Tetracycline Immobilization as Hydroquinone Derivative at Dissolved Oxygen Reduction Potential on Multiwalled Carbon Nanotube Annamalai Senthil Kumar, Sundaram Sornambikai, Shanmuganathan Venkatesan, Jen-Lin Chang and Jyh-Myng Zen J. Electrochem. Soc. 2012, Volume 159, Issue 11, Pages G137-G145. doi: 10.1149/2.061211jes Email alerting service Receive free email alerts when new articles cite this article - sign up in the box at the top right corner of the article or click here To subscribe to Journal of The Electrochemical Society go to: http://jes.ecsdl.org/subscriptions © 2012 The Electrochemical Society Journal of The Electrochemical Society, 159 (11) G137-G145 (2012) 0013-4651/2012/159(11)/G137/9/$28.00 © The Electrochemical Society G137 Tetracycline Immobilization as Hydroquinone Derivative at Dissolved Oxygen Reduction Potential on Multiwalled Carbon Nanotube Annamalai Senthil Kumar,a,z Sundaram Sornambikai,a Shanmuganathan Venkatesan,b Jen-Lin Chang,b and Jyh-Myng Zenb,z a Environmental and Analytical Chemistry Division, School of Advanced Sciences, Vellore Institute of Technology University, Vellore 632 014, India b Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan Upon continuous potential cycling of multiwalled carbon nanotube modified electrode (GCE/MWCNT) with Tetracycline antibiotic (Tet) at −0.5 to 0.4 V vs Ag/AgCl in pH 7 phosphate buffer solution, the Tet drug gets selectively immobilized as Tet-hydroquinone derivative (Tet-HQ) on the GCE/MWCNT (GCE/Tet-HQ@MWCNT) and showed a specific surface confined redox peak at E1/2 = −0.24 ± 0.02 V vs Ag/AgCl. Control potential cycling experiment with o-cresol resulted to similar electrochemical characteristic too. But with p-cresol, no such surface confined redox peak was noticed. Dissolved oxygen reduction to hydrogen peroxide (as an intermediate species) at −0.45 V vs Ag/AgCl and its chemical oxidation of the surface bound Tet@MWCNT to Tet-HQ@MWCNT is proposed as a plausible mechanism. Separate ring-disk screen-printed carbon electrode assembly, where MWCNT and a H2 O2 detection catalyst (nano-MnO2 ) modified on the ring and disk respectively, coupled with flow injection analysis showed specific current signals for oxygen reduction reaction at −0.45 V vs Ag/AgCl on the disk and subsequent H2 O2 oxidation on ring at 0.8 V vs Ag/AgCl. The surface confined redox system showed highly selective electrocatalytic reduction signal to hydrogen peroxide at ∼0.22 V vs Ag/AgCl without any interference from the ascorbic acid, uric acid, cysteine and nitrite. © 2012 The Electrochemical Society. [DOI: 10.1149/2.061211jes] All rights reserved. Manuscript submitted May 17, 2012; revised manuscript received July 17, 2012. Published September 14, 2012. Tetracycline (Tet) is a broad-spectrum antibiotic, showing activity against a wide range of gram-positive and gram-negative bacteria.1 The favorable antimicrobial property and absence of major adverse side effects have led the drug to extensive use in the therapy of human and animal infections. However, since the Tet exhibits appreciable complexation property with some metals like Al, Mg, Ca, Zn and Fe,2–4 for human, it is advised not to intake the drug along with the metal/s based supplementary drugs or foods.1 These agents bind with the Tet in the intestine and reduce its absorption into the body.1 Concerned about the mode of drug action, it is well established that Tet inhibits bacterial protein synthesis by preventing the association of aminoacylt-RNA with the bacterial ribosome.1,5 Meanwhile, as a veterinary medicine, Tet is also used for the treatment of infections in poultry, cattle, sheep, and swine.3 In some cases, e.g., for therapeutic treatment of large numbers of poultry reared on commercial farms, the antibiotic is administered directly to the feed or water.3 Note that in U.S., the annual consumption of Tet in swine and poultry husbandry in the late 1990s reached 2.3 and 0.63 million kilograms, respectively.6 Because of the extensive usage, the Tet and its byproducts were readily transported into the environment via discharge of wastewater and direct runoff, and in turn contaminating the land surface and ground water.7 Tet concentration ∼µg/L was continuously detected in the animal waste water and surface water near farms.7 In order to remove the polluted Tet antibiotic, several adsorbents such as zeolite,8 clay,9 activated sludge/carbon,10,11 titania-silica composite12 and carbon nanotubes (CNTs),6,13–15 were used. Herein, we are demonstrating a new electro-assisted methodology for the rapid uptake of Tet drug from the aqueous solution as Tet-hydroquinone (HQ) derivative using multiwalled carbon nanotube (MWCNT) modified electrode. CNT is an excellent sorbent for the adsorption of aromatic organic molecules.16 The ability to form strong pi-pi interaction between the sp2 carbon of the CNT and aromatic electrons of the organic molecules led the CNT as a superior adsorbent over the activated carbon. In 2007, Suarez et al first demonstrated the adsorption of Tet on CNT, for solid phase extraction capillary electrophoresis based mass spectroscopic Tet detection purpose.17 Couple of years later, Ji et al studied the comparative solution phase adsorption behavior of the Tet on different carbon materials; carbon nanotubes, graphene and activated charcoal materials,18 and they have concluded the order of adsorption behavior as graphite∼ =single walled carbon nanotube (SWCNT) > multiwalled z E-mail: askumarchem@yahoo.com; jmzen@dragon.nchu.edu.tw carbon nanotube (MWCNT) ≫ activated carbon. Chemically purified SWCNT (p-SWCNT, p = purified) and MWCNT (p-MWCNT) were used for the purpose. Following the above observation, recently, several research articles were published relating to the solution phase interaction between the Tet and CNTs.13–15 Meanwhile, few CNT modified electrodes were demonstrated for the electrochemical oxidative detection at +0.5 ± 0.1 V vs Ag/AgCl,19–21 possibly due to the Tet’s phenol functional group oxidation, in pH 7 phosphate buffer solution. Recently, our group demonstrated a new work on the electrochemical oxidation of phenol and o-cresol at +0.5 ± 0.1 V vs Ag/AgCl in pH 7 phosphate buffer solution and subsequent immobilization as hydroquinone (HQ) and HQ-derivative respectively on the MWCNT modified glassy carbon electrode (GCE/HQ@MWCNT) (Scheme 1, Case-1).22 Similarly, a phenolic group containing β-lactum anitibiotic, amoxicillin was also selectively immobilized on the CNT modified GCE through the phenoxy radical path way by us earlier.23 In contrast, in this work, we have observed immobilization of a phenolic functional group containing antibiotic, Tetracycline (Tet) to HQ derivative (Tet-HQ) at negative operating potential, −0.45 V vs Ag/AgCl in pH 7 PBS; where specific dissolved oxygen reduction takes place at the underlying surface (Scheme 1, Case-II). We also confirmed the HQ formation by performing several control electrochemical experiments with substituted phenols, like o-cresol, p-cresol and butylated hydroxy toluene at the negative operating potential. Interestingly, the GCE/TetHQ@MWCNT shows well-defined surface confined peak at −0.24 ± 0.02 V vs Ag/AgCl and mediate the hydrogen peroxide reduction reaction selectively. Experimental Chemicals, reagents and instrumentation.— Pristine MWCNT (outer diameter: 10–15 nm; inner diameter: 2–6 nm; length 0.1–10 µm and 90% purity) and single-walled carbon nanotube (SWCNT: 1–1.5 nm diameter) were purchased from Sigma-Aldrich. Tetracycline (Life Science Pvt.Ltd., India), as individual capsules with a labeled content value 250 mg was got from Doctor T. A. Lakshmipathy, Chennai. Manufacturer for the drug is Life Science Pvt.Ltd., India. Weight of the Drug in the capsule is 280 ± 10 mg. The rest ∼30 mg weight is due to added excipients. In our previous comparative cyclic voltammetric studies of amoxicillin as pure drug and capsule, it was confirmed that the execipient in the drug didn’t have any specific role on the electrochemical activity on the MWCNT modified G138 Journal of The Electrochemical Society, 159 (11) G137-G145 (2012) Scheme 1. Control experiments on the electrochemical oxidation of phenolic derivatives; o-cresol and p-cresol at +0.45 V vs Ag/AgCl (case-I, [22]) or −0.45 V vs Ag/AgCl (case-II, in this work) in pH 7 PBS. Case-I reaction follows electro-generated phenoxy radical as an intermediate combined with H2 O addition reaction, where case-II sequence contains electro-generated H2 O2 intermediate followed and its reaction with MWCNT surface bound o-cresol, p-cresol and butylated hydroxy toluene. electrode.23 Phenol, p-cresol, o-cresol and butylated hydroxy toluene were obtained from the SD fine chemicals, India. Other chemicals used in this work were all of ACS-certified reagent grade and used without further purification. Screen-printed gold electrodes were gifted by Zensor R&D, Taiwan. The ring and disk at the screen-printed ring disk electrode (SPRDE) separated by a gap of 0.5 mm with a geometric area of 3.14 and 7.07 mm2 , respectively obtained from Zensor R&D, Taiwan was used for the mechanistic studies. Aqueous solutions were prepared using deionized and alkaline KMnO4 distilled water (designated as DD water). Unless otherwise stated, non de-aerated pH 7 phosphate buffer solution (PBS) of ionic strength, I = 0.1 M was used as supporting electrolyte in this work. Total dissolved oxygen content in the electrolyte was measured using portable dissolved oxygen meter, Hanna Instrument (HI 9142), USA and the value is 8.3 ± 0.2 ppm. Voltammetric measurements were all carried out with CHI Model 660C electrochemical workstation (USA). The three-electrode system consists of glassy carbon or screen-printed substrate and its chemically modified electrode as working electrodes of geometric area 0.0707 cm2 , Ag/AgCl as a reference electrode and platinum wire as the auxiliary electrode. The surface of the GCE was cleaned mechanically by polishing with 0.5 micron alumina powder, washing with DD water and sonicating for 5 minutes followed by electrochemical cleaning by performing cyclic voltammetry (CV) for 10 cycles (n = 10, n = no. of cycles) in the potential window of −0.2 V to 1.2 V vs Ag/AgCl at a potential scan rate (v) of 50 mV.s−1 in pH 7 PBS. For the nanoparticle characterization and surface morphology examinations, high resolution transmission electron microscope (HRTEM, Zeiss EM902A, Germany) instrument was used. CNT chemically modified electrode preparations.— Functionalized MWCNT (f-MWCNT, where f = functionalized) sample was prepared by treating the pristine MWCNT with concentrated (13 N HNO3 ) as per the literature procedure.24 X-ray photoelectron spec- troscopy (Ulvac-PHI, PHI500, Versaprobe) of the f-MWCNT powder sample showed negligible signals at 708.6 eV due to Fe2p3/2 signal (data not enclosed). At the same time pristine-MWCNT showed marked XPS signal for iron impurity. Thermogravimetric analysis (Perkin-Elmer Pyris 1 TGA, USA) of the sample (in O2 atmosphere at a rate 10◦ C/min.) showed 7.1% residual impurities at 800◦ C due to Fe2 O3 (data not included). Removal of metal impurities from the MWCNT without activating the carbon nanotube was done similar to the earlier work by Compton group,25 where the commercial MWCNTs were stirred with 2 M dilute nitric acid for 35 h at room temperature and washed thoroughly with DD water yielding a highly purified MWCNT, designated as p-MWCNT in this work. Pristine MWCNT, f-MWCNT, p-MWCNT and SWCNT modified GCEs designated as GCE/MWCNT, GCE/f-MWCNT, GCE/p-MWCNT and GCE/SWCNT respectively were prepared by a following common procedure: 3 µL of respective CNT dispersed in ethanol (2 mg/ 500 µL) was drop coated on the pretreated GCE, and dried in air for 15 min. in room temperature. The GCE/CNT was then pretreated electrochemically by performing continuous CV for twenty cycles (n = 20) at v = 50 mV.s−1 in the blank pH 7 PBS in the potential window of −0.6 to 0.7 V vs Ag/AgCl with normal dissolved oxygen. The Tet antibiotic immobilized GCE/CNT electrodes were prepared by potential cycling method in the potential window −0.5 to 0.6 V vs Ag/AgCl in pH 7 PBS at a scan rate of 50 mV.s−1 (n = 20). The surface coverage of the Tet, Ŵ Tet (nmol.cm−2 ) immobilized on CNT was determined from the CV graph by integrating the anodic peak area (Qa ) of respective redox peak at v = 50 mV.s−1 and calculated using the equation: Ŵ Tet = Qa /nFA, where n = no. of electrons (n = 1) and A = geometrical surface area. Sample preparation procedures for the physico-chemical characterizations.— For practical convenience, samples for FTIR and TEM physicochemical characterizations were carried out using a Tet Journal of The Electrochemical Society, 159 (11) G137-G145 (2012) A. 4 C. B. GCE with Tet: 100 Electrochem. method: a. 100 50 50 A1 A2 i/µA i/µA i/µA 2 0 G139 0 GCE/Tet@MWCNT: A1 A2 A1' 0 b. -2 C2 -50 -4 C2 -50 C1 C1' -100 -0.4 0.0 0.4 E vs Ag/AgCl/V -0.4 0.0 0.4 E vs Ag/AgCl/V -0.4 C1 0.0 0.4 E vs Ag/AgCl/V Figure 1. Continuous CV responses of (A) GCE and (B) GCE/MWCNT (a) in 1 mM Tetracycline antibiotic dissolved pH 7 PBS and (b) GCE/MWCNT and (C) GCE/Tet@MWCNT in blank pH 7 PBS. Scan rate = 50 mV.s−1 . immobilized MWCNT modified gold screen-printed electrode, which was prepared by the electrochemical deposition method as mentioned in the previous section. The film was scratched out and subjected to further analysis. The Tet@MWCNT modified screen-printed electrodes showed qualitatively similar electrochemical pattern with that of the GCE/Tet@MWCNT (data not shown). Flow injection analysis of the mechanistic studies.— Flow injection analysis (FIA) coupled electrochemical detector unit compatible to SPRDE) shown in Fig. 5A was used for the oxygen reduction reaction (ORR) at negative operating potential (−0.45 V vs Ag/AgCl). For the ORR mechanistic studies, the MWCNT was coated on the disk part by droplet evaporation method. A hydrogen peroxide catalyst, nano-MnO2 was electro-deposited on the SPRDE ring electrode by cycling in the potential between 0 and 0.4 V at 20 mV.s−1 for 20 cycles in 0.1 M Na2 SO4 solution containing 0.1M Mn(II) (CH3 COO)2 as per the reported literature.26 The modified electrodes were kept at potentials, −0.45 V and +0.8 V vs Ag/AgCl respectively on the disk and ring parts corresponding to the ORR and in turn to the selective intermediate H2 O2 oxidative detection. The ORR and H2 O2 detection currents were monitored simultaneously using a bipotentiostat technique (CHI 627 electrochemical workstation (Austin, TX, USA)).27 Results and Discussion Electrochemical immobilization of Tet on CNT.— Fig. 1A shows the continuous cyclic voltammetry (CV) response (n = 20) of bare GCE in 1 mM Tet in the potential window −0.5 to 0.6 V vs Ag/AgCl in 0.1 M pH 7 PBS solution at v = 50 mV.s−1 , where no redox peak response was observed indicates the drug as such is electro-inactive on the GCE surface. Interestingly, GCE/MWCNT with 1 mM of Tet, there was growth of couple of redox peaks at apparent equilibrium potentials (E1/2 = Epa +Epc /2) −0.24 ± 0.02 (A1/C1) and 0.3 ± 0.01 (A2/C2) V vs Ag/AgCl as in the Fig. 1B, curve a. The increase in the peak current responses against the increase in the CV run indicates immobilization of the Tet drug or its by-product on the GCE/MWCNT system. After the experiment with Tet, the GCE/MWCNT was washed with deionized distilled water, transferred to a blank pH 7 PBS and twenty continuous CV was performed again. Fig. 1C is the CV response of the electrode. The redox peak at −0.24 ± 0.02 V vs Ag/AgCl (A1/C1) was retained on the surface with peak-to-peak separation, Ep = Epa Epc and Ŵ Tet values 43 mV and 1.82 nmol.cm−2 respectively. Apart from the A1/C1, feeble redox peaks at −0.4 (A1′ /C1′ ) and +0.35 V (A2/C2) vs Ag/AgCl were also noticed, which are assumed to be energetically different CNT-surface immobilized species.28 These redox species (A1′ /C1′ and A2/C2) responses are very feeble (in terms of its surface excess and peak current values) and they didn’t show any involvement in the H2 O2 electrocatalysis (see Section 3.4). We suspect fraction of the Tet-Phenolic species, which are adsorbed on the ener- getically different CNT sites, might have involved with phenoxy radical oxidation pathway reaction as illustrated in the Scheme 1, case-I, and resulted to energetically different Tet-HQ@MWCNT product. The strained and misaligned areas within the CNT are considered to be energetically different sites.28 However, it is not stable for further electrochemical measurements (see Sections 3.2−3.4). Similarly, the appearance of the A1′ /C1′ pre-peak is due to the fraction of Tet-HQ product species formed, through the H2 O2 mechanism as mentioned in the Scheme 1, case-II, on the strained and misaligned areas. In the Fig. 1C, the response of modified electrode in pure supporting electrolyte shows an incremental raise in the corresponding anodic and cathodic parts of the peaks. It is an indication that the surface confined mediator, i.e., Tet-HQ is not perfectly surface confined and it seems major part of the Tet-HQ immobilized on the MWCNTs are just entrapped into their pores as freely swimming moieties present in the supporting electrolyte consumed by nanotubes. That is why our FTIR spectrum recorded for Tet@MWCNT measured with anhydrous KBr pellet shows a huge OH peak (corresponds to water molecules) around 3395 cm−1 which was neither present originally in Tetracycline nor in MWCNT sample (see supplementary section).29 The influence of various CNTs such as the SWCNT, f-MWCNT and p-MWCNT for the Tet-HQ@CNT hybrid formation was examined (Supplementary information Fig. S1, curves b-d).29 For that, the respective CNT modified GCEs were subjected to electrochemical immobilization as mentioned above. Except SWCNT, other MWCNTs showed qualitatively similar electrochemical features for the immobilization of the Tet. The SWCNT modified electrode displayed negligible current response (Fig. S1, curve b).29 The observations depict requirement of multiwalled structure of CNT for the Tet immobilization. Possibly, the immobilized species might have staged in between the multiwalls of the CNT. The order of the redox peak response with respect to the peak current is f-MWCNT>p-MWCNT>MWCNT (pristine)>SWCNT. It has been reported that functionalization of the CNT resulted to oxygen rich groups such as carbonyl (>C=O), carboxyl (–COO–), phenolic (>C–OH) and hydroxy (–OH) on the surface.30,31 It is expected that the oxygen rich functional groups may have some positive interaction with the Tet’s carbonyl, hydroxy and amide functional groups through hydrophobic-hydrophilic and hydrogen bonding, and hence the f-MWCNT can hold larger amount of immobilized species on the hybrid surface. Note that the Tet@pMWCNT hybrid electrode showed relatively higher current signal over the pristine impure MWCNT-Tet system (Supplementary information Fig. S1, curves a and b).29 From this information, it can be concluded that the impurities such as nanometals and carbonaceous species present in the pristine MWCNT,30 have some hindrance for the incorporation of the Tet or its by-product inside the walls of the pristine MWCNT. Nevertheless, in consideration with omission of chemical treatment and qualitative similarity in the electrochemical response with other form of the CNTs, pristine MWCNT can be taken G140 Journal of The Electrochemical Society, 159 (11) G137-G145 (2012) on the Tet-HQ@MWCNT are compared, the Tet’s IR features were relatively less defined than the pristine MWCNT’s response. Possibly the amount of Tet or its by-product loaded might be relatively less and most of the immobilized species would be occupied in the innerwalls of the pristine MWCNT, and hence ill defined Tet response with the sample observed. In order to further confirm the expectation, the TetHQ@MWCNT sample was subjected to TEM studies. Figs. 2A & 2B show the comparative TEM photographs of the unmodified MWCNT (2A) and Tet-HQ@MWCNT hybrid system (2B). Empty carbon nanotube walls could be seen in the Fig. 2A; while for the Tet-HQ@MWCNT case, intense dark spots, mostly in the inner walls of the pristine MWCNT was noticed (Fig. 2B). This observation particularly evidences the predominant immobilization of the Tet drug species within the walls of the CNTs. Recently, Sun et al pointed out that the electrified interface of the MWCNT electrode can act as electrophoresis system and allow the transport of small charged species like lysozyme inside the walls.34 Hence, in the present case, during the electrochemical preparation, some electro-active Tet species might have been transferred inside the MWCNT and settled in between the walls of CNT as encapsulated species (Fig. 2B). A. MWCNT Impurity 50 nm B. Tet-HQ@MWCNT 50 nm Figure 2. TEM photographs of (A) pristine MWCNT and (B) TetHQ@MWCNT hybrid system. as model system for further electrochemical studies. To the best of our knowledge, no such well-defined and reversible type surface confined redox peak was ever reported in the literature earlier for the Tet antibiotic case. Physico-chemical characterizations of the Tet-HQ@MWCNT.— FTIR/KBr spectrum of the Tet-HQ@MWCNT in comparison with the controls, Tet and the MWCNT were carried out and displayed in supporting information Fig. S2.29 The Tet-HQ@MWCNT hybrid material shows predominant peaks at 1000–1160 cm−1 for the C–O, 1386 cm−1 for the C–OH, 1650 cm−1 for the carbonyl, 2930 cm−1 for the aromatic CH and NH peaks and 3395 cm−1 for the –OH stretching peaks.32,33 When the IR responses due to MWCNT and Tet individuals A. C. B. 20 GCE/MWCNT with Tet: b. 20 GCE/MWCNT with Tet: GCE/MWCNT: a. 0 i/µA 0 -20 0 d. c. a. i/µA 20 i/µA Mechanism for the Tet immobilization on the MWCNT.— Amongst various Tet’s functional groups, the phenolic site is expected to be specifically involved in the electrochemical reaction and to some by-product/s formation. In fact, in our previous preliminary study for the phenol electrochemical oxidation on the GCE/MWCNT, we have noticed an irreversible electrochemical oxidation peak at 0.5 ±0.1 V vs Ag/AgCl due to phenoxy radical formation, which was followed by a growth of reversible surface confined redox peak at 0.3 ± 0.05 V vs Ag/AgCl corresponding to hydroquinone formation in pH 7 PBS (Scheme 1, case-I).22 Initially, we expected the reaction mechanism similar to the above mentioned case, because of the presence of phenolic function group on the tetracycline. In aim to probe further, the GCE/MWCNT was subjected to electrochemical immobilization of Tet at restricted potential windows with starting potential (Estart ) at −0.6 and swept to 0.4 (Fig. 3A curve a) and 0 V vs Ag/AgCl (Fig. 3A, curve b) in pH 7 PBS. The reason for choosing end potential in two different positive windows, 0.4 and 0 V vs Ag/AgCl are to observe the formation and absence of the phenoxy radical intermediate, respectively. Unexpectedly, qualitatively similar redox peaks in both the cases were noticed. Thus, involvement of phenoxy radical formation pathway mechanism, as ref no. 22, was ruled-out. Possibly twisted crystal structure of the Tet and restriction in the adsorption of phenolic site on MWCNT surface, unlike to the naked phenol, is the reason for the failure. This specific observation also suggests the formation of some other intermediate species at negative potential, which might be responsible for b. a. -20 -20 H2O2 Estart Estart H2O/O2 (dissolved) -40 -0.4 0.0 E vs Ag/AgCl/V 0.4 -0.4 -0.2 0.0 E vs Ag/AgCl/V -0.4 0.0 0.4 E vs Ag/AgCl/V Figure 3. Continuous CV responses of (A and B) GCE/Tet@MWCNTs at different potential windows and (C) GCE/MWCNT in the potential window of −0.6 to +0.6 V vs Ag/AgCl. All in blank pH 7 PBS at scan rate = 50 mV.s−1 . Journal of The Electrochemical Society, 159 (11) G137-G145 (2012) G141 Scheme 2. Cartoon for the electrochemical immobilization of Tetracycline as HQ derivative (Tet-HQ) on the MWCNT at −0.45 V vs Ag/AgCl in pH 7 PBS through hydrogen peroxide intermediate reaction. A1/C1 redox behavior. In order to get more detail about the mechanism, we have performed additional experiments on electrochemical immobilization of the Tet on GCE/MWCNT at varied negative potential regions (Fig. 3B). In this case, by fixing the starting potential at +0.2 V vs Ag/AgCl, where there is no phenoxy radical complication, the final potentials were varied as −0.3(a), −0.35 (b), −0.40 (c) and −0.45 V (curve d) vs Ag/AgCl (Figs. 3B). As can be seen in the figure, when the potential was swept from 0.2 to −0.3 (a) and 0.2 to −0.35 V (b), there were no sign for the formation of the A1/C1 redox peak. Whereas for case c, a slight appearance and if further moved to −0.45 V (case d) a profound formation of A1/C1 redox peak at −0.2 V were noticed. Meanwhile, as a control experiment, unmodified GCE/MWCNT was also subjected to CV in the negative potential region in blank pH 7 PBS with normal dissolved oxygen (8.3 ± 0.2 ppm) as in Fig. 3C. Interestingly, large oxygen reduction signal at −0.45 V vs Ag/AgCl was observed due to oxygen reduction reaction (ORR) with significant amount of hydrogen peroxide and peroxy radicals (• OH) as reaction product/s.35,36 These observations suggests the formation of H2 O2 intermediate species which is responsible for the in-situ oxidation of the surface bound Tet@MWCNT to Tet-HQ@MWCNT (Scheme 2B) (i.e., A1/C1 redox peak). In order to further confirm the involvement of H2 O2 intermediate species through ORR at negative operation potential, separate qualitative electrochemical flow injection analyzes (FIA) using screenprinted ring-disk electrode (Fig. 4A and 4B) was carried out, as per the reported procedure.26,27 In the disk part, the MWCNT was coated along with 0.1% Nafion, as an over layer. The Nafion over-layer doesn’t have any electrochemical significance in the ORR. It can be used to protect the underlying MWCNT in hydrodynamic electrolyte condition. The ring part is covered with nano-MnO2 catalyst, which is selective for the H2 O2 electrocatalytic oxidative detection at 0.8 V vs Ag/AgCl in pH 7 PBS (without any interference from dissolved oxygen).26,27 During the FIA measurements, N2 purged pH 7 PBS was circulated and oxygen saturated ([O2 ] = 12 ± 0.5 ppm) pH 7 PBS was injected. As proposed earlier, specific reduction signal at disk (−0.45 V) and oxidation signal at ring (0.8 V vs Ag/AgCl), will be expected corresponding to ORR and subsequent oxidative detection of H2 O2 product respectively, in this work. Fig. 4C is the typical FIA responses of the disk (A) and ring (B) under a hydrodynamic condition. As expected, significant FIA signals were noticed both on the disk and ring parts simultaneously, when oxygen saturated pH 7 PBS was injected in the system. Control FIA experiment with injection of N2 Figure 4. (A) Photograph of a flow injection analysis (FIA) coupled electrochemical detector unit compatible to screen-printed ring-disk electrode (SPRDE), (B) Scheme of the H2 O2 oxidation and oxygen reduction reaction (ORR) at the ring and disk electrodes respectively and (C) FIA graphs of H2 O2 oxidation at nano MnO2 catalyst modified ring at an operating potential 0.8 V vs Ag/AgCl and ORR at the SPRDE/CNT coated with 0.1% Nafion disk detected at −0.45 V vs Ag/AgCl. G142 Journal of The Electrochemical Society, 159 (11) G137-G145 (2012) B. A. OH C. OH OH CH3 20 OH CH3 20 @MWCNT c. -20 Estart 0 i/µA i/µA i/µA 0 c. b. a. e. CH3 CH3 CH3 0 -20 No reaction @MWCNT OH d. CH3 20 OH @MWCNT f. H3C OH f. e. d. b. a. -20 Estart e. Estart -40 -0.4 0.0 E vs Ag/AgCl/V 0.4 -0.4 0.0 0.4 E vs Ag/AgCl/V -0.4 0.0 0.4 E vs Ag/AgCl/V Figure 5. CV responses of (A) GCE/o-cresol@MWCNT, (B) GCE/p-cresol@MWCNT and (C) GCE/butylated hydroxy toluene@MWCNT in different potential windows in pH 7 PBS at a scan rate of 50 mV.s−1 . saturated pH 7 PBS failed to show any current signals. These observations confirm the intermediate H2 O2 formation on the unmodified GCE/MWCNT at negative operative potential, −0.45 V vs Ag/AgCl and this species helps in the in-situ oxidation of surface bound Tet (phenol) to Tet-dihydroxy product. However, at this stage, it is difficult to conclude which isomeric form of the dihydroxy by-product, i.e., HQ or catechol derivative, get involved in the immobilization process. Additional control experiments were carried out using methyl substituted phenols; ortho-methyl phenol (o-cresol) and para-methyl phenol (p-cresol) for the electrochemical reduction based immobilization process. The idea behind the experiment is; when the ortho or para position of the phenol is blocked with an electro-inactive functional group (–CH3 ) and subjected for electrochemical reduction at −0.45 V vs Ag/AgCl by CV, only p-cresol will give a surface confined redox peak if o-hydroxy phenol isomeric compound get immobilized as a by-product. Similarly, if p-hydroxy phenol is immobilized, then the o-cresol will give specific electrochemical redox response in CV. On the other hand, if the phenolic compounds, o-cresol and p-cresol could give the redox peaks, which means, mixture of catechol and HQ samples get immobilized on the working electrode. Figs. 5A-5B are the CV responses of o-cresol and p-cresol on GCE/p-MWCNT in pH 7 PBS. Interestingly, p-cresol failed to show any reversible redox peak during the electrochemical treatment process (Fig. 5B); while the o-cresol selectively gave well defined reversible redox peak at E1/2 value 290 ± 10 mV vs Ag/AgCl (Fig. 5A). It is similar to the previous case of phenol oxidized by-product (through phenoxy radical pathway) immobilization at 300 ± 5 mV vs Ag/AgCl in pH 7 PBS.22 These observations authentically conclude selective electrochemical formation and immobilization of HQ on the GCE/MWCNT during the phenol electrochemical reduction in neutral pH, in this work. Apart from the above mentioned phenolic derivatives, other chemical compound, butylated hydroxy toluene (ortho and para positions were all blocked with methyl functional group) was also subjected to electrochemical reduction treatment by CV with GCE/MWCNT as shown in the Fig. 5C. Interestingly, there were no marked redox behaviors found for the both compounds. From the above information, following conclusions were drawn: (i) butylated hydroxy toluene was not involved in any electrochemical immobilization process, and (ii) HQ is the key product in the electrochemical reduction based immobilization of phenolic compounds. Extending the information to the tetracycline antibiotic case, where phenolic functional group is involved, Tet-HQ derivative is found to be responsible for the electrochemical reduction based immobilization and surface confined A1/C1 redox behavior, in this work. As an additional proof to the H2 O2 participation in the in-situ HQ formation and immobilization on MWCNT modified electrode, a control experiment relating CV responses of GCE/MWCNT and GCE with deliberately spiked 500 µM H2 O2 in 20 min. N2 purged pH 7 PBS+1 mM o-cresol (low [dissolved O2 ] = 0.5 ppm) system was carried out. As can be seen in the supporting information Fig. S3, significant HQ formation and immobilization on GCE/MWCNT, where the redox peak current value is about twice higher over the HQ immobilization with the dissolved oxygen condition ([dissolved O2 ] = 8.3 ppm, Fig. 5A curve (e)), and nil response with GCE, were noticed. These observations proved the H2 O2 involvement in this work. It is important to highlight that the E1/2 of the Tet-HQ@MWCNT (−0.240 V vs Ag/AgCl, electrode-1A) obtained in this work is not matching with the E1/2 of a control o-cresol-HQ@MWCNT (0.063 ± 0.02 V vs Ag/AgCl, electrode-1B,) prepared by the electro-chemical reduction method! Previously, we also observed non-similarity in the E1/2 values in the cases of the amoxicillin-HQ@MWCNT (0.05 V vs Ag/AgCl, electrode-2A) and its control with HQ@MWCNT (0.290 V vs Ag/AgCl, electrode-2B), which were prepared by a same phenoxy radical pathway immobilization method.22,23 Apart from that, the HQ immobilized CNT electrodes all show different electrocatalytic activities too. For instance, the Tet-HQ@CNT displays specific H2 O2 electrocatalysis (Section 3.4). But the control o-cresol-HQ@MWCNT shows electrocatalysis to hydrazine only (data not enclosed). Similarly, amoxicillin-HQ@MWCNT showed feeble electrocatalysis to hydrazine and H2 O2 (data not enclosed). Precise details for the discrepancy in the E1/2 and the electrocatalysis of the various HQ@CMEs are unknown for us now. Presumably, additional functional groups and structural interactions with underlying MWCNT might have played a key role for the varied electron-transfer and electro-catalyzes. For instance, in general, the electrocatalytic oxidation route occurring through inner sphere electron-transfer pathway, where the {catalyst (electrode)-substrate (analyte)} complex, high energy intermediate state (for example see the Scheme 3), is considered to be a critical step Scheme 3. Michaelis-Menten reaction for the GCE/Tet-HQ@MWCNT mediated H2 O2 reduction reaction. Journal of The Electrochemical Society, 159 (11) G137-G145 (2012) C. A. A1 200 5-500 mV/s 20 A1 10 87 6 5 pH i/µA i/µA A1' 0 -20 C1' C1 -200 C1 -0.4 0.0 -0.8 E vs Ag/AgCl/V B. 0 -0.4 0.0 0.4 E vs Ag/AgCl/V D. 200 log (ipa /µA) A1 peak: Slope=0.84 1 :A1' peak Slope=0.74 0 Epavs Ag/AgCl/mV A1 peak 2 Slope = -59.81 0 -200 -400 0 1 2 3 6 -1 log (v / mV.s ) 9 pH Figure 6. CV responses of GCE/Tet-HQ@MWCNT at different (A) scan rates and (C) solution pHs, and its corresponding peak parameter plots for their respective anodic peaks (B and D). Note Tet-HQ@MWCNT = Tet@MWCNT. for the electron-transfer kinetics. If functional group of the substrate hinder to form any such {catalyst (electrode)-substrate complex} at the intermediate step, then there will not be any electron-transfer and in turn no electrocatalysis. In addition, redox potential of the CME did not match with the analyte oxidation/reduction potential. Detailed theoretical electrochemical study is necessary to address the issue completely. Electron transfer behavior and electrocatalytic H2 O2 reduction of Tet-HQ@MWCNT.— Effect of CV scan rate on the electrochemical response of GCE/Tet@MWCNT in pH 7 PBS was examined (Fig. 6A). Symmetric increase both in anodic and cathodic current sides against the increase in the scan rate was noticed. Double logarithmic plots of G143 base-line corrected anodic (ipa ) currents versus scan rate yielded slope (=∂logipa /∂log (v/mV.s−1 )) values are 0.84 ± 0.05 and 0.74 ± 0.04 respectively for the A1 and A1′ peaks (Fig. 6B). These values are between the ideal values; 0.5 for diffusion controlled and 1 for adsorption controlled electron-transfer reactions. Hence, the mechanism for the surface confined redox peak at A1/C1 and A1′ /C1′ with the Tet-HQ@MWCNT is due to mixed adsorption and diffusion (within the film) controlled electron-transfer reactions. Fig. 6C is the CV responses of GCE/Tet@MWCNT at different solution pHs. A slope (=∂Epa /∂pH) value −59.8 ± 1 mV/pH was noticed for the redox peak with the Tet@MWCNT hybrid system (Fig. 6D), which is very close to Nernstian value 59 mV/pH, suggests the involvement of equal numbers of proton and electron in the redox process. Fig. 7A represents the CV responses of GCE/Tet-HQ@MWCNT with increasing concentration of H2 O2 up to 500 µM at a fixed scan rate 10 mV.s−1 in a pH 7 PBS. In presence of H2 O2 , the hybrid modified electrode showed profound reduction peaks at about −0.30 ± 0.1 V vs Ag/AgCl, which is near to the surface confined redox peak (E1/2 ∼ −0.24 V) for the Tet-HQ@MWCNT that evidence the mediated reduction of the H2 O2 by the hybrid modified electrode. Control H2 O2 reduction experiment with the unmodified electrode, GCE/ MWCNT showed shoulder like feeble reduction peak at −0.50 ± 0.05 V vs Ag/AgCl, which is about 100 times lower in the current and 300 mV over-potential than the mediated H2 O2 reduction by the hybrid system (Fig. 7A, curve b). This observation clearly depicts the unique electrocatalytic function of the GCE/Tet-HQ@MWCNT. The reduction reaction mechanism may be due to the immobilized Tet-HQ/quinone redox peak mediation (Scheme 3). The mediated reduction peaks (i.e., base-line corrected ipc ) were linear against the [H2 O2 ] upto 400 µM with a linear equation of ipc (µA) = 0.272 ± 0.007[H2 O2 ](µM)-3.61µA (R = 0.9945), after that the peak current response (linear line like response, difficulty in base-line correction) slowly tend to plateau as in Fig. 7B. A saturation in the mediation current was noticed at 3 mM of H2 O2 (data not enclosed). This observation is the typical example for the Michaelis-Menten (MM) type of reaction for the H2 O2 reduction.37 Possible reaction mechanism for the H2 O2 reduction reaction based on MM kinetics is sketched in Scheme 3. Where the H2 O2 analyst first bind to active site of the electro-catalyst and form a high-energy intermediate complex (Step-1), which further decompose to product (2OH− ) and oxidized form of the mediator (i.e., Tet-quinone@MWCNT, Step2). The Tet-quinone@MWCNT can be regenerated back to active catalyst, Tet-HQ@MWCNT at the operating potential simultaneously (Step-3). The H2 O2 concentration detection range obtained in A. B. 50 b.GCE/MWCNT +H2O2 100 ipc /µA i/µA 0 -50 -100 0 a.GCE/Tet-HQ@MWCNT +H2O2 50 y = 3.16 + 0.272x Rsqr = 0.998 500 µM -150 0 -0.6 -0.3 0.0 E vs Ag/AgCl/V 0.3 0 100 200 300 400 500 600 [H2O2]/µM Figure 7. CV responses of a GCE/Tet-HQ@MWCNT with various concentrations of hydrogen peroxide (a) and GCE/MWCNT with 500 µM H2 O2 (b) in pH 7 PBS at a scan rate = 10 mV.s−1 . (B) The calibration plot for the GCE/Tet-HQ@MWCNT in different H2 O2 concentration. G144 Journal of The Electrochemical Society, 159 (11) G137-G145 (2012) A. C. B. 0.6 UA CySH NO2 AA b. MWCNT -0.1 -0.2 0.4 i/µA At 0 V [H2O2] = 20 µM 0 c. H2O2 i/µA 0.0 i/µA 1 a. GCE 0.2 -1 -2 -0.3 c. Tet-HQ@MWCNT 100 µL each spike b. a. 0.0 -0.4 -3 0 400 800 1200 0 50 Time/s 100 150 [H2O2] /µM 200 0 200 400 600 800 Time/s Figure 8. (A) Amperometric i-t response of the GCE/Tet-HQ@MWCNT (c), GCE/MWCNT (b) and GCE (a) for n = 10 spikes of [H2 O2 ] = 20 µM at an applied potential of 0 V vs Ag/AgCl and (B) its corresponding calibration plots. (C) Amperometric i-t response for the GCE/Tet-HQ@MWCNT with 100 µM each of H2 O2 , ascorbic acid (AA), uric acid (UA), cysteine (CySH) and nitrite (NO2 − ). this work is reasonably comparable with the range reported for the myoglobin enzyme-ciprofloxacin-MWCNT hybrid modified electrode (up to 700 µM), but at a more negative applied potential of −0.327 V vs Ag/AgCl.38 The analytical applicability of the Tet-HQ@MWCNT is further examined by subjecting the electrode for amperometric i-t detection of 20 µM spikes of H2 O2 at low applied potential, 0 V vs Ag/AgCl in pH 7 PBS. Comparative experiments were also done using GCE/MWCNT and GCE. Fig. 8A, curve c is the typical amperometric i-t response of the hybrid electrode in comparison with the respective GCE and GCE/MWCNT systems (Figs. 8A, curves a and b). Respective calibration plots were given in Fig. 8B. The Tet-HQ@MWCNT hybrid electrode showed current linearity up to 200 µM of H2 O2 with current sensitivity values of 2.2 ± 0.008 nA/µM (=31.1 mA.M−1 .cm−2 ) respectively; whereas the unmodified electrodes show negligible amperometric current response at 0 V vs Ag/AgCl. Obtained current sensitivity value is relatively higher over recent reports for the H2 O2 sensors, for example; GCE/NafionCNT/Clay at −0.3 V vs Ag/AgCl in pH 7 (4 mA.M−1 .cm−2 ),39 GCE/B2 O3 -MWCNT/Horseradish peroxide(HRP)/Nafion at −0.3 V vs AgCl in pH 7 (26.54 mA.M−1 .cm−2 )40 and GCE/MWCNT-HRPPoly(neutral red)/Pt at −0.22 V vs saturated calomel electrode (0.035 mA.M−1 .cm−2 ).41 Interference from common biological samples on the amperometric i-t response of GCE/Tet-HQ@MWCNT was also examined (Fig. 8C). Interestingly, the hybrid electrode showed tolerable interferences to other biological compounds; ascorbic acid (AA), uric acid (UA), cysteine (CySH) and nitrite (NO2 − ) as shown in the Fig. 8C. The selectivity is an important advantage of the present electrode. These observations attributes enzyme-less sensing of H2 O2 by the antibiotic@CNT hybrid modified electrode in this work. Conclusions Electrochemical potential cycling treatment of MWCNT modified electrode at negative operating potential, −0.45 V vs Ag/AgCl with dilute concentration of Tetracycline antibiotic resulted to immobilization of Tetracycline as hydroquinone derivative by-product (Tet-HQ) on MWCNT. The Tet-HQ@MWCNT hybrid electrode showed specific surface confined redox peak at −0.240 ± 0.02 V vs Ag/AgCl in pH 7 PBS due to the electron-transfer behavior of the Tet-HQ/quinone redox site. Electrochemical reduction of dissolved oxygen to H2 O2 product at the negative operating potential and its chemical reaction with surface bound Tet@MWCNT to Tet-HQ@MWCNT was proposed as a possible reaction mechanism for the immobilization process. Specific experiments designed with screen-printed ring-disk electrode/flow injection analysis confirm the formation of H2 O2 intermediate. Control electrochemical experiments for the immobilization of o-cresol and p-cresol on GCE/MWCNT resulted to selective im- mobilization of o-cresol only. This observation also evidenced the immobilization of Tet drug (phenolic site) to Tet-HQ, in this work. Physicochemical characterizations of the Tet-HQ@MWCNT hybrid sample by FTIR and TEM collectively revealed the presence of TetHQ species within in pristine MWCNT. The Tet-HQ@MWCNT’s surface confined peak can electro-catalytically reduce the hydrogen peroxide at 0 V vs Ag/AgCl in pH 7 PBS without any interference from the common electro-active biochemical such as ascorbic acid, uric acid, cysteine, and nitrite. The investigation carried out in this work may be helpful to following aspects: (i) new approach for immobilization of phenolic derivatives as HQ species on MWCNT, (ii) development of enzyme-less hydrogen peroxide sensor, (iii) procedure for enhanced uptake of Tet antibiotic from polluted water samples (e.g., poultry), (iv) to get some in-vitro info about the tetracycline electrochemical interaction with hydrogen peroxide/oxygen radical species, which may happen in certain occasion/s in physiological system and (v) understanding of phenol group oxidation reaction on carbon nanotube surface. Acknowledgments The authors thank the financial support from Department of Science and Technology (DST), SERC Scheme, India. SAIF IIT Madras, Chennai for the TEM analysis. S.S thanks the Council of Scientific and Industrial Research (CSIR) for the award of Senior Research Fellowship. 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