications in diagnostic and clinical fields as biomarkers of several diseases such as Parkinson´s and Alzheimer’s diseases. 3

ACCEPTED MANUSCRIPT However, there are several limitations associated with conventional electrodes for determination of catecholamines. The critical issue connected with the quantification of catecholamine drugs is the passivation of the electrode’s surface due to the polymerization of the oxidation products of catecholamines and producing a melanin-type polymer film onto the surface of electrode [11]. This phenomenon leads to electrode poisoning or fouling,

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resulting in a poor selectivity and sensitivity.

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Graphene oxide (GO) is a precursor of graphene and is typically formed through the

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oxidation of graphite by chemical method [12]. Thus, various oxygen containing functional groups have been identified as typically in the form of hydroxyl and epoxy groups on the

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basal plane, with slighter amounts of carboxy, carbonyl, phenol and lactone at the edges of the sheet in GO. The presence of oxygen functionalities in GO strongly affects its

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mechanical, electronic and electrochemical properties. The presence of these functional groups can offer potential advantages for abundant applications.

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β-Cyclodextrin (β-CD) is a kind of cyclic oligosaccharides composed of seven

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glucose units. The β-CD is toroidal in shape with a hydrophobic inner cavity and a hydrophilic exterior. Due to the shape selectivity of the β-CD cavity and the hydrophobic

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interaction between guests and β-CD host, β-CD can selectively bind to various organic, inorganic and biological guest molecules in their cavities to form stable host-guest inclusion

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complexes. Further, the hydrophilic exterior can make β-CD as an efficient molecule to increase the dispersibility of various functional materials in solvents. Moreover, β-CDs are water-soluble, environmentally friendly, and can increase the solubility and stability of functional materials. However, GO disperses poorly in water and easily gets aggregated, causing a decrease in the surface area of GO itself and limiting its applicability [13]. Hence, attention has been paid towards dispersing GO with other materials for fabricating hybrids or composites that have good dispersibility properties. By the hydrophobic nature of graphene

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ACCEPTED MANUSCRIPT and related materials, which get well dispersion in β-CD and forms a composite as a suspension material. The composite may instantaneously hold individual properties of two materials such as good conductivity and large surface area of GO as well as supramolecular recognition and enrichment capability of β-CD, which provides good opportunities for applications in the areas of sensors, electrocatalysis and electronics etc.

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Recently, conducting polymers have drawn excessive potential to a wide range of

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applications including sensors, biosensors and transistors due to good electronic conductivity.

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The most commonly using procedure for deposition of conducting polymer films onto the electrode surfaces is electropolymerization. Electropolymerization is a flexible and

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resourceful approach due to its advantages in the detection of biomolecules such as easy preparation of uniform films, homogeneity in electrochemical deposition, inexpensive, simple

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procedure, well-controlled thickness on the electrode surfaces, high selectivity, good sensitivity, strong adherence to the active surface of the electrode and stability of the polymer

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layer onto the surface of electrode [14, 15]. Moreover, these conducting polymers can be

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considered as very effective substrates for bisomaterial immobilization [14, 15]. Hence, herein, acid yellow 9 (AY, IUPAC name: 4-Amino-1, 1′-azobenzene-3, 4′-disulfonic acid)

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dye was used as a monomer for the preparation of conducting acid yellow 9 polymer (PAY). Moreover, the integration of β-CD functionalized GO (abbreviated as β-CD-GO) with

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conducting polymers is anticipated to significantly expand the electrocatalytic activity of the composite. Therefore, the combination of β-CD-GO and PAY (abbreviated as β-CDGO/PAY) was considered as a good candidate for sensors applications. Herein, we report the development of aqueous suspension of GO using various amounts of β-CD as dispersant through a simple wet-chemical strategy. The prepared aqueous suspension of GO (β-CD-GO) was effectively used to drop-cast onto the PAY modified glassy carbon electrode (GCE). The simply and facilely constructed composite

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ACCEPTED MANUSCRIPT modified GCE (β-CD-GO/PAY/GCE) was successfully utilized for quantification of IP. To date, no report has been made on the use of β-CD-GO/PAY/GCE for the determination of IP. The β-CD-GO/PAY/GCE displayed good performance towards the quantification of IP. Additionally, it exhibited good selectivity, sensitivity, stability and reproducibility. Moreover, the analytical applicability of the sensor was successfully evaluated in real samples and good

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recoveries were attained.

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2 Experimental

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2.1 Apparatus and chemicals

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All electrochemical experiments were carried out using CHI 660E electrochemical workstation (CH Instruments, USA). The measurements were executed in a single

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compartment cell using a three-electrode system, where a GCE or a modified GCE as the working electrode, and an Ag/AgCl (3.0 M NaCl) electrode and a platinum wire as the

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reference and axillary electrodes, respectively. All potentials reported were versus the

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Ag/AgCl (3.0 M NaCl) electrode. Extech Instruments-pH 60 pH pen (FLIR Systems Inc., USA) was used to measure the pH of buffer solutions. Field emission-scanning electron

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microscope (FE-SEM)-ZEISS® FE-SEM Ultra Plus (Germany) was used to study the surface morphology of developed composites. Fourier transform infrared (FT-IR) spectra were

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recorded using a Bruker® Alpha-P ATR–FT-IR (Germany). Isoprenaline hydrochloride (Sigma, China), uric acid (≥99%, Sigma, USA), Acid Yellow 9 (95%)

graphene oxide powder (15-20 sheets, Sigma-Aldrich, USA), β-

Cyclodextrin (≥97%, Sigma, USA), potassium ferrocyanide (≥98.5%, Sigma-Aldrich, China), potassium ferricyanide (97.0%, Saarchem, South Africa), sodium chloride (≥99.5%, SigmaAldrich, USA), glucose (99%, ACE, South Africa), L-lactose (ACS grade, Sigma-Aldrich, Germany), sucrose (≥99%, Merck, South Africa), potassium chloride (99.0-100.5%,

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ACCEPTED MANUSCRIPT Merck,South Africa), magnesium sulfate (RLFCI, India) were used as received without additional pretreatment. Phosphate buffer solution (PBS) was used as a supporting electrolyte for this study. Sodium dihydrogen orthophosphate dihydrate (98.0-100.5%, Merck, South Africa) and sodium phosphate dibasic dihydrate (98.5-101%, Sigma-Aldrich, Germany) were used to prepare 0.1 M PBS of appropriate pH. Double distilled water used throughout this

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study was purified using Millipore system.

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2.2 Preparation of PAY/GCE

Prior to the modification, the GCE (3 mm diameter) was mechanically polished with

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0.3 and 0.05 micron of γ-alumina powder to get a mirror-like finish using smoothing pads,

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then thoroughly rinsed with distilled water and was allowed to dry at room temperature (22±3oC). The pre-treated GCE was then permitted for electrochemical polymerization by

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potential cycling at a range of -0.5 to 2.0 V in 0.1 M PBS (pH 7.0) containing 0.25 M AY solution at a scan rate of 0.05 V s-1. From cyclic voltammetric measurements (Fig.1), we can

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confirm that a thin polymer layer of AY was formed.

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2.3 Preparation of β-CD-GO/PAY composite modified GCE For the preparation of β-CD-GO/PAY composite, different amounts of β-CD (1.0,

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2.0, 4.0 and 6.0 mg) were added to 200 μL of GO (1 mg mL-1) aqueous solution and the mixture was sonicated for 15 min. at high frequency in a ultra-sonicator. The resultant

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composites were used for the modification of PAY/GCE. About 6 μL of β-CD/GO dispersion was drop cast onto the PAY/GCE and dried under IR lamp. The developed β-CDGO/PAY/GCE was considered as a resultant electrode. The step by step preparation mechanism of β-CD-GO/PAY/GCE is shown in Scheme 2. The resultant electrode was used for performing all the electrochemical parameters. The β-CD-GO/GCE was prepared by similar procedure on bare GCE surface. The GO modified GCE (GO/GCE) was also prepared without addition of β-CD to GO onto GCE.

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ACCEPTED MANUSCRIPT 2.4 Preparation of injection and urine sample An ampoule containing IP (Isolin®, the specified content of IP 2.0 mg mL−1) was procured from India (Samarth Life Sciences Pvt. Ltd., India). The necessary amount of sample was dissolved in the 0.1 M PBS (7.0) to prepare a stock solution. The specified content of IP in the injection sample was then cross-checked by means of DPV response of

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proposed composite based electrode. For the biological matrix, the urine sample of a healthy

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personnel was taken and diluted 100 times with 0.1 M PBS (pH 7.0). The real sample

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examination was carried out in human urine in order to evaluate the practical applicability of proposed sensor.

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3 Results and discussion

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3.1 Preparation of PAY film coated GCE and its properties

The electropolymerization of AY was carried out onto the surface of pre-treated GCE as

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mentioned in section 2.2. Fig.1 displays ten successive cyclic voltammograms of polymer

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growth onto the surface of GCE. During the process of electropolymerization, the scanning cycle started at -0.5 V and an anodic peak appeared at 1.049 V for initial scanning. The

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appeared peak corresponds to oxidation of amine group of AY [16]. Subsequently, upon continuous cycling, one cathodic peak (0.228 V) and another anodic peak (0.283 V) were

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observed indicating the growth of the polymer layer onto the surface of GCE. A thin and transparent adherent polymer film was observed onto the surface of GCE. The possible electrochemical polymerization mechanism of AY is based on nitrogen-nitrogen coupling reactions (head-to-head coupling). In the nitrogen-nitrogen coupling mechanism, the first step is oxidation of the amine group (peak a1) forming a free radical at positive potential value (1.049 V), subsequent immediate combining of the two formed free radicals to give a molecule of hydrazobenzene sulfonic acid (peak c1) and the structure of hydrazobenzene

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ACCEPTED MANUSCRIPT sulfonic acid was comparatively stable. Later, hydrazobenzene sulfonic acid and azobenzene sulfonic acid became a pair of redox couple with peaks c1 and a2 corresponding to their reduction and oxidation processes respectively. The similar kind of mechanism was reported for some azo compounds. The detailed electrochemical polymerization mechanism can be

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seen in Scheme 1.

3.2 Characterization of electrodes

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The successful preparation of the composite β-CD-GO/PAY was verified by FT-IR. The

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FT-IR spectra of (a) PAY, (b) GO, (c) β-CD and (d) β-CD-GO/PAY are displayed in Fig.S1. As depicted in Fig.S1(A), the broad stretching band observed at about 2950 to 3600 cm-1

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corresponds to amine group and water molecules in PAY. The bending vibration of PAY at

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1675 cm-1 corresponds to N-H characteristics and the absorption bands at 1642 (C=C), 1255 (C-N), 1099 (aromatic C–H bending) and 2310 (C-S) cm−1 also confirm formation of PAY.

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The layered GO material contains carboxy, carbonyl and phenol at both the edges and basal

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plane. As displayed in Fig.S1(B), the characteristic peak at 1650 cm-1 corresponding to C=O stretching vibrations of carbonyl and carboxylic groups. The peak at 1610 cm-1 corresponds to the aromatic stretching vibration of C=C group. Moreover, a peak was also observed at

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1110 cm-1 and which indicates presence of epoxy and C-O alkoxy vibrations [17]. Fig.S1(C)

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displays the IR spectra of β-CD. A broad peak appearing at around 3350 cm-1 attributes to OH stretching vibration of hydroxyl groups. Another peak appeared at 1639 cm-1 that attributes to C=O stretching vibrations. In addition, the peaks observed at 1159 and 1025 cm-1 attribute to C-O-C groups and C-OH groups, respectively [13]. It is noted that due to more oxygen functionalities onto the GO surface, it supports the initiation process for the functionalization of -CD with GO by electrostatic attraction [18]. As can be seen in Fig. S1(D), the observed absorption peaks of β-CD-GO/PAY composite are consistent with the absorption bands of PAY, GO and β-CD, which confirms that β-CD-GO/PAY composite was prepared 9

ACCEPTED MANUSCRIPT successfully. Fig.2 displays the morphology of PAY, GO and β-CD-GO/PAY composite. A layer of polymer matrix morphology can be seen clearly in Fig.2A and B. It confirms that the polymer layer of PAY was successfully prepared by the electrochemical polymerization process. Fig.2C and D show randomly distributed sheets of GO. However, from the images of β-CD-GO/PAY, the brick-like structure appeared onto the GO sheets and polymer layer in Fig. 2E

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and Fig. 2F was due the β-CD. The -CD was immobilized onto the GO surface by the

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interaction of the functional groups on GO and -CD.

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3.3 Electrochemical characterization of modified electrodes

The charge transfer resistance (Rct) of the various modified and unmodified electrodes

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was evaluated by means of EIS technique. The experiment was performed in 0.1 M KCl containing 2.5 mM [Fe(CN)6]3-/4- as a redox probe for various electrodes over the frequency

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of 1 to 105 Hz with the initial potential of 0.296 V. The resultant Nyquist plots for the unmodified GCE, PAY/GCE, GO/GCE, GO/PAY/GCE, β-CD-GO/GCE, and β-CD-

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GO/PAY/GCE are shown in Fig.3(A). Usually, Rct values are obtained from Randles

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equivalent circuit and that can be interpreted as interface ohmic resistance in the EIS technique. It is generally used to characterize and assess the material which is adsorbed onto

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the surface of the electrode, since it expresses the resistance of a charge transfer between the electrode and the solution of redox probe [19]. The charge transfer resistance (Rct), warburg

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impedance (W), solution resistance (Rs), and double layer capacitance (Cdl) are the elements of Randles equivalent circuit. This parallel connection of Rct - Cdl in Randles equivalent circuit causes the semicircle in the Nyquist plot and the Rct value is directly measured from the diameter of the semicircle.The Rct value significantly varies based on the modification of the electrode surfaces. Rct and Cdl are connected with the redox behaviour of [Fe(CN)6]3-/4- at the active surface area of the electrodes. As depicted in Fig.3(A), a semicircle with large diameter was witnessed with Rct of 872 Ω at unmodified GCE, implying poor interfacial 10

ACCEPTED MANUSCRIPT electron transfer at the surface of unmodified GCE in redox probe solution. After GCE modification with PAY, the diameter of semicircle got diminished to Rct of 464 Ω. This was due to the good conductivity of the AY polymer. The Rct value at the surface of GO/GCE decreased observably (146 Ω), indicating that the introduction of GO significantly enabled electron transfer. Further, the Rct value at the surface of β-CD-GO/GCE decreased to 109.82

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Ω, demonstrating that β-CD could significantly be influenced in facilitating better interfacial

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electron transfer rate. At GO/PAY/GCE surface, the Rct value was 17.88 Ω, which is low and was due to the combined effect of GO and PAY. However, the charge transfer resistance

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remarkably decreased to 7.828 Ω at the surface of β-CD-GO/PAY/GCE, which can be

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attributed to the synergistic effect of both PAY and β-CD-GO materials. The results confirm that the GCE was successfully modified with β-CD-GO/PAY composite.

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The electrochemical active surface area of the β-CD-GO/PAY/GCE was calculated from the cyclic voltammograms of 2.5 mM K3[Fe(CN)6] in 0.1 M KCl solution using

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Randles-Sevcik eq.

ip= (2.69 x 105) AD1/2 n3/21/2 C0

where ip is the peak current (A), n is the number of electrons that participated in the

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reaction (n=1 for K3[Fe(CN)6]), A is the electrochemical active surface area of the electrode

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(cm2), D is diffusion coefficient of the molecule in the bulk solution (7.60×10-6 cm2 s-1 for K3[Fe(CN)6]), υ is the scan rate (V s-1), and C0 is the concentration (mol. cm-3). From the slope of the plot of ip vs ν1/2 (Fig.3(B)), the electrochemical active surface area of the β-CDGO/PAY/GCE was calculated to be 0.0956 cm2. This evidence upholds that the β-CDGO/PAY/GCE has the good electrochemical active surface area. 3.4 Optimization of electropolymerization cycles

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ACCEPTED MANUSCRIPT In the electrochemical polymerization process, the electrocatalytic activity of the polymer depends on the number of cycles. In order to optimize the number of cyclic voltammogram cycles, the electropolymerization of AY was carried out onto the surface of pre-treated GCE with 5, 10, 15 and 20 consecutive cycles. As can be seen in Fig.4(A), the maximum peak current for IP oxidation was observed for 10 uninterrupted cycles of cyclic

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voltammograms, demonstrating that the polymer film was effectively prepared onto the GCE

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surface. The optimal 10 successive cycles displayed the best results in terms of peak current. Therefore, 10 cycles were chosen for electropolymerization of AY.

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3.5 Effect of β-CD in a composite of β-CD/GO towards oxidation of IP

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To estimate the influence of -CD the electrochemical response of β-CD/GO/GCE was observed in the 0.1M PBS (pH 7.0) having 0.2 mM IP at a scan rate of 0.05 V s-1. From

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Fig.4(B) reveals that the electrocatalytic oxidation of IP changes for various amounts of CD in a composite of β-CD-GO ranging from 1.0 to 6.0 mg. The amounts of -CD did not

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show any linearity with the electrocatalytic ability towards IP detection. This is due to the

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activity of -CD being considerably influenced by three key factors which are: (a) specific guest-host interaction, (b) stoichiometric ratio for the insertion complex formation and (c)

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spatial arrangement on the surface confinement [20]. From the electrocatalytic activity of IP

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at various amounts of -CD in β-CD/GO composite modified GCEs, it is expected that the lowest amount (1 mg) of -CD lack from the stoichiometric ratio for the inclusion complex formation with IP. On the other hand, the highest amount (6 mg) of -CD suffers from steric hindrance on the surface confinement. In the present study, the 1.0, 2.0, 4.0 and 6.0 mg of CD loaded GO composites were demonstrated towards the oxidation of IP. The comparative cyclic voltammetric results can be seen in Fig.4(B). The amount of -CD was optimized in terms of highest magnitude of the peak currents when CV was performed for 0.2 mM IP in

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ACCEPTED MANUSCRIPT 0.1 M PBS of pH 7.0. As can be seen in Fig.4(B), the 2 mg -CD loaded GO modified GCE manifested a good catalytic response for oxidation of IP. 3.6 Electrochemical behaviour of IP at different modified electrodes The electrochemical behaviour of IP was investigated at different modified electrodes in 0.2 mM IP in 0.1 M PBS of pH 7.0 at a scan rate of 0.05 V s-1 using cyclic voltammetry

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(CV) technique. From Fig.5, the bare GCE displayed poor current intensity of 14.61 µA at

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0.410 V with broad CV diagram which describes the lack of electrocatalytic characteristic

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with slow electrode kinetics (system ‘a’). After modification with PAY, a good oxidation peak was observed with an improved current intensity of 36.41 µA at a relatively low

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potential of 0.310 V (system ‘b’). This observation was due to the good electronic conductivity of conducting polymer of AY onto the GCE surface. Additionally, the PAY

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provided a homogeneous, stable and adherent polymeric film on the GCE. On the other hand, another better oxidation peak was observed with a relatively enhanced current intensity of

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47.11 µA whereas peak shifted to towards positive potential (0.342 V) when the GCE was

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modified with GO material (system ‘c’). This response is attributed to the good electron shuttling property of GO that enhances electron transfer rate at the interface of electrode

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surface and electrolyte. Further, GO material was functionalized with β-CD to improve the dispersion of GO in aqueous solution. At the β-CD functionalized GO/GCE, the current

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response was better and the oxidation peak shifted towards negative potential (0.323V) (system ‘d’). However, a notable improvement in the peak current of IP (79.80 µA) was observed when the GCE was modified with PAY and GO when compared to PAY/GCE, GO/GCE and β-CD-GO/GCE (system ‘e’). This performance describes that the unique combination of PAY (good electronic conductivity) and GO (excellent 2D structures and large surface area) makes good electrode material for the good sensitivity of IP. Interestingly, β-CD functionalized GO material was integrated with PAY to improve the selectivity of IP.

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ACCEPTED MANUSCRIPT Thereby, outstanding enhancement in the peak current and well-shaped oxidation peak of IP was observed when CV was executed at the GCE which was modified with PAY and β-CD functionalized GO (β-CD-GO/PAY/GCE) (system ‘f’). The result displayed considerably higher peak current with β-CD-GO/PAY/GCE compare to the rest of electrodes for the IP investigation. Additionally, β-CD also served as a dispersing agent for GO. This suggests that

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the composite of β-CD-GO integration with PAY modified GCE provides enhanced

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electrochemical activity and large surface area for analyzing IP at a physiological pH.

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Therefore, it is evident that β-CD played a significant role as a dispersant in the enhancement of surface area and electrocatalytic activity of GO in combination with PAY as an electrode

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surface modifying material. 3.7 Effect of scan rate

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The influence of potential scan rate at β-CD-GO/PAY/GCE for electrocatalytic oxidation of 0.2 mM IP in 0.1 M PBS (pH 7.0) was investigated by CV technique. A set of well-defined

. The oxidation peak potential shifts with increasing scan rates towards a more positive

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1

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anodic peaks for IP was observed at β-CD-GO/PAY/GCE from scan rate of 0.01 to 0.12 V s-

potential, approving the kinetic limitation of the electrochemical reaction. From the Fig.6, it

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is observed that a good linear relationship associated with the peak current of IP and a square root of scan rate (ν1/2). The corresponding linear regression equation can be signified as

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ip (μA) = (17.60998±0.60863) + (98.90894±2.07744)

R= 0.9978.

This result indicates that the charge transfer was under diffusion control at the surface of β-CD-GO/PAY/GCE. Moreover, with increasing scan rate, the oxidation peak potentials shifted towards the more positive potential window, confirming that the electrochemical reaction process was a kinetic limitation at higher scan rates. 3.8 Effect of solution pH

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ACCEPTED MANUSCRIPT The effect of solution pH in the range from 5.0 to 8.5 was carried out to examine the electrochemical behaviour of the IP at β-CD-GO/PAY/GCE. The differential pulse voltammograms were recorded in the range from pH 5.0 to 8.5 in 0.1 M PBS shown in Fig. 7(A). It was observed that both the oxidation peak current and the peak potential of 0.2 mM IP were affected by the pH of 0.1 M PBS at β-CD-GO/PAY/GCE. As depicted in Fig. 7(B),

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the peak potential of IP shifted towards negative potential with the increase in the pH of the

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supporting electrolyte. This behaviour indicates towards the direct participation of the protons

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in the oxidation step of IP. A good linear relationship was observed between Ep and pH of the supporting electrolyte. The linear eq. can be expressed as follows: R= 0.97702

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Ep (pH 5.0 – 8.5) = (0.72187±0.03471) - (0.06197±0.00472)pH

The resulted slope of pH vs Ep (0.06197 V pH-1) is very close to the Nernstian

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theoretical value of 0.059 V pH-1 (at 25 oC), indicating the involvement of an equal number of electrons and protons in the electrochemical oxidation of IP at β-CD-GO/PAY/GCE.

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Further, from Fig.3, the maximum current intensity was viewed at pH 7.0. Thus, pH 7.0 was

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considered as optimum pH in further study to get good sensitivity and selectivity for IP determination.

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3.9 Electrochemical determination of IP Differential pulse voltammetry (DPV) was chosen for the quantification of IP under

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optimal experimental conditions because the technique has lower detection limit and higher sensitivity and better resolution as compared to amperometric i-t curve technique. DPV is one of the most sensitive techniques for development of a simple and suitable method for determining the low levels detection of electrochemically active molecules. Under optimal experimental conditions, the electrochemical determination of IP in 0.1 M PBS (pH 7.0) was performed at β-CD-GO/PAY/GCE using DPV technique (Fig.8 (A)). It can be observed that there was no prominent peak appeared for the absence of IP. However, a sharp and well-

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ACCEPTED MANUSCRIPT defined peak appeared at 0.244 V represents the oxidation of IP when 1 µM of IP was added. The oxidation peak current of the IP was plotted against the concentration of IP and shown in Fig.8 (B). As displayed in Fig.8 (B), the peak current linearly increased upon increasing the concentration of IP from 0 to 52 μM. The observed linear regression equation can be expressed as follows R = 0.99885.

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ip (µA) = (-1.36582±0.26336) + (0.86705±0.01122)[IP] µM

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From the calibration plot, the limit of detection (S/N=3) was found to be 3.3×10-8 M, where ‘S’ is the standard deviation of the blank responses and ‘N’ is the slope of the

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calibration curve. In order to reveal the advantages of β-CD-GO/PAY/GCE, electroanalytical

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performances of IP quantification at β-CD-GO/PAY/GCE were compared with the previously reported modified electrodes for IP quantification and tabulated in Table 1. Table

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1 evidently reveals fairly comparable results with the earlier reported literature and enlightening the ability of the proposed sensor towards the quantification of IP. The good

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analytical activity of β-CD-GO/PAY/GCE is attributed to the outstanding performance by the

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synergistic property of conducting polymer and β-CD-GO. From the results, it is obvious that

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the proposed β-CD-GO/PAY/GCE is sensitive and reliable for low-level detection of IP.

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Table 1: Comparison of analytical parameters of various modified electrodes proposed for quantification of IP. Modifier 5ADB PMP-DNA

ZnONP-IL

Limit of detectiom

Electrode

pH

Technique

CPE

7.0

SWV

GCE

4.0

CV

1.6 ×10−7 M

CPE

5.0

SVW

0.09 µM

−7

2.0×10 M

Reference electrode

Ag/AgCl/ KCl (3.0 M) Ag/AgCl Ag/AgCl/ (KClsaturated)

Ref.

[21]

[22] [23]

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FCA CHF molybdenum (VI) complex

8.0

SVW

0.3 µM

SCE

CNTPE

5.0

DPV

2.0×10−7 M

Ag/AgCl/

CPE

6.0

CV

8.0×10-5 M

CNTPE

7.0

DPV

35.0 nM

DPV

0.0087 µM

CNTPE

5

CPE

9.0

DPV

0.075 µM

CPE

7.0

DPV

GCE

6.5

CA

DDE

CNPE

7.0

DPV

Graphene

GCE

4.0

GCE

7.0

Lac-SiSG/ MWCNT

β-CD-

SCE Ag/AgCl/KCl (3.0 M) Ag/AgCl/KCl (3.0 M) Ag/AgCl/(KCl, saturated)

Ag/AgCl/KCl (3.0 M)

[24]

[25] [26] [27]

[28]

[29]

[30]

0.18 µM

SCE

[31]

0.020 µM

SCE

[32]

CV

6.4 ×10-8 M

Ag/AgCl

[33]

DPV

3.3×10-8 M

Ag/AgCl/KCl

Present

(3.0 M)

work

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GO/PAY

(KClsaturated)

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NPs

0.47 µM

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CPMBD/TiO2

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DDTA/CNPs

10.

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p-chloranil

CPE

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OCNT

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PNH &

5ADB: 5-amino-3´, 4´-dimethylbiphenyl-2-ol; CPE: carbon paste electrode; SWV: Square

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wave voltammetry; PMP-DNA: Poly(1-methylpyrrole)-DNA; ZnONP-IL: Zinc oxide

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nanoparticle-ionic liquid; PNH & OCNT: 2,2′-[1,4-phenylenediyl-bis (nitrilomethyl-idene)]bis(4-hydroxyphenol) & oxidized multiwall carbon nanotubes; SCE: saturated calomel electrode; FCA: Ferrocenemonocarboxylic acid; CNTPE: carbon nanotubes paste electrode; CHF: Copper(II) hexacyanoferrate(III); DDTA: 7-(3,4-dihydroxyphenyl)-10,10-dimethyl9,10,11,12-tetrahydrobenzo[c]acridin-9 (7H)-one; CNPs: Carbon nanoparticles; CPMBD: (E)-2-((2-chlorophenylimino)methyl)benzene-1,4-diol;

TiO2NPs:

Titanium

dioxide

nanoparticles; Lac-SiSG/MWCNT: Laccase-silica sol-gel/multiwalled carbon nanotubes; CA:

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compounds under optimal experimental conditions. The substances that could potentially

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interfere were selected from a group of substances commonly found with IP in

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pharmaceuticals and/or in biological fluids. The tolerance limit was defined as the maximum concentration of the interfering substances that caused an error less than ±5% for the

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quantification of IP. The results exhibited that 500-fold of inorganic ions like Mg2+, K+, Na+, SO42−, Cl-, L-lactose, glucose, sucrose and UA do not affect the detection of IP.

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3.11 Stability, repeatability and reproducibility

The stability, repeatability and reproducibility are the key elements of a sensor’s

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performance. Hence, the repeatability of β-CD-GO/PAY/GCE was verified in 0.2 mM IP in

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0.1 M PBS (pH 7.0) for 40 repeated CV cycles at a scan rate of 0.05 V s-1 (Fig.S2). The relative standard deviation (RSD) of 2.3% was estimated at β-CD-GO/PAY/GCE and

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indicates that this composite based GCE has characteristic property of anti-surface fouling. The reproducibility of the sensor was tested in 0.02 M IP with 5 different electrodes which

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were prepared in the same way and the RSD was 1.2%. These results authenticate that the βCD-GO/PAY/GCE is superior for sensing of IP. The long term stability of the β-CDGO/PAY/GCE was examined by storing the electrode at room temperature for one month and then measuring the IP concentration. There was no significant decline in the oxidation peak current of IP. This observation reveals that the β-CD-GO/PAY composite can stably adhere to the electrode surface. 3.12 Simultaneous determination of IP and UA

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ACCEPTED MANUSCRIPT The secondary intention of the present work was simultaneous determination of IP and UA. The electrooxidation of each species in the mixture was investigated when the concentration of one species was changed, whereas the concentration of other species remained constant. Fig.9(A) displays DPV peaks with varied concentration of IP and constant concentration of UA. From Fig.9(B), it is evident that the peak currents of the IP increased

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linearly with an increase in the concentration of IP from 8 to 72 μM with a linear regression

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equation of

ip (μA) = (-2.99421 ± 0.42257 ) + (0.31208 ± 0.00995) [IP] (μM)

R = 0.99596.

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In the same way, determination of UA was carried out in presence of 8 μM IP with increasing

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concentration of UA. Fig.9 (C) displays DPV peaks with varied concentration of UA and constant concentration of IP. As displayed in Fig.9 (D), the peak currents of UA linearly

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connected with an increase in the concentration of UA from 4 to 26 μM with a linear regression equation of

R = 0.99516.

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ip (μA) = (-1.45035 ± 0.87669) + (0.11634 ± 0.00514) [UA] (μM)

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Therefore, β-CD-GO/PAY/GCE has a critical role and it is necessary for simultaneous resolution of IP and UA. It can be deduced that the sensor based on the β-CD-

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GO/PAY/GCE provides a new platform for the electrochemical sensing applications. 3.13 Real sample analysis

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In order to validate the practicability of the proposed electrode, an attempt was made to quantify IP content in an ampoule (the specified content of IP 2.0 mg mL−1) at the β-CDGO/PAY modified GCE. Standard addition method was used to determine the labelled content of IP in an ampoule in order to avoid any matrix issues. The results were found to be in good agreement with the stated amount. Thus, the proposed β-CD-GO/PAY modified GCE is sensitive, accurate, practically reliable and can be used for the analysis of IP in pharmaceutical formulations. Additionally, an attempt was made to determine IP in the urine

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Added (µM)

Found (µM)

Recovery (%)

1

0.0

Not detected

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2

5.0

4.98±0.16

3

10.0

10.12±0.25

4

20.0

19.74±0.33

1

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20.0

99.6

0.40

101.12

1.20

98.70

1.30

Not detected

-

-

4.91±0.06

98.2

1.8

9.78±0.78

97.8

2.2

19.69±0.59

98.45

1.55

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Bias (%)

-

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Buffer

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Sample

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Table 2: Recovery profile of different amounts of IP at β-CD-GO/PAY/GCE.

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In summary, a β-CD-GO/PAY composite was simply and facilely developed onto the GCE. The β-CD-GO/PAY composite was successfully characterized using FE-SEM, FT-IR and EIS. The EIS results obviously displayed that the GCE was effectively modified with β-CDGO/PAY composite and also revealed that it provides good interfacial electron transfer property. The β-CD-GO/PAY/GCE exhibited excellent electrochemical activity towards IP, because of significant electrochemical synergy between β-CD-GO and PAY. The β-CDGO/PAY/GCE provided good enhancement in analytical sensitivity, resulting in low limits of

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of IP in practical applications.

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Acknowledgements

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V.N. Palakollu and A. A. S. Gill are grateful to Nanotechnology Platform (University of KwaZulu-Natal) and College of Health Sciences, University of KwaZulu-Natal, South

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Africa for providing financial support.

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Schemes & Figures

Scheme 1. Successive cyclic voltammograms for the electrochemical polymerization of AY onto the surface of GCE.

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Scheme 2. Schematic representation of stepwise preparation of β-CD-GO/PAY/GCE.

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the surface of GCE.

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Fig. 2. FE-SEM images of PAY (A, B), GO (C,D) and β-CD-GO/PAY/GCE composite (E, F) of low magnification (A, C, E) and high magnification (B, D, F).

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Fig.3 (A). EIS spectra of 0.1 M KCl containing 2.5 mM [Fe(CN)6]3-/4- at (a) GCE, (b) PAY/GCE, (c) GO/PAY/GCE, (d) GO/GCE, (e) β-CD-GO/GCE and (f)

β-CD-

GO/PAY/GCE. Inset- Randles equivalent circuit model. (B). Calibration plot of square root of the scan rate. Vs the anodic peak current of 2.5mM K3[Fe(CN)6] at β-CD-GO/PAY/GCE at scan rates of 0.01 to 0.15 V s-1. 31

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Fig.4 (A). Plot of number of successive cyclic voltammograms for electropolymerization of AY vs peak current response towards the 0.2 mM IP in pH 7.0 at a scan rate of 0.05 V s-1. (B) Plot of loaded amount of β-CD vs. peak current and peak potential response towards the 0.02 M IP in pH 7.0 at a scan rate of 0.05 V s-1.

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Fig.5 Cyclic voltammograms at (a) GCE, (b) PAY/GCE, (c) GO/GCE and (d) β-CDGO/GCE (e) GO/PAY/GCE and (f) β-CD-GO/PAY/GCE in 0.1 M PBS (pH 7.0) containing 0.2 mM IP. 33

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Fig. 6 Calibration plot of square root of scan rate vs anodic peak current.

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Fig. 7 (A) Cyclic voltammograms of IP at different pH. (B) The plot of pH (5.0–8.5) vs Ep and ip at a scan rate of 0.05 V s-1. 35

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Fig.8 (A) DP voltammograms of IP with the different concentrations (from top to bottom (a

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ACCEPTED MANUSCRIPT Fig. 9 (A) DP voltammograms obtained for blank (a) and IP in the presence of 2μMUA at βCD-GO/PAY/GCE. Concentration of IP: (b) 0.0 μM,(c) 8.0 μM, (d) 16.0 μM, (e) 24.0 μM, (f) 32.0 μM and (g) 40.0 μM, (h) 48.0 μM, (i) 54.0 μM, (j) 60.0 μM, (k) 66.0 μM and (l) 74μM; (B) Calibration plot of IP determination; (C) DP voltammograms obtained for blank (a) and UA in the presence of 15μM IP at β-CD-GO/PAY/GCE. Concentration of UA (b) 0.0

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μM, (c) 4.0 μM, (d) 8.0 μM, (e) 12.0 μM, (f) 16.0 μM, (g)20.0 μM, (h) 22.0 μM and (i) 26.0

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights 

 Facile synthesis of β-CD-GO/PAY composite.  Excellent electrocatalytic activity towards the oxidation of Isoprenaline.  The low detection limit for Isoprenaline sensing was identified as 3.3×10-8 M.  Excellent selectivity with good stability and reproducibility of the modified electrode.

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 Good recovery results attained from real samples

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