A highly selective and simultaneous determination of ascorbic acid, uric acid and nitrite based on a novel poly-N-acetyl-l-methionine (poly-NALM) thin film

Article abstract of DOI:10.1039/c6ra18440e

This paper demonstrates the facile fabrication of an N-acetyl-l-methionine (NALM) polymer film on a glassy carbon electrode (GCE) by an electropolymerization technique. Atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and electrochemical techniques such as cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to characterize the modified electrode. This poly-NALM/GCE not only exhibits strong electrocatalytic activity towards the oxidation of ascorbic acid (AA), uric acid (UA) and nitrite with a shift in oxidation potential towards the less positive side, but also enhances peak current responses at physiological pH (7.2) conditions. Further, the overlapped anodic voltammetric peaks of the three analytes on a bare GC electrode were well-resolved into their independent oxidation peaks at the poly-NALM/GC modified electrode with a peak separation of 160 and 590 mV for AA-UA and UA-nitrite, respectively. Under the optimal experimental conditions, the anodic peak currents of AA, UA and nitrite increased linearly within the concentration ranges 10-1000 μM, 1-600 μM and 1-500 μM with correlation coefficients of 0.990, 0.996 and 0.994, respectively. The detection limits are 0.97, 0.34 and 0.75 μM for AA, UA and nitrite ion, respectively (S/N = 3). The modified electrode was successfully utilized to determine AA, UA and nitrite ion simultaneously in real samples such as human urine and tap water samples.

Full text of DOI:10.1039/c6ra18440e

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Page 1 of 35
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A highly selective and simultaneous determination of ascorbic acid, uric
acid and nitrite based on novel Poly ꢀNꢀacetylꢀLꢀmethionine (PolyꢀNALM)
thin film
a
b
a
c
Ayyadurai Kannan , Arumugam Sivanesan , Govindasamy Kalaivani , Arumugam Manivel ,
a*
Ranganathan Sevvel
a
Post Graduate and Research Department of Chemistry, Vivekananda College,
Tiruvedakam West, Maduraiꢀ625 234, Tamil Nadu, India
b
Nanotechnology and Molecular Sciences Discipline, Faculty of Science and
Engineering, Queensland University of Technology, 2 George st. Gardens Point Campus,
Brisbane 4001, QLD, Australia
c
Post Graduate and Research Department of Chemistry, Saraswathi Narayanan College,
Maduraiꢀ625 022, Tamil Nadu, India
*
Corresponding author: Tel: +91 98657 08536; Fax: +91 4543 258358
Eꢀmail address: sevvelr@gmail.com
1

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Abstract
This paper demonstrates the facile fabrication of NꢀacetylꢀLꢀmethionine (NALM)
polymer film on glassy carbon electrode (GCE) by electropolymerization technique. Atomic
force microscopy (AFM), Xꢀray photoelectron spectroscopy (XPS), Scanning electron
microscopy (SEM) and electrochemical techniques such as cyclic voltammetry (CV) and
differential pulse voltammetry (DPV) were used to characterize the modified electrode. This
PolyꢀNALM/GCE not only exhibits strong electrocatalytic activity towards the oxidation of
ascorbic acid (AA), uric acid (UA) and nitrite with shift in oxidation potential towards less
positive side, but also enhances peak current responses at physiological pH (7.2) condition.
Further, the overlapped anodic voltammetric peaks of the three analytes on bare GC electrode
were wellꢀresolved into their independent oxidation peaks at PolyꢀNALM/GC modified
electrode with a peak separation of 160 and 590 mV for AAꢀUA and UAꢀnitrite, respectively.
Under the optimal experimental condition, the anodic peak currents of AA, UA and nitrite
increased linearly within the concentration ranges 10 – 1000 µM, 1 – 600 µM and 1ꢀ500 µM
with correlation coefficients of 0.990, 0.996 and 0.994, respectively. The detection limits are
0.97, 0.34 and 0.75 µM for AA, UA and nitrite ion, respectively (S/N = 3). The modified
electrode was successfully utilized to determine AA, UA and nitrite ion simultaneously in
real samples such as human urine and tap water samples.
Keywords: Electrochemical polymerization, Poly (NꢀacetylꢀLꢀmethionine)/GCE, Ascorbic
acid, Uric acid, Nitrite.
2

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1. Introduction
Ascorbic acid (AA), Uric acid (UA) and nitrite ion are indispensable biomolecules in
1,2
human metabolism which are usually coexisting in our body fluids. AA can be found in
many biological system and food materials. Because of antioxidant nature of ascorbic acid, it
acts against free radical induced diseases. It is imperative in iron absorption, collagen
synthesis, enhancement of immunity, plays crucial role in wound healing and osteogenesis,
3
ꢀ5
treatments for scurvy and cancer. UA (2,6,8ꢀtrihydroxypurine) is the main end product of
purine metabolism. For a healthy human being, the typical concentration of UA is 0.2~0.5
mM in serum and 1.4~4.4 mM in urine. Monitoring of UA in blood and urine is one of the
parameters in clinical analysis. Variations in the concentrations of UA in human body have
been related to many diseases, such as arthritis, gout, hyperuricaemia, LeschꢀNyan disease,
6
ꢀ9
obesity, diabetes, high cholesterol, high blood pressure, kidney disease and heart disease.
10
Nitrite ion is an antioxidant that is used to cure processed meat. Nitrite is found in drinking
water due to the usage of nitrate rich fertilizers used in agricultural land and also found in
11
animal feed stuffs. On the other hand, excess exposure to nitrite can cause accelerated
pulse, dyspnea, muscle tremors, weakness, vomiting, unstable gait, and cyanosis leading to
death. Ingested nitrite causes the oxidation of haemoglobin into methaemoglobin in blood,
where the methaemoglobin cannot bind with oxygen molecule and subsequently lead to
12
methaeglobinamia or blue baby syndrome. Nitrite can also be converted into carcinogenic
13
N ꢀ nitroso compounds in the body, which may result in cancer and hypertension. Due to
above physiological significance, simultaneous and selective determination of AA, UA and
nitrite is part and parcel of diagnostic research, food analysis and their physiological function
studies. Electrochemical method is a robust and userꢀfriendly technique wellꢀsuited for the
1
4
detection of these biomolecules such as AA, UA and nitrite. However, bare electrodes like
glassy carbon and other metal electrodes fail to detect the biomolecules simultaneously due to
3

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15,16
overlapping of their voltammetric signals and fouling effect.
To overcome these
complications, chemically modified electrodes (CME) have been developed in recent
17ꢀ20
decades. Few important examples of CME’s are selfꢀassembled monolayers,
conducting
21,22
23,24
25,26
27ꢀ29
polymers,
clay modified electrodes,
metal oxides,
metal nanoparticles,
and
30ꢀ32
carbon based material modified electrodes.
Among the various methods of preparation of
CMEs, electropolymerization is the promising technique to immobilize the polymer on GCE.
The advantages of electropolymerized electrodes are precise control in thickness of the film,
wide choice of electrode materials, strong adherence power on surface of the electrode,
broad potential window, large surface area which promotes higher turnꢀover efficiency, ease
3
3
in preparation, high stability and sensitivity.
Amino acids the building block of protein and are known to be precursor for various
physiological metabolic processes. Only few reports are available in the literature using
3
4ꢀ37
amino acids as polymer to sense the biomolecules.
To the best of our knowledge, the
electropolymerization of NꢀacetylꢀLꢀmethionine has not been reported so far. In this study we
report the fabrication of poly (NꢀAcetylꢀLꢀmethionine) film modified glassy carbon electrode
i.e., PolyꢀNALM by electropolymerization using cyclic sweeps and this modified electrode is
applied to catalyse the oxidation of AA, UA and nitrite and simultaneous determination of
them by voltammetric methods. The developed sensor demonstrated high selectivity, stability
and satisfactory reproducibility in response to AA, UA and nitrite. Finally, the ability of this
methodology for the simultaneous determination of AA, UA and nitrite in human urine and
tap water samples has been evaluated with satisfactory recoveries. The main adantages of the
newly fabricated AA, UA and nitrite sensors are that the polyꢀNALM preparation and
modification (rapid and inexpensive electrodepostion method) adopted in the present study is
very simple when compared to the literature reports.
4

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2. Experimental
2.1. Chemicals
NꢀAcetylꢀLꢀmethionine (NALM), ascorbic acid (AA), uric acid (UA) and sodium
nitrite were purchased from Aldrich and were used as received. All other chemicals were
used in this experiment were of analytical grade. Phosphate buffer solutions were prepared
2 4 2 4 3 3
with 0.1 M Na HPO and 0.1 M NaH PO and adjusting the pH using 0.1 M H PO . Doubly
distilled deionized water was used for the preparation of all solutions and washing.
2.2. Live subject statement
All experiments were performed in compliances with the relevant laws and
institutional guidelines. A consent was obtained for any experimentation with human
subjects.
2.3. Instruments
Electrochemical measurements like cyclic voltammetry (CV), and differential pulse
voltammetry (DPV) were performed using CHI 6088D potentiostats (CH Instrument. Austin,
TX, USA). A conventional two compartment three electrode cell, consisting of mirror
polished BASi glassy carbon electrode (GCEꢀ3 mm diameter) as the working electrode,
platinum wire as counter electrode and a Ag/AgCl (saturated KCl) as reference electrode. All
reported potentials were reference to Ag/AgCl (saturated KCl). XPS measurements were
carried out in PHI 5000 VersaProbeTM ꢀ Scanning ESCA Microprobe™ (ULVACꢀPHI,
−
9
α
Japan/USA, 2008) instrument at base pressure <5×10 mbar. Monochromatic AlK radiation
was used and the Xꢀray beam, focused to a 100ꢀꢁm diameter, was scanned over a
2
(
250×250) ꢁm surface at an operating power of 25 W (15 kV). SEM measurements were
performed using FEI Nova™ NanoSEM Scanning Electon Microscope 450 with an
accelerating voltage of 10 kV under high vacuum. Atomic Force Microscopy (AFM) image
5

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was recorded by scanning probe microscope (Vecco, USA). All experiments were done at
room temperature.
2.4. Fabrication of the polyꢀNALM/GCE
PolyꢀNALM/GCE film electrode was prepared by the following process. The GCE
was first mirror polished mechanically with 0.05 µm alumina slurry using polishing cloth and
subsequently rinsed with deionized water. Then the electrode was sonicated in deionized
water for 15 min to completely remove any adsorbed alumina particles.
Electropolymerization of NꢀAcetylꢀLꢀmethionine (NALM) on GCE was carried out by 7
successive potential sweeps between ꢀ0.3 and 1.8 V at a scan rate of 50 mV/s in 2.5 mM of
NꢀAcetylꢀLꢀmethionine (NALM) containing 0.1 M Phosphate Buffer Solution (PBS pH 7.2).
After the electropolymerization, the modified electrode was rinsed thoroughly with distilled
water. The NꢀAcetylꢀLꢀmethionine modified electrode was denoted as PolyꢀNALM/GCE. To
optimise the concentration of NꢀacetylꢀLꢀmethionine monomer for polymerization, we
prepared polyꢀNALM from different concentration of NALM solution ranging from 0.5 mM
to 4 mM concentration. The surface coverage was evaluated from the CV at 100 mV/s. Based
on the Fig. S1, the surface coverage increases with increasing NALM concentration in the
range of 0.5ꢀ2.5 mM and starts to level off when NALM concentration is higher than 2.5
mM. Therefore, 2.5 mM NALM concentration was selected as optimum level in subsequent
studies. Throughout the studies we have used 7 cycles deposited NꢀAcetylꢀLꢀmethionine film
because it showed highest sensitivity towards biomolecules.
3. Results and Discussions
3.1. Electropolymerization of NALM on GCE
6

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Fig. 1 shows a continuous cyclic voltammogram (CV) recorded during the
electropolymerization of NALM on the surface of GCE in phosphate buffer (pH 7.2)
solution. In the first cycle, NꢀacetylꢀLꢀmethionine showed an irreversible electrochemical
oxidation peak at potential of 1.39 V. In the second cycle, the oxidation peak current was
decreased and the potential of peak was slightly shifted to less positive potential. From the
third cycle onwards the oxidation peak current was slowly increased up to seventh cycle.
Increasing of peak current suggests that the new monomer undergoes oxidation in each
oxidation potential scan and the quantity of electroactive conducting polymer increases on
GCE. Based on Fig. 1, we proposed mechanism of electropolymerization of NALM in
scheme 1. The amine group of NALM was oxidized to form free radical at the surface of the
glassy carbon electrode. Then the reactive free radicals combine with surface of GCE rapidly,
and subsequent oxidation of NꢀacetylꢀLꢀmethionine gives poly NALM film on GCE surface.
Fig. S2 A displays the XPS of PolyꢀNALM on ITO plate in C 1s and N 1s regions. The C 1s
spectrum of PolyꢀNALM was deconvoluted into three component peaks at 284.1, 285.8,
38
287.4 and 288.8 eV and were attributed to (ꢀCꢀCꢀ), (ꢀCꢀSꢀ), (ꢀCꢀNꢀ) and (ꢀNꢀC=O),
respectively. Fig. S2 B displays the N 1s region of PolyꢀNALM was deconvoluted into one
39
component peak at 399.7 eV and it was associated with (–NꢀCꢀ). The results obtained from
CV and XPS support the proposed mechanism as given in Scheme 1.
<
Fig. 1 >
<
Scheme 1 >
3.2. Surface Morphology of PolyꢀNALM film
The surface morphology of polyꢀNALM film was analysed by SEM and AFM. SEM
images (Fig. S 3 A, B) and AFM image (Fig. 2) showed the surface morphology of polyꢀ
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NALM film deposited by 7 cycles on ITO electrode. The SEM images of polyꢀNALM film
showed comparatively smooth and homogeneous surface. Similarly, the AFM image of polyꢀ
NALM clearly indicates that the homogeneous spherical like structure with increased real
surface area. It is corroborated with the increased oxidation peak currents of AA, UA and
nitrite at polyꢀNALM/GC modified electrode.
<
Fig. 2 >
3.3. Electrochemical impedance spectroscopy study
Electrochemical Impedance Spectroscopy (EIS) is an effective tool for studying
resistivity changes of electrode surface during modification process. EIS contains both
semicircle part at high frequency and linear part at low frequency which are correspond to
electron transfer limited process and diffusion process respectively. The diameter of the
semicircle in the impedance spectrum is equal to charge transfer resistance, R Fig. 3 shows
ct.
the results of impedance spectra of bare GCE (curve a) and polyꢀNALM/GCE (curve b). On
the bare GCE, the value of Rct was obtained 6960 Ω (curve a). After fabrication of polyꢀNꢀ
acetylꢀLꢀmethionine at the GCE surface, the value of Rct was obtained 834 Ω (curve b). The
decreased Rct value for polyꢀNALM/GCE shows that the conductive polyꢀNALM film has
been attached to the GCE surface and it indicates that the electron transfer process of
modified electrode is relatively fast compared to bare GCE.
<
Fig. 3 >
3.4. Electrochemical properties of the polyꢀNALM/GCE
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2 4
The CV obtained for polyꢀNALM deposited on GCE by 7 cycles in 0.1 M H SO at a
scan rate of 20 mV/s is shown in Fig. 4A. It can be seen that the polyꢀNALM film on GCE
shows redox couple about 0.338/0.370 V at low scan rate of 20 mV/s. The observed peak
separation potential, ∆ Ep = (Epa – Epc) of 32 mV indicates that the redox couple of polyꢀ
NALM film has a reversible behaviour which denotes the perfect Nernstian system. Based on
40
Nernst equation (∆ Ep is close to 2.3 RT/nF or 59/n mV at 25 °C), the number of electron in
the redox process is 2. The effect of potential scan rates (υ) on the electrochemical properties
of polyꢀNALM/GCE was studied by CV in 0.1 M H
2 4
SO solution at different scan rates (Fig.
–1
4
B). When the scan rate was increased from 50 to 1000 mVs , both the oxidation and
reduction peak currents were linearly increased without any change of redox potentials,
which confirmed that the electrode reaction is surface confined redox process and reversible
redox reaction. Plots of anodic currents linearly dependent on scan rate (υ) in the range from
–
1
5
0 to 1000 mVs (Fig. 4B inset). The surface coverage was evaluated from the cyclic
voltammogram recorded at 0.02 V/s and using the equation Г = Q/nFA, where Q is the
charge obtained by integrating the anodic peak current under the background correction, n is
the number of electron involved in the electron transfer process, F is the Faraday constant and
2
41, 42
A is the geometric area of the GC electrode (0.0707 cm ).
The surface coverage of polyꢀ
ꢀ10
ꢀ2
NALM film on glassy carbon electrode is 2.04 x 10 mol cm indicating the formation of
ultrathin polyꢀNALM film on GCE.
<
Fig. 4 >
3.5. Single electrochemical oxidation of AA, UA and Nitrite
The electrochemical behaviour of AA, UA and nitrite was investigated by cyclic
voltammetry at the surface of bare GCE and polyꢀNALM/GCE. Fig.5. (A) shows the CV of
9

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750 µM AA at bare GCE (curve a) and polyꢀNALM/GCE (curve b) in 0.1 M phosphate
buffer solution (pH 7.2), respectively. As can be seen, the bare GC electrode showed a broad
oxidation peak for AA at 310 mV (6.5 µA), which indicates that slow electron transfer
kinetic. After electrode modification, the polyꢀNALM/GCE showed a wellꢀdefined oxidation
peak for AA at 130 mV with an enhanced peak current (17.1 µA). Furthermore, a 180 mV
less positive potential shift and a threefold higher oxidation current were obtained for AA at
polyꢀNALM/GCE compared to the bare GC electrode. For 400 µM UA (in Fig. 5. B), a
sluggish oxidation peak potential of 440 mV (7.5 µA) was observed at bare electrode,
however a wellꢀdefined and sharp oxidation peak was obtained at 320 mV (14.2 µA) at polyꢀ
NALM/GC modified electrode. Moreover, a 120 mV less positive potential shift and twofold
higher oxidation current were observed at polyꢀNALM/GC electrode which indicates that
modified electrode shows high electron transfer rate. In similar way (in Fig. 5. C), for 500
µM nitrite, the oxidation peak potential of 1090 mV (13.7 µA) was observed at bare GC
electrode but a clearly defined oxidation peak potential of 870 mV (25.1 µA) at polyꢀ
NALM/GCE with 220 mV less positive potential and approximately two fold higher
oxidation current were observed. These significant increase in current responses and shifting
to less positive potential at polyꢀNALM modified electrode confirms that the modified
electrode facilitate the electrochemical oxidation and thus decreases the over potential of
analytes.
<
Fig. 5 >
3
.6. Influence of the solution pH value
The pH value of buffer solution significantly influences the oxidation peak current
and peak potential of AA, UA and nitrite ion at polyꢀNALM/GC electrode. The effect of pH
1
0

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value of the solution on peak current and peak potential has been studied by recording the
CVs of AA, UA and nitrite ion with concentrations 750 µM, 400 µM and 500 µM,
respectively, in a series of PB solutions of varying pH in the range 4.2 ꢀ9.2. The plot of the
peak currents versus varying pH for AA, UA and nitrite ion are shown in Fig. 6 A. As can be
seen, the peak currents of these three compounds increase with the pH value from 4.2 to 7.2
and reach a maximum at pH 7.2, and then they decrease gradually when the pH values were
increased. Thus pH 7.2 was selected as the optimum pH for the determination these three
compounds. It can be seen from Fig. 6 B that, the oxidation peak potentials of AA, UA and
nitrite ion shift negatively with gradual increase of pH value. Slope of the curve Ep versus pH
was obtained ꢀ0.061 V, ꢀ0.063 V and ꢀ0.059 V for AA, UA and nitrite, respectively. The
–1
obtained slopes are close to the theoretical value of 0.059 VpH and suggest that same
3
7
protons and electrons are involved in the electrode reaction.
Fig. 6 >
<
3.7. The effect of scan rate on the electrochemical oxidation of AA, UA and nitrite ion
The influence of scan rate on the electrochemical behaviour of AA, UA and nitrite ion
at polyꢀNALM/GC modified electrode was studied by CV. As shown in Fig. 7, the anodic
peak currents of AA, UA and nitrite increase linearly with the square root of scan rate in the
–
1
–1
–1
range of 50ꢀ1000 mVs , 50ꢀ1000 mVs and 50ꢀ800 mVs , respectively. The linear
1/2
1/2
equations for AA, UA and nitrite ion are ipa = 3.073 υ ꢀ2.004, ipa = 2.1964 υ ꢀ8.969 and ipa
1/2
=
2.806 υ ꢀ 4.794 with correlation coefficients of 0.9974, 0.9992 and 0.9989, respectively.
These results indicate that the electrode reactions of these three analytes at polyꢀNALM/GC
1
, 28
modified electrode are diffusion controlled processes.
<
Fig. 7 >
1
1

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3.8. Electrochemical behaviour of AA, UA and nitrite in a mixture at polyꢀNALM/GCE
Fig.8 displays the CVs of 750 µM of AA, 500 µM of UA and 500 µM of nitrite in
phosphate buffer solution (pH 7.2) at bare GC and polyꢀNALM/GC electrodes. Bare
electrode showed the oxidation potential of AA and UA at 440 mV and nitrite at 1100 mV
which means that the oxidation peaks of AA and UA completely merged and the peak
potentials for AA and UA are indistinguishable (curve a). This reveals that it was not possible
to determine AA, UA and nitrite simultaneously at bare GCE. Further, in the subsequent
cycles the decreasing of peak currents observed for AA, UA and nitrite (curve b). This
revealed that bare GC electrode is not suitable for stable detection. In contrast to bare GC
electrode, polyꢀNALM/GCE oxidizes AA, UA and nitrite at 170 mV, 330 mV and 920 mV
respectively with enhanced current response in mixture (curve c). Further the oxidation peaks
of AA, UA and nitrite were more stable at polyꢀNALM/GCE in the subsequent cycles (curve
d). The separation of oxidation peak potential for AAꢀUA and UAꢀnitrite were of 160 mV
and 590 mV respectively. The separations were good enough for simultaneous determination
of AA, UA and nitrite. This indicated that the oxidation of AA, UA and nitrite at polyꢀ
NALM/GCE is independent and therefore simultaneous measurements of these three analytes
are possible without any interference. Curve e is the CVs of modified electrode in the absence
of analytes.
<
Fig. 8 >
3
.9. Simultaneous determinations of AA, UA and nitrite
The main objective of this study is to determine AA, UA and nitrite
simultaneously using polyꢀNALM/GCE. Fig.9A shows the DPV obtained for simultaneous
1
2

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determination of AA, UA and nitrite at polyꢀNALM/GCE in 0.1 M phosphate buffer solution
pH 7.2). It shows the oxidation peak for 10 µM AA at 40 mV, 1 µM UA at 260 mV and 1
(
µM nitrite at 770 mV. We investigated the current response of AA, UA and nitrite at polyꢀ
NALM/GCE while simultaneous increment of the concentration of AA, UA and nitrite. It
showed that the oxidation current of AA, UA and nitrite were linearly increased when
simultaneously increase the concentration of them without any shift in the oxidation
potential. The linear relationships between peak currents ipa (µA) and concentrations c (µM)
of AA, UA and nitrite are: ipa, AA = 1.154 + 0.0103cAA (r = 0.990), ipa, UA = 1.818 +
0
.036cUA (r = 0.996), and i nitrite = 1.382 + 0.0218 cnitrite (r = 0.994) respectively (Fig. 9 B,
pa,
C & D). The linear concentration ranges of AA, UA and nitrite in simultaneous
determinations are 10 – 1000 µM, 1 – 600 µM and 1 – 500 µM respectively. The detection
limits of AA, UA and nitrite are found to be 0.97, 0.34 and 0.75 µM (S/N =3), respectively.
The performance of the fabricated AA, UA and nitrite’s sensors are very much comparable to
1
,2,43ꢀ48
the literature values (Table 1).
It is evident from Table 1, the newly designed polyꢀ
NALM modified electrode exhibited relatively low detection limit, high sensitivity and wide
linear range. The superiority of the present sensor can be attributed to that fact that the high
electrical conductivity and electroactive functional groups of the polymers.
<
Fig. 9>
3.10. Selective determination of AA, UA and nitrite
The other objective of this work is selective determination of one compound in the
presence of higher concentrations of other compounds. It is well known that AA, UA and
nitrite coexisted in human fluids. Therefore it is inevitable to selectively determine each
compound. Fig.10 (AꢀC) showed selective determination of AA, UA and nitrite at polyꢀ
1
3

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NALM/GCE in 0.1 M phosphate buffer solution (pH 7.2). Fig. 10 (A) showed the DPVs for
the consecutive increment of 10 µM AA in presence of 250 µM each UA and nitrite. Higher
concentrations of UA and nitrite did not affect the oxidation peak current and peak potential
of AA. The electroꢀoxidation of AA, UA and nitrite on polyꢀNALM is represented in
Schemeꢀ2. The oxidation peak current of AA linearly increased with increasing
concentration. Likewise, Fig. 10 (B) and 10 (C) showed linear increase of oxidation currents
of UA and nitrite when the concentration of other two compounds are high and constant. The
oxidation currents of AA, UA and nitrite were linearly increased with correlation coefficient
of 0.9997, 0.9996 and 0.9965, respectively. These results proved that the selective and
sensitive determination of AA, UA and nitrite is possible at polyꢀNALM/GC electrode.
<
Fig. 10 >
<
Scheme 2 >
3.11. Anti – interference of the polyꢀNALM film
The anti – interference ability of polyꢀNALM/GCE was investigated towards the
+
+
+
4
detection of AA, UA and nitrite in the presence of common existing ions Na , K , NH ,
2
+
2+
–
–
2–
2–
Mg , Ca , Cl , F , CO
3
and SO
4
and some physiological interferents such as glucose,
citric acid, cysteine and oxalate by DPV method. No change in current and shifting of
potential were observed for 30 µM AA, 10 µM UA and 10 µM nitrite in presence 500ꢀfold
excess of above mentioned species, indicating that the present modified electrode was not
affected by maximum concentration of foreign substances and highly selective towards the
AA, UA and nitrite. Moreover, Dopamine, epinephrine, serotonin and tyrosine had very small
influences on the determination of AA and UA which is shown in Table S4.
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3.12. Repeatability and reproducibility of the polyꢀNALM film
To validate the performance of polyꢀNALM/GC electrode on biosensing, repeatability
and reproducibility are very significant parameters. To check repeatability of modified
electrode, the DPVs were recorded for 50 µM AA, 20 µM and 20 µM nitrite in 0.1M
phosphate buffer solution (pH 7.2). It was found that the oxidation peak currents of AA, UA
and nitrite remain the with a relative standard deviations of 2.1 %, 1.8 % and 2.4 %
respectively for 20 repetitive measurements, indicating that this modified electrode has good
stability on repetitious measurements. The modified electrode was stored in 0.1 M phosphate
buffer solution at 4 °C in a refrigerator. After ten days, the current responses were decreased
by 4 %, 2.5 % and 3.3 % for AA, UA and nitrite respectively. In addition, to verify the
reproducibility of the results, three different polyꢀNALM/GC electrodes were prepared and
responses toward the AA, UA and nitrite recorded. The peak current showed relative standard
deviations of 2.4 %, 2.4 % and 3.1 % for AA, UA and nitrite respectively, confirming that the
results are reproducible.
3.13. Real sample Analysis
To asset practical applicability of this research work for simultaneous determination
of AA, UA and nitrite in real sample, human urine and tap water were selected for analysis
using standard addition technique. For the pretreatment of human urine, the urine sample was
centrifuged and obtained supernatent was diluted 100 times with 0. 1 M phosphate buffer
solution(pH 7.2). The tap water sample was used as such. No AA or nitrite has been detected
49
in male urine which was in line with reported literture and uric acid concentration is very
46,50
close to literature.
. The results are summerized in Table 2, which indicate that the
recoveries of the spiked samples were in the range of 97 – 106 %. Therefore, the real sample
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analysis, showed that the successful applicability of the this work for simultaneous
determination of AA, UA and nitrite.
4. Conclusions
The fast and simple procedure was adopted for the fabrication of polyꢀNALM/GC
modified electrode for the electrochemical detection of AA, UA and nitrite simultaneously.
The modified electrode exihibited good elctrocatalytic activity towards the oxidation of AA,
UA and nitrire. Further, this modified electrode not only enhanced the electrochemical
catalytic oxidation of AA, UA and nitrite also resolved the overlapping of anodic oxidation
peaks. The separation of oxidation peak potentials for AAꢀUA and UAꢀnitrite were of 160
and 590 mV, respectively, demonstrated electrode could be used to detect AA, UA and nitrite
simultaneously and selectively. Furthermore, this modified electrode showed good stability,
high reproducibility and low detection limit. In addition, this modified electrode was
employed for the simultaneous determination of AA, UA and nitrite in real samples with
satisfactory results.
Acknowledgements
Financial support from the University Grants Commission – South Eastern Regional
Office (UGC – SERO), Hyderabad, for the award of Minor Research Project (No. F MRPꢀ
5855/15(SEROꢀUGC) is gratefully acknowledged by AK. AK, GK and RS would like to
thank The Management and The Principal, Vivekananda College, Tiruvedakam West to carry
out this work.
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Figure Captions
Fig. 1. Cyclic voltammograms (7 cycles) obtained for electropolymerization of NALM on
–1
GCE in 0.1 M phosphate buffer solution (pH 7.2) at a scan rate of 50 mVs . 1, 2 and
st nd
th
7
represent 1 , 2 and 7 cycles.
Fig. 2. AFM image of PolyꢀNALM film
Fig. 3. Nyquist plots showing Faradaic impedance measurements of bare GCE (a), Polyꢀ
NALM/GCE (b) in 1mM K
3
[Fe(CN)
6
]/ K
4
[Fe(CN)
6
] (1:1) containing 0.1 M KCl.
SO at scan rate of 20
mVs . (B) Cyclicvoltammograms of polyꢀNALM/GCE in 0.1 M H SO at scan rates
Fig. 4. (A) Cyclic voltammogram of polyꢀNALM/GCE in 0.1 M H
2
4
–
1
2
4
of (a) 50, (b) 100, (c) 200, (d) 300, (e) 400, (f) 500, (g) 600, (h) 700, (i) 800, (j) 900,
–1
(
2 4
k) 1000 mVs in 0.1 M H SO . Inset: Plot of anodic peak current vs scan rate.
Fig. 5. Cyclic voltammogram of 750 µM AA (A), 400 µM UA (B) and 500 µM nitrite (C) at
bare GCE (a) and a polyꢀNALM/GCE (b) in phosphate buffer solution (pH 7.2). Scan
–1
rate 50 mVs .
Fig. 6. Effect of pH on peak current (A) and peak potential (B) for the oxidation of 750 µM
AA, 400 µM UA and 500 µM nitrite at polyꢀNALM/GC modified electrode.
Fig. 7. CVs of 750 µM AA (A), 400 µM UA (B) and 500 µM nitrite (C) at PolyꢀNALM/GCE
in 0.1 M PBS pH 7.2, under various scan rates. Scan rates for AA and UA (from inner
–
1
to outer) are 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 mVs . Scan
rates for nitrite (from inner to outer) are 50, 100, 200, 300, 400, 500, 600, 700 and 800
–
1
mVs . The insets in A, B and C are plots of peak currents vs. square root of the scan
rates.
Fig. 8. Cyclic voltammograms of 750 µM AA, 500 µM UA and 500 µM nitrite at bare GCE
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th
th
(
a), after 10 cycle (b) and at a polyꢀNALM/GCE (c) after 10 cycle (d) in phosphate
–
1
buffer solution (pH 7.2). Scan rate 50 mVs . (e) CV obtained for polyꢀNALM/GCE
in the absence of AA, UA and nitrite in 0.1 M phosphate buffer solution.
Fig. 9. Differential pulse voltammogramms for the simultaneous increase of (I) a) 10, b)
2
0, c) 40, d) 60, e) 100, f) 150, g) 200, h) 300, i) 400, j) 600, k) 800, l) 1000 µM of
AA, (II) ) a) 10, b) 25, c) 50, d) 100, e) 150, f) 200, g) 250, h) 300, i) 350, j) 400, k)
00, l) 600 µM 0f UA and (III) ) a) 1, b) 5, c) 25, d) 50, e) 75, f) 100, g) 150, h) 200,
5
i) 250, j) 300, k) 400, l) 500 µM of nitrite at a polyꢀNALM/GCE in phosphate buffer
solution (pH 7.2).(B) Plot of concentration of AA vs Current. (C) Plot of
concentration of UA vs Current. (D) Plot of concentration of nitrite vs Current.
Fig. 10. DPVs obtained at polyꢀNALM/GCE for (A) each addition increases the 10 µM of
AA from 0 to 130 µM in presence of 250 µM of each UA and nitrite, (B) each
addition increases the 5 µM of UA from 0 to 65 µM in presence of 500 µM AA and
250 µM of nitrite and (C) each addition increases the 10 µM of nitrite from 0 to 130
µM in presence of 500 µM AA 250 µM of UA. Inset: In A, B and C is plot of
oxidation current vs concentration of AA, UA and nitrite respectively.
Scheme 1. Mechanism of electropolymerization of NꢀacetylꢀLꢀmethionine on GCE
Scheme 2. Electrochemical oxidation of AA, UA and nitrite on PolyꢀNALM/GCE
Table 1. Comparison of some modified electrodes in the determination of AA, UA and nitrite
Table 2. Determination results of AA, UA and nitrite in real samples (n = 6)
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4
3
2
1
00
00
00
00
0
1
00
1
7
2
1
.2
1.3
1.4
1.5
7 1
E / V vs Ag/AgCl (KCl sat)
2
0
.0
0.5
1.0
1.5
E / V vs Ag/AgCl (KCl sat)
Fig. 1
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Fig. 2
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8000
6000
4000
2000
0
a
b
0
4000
8000
12000
16000
20000
Z'/ohm
Fig. 3
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A
2
0
2
ꢀ
ꢀ
ꢀ
0.0
0.2
0.4
0.6
E / V vs. Ag / AgCl (KCl sat)
4
2
0
0
0
k
B
a
40
2
R
= 0.9991
30
20
10
0
0
200
400
600
800
1000
Scan rates (mV / s)
ꢀ
20
0
.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
E / V vs. Ag/AgCl (KCl sat)
Fig. 4
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A
b
1
6
8
0
a
ꢀ
0.2
0.0
0.2
0.4
0.6
E / V vs. A g /A g C l (K C l sa t)
1
6
B
b
8
0
a
0
.0
0.2
0.4
0.6
E / V v s. A g/A gC l (K C l sa t)
b
C
2
0
a
1
0
0
0
.0
0.3
0.6
0.9
1.2
E / V vs. A g /A g C l (K C l sa t)
Fig. 5
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28
24
20
16
12
8
A
_
NO2
AA
UA
4
5
6
7
8
9
10
pH
1.6
1.2
0.8
0.4
0.0
B
2
Y = ꢀ0.061 x + 0.5698 (R =0.992)
_
NO2
2
Y = ꢀ0.063 x + 0.774 (R =0.989)
UA
AA
2
Y = ꢀ0.059 x + 1.298 (R =0.9949)
ꢀ0.4
3
4
5
6
7
8
9
10
11
pH
Fig. 6
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8
4
0
0
0
A
6
4
2
0
0
0
5
10
15
20
25
30
35
1/2
ꢀ1 1/2
)
υ
/(mVs
ꢀ
0.4
ꢀ0.2
0.0
0.2
0.4
E / V vs. Ag/AgCl (KCl sat)
B
8
4
0
0
0
8
6
4
2
0
0
0
0
5
10
15
20
25
30
35
1
/2
ꢀ1 1/2
)
υ
/(mVs
ꢀ0.3
0.0
0.3
0.6
E/V vs. Ag/AgCl (KCl sat)
C
8
4
0
8
0
0
0
6
4
0
0
2 0
5
10
15
2 0
1/2
2 5
30
υυ
1
/2/(m V sꢀ1
)
0
.0
0.4
0.8
1.2
E / V vs. Ag/AgCl (KCl sat)
Fig. 7
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-
c
NO2
UA
d
2
0
0
0
a
b
AA
1
e
ꢀ0.4
0.0
0.4
0.8
1.2
E / V vs. Ag/AgCl (KCl sat)
Fig. 8
2
9