Ciprofloxacin immobilization

Copyright © 2014 American Scientific PublishersAll rights reservedPrinted in the United States of America

ArticleJournal of

Nanoscience and NanotechnologyVol. 14, 1–12, 2014www.aspbs.com/jnn

Simple Electro-Assisted Immobilization of Ciprofloxacinon Carbon Nanotube Modified Electrodes: Its Selective

Hydrogen Peroxide Electrocatalysis

Sundaram Sornambikai and Annamalai Senthil Kumar∗

Environmental and Analytical Chemistry Division, School of Advanced Sciences, Vellore Institute of Technology,Vellore 632014, India

Ciprofloxacin (Cf) is a synthetic fourth generation fluoroquinolone class antibiotic used for the treat-ment of gram-positive, gram-negative and mycobacterium species infections. Electrochemical char-acteristic of the Cf antibiotic on carbon nanotube modified glassy carbon electrode (GCE/CNT) inpH 7 phosphate buffer solution has been investigated. Electrochemically oxidized radical byproductof the Cf drug, which is formed as intermediate, gets immobilized on the GCE/CNT (GCE/Cf@CNT)and showed stable and well defined surface confined redox peak at −0.220 V versus Ag/AgCl.Control electrochemical experiment with unmodified GCE failed to show any such immobilizationand redox features. Physicochemical characterizations of the Cf@CNT by transmission electronmicroscope, scanning electron microscope, infrared spectroscopy, UV-Vis and gas chromatogra-phy coupled mass spectroscopic analyses of Cf@CNT collectively revealed presence of nativeform of the Cf antibiotic molecule onto the CNT. The interaction between the Cf molecule and theCNT tubes are revealed from the decreased intensity in the Raman spectrum. The GCE/Cf@CNTshowed excellent electrocatalytic response to hydrogen peroxide reduction reaction in pH 7 phos-phate buffer solution. Amperometric i–t analysis for the detection of H2O2 showed a current linearityplot upto [H2O2]= 200 �M at an applied potential −0.1 V versus Ag/AgCl with a current sensitivityvalue 678 �A mM−1 cm−2. No interferences were noticed with ascorbic acid, uric acid, cysteine andnitrite. The present study can be highly helpful to understand the interaction between the Cf andH2O2 in physiological systems and for the removal of Cf from the antibiotic polluted water samplesespecially in the aquaculture and agricultural systems.

Keywords: Ciprofloxacin, Immobilization, Carbon Nanotube Hybrids, Electro-Catalysis,Hydrogen Peroxide.

1. INTRODUCTIONThe immobilization of antibiotic on nanomaterials andtheir enhanced physical properties such as sustained drugrelease, stabilization of active form of the drug, welldefined electron-transfer behavior and enhanced microbi-ological function gained tremendous attention in recentyears.1–4 Recently, matrices such as carbon nanotubes(CNT),5–10 core shell silica, certain polymers and goldnanoparticles were found to be efficient biocompati-ble nanomaterials those have engrossed great interestin nanobiotechnology. These matrices possess generictransporting ability of the porous material and widerange of functional properties.1–3 Some of available

∗Author to whom correspondence should be addressed.

references for the preparation and applications of hybrid-antibiotic materials are; amoxicillin (AMX) immobilizedcarbon nanotubes for enhanced antimicrobial activity,4

ciprofloxacin (Cf) antibiotic incorporated-silica nanoshellfor enhanced antibacterial action against E.coli DH5x andL.lactis MG 1363,11 Cf doped electropolymerised polypyr-role outer shell layer with poly(3,4-ethylenedioxythioene)poly(styrenesulfonate) inner core fibre for electrical con-trolled release of Cf,12�13 Cf capped gold nanoparti-cle (Cf@Au)14 and in situ precipitation of calciumphosphate and Cf crystals during the chitosan hydro-gel formation15 for drug delivery application, gentamycinantibiotic loaded silica-hydroxyapatite matrix throughpoly(lactide-co-glucolide) scaffold microspheres for drugdelivery in bone replacement,16 Hydroxyapatite-Alginate

J. Nanosci. Nanotechnol. 2014, Vol. 14, No. xx 1533-4880/2014/14/001/012 doi:10.1166/jnn.2014.9358 1

Simple Electro-Assisted Immobilization of Ciprofloxacin on Carbon Nanotube Modified Electrodes Sornambikai and Senthil Kumar

nanocomposite as drug delivery matrix for sustainedrelease of Cf,17 polyester conjugated Cf and nor-floxacin (NOR) materials for drug delivery system18 andcytochrome C and myoglobin enzymes coupled multi-walled carbon nanotube (MWCNT)-Cf hybrid modifiedelectrodes as biosensors for hydrogen peroxide sensingat − 0.3 V versus Ag/AgCl in neutral pH.19�20 In thecase of references 19 and 20 for the preparation of theworking electrodes, a Cf dissolved in 0.1 M HCl, son-icated and dispersed with MWCNT was coated on aGCE surface and dried in oven at 60 �C for 30 minutes(electrode designated as GCE/MWCNT+Cf). Followingthat, the GCE/MWCNT+Cf was electrochemically treatedwith millimolar concentration of the respective enzymes,cytochrome C or myoglobin to form a GCE/MWCNT+Cf+ enzyme biocomposite chemically modified electrodefor electrocatalytic hydrogen peroxide reduction reaction.In the above enzyme modified systems, there were noH2O2 electrocatalysis noticed without the enzyme. Herein, we are reporting the Cf immobilized carbon nano-tube modified electrode prepared by direct electrochemicalmethod without any enzyme and linker for selective H2O2

sensing in a neutral pH.Cf is a synthetic fourth generation fluoroquinolone class

antibiotic used for both gram-positive and gram-negativeand some mycobacterium species infections in human andveterinary medicines.21–23 In specific, the Cf is used totreat infections of bones and joints, endocarditis, gas-troenteritis, malignant otitis externa, respiratory tract infec-tions, cellulitis, urinary tract infections, prostatitis, anthraxand chancroid.23�24 Apart from that, Cf is also a primarydegradation product of enrofloxacin antibiotic, which isused as a drug worldwide in aquaculture and agriculturalapplications.25�26 It is readily transported into the envi-ronment via discharges of wastewater and direct runoff.Note that the Cf has been repeatedly detected at �g/Lconcentrations in untreated hospital sewage.27 Hence theremoval or uptake procedure for Cf from the polluted envi-ronmental samples is of significant environmental researchinterest off-late. Recent research revealed that there isan intercalative interaction between the Cf and DNAwhich was detected using spectroscopy and voltammetrytechnique with a GCE/MWCNT electrode.21 MeanwhileCNTs are considered to be potential biomedical materialsbecause of their unique hollow structure and functional-ization property.27–32 Recently, few CNT-therapeutic drughybrid materials such as ampotericin B modified acid-functionalized multi walled carbon nanotube,33 anticancerdrug doxorubicin modified onto p-glycoprotein functional-ized SWCNT,34 sulfamethaxazole physisorbed functional-ized CNTs35 were reported. However, most of the existingpreparation methodologies were based on solution phasecovalent and physical immobilization approaches; wherethe stability and structural identity of the immobilizeddrugs are highly questionable. For instance, preparationof doxorubicin (DOX) immobilized single walled carbon

nanotube by solution stirring method overnight.36 Sincethe DOX was reported to be less stable in aqueoussolution,37–39 it is expected that the decomposed or dena-tured species of the DOX may get attached on the CNT.In this work, we have prepared Cf immobilized multi-walled carbon nanotube (designated as Cf@MWCNT) bya simple electrochemical approach. The hybrid materialwas characterized by cyclic voltammetry (CV), Attenuatedtotal internal reflectance/Infrared spectroscopy (ATR/IR),UV-Vis spectrophotometer, Transmission electron micro-scope (TEM) and Gas chromatography coupled mass spec-troscopic (GC-MS) techniques. We have identified that theantibiotic can be immobilized without any major structuralchange. Interestingly, the Cf@CNT hybrid system showsspecific and well defined electron-transfer behavior exam-ined by CV and it can mediate peroxide reduction with-out any enzyme, unlike to the previous cases with specificenzymes.19�20 Meanwhile, it has been reported that Cf canstimulate the H2O2 production in immature rabbit articularchondrocytes;40 but the interaction between the Cf or itsbyproduct and H2O2 in the pharmacodynamics has neverbeen reported in the literature. Hence, the present studymay provide some preliminary insight about interaction ofthe Cf and H2O2 and new electrochemical procedure toremove the Cf from polluted aqueous solutions too.

2. EXPERIMENTAL DETAILS2.1. Chemicals and ReagentsPristine-MWCNT (outer diameter: 10–15 nm; inner diam-eter: 2–6 nm; length 0.1–10 �m and 90% purity) andsingle-walled carbon nanotube (SWCNT: 1–1.5 nm diam-eter) were purchased from Sigma-Aldrich. Ciprofloxacinantibiotic sample was got from Dr. T. A. Lakshmipathy,Chennai. Cyclohexanol and piperazine were obtained fromSD fine chemicals, India. Other chemicals used in thiswork were all of ACS-certified reagent grade and usedwithout further purification. Screen-printed gold electrodes(AuSPE) were gifted by Zensor R&D, Taiwan. Aque-ous solutions were prepared using deionized and alkalineKMnO4 distilled water (designated as DD water). Unlessotherwise stated, pH 7 phosphate buffer solution (PBS) ofionic strength, I = 0�1 M was used as a supporting elec-trolyte in this work.

2.2. Apparatus and InstrumentationVoltammetric measurements were all carried out with CHIModel 660C electrochemical workstation (USA). A three-electrode system consists of glassy carbon or screen-printed substrate and its chemically modified electrodesof geometric area 0.0707 cm2 as a working electrode,Ag/AgCl as a reference electrode and platinum wire as anauxiliary electrode. The surface of the GCE was cleanedmechanically by polishing with 0.5 micron alumina pow-der, washing with DD water and sonicating for 5 minutesand finally by performing cyclic voltammetry (CV) for

2 J. Nanosci. Nanotechnol. 14, 1–12, 2014

Sornambikai and Senthil Kumar Simple Electro-Assisted Immobilization of Ciprofloxacin on Carbon Nanotube Modified Electrodes

10 cycles (n = 10, n = no. of cycles) in a potential win-dow of − 0.2 to 1.2 V versus Ag/AgCl at a potentialscan rate (v) of 50 mV/s in pH 7 PBS. For the nanopar-ticles characterization and surface morphology examina-tion, high resolution transmission electron microscope(HRTEM, Zeiss EM902A, Germany) and field emissionscanning electron microscope (FESEM, Carl Zeiss SMT,Ultra plus, Germany) instruments were used. Surface func-tional group analyses were done using an FTIR spec-troscopy with ATR mode (Jasco FTIR-460 Plus, Japan).The interaction between the MWCNT and the Cf wasanalysed through surface enhanced Raman Spectroscopywith AZILTRON, PRO 532 (USA) with 532 nm laserexcitation, UV-Vis analysis was done by using UV-VisNIR spectrophotometer (JASCO V-670, Japan). Gas chro-matography assisted mass spectroscopy (GC-MS, JEOLJMS-GC Matell, Germany) was used for the antibioticsstructure analysis.

2.3. Preparation of Various Cf@CNTModified Electrodes

Functionalized and purified MWCNT (f -MWCNT and p-MWCNT, where f = functionalized and p= purified) wereprepared by treating the pristine-MWCNT with concen-trated (13 N HNO3) and diluted (2 N HNO3) acid solutionsas per the literature procedure.41�42 MWCNT, f -MWCNT,p-MWCNT and SWCNT modified GCEs designated asGCE/MWCNT, GCE/f -MWCNT, GCE/p-MWCNT andGCE/SWCNT respectively were prepared by a followingcommon procedure: 3 �L of respective CNT dispersedin ethanol (2 mg/500 �L) was drop coated on the pre-treated GCE, and was dried in air for 15 minutes in roomtemperature. Since dissolved oxygen didn’t influence theelectrochemical response, experiments were all carried outwithout any deaeration as like physiological system. Thesurface coverage of the immobilized antibiotic drug, �Cf(nmol cm−2) was determined from the CV graph by inte-grating the anodic peak area (Qa� of the respective redoxpeak at v = 50 mV/s and calculated using the equation:�Cf = Qa/nFA, where no. of electrons (n = 1) and A isthe geometrical surface area (0.0707 cm2).

2.4. Sample Preparation Procedures forPhysicochemical Characterizations

For practical convenience, Cf@CNT samples forphysicochemical characterizations such as TEM,FESEM, surface enhanced Raman spectroscopy andATR/IR were prepared on gold screen-printed electrode(AuSPE/Cf@p-MWCNT) as per the procedure reportedin the Section 2.3. The AuSPE modified samples showqualitatively similar electrochemical pattern with that ofthe GCE modified electrodes (figures not enclosed). ForTEM characterization, the film particles from the aboveAuSPE/Cf@p-MWCNT electrode that was scratched outand suspended in 500 �L ethanolic solution, was used.

For the GC-MS study, the AuSPE/Cf@p-MWCNT soni-cated for few minutes in 500 �L of ethanol solution, wasfiltered using Nupore 0.22 �m micro-syringe filter andtaken for further analysis.

3. RESULTS AND DISCUSSION3.1. CV Investigation of Cf Antibiotic on GCE/CNTSTwenty continuous CV responses of a GCE with 1 mM Cfin a potential window of − 0.4 to 0.45 V versus Ag/AgClis displayed in Figure 1(A). Absence of any redox peakresponse in the figure indicates electro-inactive nature ofthe Cf molecule in the working potential window. Interest-ingly, the same experiment repeated using GCE/MWCNTas a working electrode showed well defined cathodic peakcurrent (ipc) response at − 0.40 V and its counter partas a feeble anodic peak current (ipa) at − 0.20 V vs.Ag/AgCl, in the 1st cycle of the CV (Fig. 1(B)). Uponcontinuous CV runs, the ipc decreases up to 10th cycleafter which a decline in the response was noticed; whilefor ipa case, sharp increase in the response up to 20thcycle was noticed. The growth in ipa as shown in theFigure 1(B) may suggest incorporation of Cf or its elec-trochemically oxidized or reduced byproduct/s within theGCE/MWCNT. As a control experiment, continuous CVof GCE/MWCNT in blank pH 7 PBS was examined inthe − 0.4 to 0.45 V versus Ag/AgCl (Fig. 1(B)). A fee-ble irreversible cathodic peak at − 0.45 V versus Ag/AgCl(ipc/ipa � 1), which may correspond to oxygen reductionreaction, was observed. The GCE/MWCNT response wasstable for multiple CV cycling. Concerned about the sta-bility of the CNT on the electrode surface, we expect that,there might be existence of some micro and nano-porousvoids on electrode surface (after the mechanical and elec-trochemical pretreatments), which when CNT was mod-ified as ethanol dispersion on the surface, some of theportions of CNT’s nano-walls may occupy the porous siteof the underlying surface and thereby stabilizing the CNTmatrix. After the CV experiments with the Cf drug, theGCE/MWCNT was washed with DD water, transferredto a blank pH 7 PBS and twenty continuous CV wasperformed. Interestingly, a well defined surface confinedredox peak with a peak-to-peak separation (�Ep = Epa−Epc), apparent standard electrode potential (Eo′ = Epa +Epc/2) and anodic/cathodic peak current ratio (ipa/ipc)values of 50± 5 mV, −220±5 mV versus Ag/AgCl and∼ 1 respectively, were obtained (Fig. 1(C)). The relativestandard deviation (RSD) value for last 10 cycles of theCV response was 2.1%; which evidence the appreciablestability of the surface confined peak with the Cf mod-ified GCE/MWCNT (designated as GCE/Cf@MWCNT)system. These observations suggest selective immobiliza-tion of the oxidized or reduced form of the Cf and on theMWCNT.Some of the surface confined Cf materials reported

in the literature are: Cf@SiO2 and Cf@nano gold

J. Nanosci. Nanotechnol. 14, 1–12, 2014 3


–0.4 0.0 0.4

–80

–40

0

40 A1

C1

(B)

1st20th cycle

(A)

E/V (vs Ag/AgCl) E/V (vs Ag/AgCl) E/V (vs Ag/AgCl)–0.4 0.0 0.4

i/µA

i/µA

i/µA

–0.4

0.0

0.4

only CNT

with Cf(C)

–0.4 0.0 0.4

–40

0

40 A1

C1

NNHN

OHFO O

Ciprof loxacin

1st

20th cycle

Figure 1. Continuous CV responses of (a) unmodified GCE and (b) GCE/MWCNT in 1 mM Cf (a), unmodified GCE/MWCNT in blank pH 7 PBS(b) and (c) GCE/Cf@MWCNT. Electrolyte= pH 7 PBS and scan rate= 50 mV/s. Inset (B) is the structure of Cf.

adsorbed gold electrode systems.11�14 Nevertheless, theCV of these reported systems showed irreversible,feeble and unstable oxidation peaks at about 0.8and 1.2 V versus Ag/AgCl that corresponds to thereduction of piperazinyl and pyridone moieties respec-tively in dimethyl sulphoxide+ tetrabutylammonium hex-aflurophosphate medium.4�43 Meanwhile, Cf stabilizedMWCNT colloidal solution was also reported,44 wherethe colloidal solution modified on a GCE surface showedabsence of any surface confined redox peak at ∼ 0 mVversus Ag/AgCl.44 Hence the new observation of a well-defined surface confined redox peak at − 220 mV versusAg/AgCl in this work is a unique observation.Initially, we speculated that the oxygen reduction

reaction response at − 0.45 V versus Ag/AgCl onGCE/MWCNT (Fig. 1(B)), may influence the Cf oxidationor reduction and for the immobilization process. In orderto verify the observation, we have repeated the experimentwith 20 min N2 purged pH 7 PBS solution. Qualitativesimilar CV pattern for the GCE/Cf@p-MWCNT formationwas noticed (data not enclosed). This observation ruled outthe possibility of the oxygen reduction reaction involve-ment in the Cf immobilization on the GCE/MWCNT.We also prepared the GCE/Cf@MWCNT by mix-

ture coating method. In this method Cf+MWCNT sus-pension in ethanol solution was coated on a cleanedGCE. Figure 2(a) is the typical CV response ofGCE/Cf@MWCNT. We have noticed a redox pattern atEo′ −0�220 V versus Ag/AgCl which is qualitatively sim-ilar to that of the electrochemical deposition method asdisplayed in the Figure 1(C). Though the mixture coatingmethod showed the peak current value 3 times lesser thanthe electrochemical deposition (Edep) method, the proce-dure is relatively easier, avoids the extra oxygen reduc-tion reaction complication at − 0.45 V versus Ag/AgCland allows rapid preparation of the GCE/[email protected], we have chosen the mixture coating method asoptimal method for further electrochemical studies.Apart from the pristine-MWCNT, other forms of CNTs

such as f -MWCNT, p-MWCNT and SWCNT were alsoexamined for the GCE/Cf@CNT hybrid formations bythe mixture coating preparation method (Fig. 2). It is

surprising that the SWCNT modified electrode failedto show any defined surface confined Cf redox peak(Fig. 2(b)). This particular observation suggests the needfor multiwalls for immobilization of the Cf. The MWCNThas relatively higher number of graphene sheets than theSWCNT which further enables more immobilization of theCf molecules onto interface. CV of the Cf@f -MWCNTshows a redox couple with huge background current sig-nal (Fig. 2(c)). The rich surface functional groups suchas carbonyl, phenolic and carboxylic sites present withinthe f -MWCNT,42 are the specific reason for this observa-tion. The Eo′ value of the GCE/Cf@f -MWCNT is −95±3 mV versus Ag/AgCl, which is 125 mV lesser thanthe Eo′ value observed with other Cf@CNTs (Fig. 2).This observation may indicate the change in immobiliza-tion mechanism, probably rather than the pi–pi interactionbetween aromatic carbons of the Cf and CNT’s sp2 car-bons (Scheme 1(F)), a covalent immobilization may occur(the >NH of piperazine interacts with >C O of thef -MWCNT) and hence reduction in the redox behavior.On the other hand, p-MWCNT shows well-defined and

E/V (vs Ag/AgCl)–0.4 0.0 0.4

–40

0

40

d. p-MWCNT

a.MWCNT

c. f-MWCNT

b. SWCNT

A1

C1

i/µA

Figure 2. Continuous CV response of different GCE/Cf@CNTs, pre-pared by the mixture coating procedures, in pH 7 PBS at v = 50 mV/s.a = GCE/Cf@MWCNT; b = GCE/Cf@SWCNT; c = GCE/Cf@f -MWCNT; d = GCE/Cf@p-MWCNT.



Scheme 1. Cartoon for the electrochemical oxidation of Cf on GCE/MWCNT and other Cf transformations.

sharp redox peak (Eo′ = −220± 3 mV versus Ag/AgCl)even better than the pristine-MWCNT-Cf hybrid electrode(Cf@MWCNT) system (Fig. 2(d)). Note that, since the>C O concentration on p-MWCNT is very less, cova-lent bonding of the Cf with p-MWCNT is not significantin this work. Calculated �Cf value with Cf@p-MWCNT is(7.2 nmol cm−2) about 25% higher than the Cf@MWCNT(pristine-MWCNT). It has been reported that the pristine-MWCNT contains some residual impurities such as nanometals (Fe, Co and Ni) and carbonaceous species.43 Fromthe above CV observations it can be concluded that theresidual impurities have some hindrance for the immobi-lization of Cf molecular species into the CNT, that’s whythe Cf@MWCNT sample would have showed about 25%lesser in the �Cf value than the Cf@p-MWCNT sample.

3.2. Physico-Chemical CharacterizationsATR/IR responses of AuSPE/Cf@p-MWCNT hybridmaterial in comparison with AuSPE, AuSPE/Cf(Cf/ethanol suspension drop coated and dried) andAuSPE/p-MWCNT were displayed in Figure 3. Asseen in the Figures 3(a)–(c), the AuSPE and AuSPE/p-MWCNT didn’t show any defined IR responses, whereasthe AuSPE/Cf sample gives marked IR responses as fol-lows: �max/cm

−1 1076 (–C–F),45 1650 (–COOH,46 weak),1714 (>C O,47 weak), 2354 (–C N)48 and 2933/2852(sp2/sp3 carbon).49 Figure 3(d) is the ATR/IR response ofthe Cf@p-MWCNT modified electrode, where IR peaks at�max/cm

−1 1055, 1641, 1991, 2323 and ∼ 2936 cm−1 werenoticed, as like AuSPE/Cf, but with ∼ 20–30 cm−1 shift inthe wave number. The data evidence immobilization of theCf antibiotic units within the MWCNT hybrid material,possibly due to the pi–pi interactions between the aromaticring of the Cf and sp2 hexagonal carbon on the MWCNT(Scheme 1(F)). Further TEM and SEM analyses of thehybrid samples will provide detailed surface morphologyand position of the drug present in the matrix.

Figure 4(A) is the TEM picture of the pristine-MWCNT,showing long nanosized tube like structures with few blackspots into the tube which is due to the impurities presentin the CNT. Figures 4(B)–(E) show some of the TEMand SEM photographs of the Cf@p-MWCNT at differ-ent magnifications. Clear black spots both on the sur-face (∼ 70 nm) and inner walls (continuous black spots)of the p-MWCNT were noticed in the TEM pictures forthe Cf@p-MWCNT sample (Figs. 4(B) and (C)). SEMof the same sample displayed in Figure 4(E), gives clear

Wavenumber/cm–1

1000200030004000

a

c

b

d

33663748

2933

2354 1076

6392

3232

0382

1055

2852v(C-F)

v(OH)

v(C-N)

26633973

sp2 and sp3 carbon stretching

1461

0961

-COO>C=O

0561

4171

Figure 3. ATR/IR responses of the (a) AuSPE, (b) AuSPE/p-MWCNT,(c) AuSPE/Cf (d) AuSPE/Cf@p-MWCNT.



Cf@p-MWCNT 50 nm Cf@p-MWCNTMWCNT50 nm 50 nm

(A) (B) (C)

(E)(D)

p-MWCNT100 nm 100 nm Cf@p-MWCNT

Figure 4. TEM pictures of (A) MWCNT, ((B) and (C)) Cf@p-MWCNT and SEM pictures of (D) p-MWCNT and (E) Cf@p-MWCNT. For the SEMthe underlying surface used is AuSPE.

white spots of average size 100 nm may corresponds tothe surface bound drug molecule on the CNT; whereasthe unmodified MWCNT (Fig. 4(D)) resulted in onlynanotube morphology without any cloudy white spots asin Figure 4(E). All these observations clearly evidencethe immobilization of the drug onto the CNT networkin this work. Recently, Sun et al. pointed out that theelectrified interface of the MWCNT electrode can act aselectrophoresis system and allow the transport of smallcharged species like lysozyme inside the walls.50 Hence,in the present case during the electrochemical preparationprocedure the electro-active Cf species might have beentransferred inside the MWCNT and settled in between thewalls of CNT. Hence, black spots on the Cf@p-MWCNTin the TEM (Fig. 4(C)) were observed.Concerned about the redox feature of the Cf@p-

MWCNT, we initially suspected that the presence ofoxidized forms of drug within the hybrid matrix maybe responsible for the electro-activity. In order to con-firm the incorporated species; we have examined theorganic extract of the hybrid materials by GC-MS anal-yses (Fig. 5(A)) in comparison with the unmodified Cfextract (Fig. 5(A)). Interestingly the results showed singleGC peak response of intensity ∼ 98% (Fig. 5(B)).In addition, the Cf and organic extract showed the fol-

lowing m/z fragmentation patterns: 306 (90%, M-C2H),288 (70, M-C2H–F), 245 (100, M-C2H–F–C2H5N) and 203(65, M-C2H–F–C2H5N–C2H4N). Corresponding chemicalstructures of the fragments are displayed in the Scheme 2,which is in agreement with that of the literatures.51�52

Though the m/z values were qualitatively similar, peak

intensity ratios’ observed are significantly different. It isexpected that fraction of organic impurity, may be fromMWCNT, might have combined with the organic extractof the hybrid and caused the deviation in the peak inten-sity of the Cf species. In order to confirm the observation,the above samples were subjected to parallel UV-Vis char-acterization as in Figure 6.Control sample from p-MWCNT + ethanol mixture,

sonicated and filtered using micro-syringe filter (Nupore0.22 �m), was also subjected to the UV-Vis examination(Fig. 6(c)). As can be seen the bare p-MWCNT-ethanolextract (Fig. 6(a)) shows three distinct absorbance peaks atwavelength values of 206, 225 and 279 nm. These peaksare assigned due to the presence of colloidal residues ofCNT as per the literature reports.53–55 Meanwhile, for Cfas such (Fig. 6(b)), the characteristic peaks are found at281 nm and 318 nm, which is in agreement with the lit-erature values.43 Further, for the Cf@p-MWCNT hybridmaterial case, mixture of the characteristic peaks for theindividual was observed. This observation authenticallyconfirms the immobilization of native form of the Cfantibiotic on the Cf@p-MWCNT hybrid material.Raman spectroscopic characterization results of screen-

printed gold (SPEAu)/Cf@p-MWCNT and unmodifiedSPEAu/p-MWCNT showed significant variations in theintensity ratios, ID/IG of the p-MWCNT’s D (defect-related mode) and G (graphitic hexagonal pinch mode)bands (Fig. 7).56–58 In general, graphitic materials displayspecific D and G bands at ∼ 1330 and 1570 cm−1 dueto the disordered (sp3 bonded sites) and ordered hexogancarbon sp2 structure respectively and the intensity ratio of



(A) Cf ( >99% by GC)

(B) Organic extract of the Cf@p-MWCNT hybrid (98% by GC)

Inte

nsity

Inte

nsity

m/z

Figure 5. Mass spectrum of (A) Ciprofloxacin (Cf) and (B) the organicextract of the Cf@p-MWCNT.

D and G bands, i.e., ID/IG can be taken as a measurefor the graphitic disorderness.57�58 Here, we observed ashift in the D and G bands towards the lower wavenum-bers and in addition, the ratio between the bands decreasedfor the Au/Cf@p-MWCNT. This observation reveals that,after the formation of Cf@p-MWCNT, there is an increasein the sp2 carbon strength on the hybrid material, whichmay be due to the Cf aromatic structure.56 The upshift and

O

N

F

NNH

O

HO

Ciprofloxacin

C17H18FN3O3Mol. Wt.: 331.3415

HN

F

NNH

O

m/z = 288

HN

F

N

O

m/z = 245

HN

FO

m/z = 203

O

N

F

NHNH2

O

HO

m/ z = 306

Scheme 2. Mass fragmentation pathway of the Ciprofloxacin (Cf).

Wavelength/nm200 400 600 800

Abs

/AU

206

225

276

318

281

0.2

0.2

0.2

b.

c.

a.

318

Figure 6. UV-Vis spectrum of ethanolic solutions of (a) unmodifiedp-MWCNT extract, (b) Cf and (c) Cf@p-MWCNT hybrid extract.

the alteration in the band ratio collectively confirms theinteraction between the Cf and p-MWCNTs.

3.3. Mechanism for the Incorporation and the RedoxFor the incorporation of the Cf into the CNT, either piper-azine or pyridone moiety get oxidized and subsequentlystabilizes within the CNT (Scheme 1). Cartoon for

ν/cm–1

1200 1400 1600 1800

Inte

nsity

200

250

300

350

400

D bandG band

ID/IG = 0.7048

ID/IG = 0.6504

a. p-MWCNT

b. Cf@p-MWCNT

Au@

Figure 7. Comparative surface enhanced Raman spectroscopyresponses of p-MWCNT (a) and Cf@P-MWCNT (b).



the electrochemical oxidation of Cf (for example) onGCE/MWCNT and other Cf transformations were given inthe Schemes 1(A)–(D). Initially, we expected that the alco-holic functional group (cyclohexanol derivative), whichis formed by the keto-enol tautomerism with the pyri-done unit (Scheme 1(D)), oxidizes and the product mayget attached on MWCNT. However, a control experi-ment for the electrochemical oxidation of cyclohexanol onGCE/MWCNT failed to show any redox behavior both incyclohexanol solution and after the electrochemical oxida-tion (Fig. 8(A)). Hence the option was ruled out. Alter-nately, we propose electrochemical oxidation of tertiaryamine (more basic) corresponding to the piperazine quater-nary ammonium unit �>N�··)-→>N�+·�−+1e−) or itschemically equilibrated ketamine with –C N(+)- groupmay get immobilized on the MWCNT as in Scheme 1(E).In order to confirm the proposal, electrochemical oxidationof piperazine on GCE/MWCNT was performed as a con-trol experiment as shown in Figure 8(B). Unexpectedly,the GCE/MWCNT showed feeble oxidation peak at 0.4 Vand an irreversible and ill defined peak at − 0.3 V ver-sus Ag/AgCl. This observation disproves the alternate pro-posal. Meanwhile, Thabaj et al. reported alkaline KMnO4

oxidation of the tertiary amine of Cf to its correspond-ing nitrogen radical species as an intermediate and sub-sequently to other oxidized products.59 Since the GC-MSand UV-Vis characterizations evidence the immobilizationof the native form of the Cf antibiotic within MWCNT,possibly a radical intermediate through piperazine groupresonance illustrated in the Scheme 1(B) may be gener-ated by the electro-oxidation of the fluoroquinolone (forexample Cf in the Scheme 1(B)) and might get stabi-lized within the MWCNT. This radical intermediate stabi-lized MWCNT might be involved in the redox behavior atabout − 0.2 V versus Ag/AgCl in this work. But for theGCE/MWCNT+Cf+ enzyme, the enzyme redox activesite is the responsible species for the H2O2 reduction.18�19

Schemes 1(B), (C) and (F) show typical electrochemical

–0.4 0.0 0.4–40

0

40

80

NH

HN

OH

–0.4 0.0 0.4–20

–10

0

10(A) (B)

E/V (vs Ag/AgCl) E/V (vs Ag/AgCl)

i/µA

i/µA

Figure 8. Control CV responses of (A) GCE/cyclohexanol@p-MWCNT and (B) GCE/piperazine@p-MWCNT, prepared by the mixture depositionmethod, in a blank pH 7 PBS for n= 20 and v = 50 mV/s. Insets are structures of the cyclohexanol and piperazine.

formation, stabilization and electrocatalytic reaction of theCf@MWCNT in this work.

3.4. Electrochemical Features of theGCE/Cf@p-MWCNT

Effect of scan rate (Fig. 9(A)) and solution pH(Fig. 10(A)) on the electron-transfer behaviors of theGCE/Cf@p-MWCNT were investigated individually. Sys-tematic increase in the peak currents against the scanrates was noticed. For the GCE/Cf@p-MWCNT case anadditional peak at ∼ 0.1 V versus Ag/AgCl appearedspecifically at high scan rates. The peak may be dueto the energetically different sites adsorbed Cf species(strained and misaligned areas) on the MWCNT. Base-line corrected anodic peak current values for the A1/C1and A2/C2 processes were plotted against the scan rateas double logarithmic plots (Fig. 9(B)). Calculated slope(= log ipa/ logv) values for the A1 and A2 peaks are1�07± 0�003 and 0�96± 0�003 respectively for the redoxprocesses and the corresponding linear equations are:log�ipa/�A� = 1�07± 0�002 · log�v/mV−1 s−1)− 1�38±0�005 (R = 0�9937) and log�ipa/�A� = 0�96± 0�0032 ·log�v mV−1 s−1)−0�30±0�0061 (R= 0�9899). In consid-eration with the ideal slope values of 1 and 0.5 respec-tively for the adsorption and diffusion controlled electron-transfer processes4 the electron-transfer behaviors with thepresent system can be ascribed as an adsorption controlledmechanism. From the plots of Epa versus pH (Fig. 10(B)),calculated slope (Epa/pH) values are −65�5± 2 and−64�9± 2 mV/pH respectively for the A2 and A1 redoxpeaks respectively. This observation indicates ideal Nern-stian type behavior of the redox system with involvementof equal numbers of protons and electrons.4

3.5. Electrocatalytic Feature of theGCE/Cf@p-MWCNT

Hyrdogen peroxide reduction reaction was taken as amodel system to examine the electrocatalytic feature of



E/V (vs Ag/AgCl)–0.4 0.0 0.4

–200

0

200A2

A1

C1

C2

log (v/mV s–1)

log

(i pa/

µA)

i/µA

1 2 3–1

0

1

2

3

(A1 peak: Slope = 1.07)

(A2 peak: Slope = 0.96)

(A) (B)

5 – 500 mV · s–1

Figure 9. CV response of GCE/Cf@p-MWCNT at different scan rates (A) and its corresponding plots of log ipa versus log scan rate (B).

the GCE/Cf@MWCNT in neutral pH. Figure 11(A) rep-resents the CV responses of GCE/Cf@p-MWCNT with-out (a) and with 100 �M H2O2 (b) at v = 10 mV/sin a pH 7 PBS. With H2O2, the electrodes showedprofound reduction response at about − 0.25 V versusAg/AgCl, which is about five times higher the cur-rent and about 300 mV reduction in the peak poten-tial than the respective drug unmodified electrode (i.e,GCE/p-MWCNT) as in Figure 11A(c). Note that theH2O2 reduction peak potential appeared is same withthe redox potential of the surface confined peak. Thisobservation indicates the eligibility for electro-catalysisof the incorporated Cf species for the H2O2 reductionreaction.Comparative amperometric current–time (i–t) sensing

behaviors of the GCE, GCE/p-MWCNT and GCE/Cf@p-MWCNT were demonstrated at an applied potential of− 0.1 V versus Ag/AgCl by continuously spiking 20 �Mof H2O2 in to 10 mL pH 7 PBS. Figure 11(B) showsthe typical amperometric responses of the hybrid elec-trode (c) in comparison with the respective GCE (a) andGCE/MWCNT (b) systems. Figure 11(C) is the corre-sponding plots of current versus H2O2 concentration of

E/V (vs Ag/AgCl)

–0.4 0.0 0.4 0.8

–40

0

40

pH

3 6 9

–300

0

300(A2 peak: Slope = –65.5)

(A) (B)

A1/C1

A2/C2

(A1 peak: Slope = –64.9)

pH 9-3

Epa

/mV

(vs

Ag/

AgC

l)

i/µA

Figure 10. CV response of GCE/Cf@p-MWCNT at different pHs (A) and its corresponding plots Epa versus pH (B).

the hybrid (c), GCE/MWCNT (b) and GCE (a). TheGCE/Cf@p-MWCNT hybrid electrode displayed currentlinearity up to 200 �M of H2O2 at an applied potentialof − 0.1 V versus Ag/AgCl. Obtained linear equation is i(�A) = 0�048± 0�002 [H2O2] (�M)+ 1�77± 0�002 (R =0�9882). Calculated surface area normalized current sensi-tivity value is 678 �A mM−1 cm−2. At the same time, thetwo control electrodes, GCE/p-MWCNT and GCE showsignificantly less amperometric signals at the operatingpotential. The analytical parameters obtained in this workis reasonably comparable with the myoglobin enzyme-Cf-MWCNT hybrid modified electrode for the H2O2 sens-ing up to 200 �M at a more negative applied potentialof − 0.327 V versus Ag/AgCl. However, current sensi-tivity of the present system (678 �A mM−1 cm−2) ismarkedly higher than the recently reported H2O2 chemicaland biochemical sensors as displayed in the Table I.60–70

Sensitivity value of the present system can be increasedfurther, if the applied potential is moved towards nega-tive side. Apart from that, the GCE/Cf@MWCNT hybridelectrodes show tolerable interferences to other biologicalcompounds; ascorbic acid (AA), uric acid (UA), cysteine(CySH) and nitrite (NO−

2 ) as shown in Figure 9(D). These



E/V (vs Ag/AgCl)

–0.4 0.0 0.4–200

–100

0

c.

b.

0 400 800 1200–20

–15

–10

–5

0

[H2O2] (mM)

0 100 200

0

4

8

12

b.

a.

a.

b.

c.

a.

c.

(A) (B)

t/s

0 400 800

–10

–5

0

(C)

50 mM each at –0.1 V

a. AA

b. UA

c. CySH

d. NO–2

e. H2O2

[H2O2] = 20 mM at –0.1 V

(D)a b c d e

t/s

i/µA

i/µA

i/µA

i/µA

Figure 11. CV responses of (A) GCE/Cf@p-MWCNT in the presence (b) and absence (a) of 100 �M H2O2 in a pH 7 PBS at a v = 10 mV/s.Curve c is the control H2O2 reduction on a GCE/MWCNT. (B) Amperometric i–t responses of GCE (a), GCE/p-MWCNT (b) and GCE/Cf@MWCNT(c) for successive spikes of 20 �M H2O2 at an applied potential of − 0.1 V versus Ag/AgCl in a pH 7 PBS under hydrodynamic condition (no. ofspikes = 10). (C) Corresponding calibration plots of the Amperometric i–t responses given in the figure (B). (D) Amperometric i–t response of theGCE/Cf@p-MWCNT with successive spikes of 50 �M of AA, UA, CySH, and NO−

2 along with H2O2.

observations attributes enzyme-less, sensitive and selec-tive sensing of H2O2 by the Cf@CNT hybrid modifiedelectrode in this work. On the whole, the GCE/Cf@p-MWCNT can provide efficient electrocatalytic reduction

Table I. Sensitivity values achieved by various chemically modified electrodes.

Chemically modified electrode pH Eapp/V Linear range Sensitivity (�A ·mM−1 · cm−2) Ref.

1 Au/HRP/PPy/SWCNT 6.8 −0�10 a� 0.93–4.93 mM 416�6 [60]2 Au/HRP/PAn/f -MWCNT 7.0 −0�35 a� 86 �M–10 mM 194�9 [61]3 Au/HRP/f -MWCNT 7.0 −0�35 a� 26 �M–8 mM 174�5 [61]4 GCE/HRP/PAn/MWCNT 7.4 −0�10 a� 0.2–19 �M 156�7 [62]5 Nanowire Au array electrode/HRP 7.0 −0�10 a� 0.74 �M–15 mM 45�9 [63]6 GCE/HRP/Methylene Blue 7.0 −0�30 a� 10 �M–1.2 mM 8�7 [64]7 GCE/GO/PB 6.0 0�10 b� 5 �M–1.2 mM 480�7 [65]8 GCE/Sol–gelAunano 7.0 −0�50 b� 2.5–45 �M 424�0 [66]9 GCE/Thionine/MWCNT 7.0 −0�30 a� 20 �M–3 mM 163�0 [67]10 CPE/PMo12-PPy 2.0 0�18 a� 0.2–30 mM 12�2 [68]11 �2-K7P2W17VO62/graphite/organoceramic 2.0 0�44 a� 0.1–20 mM 8�4 [69]

composite electrode12 GCE/Nf-CNT/Clay-Au 7.0 −0�30 a� 0.05–5.46 mM 4�0 [70]13 GCE/Cf@p-MWCNT 7.0 −0�10 a� 20–200 �M 678�0 This work

Notes: HRP-Horseradish peroxidase, PPy-polypyrrole, SWCNT-Single walled carbon nanotube, f -MWCNT-Functionalised multiwalled carbon nanotube, p-MWCNT-purifiedmultiwalled carbon nanotube, PAn-Polyaniline, GO-Graphene oxide, PB-Prussian blue, PMo12-Phosphomolybdate, CPE-Carbon paste electrode. a� = E versus Ag/AgCl;b�= E versus Saturated calomel electrode; Eapp = applied potential.

and sensing of hydrogen peroxide without any enzyme atlow working potential in the physiological solution unlikethe reports71�72 available with the Cf incorporated CNTmodified with enzymes.



4. CONCLUSIONSCiprofloxacin antibiotic was electrochemically immobi-lized specifically on a carbon nanotube modified electrodesurface and showed stable and well defined redox peakat −220± 5 mV versus Ag/AgCl in pH 7 PBS. No suchCf adsorption and electrochemical feature were observedon conventional unmodified glassy carbon surface. Physic-ochemical and electrochemical characterization of theCf@CNT chemically modified electrode by SEM, TEM,ATR/IR, UV-Vis and GC-MS collectively revealed immo-bilization of the Cf drug onto the CNTs. Amongst variousCNT viz., pristine-MWCNT, p-MWCNT, f -MWCNT andSWCNT, for the preparation of Cf@CNT by the mixturecoating way of preparation method, the Cf@p-MWCNTshowed best electrochemical redox feature. The graphenesheets in the MWCNT played a significant role in theuptake of the antibiotic molecule. In addition, the car-bonaceous and nano-metals impurities with the MWCNThad some hindrance for the immobilization of the Cfon the CNT. The redox peak has adsorption controlledelectron-transfer mechanism along with Nernstian typeof electrochemical response. The GCE/Cf@p-MWCNTelectrode showed electrocatalytic H2O2 reduction signalat − 0.250 V versus Ag/AgCl, where the surface con-fined redox peak exists, which is about 300 mV reductionin the over-potential and about five times higher in thepeak current over the GCE/MWCNT. Finally amperomet-ric i–t detection of the hydrogen peroxide was successfullydemonstrated without any interference from common bio-chemicals such as ascorbic acid, uric acid, cysteine andnitrite. Measured current sensitivity value was significantlyhigher over the several enzyme and non-enzyme basedamperometric sensors.

Acknowledgment: The authors thank the financial sup-port from Department of Science and Technology (DST),SERC, India and SAIF, IIT Madras for the GC-MS anal-yses. Sundaram Sornambikai thanks the Council of Sci-entific and Industrial Research (CSIR) for the SeniorResearch Fellowship. The authors also thank Dr. N. S.Karthikeyan for his valuable discussion on the GC-MSdata analyses.

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Received: 3 May 2013. Accepted: 14 June 2013.


Ciprofloxacin immobilization

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