Selective sensing of dopamine by sodium cholate tailored polypyrrole-silver nanocomposite

Synthetic Metals 260 (2020) 116296

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Selective sensing of dopamine by sodium cholate tailored polypyrrole-silver nanocomposite

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Arpita Adhikaria, Sriparna Def, Dipak Ranab, Jyotishka Natha, Debatri Ghoshc, Koushik Duttaa, Subhadip Chakrabortyd, Sanatan Chattopadhyayd, Mukut Chakrabortye, Dipankar Chattopadhyaya,* a

Department of Polymer Science and Technology, University of Calcutta, 92 A.P.C. Road, Kolkata 700 009, India Departments of Chemical and Biological Engineering, Industrial Membrane Research Institute, University of Ottawa, 161 Louis Pasteur St., Ottawa, ON, K1N 6N5, Canada c Institute of Post Graduate Medical Education & Research (IPGMER), SSKM Hospital, Kolkata 700 020, India d Department of Electronic Science, University of Calcutta, 92 A.P.C. Road, Kolkata 700 009, India e Department of Chemistry, West Bengal State University, Barasat, Kolkata 700 126, India f Brainware University, Department of Allied Health Sciences, Barasat, Kolkata-700125, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Sodium cholate biosurfactant Polypyrrole-silver nanocomposite Dopamine sensing Glassy carbon electrode Antimicrobial study Cytotoxicity study

Conducting polymer-noble metal nanocomposites have attracted considerable interest as electrochemical sensors because they provide better sensing responses to different analytes. We report the synthesis of rod like polypyrrole (PPY)-Ag nanocomposite using sodium cholate as soft-template for sensing nanomolar concentration of dopamine (DA). The nanocomposites were characterized by employing FT-IR, XRD, scanning and transmission electron microscopic analysis. Electrochemical impedance spectroscopy and cyclic voltammetry experiments were performed for electrochemical characterization of the PPY-Ag nanocomposite deposited on glassy carbon electrode (GCE). Electrochemical detection of DA using this nanocomposite deposited on GCE was done following linear sweep voltammetry. The PPY-Ag nanocomposite deposited electrode showed improved sensitivity value of 8.22 mAμM−1 for DA and a detection limit of 0.00005 μM. Biocompatibility assay of the developed PPYAg nanocomposites have exhibited less toxicity to mouse fibroblast cell even in comparison to that of PPY without silver. Antimicrobial property of the PPY-Ag nanocomposites towards E.coli and S. Aureus have also been observed.

1. Introduction Conducting polymers are excellent materials by virtue of their unique electronic charge conduction properties and are used in various electronic devices such as sensors and power sources, etc. [1]. Following the discovery of polyacetylene, various conducting polymers, viz., polypyrrole (PPY), polyaniline (PANI), poly(3,4-ethylene dioxythiophene) (PEDOT), polythiophene (PTH),etc. have been developed and used to modify electrodes for application as sensors [2], supercapacitors [3,4], for their excellent electrochemical performances. Achieving the optimum stability of such conducting polymer fabricated sensor devices for sensing nanomolar level of analytes is challenging. As a result, researchers are tirelessly working to develop stable and ultrasensitive electrode devices using delocalized extended π-electron conjugated conducting polymers along with different nanostructured

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materials [5,6]. A promising trend has been observed with the incorporation of noble metal nanoparticles in such conducting polymer composite electrodes resulting in increased electrochemical performance of the sensor devices [7–9]. Such types of composite electrode devices are being developed for electrochemical sensing of different neurotransmitters such as dopamine, serotonin, histamin, epinephrine, etc. [10–13]. Among the conducting polymers, PPY and its derivatives including PEDOT, PANI have been extensively used in making composites with materials like metal nanoparticles, carbon nanotube, graphene, nafion, etc. and their subsequent deposition on bare glassy carbon electrodes (GCE) for the sensing of neurotransmitters in general, and dopamine(3,4-dihydroxyphenyl ethylamine)(DA) in particular [14–17]. It has been reported that the metal nanoparticles coated electrodes exhibit enhanced electrocatalytic performance towards analytes having feeble redox activity in comparison to those of bare

Corresponding author. E-mail address: [email protected] (D. Chattopadhyay).

https://doi.org/10.1016/j.synthmet.2020.116296 Received 1 May 2019; Received in revised form 27 December 2019; Accepted 3 January 2020 0379-6779/ © 2020 Elsevier B.V. All rights reserved.

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hydroxytryptamine and dopamine with the nanomolar detection limit [32]. Nanomolar concentrations of dopamine and uric acid were determined by using electrochemically deposited copper nanoparticles on glassy carbon electrode coated with oxidized polypyrrole. Simultaneous electrocatalytic oxidation of dopamine and uric acid was observed in CV experiments with the same electrode [33]. Interference free sensing of DA was done by Saha et al. [34] using p-toluene sulfonic acid (PTSA) doped PPY, deposited on a Pt electrode. Such doped PPY, Ag-nanoparticles were deposited and stabilized with chitosan for obtaining superior electrocatalytic activity. Martí et al. [35] fabricated free standing hollow ∼30 nm thick microspheres of poly(N-methyl pyrrole) (PNMPy) nanolayers using polystyrene particle templates. Hollow PNMPy microspheres deposited over GCE was used to prepare one type of sensor and in another case, gold-PNMPy nanomembrane deposited over GCE was used for the detection of DA at nanomolar concentrations. Ultrathin composite films of gold nanoparticles deposited PPY derivatives including poly[N-(2-cyanoethyl)pyrrole] and PNMPy were used for the detection of DA in small concentration. Out of the two PPY derivatives, poly[N-(2-cyanoethyl)pyrrole] was reported to be more effective in sensing than the other due to the flexibility of cyanoethyl group and dipolar interaction between cyano group and C]O bond of oxidized DA [36]. For the determination of DA in human blood in presence of AA and UA as interfering elements, reduced GO doped PPY thin film deposited electrode was used [37]. Dopamine imprinted PPY-carbon nanotube electrochemical sensor was prepared for in vivo sensing of DA with a sensitivity of 16.18 μA/μM and a detection limit of 1 × 10−11 M [38]. Lin [39]used ZnO nanowire template for preparing an over-oxidized PPY-gold nanoparticle composite array by electrodeposition, for the selective measurement of DA without any interfering effect of ascorbic acid. A durable field effect transistor (FET) sensor containing Pt deposited 3-carboxylate polypyrrole conducting polymer was effectively used for the determination of DA present in very low concentration with high sensitivity without interference of other biomolecules in biological samples [40]. In a separate work, Ghanbari and Hajheidari [41]fabricated Ag nanoparticle deposited PPY nanofiber electrode, coated on GCE, by in situ electrodeposition and electrochemical oxidation for the purpose of simultaneous determination of DA, AA and UA within the detection limits of 0.1 μM, 1.8 μM and 0.5 μM, respectively. Such Ag/PPY nanocomposite was applied for the determination of dopamine in injectable medicine and UA in urine sample. Mahmoudian et al. [42] have reported the use of PPY coated palladium-silver nanosphere, for electrochemical detection of dopamine. The PPY coated Pd-Ag nanospherical composite was prepared by the reduction of aqueous alkaline Pd2+ and Ag+ solution in presence of pyrrole. DA is well known as an important neurotransmitter and its presence in brain tissue, even in very low concentration, causes different neural disorders like Parkinson’s disease, Schizophrenia, etc. This calls for the development of simple method of sensing DA at low concentration. Owing to better selectivity and sensitivity, the electrochemical methods have received much attention in comparison to the other methods. A review of scientific literature shows that different conducting polymers, metal nanoparticles, graphene, carbon nanotubes, etc., have been used for the improvement of electrochemical function of DA sensing electrodes. But the problem of interfering effects of AA and UA, which are present along with DA in biological sample, is still a challenge to the researchers, due to the low detection efficiency of DA as a result of their very close oxidation potentials. Silver nanoparticle (AgNP) dispersed conducting polymer electrode provides excellent electrocatalytic redox reaction with dopamine due to high surface area and better mass transport propertyof the electrodes. Although many approaches and advanced methods have been reported for developing PPY-Ag nanocomposites, still the preparation of PPY-Ag nanocomposite in a simple and cost-effective way for nanomolar level sensing of analytes with feeble electrocatalytic output is a challenge. We anticipated that the soft template-oriented preparation of conducting polymer silver

electrodes [18]. Recently, Naveen et al. [19] have extensively reviewed the synthesis, functionalization and applications of different conducting polymer nanocomposites using metals, metal oxides as well as carbonbased materials for the fabrication of biosensors. Moon et al. [20] have also reviewed the research outcomes of biosensing of a wide category of neurotransmitters with the help of conducting polymer based electrochemical sensors. Metal containing conducting polymer nanocomposites are advantageous since the polymer molecules restrict crystallization of the metal atoms due to steric hindrance. Presence of metal in polymer backbone creates electrostatic interactions which also prevents aggregation of the polymer molecules. Thus, the electrodes coated with such conducting polymer metal nanocomposites provide increased electron transport to the electrolyte [21].Graphene oxide (GO)doped PEDOT nanocomposite was prepared by electrochemical deposition for selective sensing of DA [22].Coating of carbon fiber microelectrode with PEDOT-Nafion composite is also reported for in vivo detection of DA [23]. In another work, poly(ionic liquid) (PIL) was used for the functionalization of PPY-GO composite coated over GCE for sensing of DA with excess ascorbic acid(AA) as an interfering agent. From the cyclic voltammogram analysis it was claimed that PIL played an important role for the separation of oxidation potentials between DA and AA, enabling easy DA detection in biological samples as reported by Mao et al. [24].Similarly, DA secretion from pheochromocytoma (PC12) cells was detected selectively in presence of AA by PPY-graphene nanocomposite without any interference due to the electrocatalytic properties of negatively charged graphene structure [14]. For the detection of dopamine, Qian et al. [25] reported the use of Au nanoparticles deposited PPY/reduced GO in the form of hybrid sheets.The fabricated PPY/reduced GO sheet with flower-like Au-nanoparticles was used as the dopamine sensor at nanomolar levels. As reported by Mir et al. [26], a sensor probe, made of GO, Au nanoparticles (AuNP) and EDTA immobilized poly(1,5-diaminonaphthalene), was used for the monitoring of DA release in nanomolar to millimolar range from the real sample (PC12 cells). Fabregat and coworkers [27] synthesized conducting polymers like poly(N-methylpyrrole), poly(N-cyanoethyl pyrrole) and PEDOT on glassy carbon electrode followed by Au-nanoparticle deposition from a colloidal solution for selective determinations of DA, AA and uric acid (UA) in ternary mixtures. Out of the three polymers, PEDOT exhibited the best levels of detection amongst these three analytes. An in-vivo detection of DA in rat has been reported with a biocompatible carbon fiber microelectrode, which was coated with PEDOT-Nafion composite [23].Weaver et al. [28] also claimed that PEDOT doped GO nanosheet on GCE surface provided improved and selective sensitivity to DA in presence of AA and recommended it as a good biosensor for biological samples.Wang et al. [22] have reported the electrochemical sensing of DA by using reduced GO doped PEDOT sensor and observed no interfering effects of AA and UA. A cost-effective method of fabrication of PEDOT/(IL) nanocomposite using1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl) imideas IL has also been reported [29].This electrodeposited PEDOT/IL nanocomposite on GCE with nanoporous structure showed very good electrocatalytic function to the oxidation of DA. Scavetta et al. [30] coated indium tin oxide (ITO) with PEDOT by polymerizing EDOT in presence of poly(styrene-4-sulfonate) (PSS) as emulsifier followed by functionalization with ferrocene for use of this coated electrode in the amperometric detection of DA. The PEDOT-PSS system provided a good electrochemical performance due to the interaction of positive charges of PEDOT with the PSS anion. Recently, Devaramani et al. [31] have reported a selective DA sensing electrode, which was prepared by covalently binding 4-aminobenzene sulfonic acid (4-ABSA) through electrografting on graphite pencil lead. Such electrodes show a linear detection response in millimolar range in presence of AA and UA. A screen-printed carbon sensor was developed from graphene (GR) and conducting poly (4-amino-3hydroxy-1-naphthalenesulfonic acid) for the determination of 52

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nanocomposites and subsequent electrode fabrication could be a suitable approach for application, in selective sensing of dopamine. Therefore, in the present work we report a novel approach for preparing Ag NP deposited PPY-nanorod by template polymerization of pyrrole, using sodium cholate bio-surfactant micelle, to obtain improved sensitivity of DA. Since biological samples are the primary source of DA, therefore, the evaluation of biocompatibility as well as antimicrobial activity of the prepared PPY-Ag nanocomposite electrode material has also been taken into consideration.

2.3.3. Morphological analysis by scanning and transmission electron microscopy (SEM and TEM) The surface morphology of the PPY-Ag nanocomposite samples was studied in a scanning electron microscope (Model: ZEISS EVO 18). Before scanning, the samples mounted on the specimen stub were sputter-coated with platinum. The bulk morphology of the nanocomposite samples were visualized under a transmission electron microscope (Model: TEM, JEOL-JEM-2100). For this purpose, PPY-Ag nanocomposite samples, dispersed in distilled water, were drop cast on a copper grid coated with carbon and dried for TEM analysis.

2. Experimental

2.3.4. Thermogravimetric analysis (TGA) The TGA analysis of rodlike PPY-Ag and PPY-Ag was carried out in a thermogravimetric analyzer (TA Instruments Model Q 50) in air atmosphere at a heating rate of 10 °C min from ambient to 650 °C.

2.1. Chemicals Pyrrole (supplied by Sigma-Aldrich Inc.)was distilled under vacuum and preserved carefully at 4 °Cundernitrogen atmosphere prior to its use. Ferric chloride, hydrochloric acid and silver nitrate were purchased from Merck India Ltd. and dopamine hydrochloride was procured from Sigma-Aldrich Inc. The phosphate buffer saline as supporting electrolyte and sodium cholate used as surfactant during polymerization of pyrrole monomer were purchased from SRL, Mumbai, India. The entire experimental work was performed using water after three times repeated distillation.

2.4. Electrochemical characterization 2.4.1. Electrode preparation with rodlike PPY-Ag nanocomposite Electrochemical properties of PPY-silver nanocomposite, deposited over glassy carbon electrode(GCE), were evaluated in an electrochemical analyzer (Model:C–H 660 instrument) using 10 mL potassium ferrocyanide solution (5 mM) containing 0.1 M potassium chloride and 1 M phosphate buffer solution of pH 7. Electrochemical sensing performance of the as prepared PPY-Ag nanocomposite deposited over GCE for dopamine in presence or absence of AA and UA was done with the same instrument. Electrochemical properties were measured using GCE as the working electrode, Ag/AgCl as the reference electrode and Ptwire as the counter electrode. The preparation of PPY-Ag nanocomposite deposited GCE was done in the following manner as described.

2.2. Polypyrrole-silver nanocomposite preparation For the sodium cholate (surfactant) tailored polymerization of pyrrole, first an aqueous dispersion of sodium cholate (16 mM) was prepared by mixing it in ice cold water containing 1.25 M HCl. Then pyrrole monomer (0.058 mM) was added to this dispersion followed by addition of 13 mL 10−2 M aqueous AgNO3under vigorous stirring over a period of 15 min. Polymerization of pyrrole was initiated subsequent to addition of aqueous FeCl3 solution (0.058 mM) to the above mixture and the polymerization was allowed to continue for 5 h at 0–4 °C. The molar ratio of pyrrole to FeCl3 was kept at 1:1. After completion of the reaction, an aqueous dispersion of black solid particles of PPY-Ag nanocomposite was obtained. The PPY-Ag nanocomposite so formed was then isolated by filtration of the dispersion followed by repeated washing with distilled water to remove traces of residual monomer, silver nitrate, FeCl3and sodium cholate surfactant. In order to ensure acid doping of the composites, the PPY-silver nanocomposite was further washed with 1.25 M aqueous HCl. Finally, the acid doped polymer was washed with acetone, dried and stored in a desiccator for characterization and sensing measurements. PPY-Ag nanocomposite without sodium cholate was also prepared following the above method under identical condition for the sake of comparison of its characteristics with those of PPY-Ag nanocomposite prepared in presence of sodium cholate.

2.4.2. Polishing of GCEsurface The GCE surface was polished to obtain a mirror finish by first cleaning with a polishing paper and then by rubbing with alumina powder. The polished GCE surface was subsequently cleaned by ultrasonication in water-ethanol mixture to remove any traces of alumina powder used in the previous step. The polished and washed GCE surface was then used for drop casting of PPY-Ag nanocomposite suspension. 2.4.3. Preparation of PPY-Ag nanocomposite suspension for drop casting on GCE The prepared PPY-Ag nanorod (∼ 2 mg) was dispersed in distilled water under sonication for 1 h. 2.4.4. GCE-PPY-Ag nanocomposite working electrode preparation by drop casting A drop (∼15 μL) of the well-dispersed PPY-Ag nanocomposite in water, containing approximately 0.03 mg of PPY-Ag nanocomposite was placed carefully on the previously polished, dry working area (3 mm diameter) of the GCE surface. Air-drying in order to obtain a stable composite film prior to electrochemical measurement slowly evaporated the water in the drop cast film on GCE surface. The thickness of the dry PPY-Ag nanocomposite film was measured to be about 0.04 mm.

2.3. Characterization of PPY-silver nanocomposite 2.3.1. XRD analysis X-Ray diffraction analysis of PPY-Agnanocomposite samples was performed in an X-Ray diffractometer (Model: X-PERTPRO Panalyticaldiffractometer)usingCu Kα as X-ray source(λ = 1.5406) at a scanning rate of 1° per minutemaintaining 40 kV and 30 mA voltage and current, respectively.

2.5. Biocompatibility evaluation of rodlike PPY-Ag nanocomposite Cytotoxicity assay of the rodlike PPY-Ag nanocomposite was done following the direct contact method according to MTT [3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, using fibroblast cell line. Mouse Fibroblast cells (L929) were cultured using a cell culture medium of Dulbecco’s Modified Eagle’s Medium (DMEM, Hi-Media, India).The cell growth was performed in a 96-well polystyrene tissue culture plate in a controlled atmosphere of 5 % CO2 incubator at 37 °Cusing DMEM cell culture medium, containing 10 % Fetal Bovine Serum and penicillin-streptomycin antibiotic solution.

2.3.2. FT-IR analysis FT-IR analysis of polypyrrole-Ag nanocomposite samples were performed in a FT-IR spectrophotometer (Model: Perkin-Elmer Spectrum Two) using pellet of polymer dispersed in KBr over 400–4000 cm−1 wave number. 3

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Then 200 μL mouse fibroblast cells were seeded into 96-well culture plate at a density of 8 × 103 cells/well and incubated for 24 h at 37 °C in CO2 atmosphere when the cells attained confluency. After removal of the cells, fresh medium having various concentrations ofPPY, rodlike PPY and rodlike PPY-Ag nanocomposite (2.5–30 μg/mL) were added into each of the96 wells and incubated for a further period 24 h. This was followed by the addition of 20 μL MTT solution to each well, containing added polymer samples and cultured cells, followed by incubation at 37 °C for 4 h. After incubation,150 μL DMSO solution was mixed (when insoluble purple product transformed into colored solution) followed by continuous shaking for 20 min in a shaker incubator by wrapping with an aluminium foil. Finally, the absorbance intensity was recorded on an ELISA microplate reader at a wavelength of 480 nm. Simultaneously the bright field optical micrographs of PPY, rodlike-PPY and PPY-Ag nanocomposite treated cells were also taken under fluorescence microscope (Model: Nikon Eclipse Lv100pol).

PPY-Ag nanorod separates out from solution as black solid particles. For the complete removal of iron chloride and sodium cholate the PPY-Ag nanorod was thoroughly washed with water. It is also expected that as a result of rigorous washing, sodium cholate has been completely removed. Subsequent to the removal of iron and sodium cholate, the wet PPY powder was washed by aqueous acid (1.25 M HCl) for doping, prior to drying and storage in a desiccator. Fig.1 shows a schematic description of polypyrrole synthesis in presence of sodium cholate. The yield of polymerization remained within the range of 75–80 % for different batches of the synthesized polymer. A similar synthesis under identical condition was carried out in absence of sodium cholate to prepare PPY-Ag nanocomposite without cholate. In absence of sodium cholate the yield of PPY-Ag nanocomposite was comparatively low (∼ 40 %).

2.6. Characterization of bacterial cell morphology

The XRD pattern of PPY-Ag nanocomposites with and without sodium cholate is shown in Fig. 2. Fig. 2 shows typical characteristic peaks which match the face centred cubic structure of bulk silver with sharp peaks at 28.16, 32.6, 46.56, 55.16, and 77.01° corresponding to the (210), (113), (124), (240), and (300) planes, respectively. Other groups have reported similar peaks corresponding to the above planes, however, with slight shift in the peak positions, viz., 27.51, 31.87, 45.57, 56.56, and 75.25° [44,45].The small broad diffraction peak at around 23° is ascribed to the partial crystalline morphology of PPY in the nanocomposite. The XRD pattern indicates the existence of metallic Ag in the composite and suggests that Ag+ has been reduced to Ag during polymerization of pyrrole. The XRD peak positions of PPY-Ag nanocomposite are similar to those of PPY synthesized without silver in our earlier research [6]. However, the broad hump showed earlier is absent which suggests an improvement in crystallinity of the nanocomposite. The XRD pattern of PPY-Ag nanocomposite without sodium cholate (Fig. 2) shows similar diffraction peak positions.

3.2. XRD analysis

For the characterization of bacterial cell morphology, two types of bacterial cells (E.coli and S. aureus) were incubated with different nanocomposite samples for 24 h. Thereafter the incubated cells were washed with PBS buffer and 20 μL solution of washed cells were diluted in an eppendorf tube, containing 2 mL water. Subsequently, the diluted cells were placed onto cover slips and fixation was done for 2 h by adding 2.5 % glutaraldehyde solution. The sequential dehydration treatment on the fixed cells was done, using aqueous ethanol solutions of different concentrations, viz. 50, 70, 85, 90, and 100 % for 10 min. Finally; all of the samples were dried under vacuum for SEM analysis. For the preparation of SEM specimens, the coverslips with bacterial cells were gold sputter coated and inserted into the chamber of the scanning electron microscope (Model: ZEISS EVO-MA 10) for imaging. 3. Results and discussion

3.3. FTIR analysis

3.1. PPY-silver nanocomposite synthesis

For ascertaining the chemical structure of the synthesized PPY-Ag nanocomposite, the FTIR spectrum of rod like PPY-Ag nanocomposite in presence of sodium cholate and PPY-Ag nanocomposite in absence of sodium cholate is shown in Fig. 3. The different characteristic bands for the nanocomposites are due to the in-plane deformation vibration of –C–H at 1050 cm−1 and antisymmetric stretching vibration of (C]N) group at 1462 cm−1. The antisymmetric stretching vibration of (C]N) in pyrrole ring, which is generally observed at 1454 cm-1 is shifted to 1462 cm−1 probably due to the influence of Ag nanoparticles. The peaks appearing at 1050 and 908 cm−1 are due to the in and out of plane deformation of CeH bond, respectively. The band observed at 3444 cm−1 is attributed to the NeH stretching vibrations. The peak near 1185 cm-1 is associated to the breathing vibration of pyrrole ring [46]. The PPY-Ag nanocomposite in absence of sodium cholate exhibits an FT-IR spectrum similar to those of PPY without silver as reported earlier [6], where the bands at 1544, 1454, 1302, 1165 and 897 cm-1 are shifted to 1555, 1462, 1308, 1185 and 908 cm−1 respectively, which may be attributed to the interaction of the incorporated silver on the PPY surface. Such observations also confirm the incorporation of Ag NPs into the PPY surface [47,48].

In this study, PPY was prepared in an aqueous acidic medium in presence of sodium cholate as surfactant(soft template) and FeCl3 as an oxidant. The role of sodium cholate surfactant (Image 1), which acts as a soft template and guides the formation of PPY nanorod morphology, has been reported earlier by us [6]. Before the addition of the oxidant, AgNO3 solution was mixed with pyrrole to facilitate deposition of silver nanoparticles on the PPY nanorods. It is known that sodium cholate forms self-associated aggregates in aqueous solutions [43]. Sodium cholate helps the solubilization of hydrophobic pyrrole monomer through encapsulation within the micellar aggregates and thus favors oxidative polymerization by FeCl3. Micellar aggregates of sodium cholate also favored the formation of nanorod morphology of PPY in presence of silver nitrate. During oxidative polymerization of pyrrole, silver nanoparticles were deposited in-situ on PPY nanorod due to the reduction of silver nitrate and subsequently, the

3.4. Evaluation of morphology by SEM and TEM analyses Surface morphology of the as prepared PPY-Ag nanocomposite was visualized under scanning electron microscope, as shown in Fig. 4a and c. In-situ deposition of Ag nanoparticles was found to adhere on the surface of the PPY nanorods, probably as a result of redox reactions among pyrrole, FeCl3 and AgNO3. Apart from the Ag nanoparticles deposited nanorods, there are clusters of μm range spherical particles of

Image 1. 2D Structure of sodium cholate surfactant.. 4

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Fig. 1. Schematic presentation of synthesis of rodlike PPY-Ag nanocomposite using sodium cholate micelles as soft template.

Fig. 3. FTIR spectra of rodlike PPY-Ag nanocomposite and PPY-Ag nanocomposite.

such adherence of silver nanoparticles on PPY nanorod might help in sensing of dopamine. Fig. 4c shows the surface morphology of PPY-Ag nanocomposite prepared without sodium cholate. It clearly shows that the particles are spherical in nature. The change in particle morphology is presumably due to the absence of sodium cholate micellar structure which acts as a template. Transmission electron micrograph of silver deposited PPY (synthesized in absence of sodium cholate) is shown in Fig. 4d. The inset of TEM image in Fig. 4d reveals the spherical PPY morphology (∼ 311 nm dia) with silver particle (∼ 63 nm dia) deposition as dark spot along with other smaller silver particles.

Fig. 2. X-Ray diffraction patternsof rodlikePPY-Ag and PPY-Ag nanocomposites.

silver [Fig. 4a]. Such deposition in bulk of silver particle clusters occured probably, as a result of coalescence of silver particles formed during redox reaction subsequent to deposition on PPY nanorods. On the contrary, when PPY was synthesized in absence of AgNO3 no such clusters of spherical silver particles were formed [6]. In order to study the bulk morphology of PPY-Ag nanocomposite TEM analysis of the samples was also done. TEM micrograph of rodlike PPY-Ag nanocomposite is shown in Fig. 4b. TEM image of a representative portion of the sample (Fig. 4b) reveals that the diameter of PPY rod is ∼142−148 nm and the diameter of the deposited Ag NPs, seen as dark spots, is ∼3.50-3.98 nm (calculated by image analyzer). We anticipated that

3.5. TGA analysis The analysis of rodlike PPY-Ag (synthesized in presence of sodium cholate) and PPY-Ag (synthesized in absence of sodium cholate) samples by TGA was carried out for estimating the amount of silver as residue since the residue is related to the amount of metallic silver [49]. From TGA analyses shown in Fig. 5 we obtained 13 % Ag in the rodlike PPY-Ag and 9 % Ag in PPY-Ag samples. 5

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Fig. 4. Scanning electron micrographs of (a) silver deposited rodlike PPY (synthesized in presence of 16 mMsodium cholate), (c) silver deposited PPY (synthesized in absence sodium cholate). Transmission electron micrographs of (b) silver deposited rodlike PPY (synthesized in presence of 16 mM sodium cholate) (Inset: higher magnification of silver deposited PPY), (d) silver deposited PPY (synthesized in absence of sodium cholate).

redox couple. The semi-circular nature of the plot at lower impedance value and the EIS line signify frequency dependent electrochemical reaction in the vicinity of the electrode surface. At lower impedance side of the semicircle, which is in higher frequency zone, the electrode process is governed by interfacial charge transfer phenomenon. At higher impedance side (lower frequency zone), the electrode process is governed by the diffusion process [50]. The diameter of the semi-circular region is related to charge transfer resistance (Rct). The value of Rct of bare GCE is 1200 Ω which is relatively higher than that of PPY modified GCE [6]. Our synthesized rodlike PPY-Ag nanocomposite exhibits a Rct value of 98.6 Ω which is the lowest when compared with the other PPY modified electrodes. This is possibly due to the presence of silver, which is a good conductor of electricity. The silver nanoparticles, uniformly distributed on the rod like PPY matrix, facilitate the electron switching at the interface of electrode surface. This behavior indicates that, metal nanoparticles in the polymer matrix promote ion diffusion rate by improving the electron transfer kinetics. However, in absence of sodium cholate, the particle morphology becomes spherical which gives rise to a higher Rct value (∼ 250 Ω) of PPY-Ag nanocomposite. The lower value of Rct for rodlike PPY-Ag nanocomposite was the deciding factor to carry out further studies using this sample.

Fig. 5. Thermogravimetric analyses of rodlike PPY-Ag (synthesized in presence of sodium cholate) and PPY-Ag (synthesized in absence of sodium cholate) samples.

3.6. Electrochemical analysis by cyclic voltammetry (CV) 3.6.2. Cyclic voltammetry study Cyclic voltammetry study was performed for investigating charge transfer properties of the rodlike PPY-Ag nanocomposite deposited GCE surface. The electrodes were immersed into a solution of 5.0 mM of [Fe (CN)6]3−/4− as a redox couple mixed with 0.1 M KCl. The cyclic voltammograms recorded for PPY-Ag nanocomposite is shown in Fig. 7. The current-voltage characteristics demonstrated two clear reduction

3.6.1. Electrochemical impedance spectroscopic (EIS) analysis Fig.6 exhibits EIS in the form of Nyquist plot of real (zʹ) and imaginary (zʺ) parts of impedance data for rodlike PPY-Ag nanocomposite deposited on GCE electrode [6] obtained by dipping the electrode in an electrolytic solution of 0.1 M KCl containing5.0 mM [Fe(CN)6]3−/4− as 6

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Fig. 6. Impedance analyses of (a) rod like PPY-Ag and (b) PPY-Ag nanocomposites.

Fig. 7. Cyclic voltammogram of rodlike PPY-Ag nanocomposite.

Fig. 8. Change of current and potential due to variation of scanning rate from 10 to 60 mVs−1.

peaks, one for PPY (0.326 V) and the other for Ag+(0.202 V) with peak currentsof-1.350 × 10 -4 A and -2.472 × 10 -4 A, respectively. Two oxidation peaks, one for PPY(0.235 V) and the other for Ag°(-0.20 V) arise as a result of the combined effect of PPY-Ag nanocomposite and redox couple [51].The PPY-Ag nanocomposite modified electrode, exhibit large enhancement in current response compared to bare GCE, GCE modified with PPY synthesized without surfactant and rodlike PPY synthesized with surfactant in absence of silver, which were reported in our earlier work [6]. Such observation indicates that the PPY-Ag nanocomposite provides enhanced electroactive surface, ensuing more electron transfer between the electrolyte and the electrode surface. From this observation it may be said that the higher current value obtained for PPY-Ag nanocomposite electrode is due to the combined effect of metallic NPs (Ag) and conducting PPY. Furthermore, the total number of electrons involved in the electrochemical reaction forthe redoxcouple can be evaluated by using the following equation: N= (Epa-Epc)/ΔEp ≈0.059/n Where, ΔEprepresents the separation of peak potential, Epaand Epc denote anode and cathode peak potentials, respectively. The value of ‘n’ is associated with the number of electrons involved in the redox process. This value is found to be 0.102 for silver and 0.269 for PPY which suggest the transfer of one electron during redox reaction [52].

3.7. Effect of variable scan rate The effect of scanning rate variation on CV of the PPY-Ag nanocomposite electrode isshown in Fig.8. The CV data as a function of scanning rate in the range 10-60 mVs−1 were recorded. It is seen from Fig.8, that with the increase of scan rate, the current magnitude increases along with shifting of peak potential to a higher value as a result of electron transfer between the analyte and electrode surface. This property presumably can lead to better sensing behavior. 3.8. Electrochemical sensing of dopamine by CV Electrochemical performances of the PPY-Ag nanocomposite deposited on GCE towards dopamine sensing in PBS buffer (pH 7) have been measured by linear sweep voltammetry (LSV) technique at a scanning rate of 100 mVs−1 and the results are shown in Fig. 9. From Fig. 9, it is observed that the current values vary linearly in the range of −0.15 to −0.175 mA and −0.05 to −0.7 mA for lower [0-0.003 μM] and higher [0.78125–50 μM] concentrations of DA, respectively. Similarly, Fig. 10 exhibits that conductance values also vary linearly in the range of 0.64 mS to 0.77 mS and 0.17 mS to 0.9 mS for lower and higher concentrations of DA, respectively. From Fig. 9 it can also be 7

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Fig. 9. Current voltage characteristicsof PPY-Ag nanocomposite obtained fromlinear sweep voltammetry of varying concentrations of dopamine (a and c)and linear dependence of current on dopamine concentration (b and d)for the concentration ranges:0.00005–0.003 μM, and.0.78125–50 μM.

to signify the sensitivity of the system:

said that the increase of overall current with the increase of dopamine concentration may be attributed to the increase of number of electrons as a result of electrochemical reaction of dopamine. The slopes of the curves depicted in Fig. 9(b and d) and Fig.10 demonstrate the changes in current and conductance for a small change in DA concentration. Hence, the PPY-Ag nanocomposite has shown a detection limit of 0.00005 μM. A factor in terms of current/conductance may be defined

β=

∑ (α − 〈a〉)(γ − 〈γ 〉) ∑ (α − 〈a〉)2

where, α and γ symbolize the DA concentration and current/conductance of the system, respectively. Parameters under < > notations signify their respective average values [53].Fig. 9 has shown a linear

Fig. 10. Linear calibration curve for conductance vs. dopamine concentration. 8

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Table 1 Comparative picture of dopamine sensing with PPY-Ag nanocompositedeposited on GCEvsdifferent modified electrodes reported in the literature. Electrode

Linear concentration range (μM)

Limit of detection (μM)

Rct (ohm)

References

GO-PEDOT/GC,CV PEDOT/Pt electrode, CV PNMPy/PS,CV PPy/CNTs-MIPs/GC,DPV Au@PPy/GS,DPV nano-Cu/PPy/GC, DPV PILs/PPy/GO,DPV PPyox-PTSA/Ag-NP/Pt, DPV PPy/eRGO, DPV Ag-PPy/GCE PdAgNsPs-PPY PPY-Ag nanocomposite/GCE

1.0–40.0 0.5–25.0 10000–20000 0.0000005–5.0 0.0001–5.0 0.001–0.1 4–18 0.001–0.12 0.1–150 0.5-155 0.001-200 0.00005–0.003 & 0.78125–50

0.083 0.061 human urine 1.5 0.00001 0.00001829 0.00085 0.073 0.00058 0.023 0.1 0.0258 0.00005

– – – – – – – 20.8 – – 10.55 98.6

[28] [54] [35] [38] [25] [33] [24] [34] [37] [41] [42] This Work

3.9. Interference

oxidation peak current for every addition of dopamine within the ranges of 0.00005 to 0.003 and 0.78125–50 μM.Linear calibration plots of conductance (calculated from current and potential values) versus dopamine for the two different experimental ranges of concentrations are also presented in Fig. 10. From the calibration plots the sensitivities have been calculated to be 8.2 mAμM−1and 1.28 × 10-2 mAμM−1for the two different concentration ranges used in the experiment. The coefficient of sensitivity (sensitivity in terms of conductance) has been calculated to be 39.7 mSμM−1for lower concentration region and 0.015 mSμM−1for higher concentration region. The reason for the lower sensitivity value at higher DA concentration maybe the saturation effect as most of the interaction sites are already filled up.The experiments were performed in triplicates and the measurement uncertainty was found to be ≤1.5 %.Froma comparative LOD values of different electrodes, included in Table 1, it is seen that the LOD value of DA sensing in case of our PPY-Ag nanocomposite deposited GCE electrode is also very low. It is seen from Fig. 11 that the magnitude of peak current response for DA oxidation is dependent on variation of scanning rate from 10 to 100 mVs−1. As the scan rate increases in a given time interval, the total number of electrons responsible for redox reaction will increase. Also, the improved sensitivity is ascribed to the fact that PPY-Ag nanocomposite modified electrode provides better electroactive surface which facilitates the tunnelling of electrons within the redox couple.

In order to scrutinize the impact of various interfering elements present in biological fluid, viz., glucose, AA, and UA, LSV experiment was carried out with PPY-Ag nanocomposite deposited GCE electrode. The LSV response curves are depicted in Fig. 12. DA selectivity of the modified PPY electrode was investigated using 50 μM DA solution containing PBS buffer (pH 7) in presence of interfering substances AA, glucose and UA. The variation of current response was monitored by adding 10 μM interferents in equal proportion with that of dopamine. Hence, with the addition of AA (10 μM), no additional peak in the current response plot of DA was noticed along with DA. Also, there is no obvious peak shift in case of AA addition. Similar behavior was noticed with the addition of UA and glucose solution along with DA, implying that these interferents did not show any substantial change in peak current response. The above phenomenon manifests that the co-existence of various physiological biomolecules have not affected the sensing performance of DA solution. 3.10. Electrode stability and reproducibility In order to evaluate the stability of the modified electrode, LSV experiment was done using a fixed concentration of DA (0.00005 μM). The response to DA sensing has been studied at intervals of 25 min for the same modified electrode within a fixed time span (∼125 min). It is observed that the DA response remained same at different time intervals (Fig. 13). The result signifies that there is no diffusion of material to the electrolyte solution from the PPY modified electrode. The shelf life of the modified PPY electrode was estimated over a period of 5 weeks at intervals of 1 week by checking the reproducibility of the electrode performances (Fig.13). It has been seen that the modified electrode exhibits only about ∼2.3 % loss of activity after 5 weeks when it has been properly stored under controlled environment. These results clearly signify that the proposed biosensor has sufficient reproducibility and is stable without any significant drift. 3.11. Cytotoxicity assessment To apprehend the utility as well as biocompatibility of the developed PPY-Ag nanocomposite, cytotoxicity analysis by MTT assay was performed and the results of mouse fibroblast cell viability exposed to varying concentrations of the different electrode materials have been plotted in Fig. 14. It is evident from Fig. 14, that the cytotoxicity varies in a dosage dependent manner. In case of PPY treated cells, the cell viability is constantly decreasing which can be attributed to the cytotoxic nature of PPY sample. On the contrary, PPY-Ag nanocomposite treated cells have shown comparatively more cell viability implying the less toxic nature of PPY-Ag nanocomposite sample.Further,the bright field microscopic images shown in Fig. 15 were also analyzed to assess the cytomorphological structure of the fibroblast cell lines. No obvious

Fig. 11. CV performance of the PPY-Ag nanocomposite deposited GCE during DA sensing at different scan rates: 10–100 mVs−1 at 50 μM concentration of dopamine. 9

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Fig. 12. Interference of UA, AA, glucose and mixture of UA, AA and glucose in DA sensing.

3.12. Antimicrobial study

characteristic alteration in cellular morphology was observed in case of cells treated with PPY-Ag nanocomposite.However, after incubating with PPY samplewithout silver, few round-shaped cellular morphology have been noticed which is possibly due to the induced toxicity of PPY sample.

3.12.1. Zone of inhibition study The zone of inhibition study has been conducted to evaluate the antimicrobial efficacy of the synthesized PPY-Ag nanocomposite. There is no observed zone of inhibition against E.coli and S. aureus when incubated for 24 h with rod-like PPY and PPY. This can be ascribed to the absence ofany bactericidal effect of PPY sample, which could not

Fig. 13. Stability study of rodlike PPY-Ag modified GCE electrode at various time intervals. 10

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further hampers internal osmotic imbalances and restricts the bacterial growth. Further morphological studies have been carried out to corroborate these findings. 3.12.2. Morphological pattern analysis To scrutinize the antimicrobial efficacy of rodlike PPY-Ag nanocomposite, the change of morphology of the bacteria has been monitored using SEM. It is seen from Fig. 17 that untreated E. coli and S. aureus show typical rod and round shaped morphological patterns, respectively. In addition, smooth and even cell wall architectures are also noticed. Whereas in case of E. coli exposed to PPY-Ag nanocomposite, the significant alteration of cell morphology is noted with ruptured cellular membrane. Besides that, the membrane is crumpled/wrinkled and a total loss in cell integrity is also observed. For S. aureus the leaking of intracellular contents is noticed in cells resulting in the quenching of cell surfaces. Unlikely few survived cells are spotted with round shaped and intact surface morphology. This can possibly be due to the different chemical and structural cell wall compositions of G− and G+ bacteria. For G− bacterial cell wall only one thin peptidoglycan layer is visible which assists easy internalization of nanoparticles resulting in the cell membrane damage. On the other hand, G+ bacteria inhibits nanoparticles uptake due to its multi-layered peptidoglycan cell wall showing protective nature against PPY-Ag nanocomposite. The inhibitory mechanism of silver nanoparticles on bacterial cells is well documented. It is pertinent that upon Ag ion treatment, DNA loses its replication ability, which leads to cellular protein inactivation within the cell [55]. Additionally, it is also revealed that Ag ion binds to functional proteins, resulting in protein denaturation and catalyzes the cell death mechanism. But, still the interaction between the cell membrane and the building element is quite unresolved. Stoimenov et al. [56] described that silver nanoparticle penetrates into bacterial cell

Fig. 14. Cytotoxicity of rodlike PPY-Ag nanocomposite, rodlike PPY, PPY in terms of cell viability (%) and concentration of electrode materials.

restrict the bacterial growth on the agar plate. On the other hand, PPYAg nanocompositetreated disc shown in Fig. 16, shows higher zone of inhibition as compared to the other two samples (rod like PPY and PPY). With the increase of PPY-Ag nanocomposite concentration the zone of inhibition also increases suggesting that nano-silver governs the antimicrobial efficacy of the synthesized PPY-Ag nanocomposite. Interestingly, in case of E.coli,the zone of inhibition is quite larger as compared to S. aureus. The underlying concept is theformation of pits on the cell membrane induced by Ag nanoparticles, which in turn increase the permeability of the cell membrane and favors cell death. This

Fig. 15. Bright field image of fibroblast cell: a) control, b) PPY, c) rodlike PPY, and d) rodlike PPY-Ag nanocomposite. 11

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Fig. 16. Photographs of the zone of inhibition of PPY, Rodlike PPY, Rodlike PPY-Ag nanocomposite against (a) E. coli and (b) S. aureus.

membrane through the formation of ‘pits’ which restricts the transportation circle causing cell death. In another study [57]irregular shaped pits were monitored into outer cell lipopolysaccharide (LPS) membrane that induces membrane permeability causing by progressive release of LPS molecules and membrane proteins. Based on this hypothesis it can be concluded that engulfment of nanoparticles stimulates various bactericidal effect which promotes the cell death phenomenon by altering their structural integrity.

0.05–3 nM with detection limit of 0.05 nM.These findings reveal the fact that composite modified GCE imparts excellent electrocatalytic activity towards DA detection as compared to only rodlike PPY modified GCE. Based on the above outcomes, it can be concluded that the use of silver nanoparticle in PPY nanorod has led to more effective DA sensing.

4. Conclusions

All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication before its appearance in the Synthetic Metals. Author1: Arpita Adhikari: Conceptualization and designed the synthesis scheme, planned the overall work, collected data with analysis, wrote the paper Author2: Sriparna De:

Author statement

Silver nanoparticle depositedrodlike PPY nanostructure was synthesized with the help of sodium cholate surfactant as soft template to detect dopamine in presence of ascorbic acid, uric acid and glucose in trace amounts. The rodlike PPY-Ag composite exhibits a low Rctvalue of 98.6 Ω which is obtained from impedance analysis. A large enhancement of peak current in CV experiment is observed in case of PPY-Ag composite modified electrode with respect to only rodlike PPY modified electrode. This composite modified electrode exhibits significant increase in sensitivity value of 8.22 mAμM−1 within the linear range of 12

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Fig. 17. SEM Images of a) E. coli without treatment, b) E. coli after treatment with rodlike PPY-Ag nanocomposite, c) S. aureus without treatment, d) S. aureus after treatment with rodlike PPY-Ag nanocomposite.

References

Helped in writing the biological part of this work and did the schematic presentation Author3: Dipak Rana: Involved in supervision of this project. Author4: Jyotishka Nath: Helped in synthesis Part Author5: Debatri Ghosh: Did cytotoxicity and Antimicrobial study Author6: Koushik Dutta: Did the XRD Author7: Subhadip Chakraborty: Helped to write the data analysis in sensing part Author8: Sanatan Chattopadhyay: Involved in supervision of Electrochemical Part Author9: Mukut Chakraborty: Involved in supervision of synthesis and biological part Author 10: Dipankar Chattopadhyay: Investigated the overall work

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Declaration of Competing Interest None.

Acknowledgements The first author A. Adhikari likes to thank the DST, West Bengal for her fellowship. S. De gratefully acknowledged DST-SERB, Govt. of India, for providing Post-Doctoral fellowship under NPDF Scheme. K. Dutta likes to thank DST, West Bengal, for fellowship. The authors would like to thank DST-FIST, Govt. of India, for funding towards instrumental support for the electrochemical studies. We also acknowledge the Center for Research in Nanoscience and Nanotechnology (CRNN), University of Calcutta, for providing instrumental facilities.

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