An unusual electrochemical oxidation of phenothiazine dye to phenothiazine-bi-1,4-quinone derivative (a donor-acceptor type molecular hybrid) on MWCNT surface and its cysteine electrocatalytic oxidation function

Article abstract of DOI:10.1016/j.electacta.2015.11.032

Phenothiazine (PTZ), a thiazine class heterocyclic compound, is a well-known electron donating system and has been widely used as a starting compound to prepare various phenothiazine dyes and pharmaceutically important compounds. Quinones and its derivatives are constituents of biologically active molecules serve as excellent electron-acceptor systems. Oxidation of PTZ by chemical and electrochemical methods often resulted into monohydroxylation of benzene ring moiety, S-oxidized and polymerized compounds as end products. Electrochemical oxidation of PTZ on a multiwalled carbon nanotube (MWCNT) modified glassy carbon electrode in pH 7 phosphate buffers solution (PBS) has been investigated in this work. A highly redox active surface confined PTZ-bi-1,4-quinone derivative (PTZ-biQ) on MWCNT modified glassy carbon electrode, designated as GCE/MWCNT@PTZ-biQ, as a product was unusually observed. The GCE/MWCNT@PTZ-biQ showed well-defined redox peaks at E_1/2 = -0.07 and +0.29 V vs Ag/AgCl corresponding to surface confined electron-transfer behavior of the bi-quinone (acceptor) and PTZ-cationic radical species (donor) respectively. No such electrochemical characteristics were noticed when unmodified GCE was subjected to the electrochemical oxidation of PTZ. Existence of PTZ-biQ was confirmed by XRD, Raman spectroscopy, FT-IR and GC-MS (methanolic extract of the active layer) analyses. Position of biQ in PTZ-biQ as 1,4-quinone isomer was confirmed by observation of absence of copper-complexation with 1,4-quinone and H₂O₂ electrochemical reduction reactions at -0.1 V vs Ag/AgCl unlike to the specific copper-complexation and H₂O₂ reduction with 1,2-quinone isomer in pH 7. Cysteine (CySH) oxidation was studied as a model system to understand the electron-transfer function of the MWCNT@PTZ-biQ. A highly selective electrocatalytic oxidation and sensing by amperometric i-t and flow injection analysis of CySH at low oxidation potential, 0.3 V vs Ag/AgCl in pH 7 PBS with detection limit values (signal-to-noise ratio = 3) of 11.10 μM and 110 nM respectively, without any interference from other biochemicals like uric acid, dopamine, nitrite, citric acid and H₂O₂, unlike the conventional chemically modified electrodes with serious interference's, have been demonstrated.

Full text of DOI:10.1016/j.electacta.2015.11.032

Accepted Manuscript
Title: An unusual electrochemical oxidation of phenothiazine
dye to phenothiazine-bi-1,4-quinone derivative (a
donor-acceptor type molecular hybrid) on MWCNT surface
and its cysteine electrocatalytic oxidation function
Author: Ranganathan Shanmugam Palani Barathi Jyh-Myng
Zen Annamalai Senthil Kumar
PII:
S0013-4686(15)30805-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.electacta.2015.11.032
EA 26032
To appear in:
Electrochimica Acta
Received date:
Revised date:
Accepted date:
29-5-2015
6-11-2015
7-11-2015
Please cite this article as: Ranganathan Shanmugam, Palani Barathi, Jyh-Myng Zen,
Annamalai Senthil Kumar, An unusual electrochemical oxidation of phenothiazine dye
to phenothiazine-bi-1,4-quinone derivative (a donor-acceptor type molecular hybrid) on
MWCNT surface and its cysteine electrocatalytic oxidation function, Electrochimica
Acta http://dx.doi.org/10.1016/j.electacta.2015.11.032
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1
An unusual electrochemical oxidation of phenothiazine dye to phenothiazine-
bi-1,4-quinone derivative (a donor-acceptor type molecular hybrid) on
MWCNT surface and its cysteine electrocatalytic oxidation function
Ranganathan Shanmugam,a Palani Barathi,a Jyh-Myng Zen,b Annamalai Senthil Kumara*
aEnvironmental and Analytical Chemistry Division, School of Advanced Sciences, Vellore
Institute of Technology University, Vellore 632 014, India
bDepartment of Chemistry, National Chung Hsing University, Taichung 40227, Taiwan
*Corresponding author’s Fax: +91-416-2243092; Tel: +91-416-2202754
E-mail: askumarchem@yahoo.com

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Graphical abstract

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Highlights
 Phenothiazine gets electro-oxidized to phenothiazine-bi-1,4-quinone on MWCNT
 A phenothazine-quinone surface-confined MWCNT modified GCE was prepared
 This new single molecular unit behaves as electron donor-acceptor type system
 The modified electrode was characterized by XRD, FTIR and Raman spectroscopy
 Cysteine electro-catalytic oxidation was demonstrated at low potential
 Flow injection analysis of cysteine was studied.

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Abstract
Phenothiazine (PTZ), a thiazine class heterocyclic compound, is a well-known electron
donating system and has been widely used as a starting compound to prepare various
phenothiazine dyes and pharmaceutically important compounds. Quinones and its derivatives are
constituents of biologically active molecules serve as excellent electron-acceptor systems.
Oxidation of PTZ by chemical and electrochemical methods often resulted into
monohydroxylation of benzene ring moiety, S-oxidized and polymerized compounds as end
products. Electrochemical oxidation of PTZ on a multiwalled carbon nanotube (MWCNT)
modified glassy carbon electrode in pH 7 phosphate buffers solution (PBS) has been investigated
in this work. A highly redox active surface confined PTZ-bi-1,4-quinone derivative (PTZ-biQ)
on MWCNT modified glassy carbon electrode, designated as GCE/MWCNT@PTZ-biQ, as a
product was unusually observed. The GCE/MWCNT@PTZ-biQ showed well-defined redox
peaks at E1/2 = -0.07 and +0.29 V vs Ag/AgCl corresponding to surface confined electron-
transfer behavior of the bi-quinone (acceptor) and PTZ-cationic radical species (donor)
respectively. No such electrochemical characteristics were noticed when unmodified GCE was
subjected to the electrochemical oxidation of PTZ. Existence of PTZ-biQ was confirmed by
XRD, Raman spectroscopy, FT-IR and GC-MS (methanolic extract of the active layer) analyses.
Position of biQ in PTZ-biQ as 1,4-quinone isomer was confirmed by observation of absence of
copper-complexation with 1,4-quinone and H2O2 electrochemical reduction reactions at -0.1 V
vs Ag/AgCl unlike to the specific copper-complexation and H2O2 reduction with 1,2-quinone
isomer in pH 7. Cysteine (CySH) oxidation was studied as a model system to understand the
electron-transfer function of the MWCNT@PTZ-biQ. A highly selective electrocatalytic
oxidation and sensing by amperometric i-t and flow injection analysis of CySH at low oxidation
potential, 0.3 V vs Ag/AgCl in pH 7 PBS with detection limit values (signal-to-noise ratio = 3)

5
of 11.10 µM and 110 nM respectively, without any interference from other biochemicals like
uric acid, dopamine, nitrite, citric acid and H2O2, unlike the conventional chemically modified
electrodes with serious interference's, have been demonstrated.
Key words: Phenothiazine dye; phenothiazine-bi-1,4-quinone; donor-acceptor; multi-walled
carbon nanotube; cysteine.

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1. Introduction
Phenothiazine (PTZ) is a thiazine class heterocyclic compound and has been widely used
in the synthesis of various organic dyes such as methylene blue (MB), methylene green, thionine
(Th), azure dye (AZ), toludine blue (TB) and certain pharmaceutical drugs like chlorpromazine,
prochlorperazine and promethazine [1-3]. It has diverse pharmacology activities which include
antituberculosis, antitumor, bactericides etc. It is even helpful in treating Creutzfeldt-Jacob
(ncCJD) and HIV diseases [4]. Because of its pharmacological importance, oxidation/reduction
mechanisms of PTZ are paramount interest in understanding its pharmacokinetics. It has been
reported that PTZ is metabolized by cyctrochrom P450 isoforms in human liver and giving rise
to ring-hydroxylated, S-oxidized and N-demethylated metabolities [5,6]. Similar kinds of
compounds were obtained as reaction products when PTZ was subjected to chemical and
electrochemical oxidation in a mixture of aqueous and non-aqueous solvents [7]. However,
discreet electrochemical oxidation of PTZ on conventional electrodes (Pt, glassy carbon
electrode (GCE) and Au) either in organic or in aqueous acidic medium or multiwalled carbon
nanotube modified electrode in acid medium [8-11], yielded polymeric PTZ as a sole product.
For instance, poly-phenothiazine and poly(toluidine blue) were obtained as final products upon
the electrochemical oxidation of PTZ and toluidine blue on GCE in DMSO and 0.01 M borate
buffer (pH 9.1) + 0.1 M NaNO3 respectively [8,9]. Herein, we report an unusual electrochemical
oxidation of PTZ to a highly redox active and surface confined PTZ-bi-1,4-quinone derivative
(PTZ-biQ, biQ=bi-quinone (bi-1,4-quinone)) on a MWCNT modified glassy carbon electrode
(GCE/MWCNT@PTZ-biQ) in pH 7 phosphate buffer solution (PBS). To the best of our
knowledge, selective electrochemical oxidation of PTZ to PTZ-biquinone like product was never

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reported in the literature. However, anthraquinone-functionalized PTZ based electron acceptor-
donor compounds, prepared by discrete chemical synthetic route, were reported [12].
The combination of donor (D)-acceptor (A) like host-guest pair serves as a good charge-
transfer (CT) complexes with fascinating electrochemical and photochemical properties [13].
Quinone acceptor based CT systems are considered to be a best model owing to its mimicking
property of the Photosystem I [14]. In past, several fused molecular compounds based on
derivatives of Ferrocene (fc) and quinone (Q), as a D-A as self-assembled monolayers (SAM)
have been reported and studied as CT [15-17]. Unfortunately, most of the reported D-A systems
are redox active either in organic or in acidic medium only. In addition, degradation of the SAM
was noticed in the neutral pH solution. The new MWCNT@PTZ-biQ prepared in this work is
found to be highly redox active and stable in neutral pH. It is note-worthy that the D-A activity
observed with MWCNT@PTZ-biQ is merely due to the PTZ-biQ single molecular unit system,
unlike to the previous D-A reports with multi-molecular analogues separated by suitable spacer
[15-17]. MWCNT matrix is found to stabilize the D-A single molecular unit unusually in this
work.
MWCNT has unique surface feature. The sp2 carbons of CNT (graphitic carbon) can
interact with sp2 carbons of aromatic organic molecules through pi-pi interaction and make the
{CNT-organic compound} hybrid so facile for the low potential electrochemical oxidation to
highly redox active compounds [18]. It was found that different isomers of aromatic organic
compounds interact differently with CNT’s graphitic layer and further to different electro-
oxidative products. For example, anthracene and 1,10-phenonthroline get oxidized as
anthraquionone and 1,10-phenonthroline-quinone respectively, whereas phenthrene was found to
be electro-inactive on MWCNT surface [18,19]. Our interest in this work is to examine the

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electrochemical behavior of heteroatom substituted anthracene analogue, PTZ on MWCNT
surface. As per our previous studies [18,19], it is expected that PTZ will electro-oxidize to PTZ-
sulphone, since, the 9,10 positions of the PTZ are occupied by N and S atoms and the S atom is
prone to oxidize as sulphone derivative. On the contrary, highly redox active surface confined
molecular species (PTZ-biQ) was unusually obtained upon electrochemical oxidation of PTZ on
MWCNT in this work. Physicochemical characterizations of GCE/MWCNT@PTZ-biQ by FT-
IR, Raman spectroscopy, XRD, GC-MS (methanolic solution) and in-situ electrochemical quartz
crystal microbalance (EQCM) confirmed the presence of PTZ-biQ in GCE/MWCNT@PTZ-biQ
system. Electrochemical oxidation cysteine (CySH) was taken as a model system to study the
heterogeneous electro-transfer function of the GCE/MWCNT@PTZ-biQ. This new
MWCNT@PTZ-biQ system showed highly efficient electro-catalytic oxidation and
electrochemical flow-injection analysis of cysteine (CySH) at low operating potential, 0.3 V vs
Ag/AgCl in neutral pH solution.
CySH is an important thiol-containing amino acid that plays crucial role in the
metabolism of a number of essential biochemicals mainly biotin, heparin and glutathione etc
[20]. Thus, sensitive and selective determination of cysteine in neutral pH is an important
research interest in clinical, pharmaceutical and food chemistry. Due to lack of chromophore,
direct detection of CySH by spectroscopic techniques like UV-Vis and fluorescence are highly
difficult. Alternately, derivatization based analytical methods were often reported for this
purpose [21-25]. For instance, phenylisothiocyanate (PTC) [21,22], 9-fluorenylmethoxycarbonyl
chloride [23], oxycarbonyl chloride [24], ortho-phthaldialdehyde (OPA)/2-mercaptoethanol
[26,27] and ninhydrin [28] based UV-Vis, HPLC and fluorescence methods were employed for
CySH detection. Although, the above mentioned methods allow to sense cysteine at ~µM

9
concentration, interference by other organic functional groups such as primary amine and sulfur
containing compounds was serious. On the other hand, electrochemical methods offer
derivatization-less and selective detection for CySH [29-31]. Nevertheless, problems such as
unstable detection current signal and high-over potential are restricting the working electrodes
(GCE, Pt, Au, boron doped diamond) for extend to real sample analysis [32-36]. To avoid such
problems, specific electrochemical-mediator immobilized chemically modified electrodes were
introduced. Following are some of the representative examples for the mediator modified
electrodes and its electrochemical-oxidation conditions; prussian blue (PB) [34, 35], manganese
hexacyanoferrate [36] and copper-cobalt hexacyanoferrate at 0.7-0.9 V vs Ag/AgCl in pH 2 [37,
38], octabutylthiophthalocyaninato-cobalt(II) at 0.7 V vs Ag/AgCl in pH 4 [39], metal
porphyrins at 0.8 V vs Ag/AgCl in pH 14 [40], lead ruthenate pyrochlore at 0.9 V vs Ag/AgCl
[41], indigo carmine dye at 0.31 V vs SCE in pH 8 [42] and quinzarin dye at 0.8 V vs Ag/AgCl
in pH 2 [43]. Indeed, above mentioned electrodes showed several drawbacks such as high
oxidation potential [41,43], tedious electrode preparation procedures [40,42], instability of the
immobilized-mediators in neutral pH [33, 34] and hydrodynamic condition (which is a highly
desirable condition for amperometric i-t and flow injection analysis of CySH).
Flow injection analysis coupled with electrochemical detection (FIA-ECD) method is one
of the advanced electro-analytical techniques in the analytical chemistry for low volume
detection of electro-active species (5-50 µL). Previously, several electrochemical detector
systems like vitamin B12 adsorbed GCE at 0.8 V vs Ag/AgCl [44] and NPyCME at 1 V vs
Ag/AgCl in pH 7 phosphate buffer [41,45], poly(ethylenedioxythiophen)-PEDOT-AuNPs
modified electrode at 0.6 V vs Ag/AgCl in pH 4 citric acid buffer [46], nickel(II)
polytetrasulfophthalocyanine system at 0.6 V vs Ag/AgCl in pH 9 [47]and PbO2-MnO2-nafion at

10
0.8 V vs Ag/AgCl [48] were introduced for the FIA of CySH. Note that the above mentioned
detectors were operated either at high potentials (0.61 V) or at non-physiological pH (acid or
alkaline pH). Interestingly, a new electrochemical detector, GCE/MWCNT@PTZ-biQ developed
in this work has shown low potential (0.3 V vs Ag/AgCl) and physiological pH based
electrocatalytic oxidation and detection of CySH. Selective FIA-ECD of CySH in a couple of
pharmaceutical samples was finally demonstrated as a proof concept in end part of this work.
2. Experimental Section
Phenothiazine (98% purity), pristine multiwalled carbon nanotube (MWCNT, >90% of
carbon basis), single walled carbon nanotube (SWCNT, 50-70% carbon basis) and graphite
nanopowder (GNP) were purchased from Aldrich. Cysteine (extra pure grade, 99%) was
obtained from Himedia Laboratories PVT Ltd, India. Other chemicals used in this work were all
ACS-certified reagent grade and used without further purification. Aqueous solutions were
prepared using deionized and alkaline KMnO4 distilled water. Unless otherwise stated, pH 7
phosphate buffer solution (PBS) of ionic strength, I = 0.1 M was used as a supporting electrolyte
in this work. Caution! Because phenothiazine is toxic, proper care must be taken during
handling.
Voltammetric measurements were all carried out with two CHI instruments (USA), 660C
(for CV) and 440B (for electrochemical quartz crystal microbalance (EQCM)). The three-
electrode system consists of glassy carbon or its chemically modified electrodes (CMEs) as a
working electrode (0.0707 cm2), Ag/AgCl as a reference electrode and platinum wire as an
auxiliary electrode. X-ray diffraction (XRD) analysis was carried out using Bruker D8 Advanced
diffractometer (Germany). Fourier transform infrared spectroscopy (FTIR) analysis was carried

11
out with JASCO 4100 (Japan). AZILTRON, PRO 532 (USA) instrument with a 532 nm laser
excitation source was used for Raman spectroscopy analysis. Continuum source electrothermal
atomic absorption spectroscopic (CS-ETAAS) measurements were done using ZEEnit 65 model,
Analytika Jena AG, Jena, Germany.
Purified and functionalized MWCNT (p-MWCNT and f-MWCNT, f= functionalized and
p= purified) powder samples were prepared as per our previous reported procedure [49], where a
mixture of 200 mg of MWCNT powder + 35 mL of 6 M or 15 M HNO3 (fuming acid) was
refluxed for 12 h in a silicone oil bath at T = 413 (±2) K (140 °C), filtered, washed with copious
amount of DD H2O until the pH of the filterant solution became neutral and finally dried at T =
353 (±2) K (80 °C) in a vacuum oven. Following trace metal impurities were identified in the
CNTs samples by TGA, XRD and CS-ETAAS analyses collectively; MWCNT-Fe, 2.1 wt.%; Ni,
0.0014 wt.%; Co, 0.00016 wt.%, f-MWCNT-Fe, 0.6 wt.%; Ni, 0.0011 wt.%; Co, 0.00016 wt.%
and p-MWCNT-Fe, 0.6 wt.%; Ni, 0.0029 wt.%; Co, 0.0007 wt.% [49].
Different CNTs such as pristine-MWCNT, f-MWCNT, p-MWCNT and SWCNT were
modified on a glassy carbon electrode (GCE/pristine-MWCNT, GCE/f-MWCNT, GCE/p-
MWCNT and GCE/SWCNT) by drop casting 3 L of respective ethanolic dispersed CNT
solutions (2 mg/mL) on a cleaned GCE surface and allowed 5±1 minutes for complete air-dry.
PTZ adsorbed CNT modified glassy carbon electrodes (designated as GCE/CNT@PTZads; ads =
physically adsorbed) were prepared by drop casting 3 μL of dilute PTZ solution (2 mg PTZ
dissolved in 500 L ethanol) on different GCE/CNTs, followed by 3±0.5 minutes air-drying at
room temperature. The freshly prepared modified electrodes were electrochemically pretreated
in a potential window -0.6 to 0.6 V vs Ag/AgCl at a scan rate (v) 50 mV s-1.

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Two commercially available cysteine tablets, Tablet #1 and #2 and a soyabean juice,
sample #3, purchased from local departmental store in Vellore, were subjected to real sample
analyses. These samples were stored in the refrigerator until used. Prior to the FIA, the real samples
were suitably diluted with pH 7 PBS. Standard addition method was adopted for real sample
analysis.
3. Results and Discussion
3.1. Electrochemical behavior of PTZ on MWCNT surface
Fig. 1A is a thirty continuous CV responses of a PTZ adsorbed GCE/MWCMNT
modified electrode, GCE/MWCNT@PTZads at a potential window -0.6 to +0.6 V vs Ag/AgCl in
pH 7 PBS. As can be seen, in the first cycle, a huge irreversible anodic peak at 0.55 V vs
Ag/AgCl and in subsequent cycles generation of two well defined redox peaks at equilibrium
potential, E1/2 = Epa+Epc/2, -70±5 mV (A1/C1) and 290±5 mV (A2/C2) vs Ag/AgCl with peak-
to-peak separation, ΔEp = Epc-Epa, 47±5 and 43±5 mV respectively, were noticed. Redox peak
current of the A2/C2 decreases, where as the A1/C2 increases, against increase in the CV cycling
number up to 30 cycles. Later on, saturation in the redox peak currents was noticed. When the
above PTZ exposed electrode was gently washed with water and repeated the CV in a fresh pH 7
PBS, a stable redox peak behaviors without any alteration in the peak current was noticed (Fig.
1B, curve a). This point onwards the above PTZ modified electrode is designated as
GCE/MWCNT@PTZ-oxid, where PTZ-Oxid= oxidized form of PTZ.
Effect of scan rate (v) on the CV response of GCE/MWCNT@PTZ-oxid showed a
systematic increase in the peak currents against increase in the scan rate (Fig. 2A). A double
logarithmic plot of anodic and cathodic peak currents (ipa and ipc) versus scan rate yielded a slope
value (logipa or ipc/logv), ~0.8 for both A1/C1 and A2/C2 redox peaks (Fig.2B). Since it is in

13
between the values of 1 and 0.5 respectively for the ideal adsorption and diffusion controlled
electron-transfer of electrochemical reactions [50], a mixed adsorption and diffusion controlled
electron transfer behavior is proposed as a possible mechanism for the redox peaks. In further,
the redox peaks showed non-Nernstian type pH dependent behavior (logE1/2/logpH for A1 = -
74 and A2 = -67 mV pH-1, Fig. 2C and D). Interestingly no such electrochemical feature was
noticed when PTZ adsorbed GCE, GCE/PTZads was subjected to the electrochemical reaction
(Fig. 1B, curve b). At this stage, it is highly difficult to conclude what kind of redox active
organic species formed in MWCNT@PTZ-Oxid. In further, since the amount of the electro-
active species formed on the modified electrode is very low, probably about nano-gram quantity,
it is not easy to characterize the organic species with conventional characterization tool like
nuclear magnetic resonance (NMR) which usually requires about milligram quantity of sample
for the analysis. Thus several elegantly designed control experiments were performed to identify
the redox active organic species. The details were given below.
Initially, the effect of potential window on the preparation of GCE/MWCNT@PTZ-oxid
was examined as in Fig. 3A and B. For that freshly prepared GCE/MWCNT@PTZads systems
were subjected to CV studies. In first, starting potential of the modified electrode was fixed at
+0.6 V (Fig. 3A) and varied the negative potentials as -0.4 (curve a), -0.6 (curve b), -0.8 (curve
c) and -1.0 V (curve d) vs Ag/AgCl (Set-I). Similarly in the 2nd batch experiments, the starting
potential was set at negative potential, -0.6 V (Fig. 3B) and varied the positive potentials as +0.3
(curve a), +0.4 (curve b), +0.6 (curve c) and 0.8 V (curve d) vs Ag/AgCl (Set-II). As can be
shown in Fig. 3A, the negative potentials variation (1st set) didn’t show any alternation in the
A1/C1 and A2/C2 redox peaks formation. However, a new irreversible cathodic peak at -0.6 V
vs Ag/AgCl was noticed uniformly in all the above voltammograms, which may be due to

14
MWCNT assisted oxygen reduction reaction (ORR) to H2O2 formation [51]. Results of the
above potential segment experiments indicated that there is no influence of negative potential
sweep on the electrochemical preparation of GCE/MWCNT@PTZ-Oxid. On the other hand, the
2nd set of experiments showed contrast results. The A1/C1 peak was disappeared when the
GCE/MWCNT@PTZ was E-cycled from -0.6 to +0.3 V and +0.4 V vs Ag/AgCl. Specific
formation of the A1/C1 peak at -0.1 V vs Ag/AgCl was noticed only when the positive potential
was swept to 0.6 V and/or beyond the limit (Figure 3B, curves c & d). Previously, it was reported
that PTZ undergoes one quasi reversible and irreversible electron-transfer reactions at +0.5 V
and +0.8 V vs Ag/AgCl in a mixture of non-aqueous+aqueous solution, due to the formation of
PTZ-cation radical species and sulphone derivative, respectively [52,53]. Meanwhile,
hydroquinone derivatives immobilized MWCNT modified electrodes showed a redox peak at
approximately at about 0 V vs Ag/AgCl in pH 7 PBS [18]. Based on the above information, it
can be speculated that the redox peak obtained at E1/2= 0.29 V (A2/C2) is due to
MWCNT@PTZ-cationic-radical species and another redox peak noticed at -0.07 V (A1/C1) vs
Ag/AgCl is due to MWCNT@PTZ-biquinone derivative. The precise structure of the PTZ-Oxid
was further identified by subjecting the MWCNT@PTZ-Oxid to various physicochemical
characterizations as follows.
3.2. Physicochemical characterization
Fig. 4A, curves a-c are a comparative powder XRD pattern of MWCNT, PTZ and
MWCNT@PTZ-Oxid samples. The MWCNT@PTZ-Oxid system showed specific XRD peaks
at 2θ= 16.84, 19.21, 22.1, 25.4, 27.0 and 43.2o. Except 2θ=16.84o other peaks observed were all
,
closely matching with the 2θ of individuals of MWCNT (25.7 and 43.03) and PTZ (19.3, 22.5

15
and 27.2o), but with slightly shifted 2θ values. The new peak at 2θ=16.84o is correlatable to our
previous report on a quinone immobilized MWCNT system (2θ=14.6o) [54], which ascribes
presence of 1,4- or 1,2-quinone derivative on MWCNT@PTZ-Oxid. The shift observed in the 2θ
peaks between the individual and composite is due to the - interaction of the organic moiety
with graphic structures of CNTs [54,55]. Raman spectroscopy characterization further
substantiates the existence of quinone like structure with in the matrix. In general, graphitic
materials display specific D and G bands at ~1330 cm-1 and 1570 cm-1 due to the disordered (sp3
bonded sites) and ordered hexagonal sp2 carbon structures respectively in Raman spectroscopy
[55]. The intensity ratio of D and G bands, i.e., ID/IG can be taken as a measure for the graphitic
disorder-ness. Oxygen rich functionalities such as quinone and hydroxyl functional groups are
responsible for the D band. Fig. 4B is a comparative Raman spectroscopic response of screen-
printed gold (SPE-Au)/MWCNT@PTZ-Oxid and unmodified SPE-Au/MWCNT showing
specific peaks corresponding to D and G bands at different magnitudes. Calculated peak intensity
ratios, ID/IG (based on the area under the peak) are 0.53±0.02 and 0.46±0.01 respectively. The
above observation, higher ID/IG value with MWCNT@PTZ-Oxid sample over the MWCNT, is
substantiating the presence of oxygen rich functional groups such as quinones in the
MWCNT@PTZ-Oxid. Additionally, comparative FT-IR experiments also support the observed
result (Fig. 5, curve a). For FTIR characterization, MWCNT@PTZ-Oxid layer, which has been
scratched out from the glassy carbon chemically modified electrode using doctor needle
(~1mm×40 mm), mixed with KBr as a pellet was used. A specific peak at 1735 cm-1 was
selectively noticed with MWCNT@PTZ-Oxid sample and the above value is due to presence of
aromatic ketone, presumably hydroquinone in the system (Fig. 5, curve a). No such FT-IR
observation was obtained with MWCNT, PTZ and MWCNT@PTZads (data not enclosed)

16
samples (Fig. 5, curve b&c). In order to authentically confirm the structure of PTZ-Oxid, a
methanol extract of MWCNT@PTZ-Oxid obtained by sonicating the chemically modified
electrode in 500 uL of methanol followed by membrane filtration (membrane pore size 220 nm,
Nupore) was subjected to GC-MS analysis (Fig. S1(A)). One of the major chromatographic
peaks observed at retention time (RT) 19.0 min showed a molecular weight (Mw) value 263.01
corresponding to a molecular structure of approximately four oxygen atoms added PTZ system,
{PTZ+4O+4H+}, which is nothing but PTZ-bihydroquinone moiety (Fig. S1(B)). The above
observations reveal that four oxygen atoms added PTZ is the possible structure for the PTZ-
Oxid.
Next, to probe the mechanistic feature of the electro-oxidation reaction, separate in-situ
CV and EQCM experiments were carried out, by repeating the Fig. 1A’s experiment with
EQCM-Au electrode. Fig. 6A and B is the typical in-situ CV and EQCM (10 cycles) responses
of the EQCM-Au/MWCNT@PTZads in pH 7 PBS at scan rate 50 mV s-1. About 4.5 µg mass
was found to gain by EQCM-Au/MWCNT@PTZads system upon the electrochemical oxidation
reaction. While a control experiment with unmodified EQCM-Au/MWCNT electrode showed a
mass gain value about 1 ng only (Fig. 6B curve a). Since, EQCM technique is a powerful method
to indirectly find out the molecular mass (Mw, g mol-1) of electro-active intermediates, for
convenience, 11th cycle CV-EQCM response was further taken as a model to subject to the Mw
analysis. Figure 6D is a plot of Δm (EQCM) vs Q (C). From the slopes of various region plots,
Mw values of electro-active species involved in different potential windows were calculated
using an equation, Mw = F×m/Q, where, Q is the charge passed, F is Faraday’s constant [18,55],
as 64.1 ± 0.8; 32.2 ± 0.1; 48.5 ± 0.7; 64.5 ± 0.2; 48.1 ± 0.8; 63.6 ± 0.9; 33.7 ± 0.4 and 19.3 ± 0.2
g mol-1. These values are corresponding to molecular species of 2O2, O2, {O2 + H2O}, 2O2, {O2 +

17
H2O}, 2O2, O2 and H2O respectively ascribing involvement of water and oxygen molecules upon
the electrochemical oxidation of PTZ to PTZ-Oxid (Fig. 6E). Indeed, exact structure of PTZ-
biquinone derivative, i.e., PTZ-bi-1,4-quinone (1,4-biQ) or PTZ-bi-1,2-quinone (1,2-biQ), is
unclear now.
In order to get detailed information about the position of hydroxyl functional groups,
whether it is 1,2 or 1,3 position, in PTZ-biquinone, the GCE/MWCNT@PTZ-Oxid is subjected
to copper complexation reaction as per our previous studies [55]. It was found that 1,2-quinone
benzene isomer can complex with copper ion and can mediate hydrogen peroxide
electrochemical reduction reaction selectively at -0.1 V vs Ag/AgCl in pH 7 PBS; whereas the
1,4-quinone benzene isomer can’t complex with copper and can’t mediate H2O2 at that low
potential [55]. This idea can be used to identify position of the dihydroxy benzene in a given
unknown system. In our recent work with pyrene-tetrone immobilized MWCNT modified
electrode, the position of the tetrone as 1,2-dihydroxy benzene derivative was identified by this
approach, wherein, the pyrene-tetrone-copper complex mediated H2O2 electrochemical reduction
specifically at -0.1 V vs Ag/AgCl [55]. If 1,4-dihydroxy benzene derivative would have been
existed in the targeted sample, then no such Cu-complexation and H2O2 reduction at -0.1 V
would have observed [55]. Fig. 7A-C are typical comparative CV responses of various copper
modified systems, GCE/MWCNT@PTZ-Oxid-Cu1+/2+ (Case I), GCE/MWCNT@PTZads-Cu1+/2+
(Case II) and GCE/MWCNT-Cu1+/2+ (Case III) without (curve a & b) and with 500 µM of H2O2
(curve c) in pH 7 PBS (Scheme 1D-F). The copper complexation reaction was carried out by
immersing the respective chemically modified electrode in 2 mM CuSO4 at open-circuit
condition at pH 7 PBS for 300 ± 0.3 sec. Qualitatively similar H2O2 reduction electrochemical
responses at more negative potential (-0.25 V vs Ag/AgCl; due to the nude copper ion

18
immobilization) rather than at -0.1 V (due to complexation of copper with 1,2-dihydroxy
benzene functional groups), were noticed. These results reveal the presence of bi-1,4-quinone
derivatives (1,4-biQ), in the MWCNT@PTZ-Oxid. It is likely that N and S atoms of PTZ or
oxygen functional groups of MWCNT@PTZ-biQ complexed with copper metal ion and further
mediated the H2O2 electrochemical reduction reaction (Fig. 7 A-C, Scheme 1 D-F). Hereafter,
the MWCNT@PTZ-Oxid is re-designated as MWCNT@PTZ-biQ.
Based on the electrochemical and physico-chemical characterization results, overall
mechanism for the conversion of PTZ to PTZ-biQ is proposed as electrochemical oxidation
sulfur atom in PTZ to sulphur radical at about +0.6 V vs Ag/AgCl, followed by radical
rearrangement to benzene rings and subsequent electro-oxidation in presence of O2/H2O to PTZ-
biQ as sketched in Scheme 2. Surface of the MWCNT and its - interaction with PTZ are found
to be very unique for the electrochemical transformation. Further theoretical study in relation to
the interaction of MWCNT with PTZ is necessary to understand the mechanistic feature of the
electrochemical conversion reaction.
Different carbons viz., f-MWCNT, p-MWCNT, SWCNT and graphite nanopowder
(GNP) modified GCEs were also examined for the PTZ electrochemical oxidation reaction. With
respect to the surface excess (Γ) value of the A1 peak, the order of PTZ-biQ formation observed
is; p-MWCNT (21.1 n mol cm-2)>f-MWCNT (19.9 n mol cm-2)>pristine-MWCNT (12.9 n mol
cm-2)>SWCNT (10.5 n mol cm-2)>GNP (5.3 n mol cm-2) (Fig. 1C). Following interim
conclusions can be drawn from the above observations: (i) A dense graphitic structure is
necessary for the electrochemical oxidation; (ii) metal and carbonaceous impurities in the
pristine-MWCNT and SWCNT are hindering the electrochemical oxidation of PTZ; presumably
the N and S atoms of PTZ form a weak complex with metal impurities such as Fe, Ni and Co and

19
further avoid the electro-oxidation of benzene ring part, (iii) oxygen rich functional groups such
as carbonyl (>C=O), carboxyl (–COO–), phenolic (>C–OH) and hydroxyl (–OH) on the f-
MWCNT are assisted the electrochemical oxidation of PTZ to PTZ-biQ and further helped to
stabilize the product through intra-molecular hydrogen bonding. (iii) A strong π-π interaction
between MWCNT and PTZ is facilitated the unique electrochemical oxidation reaction. In
consideration with easy preparation step, pristine-MWCNT is chosen as model system for further
electro-analytical studies.
3.3 Electrocatalytic oxidation and FIA of cysteine
Fig. 8A is a CV response of GCE/MWCNT@PTZ-biQ without and with 1 mM cysteine
in pH 7 PBS. A profound oxidation current signal at 0.3 V vs Ag/AgCl, where the A2/C2 redox
peak exist, was noticed. Schemes 1C&3 illustrate the possible mechanism for the electrocatalytic
oxidation of CySH through A2/C2 redox site in MWCNT@PTZ-biQ, wherein, the oxidized form
of the active site, PTZ-cationic radical species mediates the CySH oxidation to CyS-SCy. Five
continuous potential cycling (n =5) of the modified electrode with 1 mM CySH solution did not
show alteration in the peak current and potential values, unlike to the continuous decrement in
the anodic peak current with the unmodified electrodes [29,30]. Control experiments with
unmodified GCE and GCE/MWCNT have shown about fifty times lesser oxidation current
signal over the optimal electrode at +0.4 V vs Ag/AgCl (Fig. 8A curves a &b). These
observations denote efficient electrocatalytic function of the GCE/MWCNT@PTZ-biQ for
CySH oxidation reaction. Effect of scan rate on the oxidation of 1 mM CySH yielded linear
increase in the oxidation peak against increase in the scan rate (data not shown). A double
logarithmic plot of anodic peak current versus scan rate displayed a slope (logipa/logv) ~0.5

20
indicating diffusion controlled electron-transfer behaviour of the CySH. Effect of CySH
concentration on the CV of the GCE/MWCNT@PTZ-biQ was studied. The oxidation peak
current increases its current magnitude upon increase the CySH concentrations, i.e., [CySH], in a
range 0-4 mM with current sensitivity and regression coefficient values of 3.60±0.05 µA/mM
and 0.998 respectively (Inset plot Fig. 8b).
Fig. 9A shows comparative amperometric i-t responses of GCE/MWCNT@PTZ-biQ,
GCE/MWCNT and GCE for successive spikes of 50 µM CySH in to 10 mL of pH 7 PBS at an
applied potential of 0.3 V vs Ag/AgCl. As can be seen, GCE/MWCNT@PTZ-biQ showed linear
increase in the current signals (Fig. 9A, curve a); whereas the unmodified electrodes,
GCE/MWCNT (Fig. 9A, curve b) and GCE (Fig. 9A, curve c) failed to show any such marked
increase in the current response. A plot of amperometric currents against the concentrations of
CySH, [CySH] is linear up to 500 µM CySH with current sensitivity and regression coefficient
values 6.21 nA µM-1 (µA mM-1) and 0.998 respectively. The sensitivity value obtained in this
work is better than that of some previously reported modified electrodes such as MnHCF (1.55
µA mM-1) [36], pencil graphite modified CuHCF (4.24 µA mM-1) [38], NPyCME (2.4 µA mM-1)
[41], indigo carmine (2.958 µA mM-1) [42], and AuNPs with Poly (3,4-ethylenedioxythiophene)
(1.15 µA mM-1) [46] (Table 1 [34-48]). Interference due to various biochemicals such as uric
acid (UA), ascorbic acid (AA), dopamine (DA), citric acid (CA), nitrite, hydrazine (Hyd) and
hydrogen peroxide (H2O2) were also tested by spiking the chemicals in different sequences as in
Fig. 9B curves a and b. Interestingly, the GCE/MWCNT@PTZ-biQ found to be highly tolerable
to the above mentioned biochemicals, unlike to the previously reported PTZ derivatives
(Thionine), prussian blue modified with FTO electrode, metal hexacyanoferrate modified
electrodes and Pb-RuO2/NPyCME with serious interference of H2O2 [56- 58], Hyd [59-61] nitrite

21
[62], and DA [63]. These observations clearly indicate the efficient and selective electrocatalytic
function of GCE/MWCNT@PTZ-biQ to cysteine detection.
MWCNT@PTZ-biQ system was further extended to FIA, where a FIA-
GCE/MWCNT@PTZ-biQ system was used as an electrochemical detector along with 0.1 M pH
7 PBS as a carrier system. In order to make the system stable in hydrodynamic condition, a dilute
solution of Nafion (0.1%) was drop-casted on the FIA-GCE/MWCNT@PTZ-biQ surface. FIA
condition was systematically optimized for detection of 100 µM of CySH as applied potential
(Eapp) = 0.3 V vs Ag/AgCl and hydrodynamic flow rate (Hf) = 1 mL min-1 (Fig. S2 A & B).
Under the optimal condition, the electrochemical detector showed linear increase in the FIA
current signal again increase in the CySH concentration in a range 5 to 1000 µM (Fig.10A).
Constructed calibration plot is linear up to 100 µM (1st linearity) with sensitivity and regression
coefficient values 10.47 nA µM-1 (or µA mM-1) and 0.999 respectively. Fourteen successive
injections (n =14) of 5 µM CySH showed a relative standard deviation (RSD) value 4.3 % (Inset
Fig. 10A curve b). Calculated detection value (signal-to-noise=3) is 110 nM. The sensitivity and
detection limit values obtained in this work are superior over the some of the literature reports
(Table 1) [34-43,46-48]. In further, FIA-GCE/MWCNT@PTZ-biQ was also subjected to
detection of various interfering biochemicals like nitrite, nitrate, hydrogen peroxide, glucose and
citric acid (Fig. 10 B). No interference due to the above chemicals was noticed with the optimal
electrode. This observation highlights the selective electrochemical behavior of MWCNT@PTZ-
biQ for CySH in this work. Fig. 11 shows typical real sample analysis of CySH in
pharmaceutical (Samples #1 & #2) and food (Soyabean, #3) samples using FIA-
GCE/MWCNT@PTZ-biQ detector system. Table 2 shows the detailed information for the
analytical assays. The original detected real sample CySH concentration values in tablet #1,

22
tablet #2 and Soyabean #3 are 21.8, 30.17 and 15.6 µg respectively. Calculated recovery values
are closer to 100%.
4. Conclusions
Phenothiazine gets electro-oxidized to Phenothiazine-bi-1,4-quinone derivative (PTZ-
biQ) on a MWCNT surface unusually in a neutral pH solution. This PTZ-biQ behaved as a
electron donor-acceptor type molecular system. Control cyclic voltammetric experiment with an
unmodified GCE failed to show any such response. The new chemically modified electrode
(MWCNT@PTZ-biQ) showed highly stable redox signals at -0.07 V and +0.29 V vs Ag/AgCl
due to surface confined electron-transfer behaviors of biQ and PTZ-cationic radical species in
pH 7 phosphate buffer solution. The redox reactions followed Nerstian type potential-pH
relation. Collective physicochemical characterization using XRD, Raman and IR spectroscopic
techniques confirmed presence of the PTZ-biQ in the hybrid matrix. The GCE/MWNT@PTZ-
biQ showed highly efficient electrocatalytic response to CySH oxidation reaction at low
potential, 0.3 V vs Ag/AgCl in pH 7 PBS. Highly selective and sensitive FIA of CySH in a
couple of real samples was successfully demonstrated using the MWCNT@PTZ-biQ electrode
as a detector.
Acknowledgments
We gratefully acknowledge the Department of Science and Technology-Science and
Engineering Research Council Scheme (DST-SERC) for the financial support. We also thank
DST-Technology System Development (DST-TSD) for the FIA-ECD. Authors also thank
Professors Sriraghavan Kamaraj and Desigan Rajagopal, Organic Chemistry Division, VIT for

23
his valuable comments on the mechanism part of the MWCNT@PTZ-biQ preparation in this
work. ASK acknowledged National Science Council (NSC) of Taiwan for his visiting
professorship.
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29
Figure Captions
Figure 1
(A) Thirty continuous CV responses of GCE/MWCNT@PTZads in pH 7 PBS at
scan rate = 50 mV s-1. (B) Comparative CVs of MWCNT@PTZ-Oxid (a) and
bare GCE/PTZads (b) in pH 7 PBS. (C) Plot of PTZ versus different carbon
materials on the formation of carbons@PTZ-biQ in pH 7 PBS. . Note: PTZ-
Oxid=PTZ-biQ, GNP= Graphite nano-powder, f-MWCNT=functionalized-
MWCNT and p-MWCNT= purified MWCNT.
Figure 2
Figure 3
Effect of scan rate in pH 7 PBS (A) and solution pH at v = 50 mV s-1 (B) on the
CV of GCE/MWCNT@PTZ-biQ. Plots of logipa vs logv and E1/2 vs pH were
given as insets.
Effect of CV potential window on the conversion of GCE/MWCNT@PTZads to
GCE/MWCNT@PTZ-Oxid in pH 7 PBS at v=50 mV s-1. (A, Set-I) CVs at fixed
starting potential, 0.6 V (Set-I) and varying end potentials, -0.4, -0.6, -0.8 and -1.0
V vs Ag/AgCl (cathodic scans). (B, Set-II) CVs at fixed starting potential, -0.6 V
(Set-I) and varying end potentials, as 0.3, 0.4, 0.6 and 0.8 V vs Ag/AgCl (anodic
scans). ORR= Oxygen reduction reaction.
Figure 4
Figure 5
Figure 6
XRD (A) and Raman spectroscopic (B) responses of MWCNT@PTZ-biQ and its
respective control samples. Note PTZ-biQ=PTZ-Oxid.
Comparative FT-IR responses of MWCNT (a), PTZ (b) and MWCNT@PTZ-biQ
(c). Note PTZ-biQ=PTZ-Oxid.
(A and B) In-situ CV and EQCM response of EQCM-Au/MWCNT@PTZads in
pH 7 PBS at v=50 mV s-1. (C) A 11th cycle CV and EQCM response. (D) Plot of
m vs Q for the curve (C). (E) Illustration for involvement of various molecular
species at different CV preparation potentials.
Figure 7
Figure 8
CV responses of GCE/MWCNT@PTZ-biQ-Cu1+/2+ (A), GCE/MWCNT@PTZads-
Cu1+/2+ (B) and GCE/MWCNT-Cu1+/2+ (C) without (b) and with (c) 1 mM H2O2 in
pH 7 PBS. Curves denoting (a) are CVs of respective copper unmodified
electrodes. Scan rate= 50 mV s-1.
(A) CV responses of GCE/MWCNT@PTZ-biQ without (c) and with (d) 1 mM
CySH in pH 7 PBS at 10 mV s−1, and curves (a) and (b) are the responses of
GCE/MWCNT and bare GCE with 1mM CySH respectively. (B) CV responses of
GCE/MWCNT@PTZ-biQ with various concentration of CySH from 0 to 4 mM at
v=10 mV s-1. Inset plot is [CySH] vs ipa.
Figure 9
Comparative amperometric i-t responses of (A) GCE/MWCNT@PTZ-biQ (a),
GCE/MWCNT (b) and GCE (c) for detection of 50 µM of CySH and (B)