A novel gold nanoparticle decorated nanocrystalline zeolite based electrochemical sensor for the nanomolar simultaneous detection of cysteine and glutathione

Article abstract of DOI:10.1039/c5ra19249h

In this work, highly dispersed gold nanoparticle decorated nanocrystalline zeolite was synthesized by the electrostatic interaction between the functionalized gold nanoparticles and functionalized nanocrystalline zeolite. An electrochemical sensor based on the gold nanoparticle decorated nanocrystalline zeolite was developed for the nanomolar simultaneous detection of cysteine and glutathione with high sensitivity, selectivity, and remarkably low detection limit. A wide linear range was obtained from 2 nM to 800 μM and 3 nM to 800 μM with a limit of detection of 0.3 nM and 0.6 nM for cysteine and glutathione, respectively. The analytical performance of the developed sensor was demonstrated in the determination of cysteine and glutathione in commercial pharmaceutical preparations with satisfactory results even in the presence of several amino acids. The proposed methodology provides promising application in clinical diagnostic and drug analysis.

Full text of DOI:10.1039/c5ra19249h

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A novel gold nanoparticles decorated nanocrystalline zeolite based
electrochemical sensor for the nanomolar simultaneous detection of cysteine
and glutathione
a
a
b
Balwinder Kaur , Rajendra Srivastava* , and Biswarup Satpati
a
b
Department of Chemistry, Indian Institute of Technology Ropar, Rupnagarꢀ140001, India
Surface Physics and Material Science Division, Saha Institute of Nuclear Physics, 1/AF,
Bidhannagar, Kolkata 700 064, India
_____________________________________________________________________________
_
Eꢀmail: rajendra@iitrpr.ac.in
Phone: +91ꢀ1881ꢀ242175; Fax: +91ꢀ1881ꢀ223395
1

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_
____________________________________________________________________________
Abstract
In this work, highly dispersed gold nanoparticles decorated nanocrystalline zeolite was
synthesized by the electrostatic interaction between the functionalized gold nanoparticles and
functionalized nanocrystalline zeolite. An electrochemical sensor based on gold nanoparticles
decorated nanocrystalline zeolite was developed for the nanomolar simultaneous detection of
cysteine and glutathione with high sensitivity, selectivity, and remarkably low detection limit. A
wide linear range was obtained from 2 nMꢀ800 µM and 3 nMꢀ800 µM with a limit of detection
of 0.3 nM and 0.6 nM for cysteine and glutathione, respectively. Analytical performance of the
developed sensor was demonstrated in the determination of cysteine and glutathione in the
commercial pharmaceutical preparations with satisfactory results even in the presence of several
amino acids. The proposed methodology provides promising application in clinical diagnostic
and drug analysis.
Keywords: Gold nanoparticles; Nanocrystalline zeolite; Electrocatalytic oxidation; Cysteine;
Glutathione
__________________________________________________________________________
2

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Introduction
Human body is composed of large number of biomolecules (such as amino acids,
proteins, carbohydrates, nucleic acids etc.) that govern the physiological function of the body.
Abnormal concentration of these biomolecules is responsible for several diseases. Therefore,
selective and sensitive detection of these biomolecules is very important in clinical diagnosis and
pharmaceutical preparations. One such biomolecule is glutathione (GSH). GSH is synthesized in
1
the body from the amino acids Lꢀcysteine, Lꢀglutamic acid, and glycine. Cysteine (CySH)
concentration is the rate limiting factor in glutathione biosynthesis because Lꢀcysteine is
1
relatively rare in food supplement and is usually synthesized in the human body. Glutathione
1
exists in reduced (GSH) and oxidized states (GSSG) in the body. In the reduced state the –SH
group of GSH can donate proton and electron to other unstable molecules such as reactive
oxygen species. After donating proton and electron, two reactive glutathione molecules combine
2
and form GSSG. GSH can be regenerated from GSSG by the enzyme glutathione reductase. In
healthy cells and tissues, more than 90% of total glutathione is in the reduced (GSH) form and
3
less than 10% exists as GSSG. An increased GSSG/GSH ratio is considered indicative of
oxidative stress. Therefore, GSH acts as an important antioxidant and prevents damage to
important cellular components caused by reactive oxygen species such as free radicals,
4
peroxides, and heavy metals. GSH is used in metabolic and biochemical reactions such as DNA
4
synthesis and repair, protein synthesis, amino acid transport, and enzyme activation. GSH also
plays an important role in iron metabolism. Hence, every system in the body can be affected by
the glutathione state especially the immune system, nervous system, and the lungs. GSH is also
an essential antidote to the overdose of paracetamol (analgesic and antipyretic drug). The
abnormal concentration of GSH in humans is directly related to various diseases such as
Alzheimer’s disease, Parkinson’s disease, diabetes, macular degeneration, cancer and
4
ꢀ8
HIV/AIDS. CySH is very important in GSH biosynthesis as discussed above and serves as an
9
ꢀ11
important amino acid in humans.
Since the GSH is related to the concentration of CySH,
therefore, simultaneous detection of these biomolecules is very important in clinical diagnosis
and pharmaceutical formulations. Since the ꢀSH bond in GSH and CySH can be oxidized, so
electrochemical technique can be employed for the detection and measurement of GSH and
CySH. Moreover, the electrochemical methods are simple, cost effective, highly sensitive, and
have capacity of being readily integrated with other techniques for multiꢀanalysis. However, the
3

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electrochemical oxidation of thiol group (ꢀSH) at the conventional electrodes (gold, platinum,
carbon) is very slow and requires very high potential (overpotential). In order to overcome these
drawbacks, several chemically modified electrodes based on carbon, boron doped diamond,
metal oxides, polymers, and layered double hydroxides with improved response have been
9
, 11ꢀ19
developed.
These modified electrodes were developed for the single determination of
CySH or GSH. However, developing electrode materials for the simultaneous determination of
these coꢀexisting thiol containing biomolecules from their mixture solution with low limit of
detection and high sensitivity is still a challenge. The present study is focused to achieve this
motive.
Our research is mainly focused on the synthesis of nanocrystalline zeolites and explores
2
0ꢀ23
their applications in electrocatalysis.
Through our recent studies we have shown that
nanocrystalline zeolites remarkably improves the electrochemical sensing ability compared to the
24
bulk zeolite. For the electrochemical oxidation of GSH and CySH, some suitable redox active
metal is also required. It is reported in the literature that gold can be used for the electrochemical
1
6, 25, 26
oxidation of thiol group.
Hence, gold nanoparticles decorated nanocrystalline zeolite was
synthesized and investigated in the electrochemical determination of CySH and GSH.
In this study, we present the synthesis of highly dispersed gold nanoparticles decorated
nanocrystalline zeolite (AuNPsꢀNanoꢀZSMꢀ5) as an electrode material for the fabrication of
electrochemical sensor for simultaneous determination of CySH and GSH. The developed
AuNPsꢀNanoꢀZSMꢀ5 based sensor exhibited remarkably high activity, sensitivity, and
selectivity. To the best of our knowledge, this is the first report which deals with the
simultaneous electrochemical determination of CySH and GSH based on gold nanoparticles
decorated NanoꢀZSMꢀ5 as an electrode material.
Experimental Section
Materials
All chemicals were of analytical reagent grade and used as received without further
purification. Tetraethylorthosilicate (TEOS, 98%), tetrapropylammonium hydroxide (TPAOH),
propyltriethoxy silane (PrTES, 97%), 3ꢀaminopropyl trimethoxysilane, and gold (III) chloride
trihydrate (HAuCl .3H O) were purchased from Sigma Aldrich, India. Sodium borohydride was
4
2
obtained from Spectrochem Pvt. Ltd., India. Trisodium citrate was obtained from Loba Chemie
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Pvt. Ltd., India. LꢀCysteine was obtained from Himedia, India. Reduced glutathione was
obtained from Sisco Research Laboratories Pvt. Ltd., India. Deionized water from Millipore
MilliꢀQ system (Resistivity 18 MΩcm) was used in the electrochemical studies. Electrochemical
measurements were performed in 0.1 M phosphate buffer (Sorenson's buffer) solution, which
was prepared by mixing potassium monohydrogen phosphate (Na HPO ) and potassium
2
4
dihydrogen phosphate (NaH PO ). All electrochemical experiments were performed in 0.1 M
2
4
phosphate buffer solution (PBS) at pH 7.4.
Synthesis of AuNPs decorated Nano-ZSM-5 nanocomposite
Nanocrystalline ZSMꢀ5 zeolite (NanoꢀZSMꢀ5) was prepared using molar composition
TEOS/10 PrTES/2.5 Al O /3.3 Na O/25 TPAOH/2500 H O by following the reported
2
3
2
2
2
0
procedure. Propyl amine was then covalently anchored on the external surface of NanoꢀZSMꢀ5.
NanoꢀZSMꢀ5 (2 g) and (3ꢀaminopropyl)trimethoxysilane (1.43 g) were taken in 50 mL toluene
and the reaction mixture was refluxed for 12 h. After the reaction, the reaction mixture was
filtered and solid sample was washed with toluene followed by acetone and dried at 323 K for 24
h in oven to obtain propyl amineꢀfunctionalized NanoꢀZSMꢀ5 (hereafter represented as Nanoꢀ
ZSMꢀ5ꢀPrꢀNH2).
For the synthesis of gold nanoparticles decorated NanoꢀZSMꢀ5 nanocomposite (hereafter
represented as AuNPs(0.5%)ꢀNanoꢀZSMꢀ5), NanoꢀZSMꢀ5ꢀPrꢀNH (300 mg) was dispersed in
2
ethanol (30 mL). Then acetic acid (3 mL) was added into it at ambient temperature to induce the
positive charge on the surface of NanoꢀZSMꢀ5ꢀPrꢀNH . The solution was then sonicated at room
2
temperature for 1 h. In another beaker, an aqueous solution (30 mL) containing HAuCl (3 mg,
4
ꢀ4
ꢀ4
2
.5 x 10 M) and trisodium citrate (2.24 mg, 2.5 x 10 M) was prepared and maintained at
temperature 276ꢀ277 K. Then, cold NaBH solution (0.9 mL, 0.1 M) was added with vigorous
4
stirring to this HAuCl solution at a temperature 276ꢀ277 K. The solution turned wine red
4
immediately after the addition of NaBH , indicating the formation of gold nanoparticles. Finally,
4
the solution of positively charged NanoꢀZSMꢀ5ꢀPrꢀNH was added to the gold solution under
2
sonication at room temperature and sonication was continued for further 30 min. The resulting
product was centrifuged, washed with ethanol and water followed by drying at 333 K. AuNPsꢀ
NanoꢀZSMꢀ5 nanocomposites with different weight ratios (0.1, 1, and 3, denoted as
AuNPs(0.1%)ꢀNanoꢀZSMꢀ5, AuNPs(1%)ꢀNanoꢀZSMꢀ5, and
AuNPs(3%)ꢀNanoꢀZSMꢀ5,
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respectively) were also synthesized using the similar procedure with varying the amount of
HAuCl4.
Instrumentation
Xꢀray diffraction (XRD) patterns were recorded in the 2θ range of 5ꢀ80° with a scan
speed of 2°/min on a PANalytical X’PERT PRO diffractometer, using Cu Kα radiation
(λ=0.1542 nm, 40 kV, 40 mA) and a proportional counter detector. Nitrogen adsorption
measurements were performed at 77 K by Quantachrome Instruments, AutosorbꢀIQ volumetric
adsorption analyzer. Sample was outꢀgassed at 473 K for 3 h in the degas port of the adsorption
apparatus. The specific surface area of zeolites was calculated from the adsorption data points
obtained at P/P between 0.05ꢀ0.3 using the BrunauerꢀEmmettꢀTeller (BET) equation. The pore
0
diameter was estimated using the Barret–Joyner–Halenda (BJH) method. TEM investigations
2
were carried out using FEI, Tecnai G F30ꢀST microscope operated at 300 kV. Highꢀangle
annular dark field scanning transmission electron microscopy (HAADFꢀSTEM) was used here
using the same microscope, which was equipped with a scanning unit and a HAADF detector
from Fischione (model 3000). The compositional analysis was performed using energy
2
dispersive Xꢀray spectroscopy (EDS, EDAX Inc.) attachment on the Tecnai G F30. The sample
was dispersed in ethanol using ultrasonic bath, and dispersed sample was mounted on a carbon
coated Cu grid, dried, and used for TEM measurement. Fourier transform infrared (FTꢀIR)
ꢀ1
spectra were recorded on a Bruker spectrophotometer in the region 600ꢀ4000 cm (spectral
ꢀ1
resolution = 4 cm ; number of scans = 100). Thermogravimetric analysis (TGA) was performed
e
on a TGA/DSC 1 STAR SYSTEM from Mettler Toledo instrument with temperature increments
of 10 K/min in air stream.
Electrode fabrication
Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and
chronoamperometry studies were performed using PotentiostatꢀGalvanostat BASi EPSILON,
USA. A threeꢀelectrode electrochemical cell was employed with Ag/AgCl as the reference
electrode (3M KCl), AuNPsꢀNanoꢀZSMꢀ5 mounted glassy carbon (3 mm diameter) as the
working electrode and Pt foil as the counter electrode. Before modification, the glassy carbon
electrode (GCE) was first polished to a mirror like surface with alumina slurry and then
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ultrasonicated in ethanol and deionized water for 5 min, respectively. 10 µL aliquot of AuNPsꢀ
NanoꢀZSMꢀ5 suspension (a homogenous sonicated solution of 2 mg of AuNPsꢀNanoꢀZSMꢀ5, 10
µL of Nafion and 1 mL of deionized water) was placed onto the GCE surface. The electrode was
dried in air leaving the material mounted onto the GC surface. For comparison, the other
modified glassy carbon electrodes were also fabricated in a similar way. Electrochemical
impedance spectroscopy (EIS) was performed using Autolab PGSTAT302N.
Real sample preparation
CySH syrup (Syp Amino Drip, Wockhardt (labeled value CySH = 5.6 mg/unit)) were
obtained from Kailash Medical Store, Ropar, Punjab, India. Ropar. The CySH syrup also
contained arginine, lysine, histidine, tyrosine, phenylalanine, tryptophan, methionine, glutamic
acid, threonine, valine, alanine, proline, serine, and glycine along with CySH. 10 mL of 0.1 M
stock solution of CySH was prepared using deionized water. Required amount of this solution
was added to the electrochemical cell containing 10 mL of 0.1 M PBS (pH 7.4) for the
determination of CySH. GSH injection (Injection Hepafresh, Macleods (labeled value GSH =
600 mg/injection)) was obtained from Kailash Medical Store, Ropar. 10 mL of 0.1 M stock
solution of GSH injection was prepared using deionized water. Required amount of this solution
was then added to the electrochemical cell containing 10 mL of 0.1 M PBS (pH 7.4) to record the
DPV voltammogram.
Results and discussion
Mesoporous zeolites exhibit all the desired criteria to act as an ideal support materials.
For the synthesis of AuNPsꢀNanoꢀZSMꢀ5, NanoꢀZSMꢀ5 was first surface functionalized with the
propyl amine group (–PrꢀNH ). NanoꢀZSMꢀ5 possesses large external surface area and abundant
2
surface silanol groups, therefore large amount of –PrꢀNH was incorporated on the surface of
2
nanocrystalline zeolite. Then the –PrꢀNH group on NanoꢀZSMꢀ5 surface was protonated with
2
acetic acid to induce the positive charge. Finally, freshly prepared citrate coated negatively
charged gold nanoparticles were immobilized on NanoꢀZSMꢀ5 surface by electrostatic
interactions as shown in Scheme 1.
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Physico-chemical characterization
NanoꢀZSMꢀ5 exhibited XRD pattern corresponding to a highly crystalline MFI
framework structure with high phase purity (Fig. 1a). MFI is a three letter code suggested by the
International Zeolite Association for ZSMꢀ5 framework topology. No change in the XRD pattern
of NanoꢀZSMꢀ5ꢀPrꢀNH was observed after the functionalization (Figure not shown), which
2
confirms that the framework structure was not affected after the functionalization. XRD pattern
of AuNPs(0.5%)ꢀNanoꢀZSMꢀ5 did not exhibit any diffraction peak corresponding to AuNPs
because of very low Au content in AuNPs(0.5%)ꢀNanoꢀZSMꢀ5 (Fig. 1a). AuNPs(3%)ꢀNanoꢀ
ZSMꢀ5 exhibited the diffraction peaks corresponding to both, NanoꢀZSMꢀ5 phase and Au
nanoparticles. Four diffraction peaks located at 2θ; 38.4, 44.5, 65, and 77.9°, which can be
assigned to (111), (200), (220), and (311) planes of the fcc structure of gold (JCPDS, File No. 4ꢀ
0784) were observed for AuNPs(3%)ꢀNanoꢀZSMꢀ5 (Fig. 1a). This confirms the formation of
AuNPs on NanoꢀZSMꢀ5. Textural properties of the zeolite materials were investigated using
nitrogen adsorptionꢀdesorption measurements. NanoꢀZSMꢀ5 and NanoꢀZSMꢀ5ꢀPrꢀNH exhibited
2
typeꢀIV isotherm similar to that of mesoporous materials (Fig. 1b). A sharp increase in the
volume of N adsorption in the region 0.4<P/P <0.9 is characteristic of the capillary
2
0
condensation within the intercrystalline mesopore void spaces. A pore size distribution in the
range of 2–10 nm was obtained in the case of NanoꢀZSMꢀ5. Textural properties of zeolite
materials are summarized in Table 1. The N ꢀadsorption study confirms that the surface area and
2
total pore volume decreased after the functionalization (Table 1). NanoꢀZSMꢀ5 has large external
surface area with large number of surface silanol groups, which makes it ideal support for
surface functionalization. Fourier transform infrared (FTꢀIR) spectroscopy was used to confirm
the functionalization of ꢀPrꢀNH on the surface of NanoꢀZSMꢀ5 (Details are provided in
2
supporting information, Fig. S1). TGA analysis confirmed that 11 wt% organic group (ꢀPrꢀNH2)
was functionalized on NanoꢀZSMꢀ5 (Details are provided in the supporting information, Fig.
S2). The amounts of Au incorporated in AuNPs(0.1%)ꢀNanoꢀZSMꢀ5, AuNPs(0.5%)ꢀNanoꢀ
ZSMꢀ5, AuNPs(1%)ꢀNanoꢀZSMꢀ5, and AuNPs(3%)ꢀNanoꢀZSMꢀ5 were found to be 0.097,
0.485, 0.982, 2.810 wt %, respectively, which were estimated by atomic absorption spectrometer.
To obtain the inꢀdepth information for AuNPs decorated NanoꢀZSMꢀ5, TEM
investigation was made. Spheroid NanoꢀZSMꢀ5 aggregated morphology (having dimension of
300ꢀ500 nm) are actually composed of very small size nanocrystals of 10ꢀ20 nm as observed in
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the high resolution TEM micrograph (Fig. S3, Supporting Information). Mesopores are created
by the crystal packing of these zeolite nanocrystals. TEM investigations clearly show that highly
dispersed Au nanoparticles were supported on the surface of NanoꢀZSMꢀ5 (Fig. 2a,b). HRTEM
analysis confirmed that 3ꢀ5 nm spherical gold nanoparticles were formed on the surface of Nanoꢀ
ZSMꢀ5 (Fig. 2c). The HRTEM image in Fig. 2d clearly showing lattice fringes indicates the
crystalline phase with a dꢀspacing of 2.33 Å. The measured dꢀspacing is very close to the (111)
interꢀplanar spacing of fcc Au (2.35 Å). To confirm the presence of Si, Al, O, and Au elements in
the material, EDX analysis was performed in STEM mode. EDX spectrum (Fig. 3b) from the
high angle annular dark field (HAADF) image (Fig. 3a) clearly shows the incorporation of Au
nanoparticles on NanoꢀZSMꢀ5 matrix.
Electrochemical characteristics of modified electrode
The electrochemical behavior of AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE was investigated
using CV. Fig. 4 shows the CVs at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE and bare GCE in 0.1 M
PBS (pH 7.4) at a scan rate of 50 mV/s. The results show that no peak was observed at bare
GCE. However, the CV at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE exhibited two distinct and sharp
peaks corresponding to the formation of gold oxide during the anodic sweep and its reduction in
+
ꢀ 27, 28
the reverse scan (2Au + 3H O
Au O + 6H + 6e ).
2 3
2
The electrochemical behavior of AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE, NanoꢀZSMꢀ5/GCE
and bare GCE was investigated using potassium ferricyanide as electrochemical probe by CV.
Study was performed in 0.1 M KCl solution containing 10 mM K [Fe(CN) ]/K [Fe(CN) ] at a
3
6
4
6
scan rate of 10 mV/s at different modified electrodes and bare GCE. The CV of various modified
3
ꢀ/4ꢀ
electrodes and bare GCE exhibited a pair of redox peaks corresponding to Fe(CN)6
redox
couple (Fig. S4, Supporting Information). Fig. S4, Supporting Information shows that
AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE exhibited much higher peak current when compared to Nanoꢀ
ZSMꢀ5/GCE and bare GCE. This may be correlated to the synergistic contribution provided by
electrochemically active gold nanoparticles and porous NanoꢀZSMꢀ5, which enhances the
diffusion of analyte.
EIS is an effective technique to study the interfacial properties of surface modified
electrodes (Fig. 5 and Fig. S5 & Fig. S6, Supporting Information). The Nyquist plot of
impedance profile include a semicircle portion at higher frequencies corresponding to the
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electron transferꢀlimited process and a linear part in the lower frequency range representing the
diffusion limited process. The semicircle diameter in the impedance profile is a measure of the
electron transferꢀresistance (R ). This resistance controls the electronꢀtransfer kinetics of the
et
redox couple at the electrode interface. Therefore, R can be used to describe the interfacial
et
properties of the modified electrode. The impedance profile was recorded at three different
conditions (Fig. 5 and Fig. S5 & Fig. S6, Supporting Information). Please note that R is highly
et
depended on the applied frequency range (Compare Fig. 5 and Fig. S6, Supporting Information ).
It also depends on the electrolyte used in the study (KCl vs KNO ) (Compare Fig. 5 and Fig. S5,
3
Supporting Information). Mesoporous silica materials (eg SBAꢀ15, MCM family etc) are
insulator but zeolites are semiconductor. Zeolites are crystalline aluminosilicates build from TO4
tetrahedra (T = Si, Al). Upon incorporation of Al into the silica framework, the +3 charge of the
Al makes the framework negatively charged, and requires the presence of extraꢀframework
cations (inorganic and organic cations) within the structure to keep the overall framework
neutral. The extraꢀframework cations present in the zeolites have considerable mobility, which
provides the conductivity in zeolites. In this study, we prepared NanoꢀZSMꢀ5 with silica/alumina
+
ratio 20, thus it has sufficient number of Na ions in its matrix. Due to the conducting nature of
+
NanoꢀZSMꢀ5 (because of Na ions present in the matrix), high surface area, and intercrystalline
mesopores, diffusion of analyte is facilitated. Therefore, R for NanoꢀZSMꢀ5 was somewhat
et
smaller than bare GCE. The modification of GCE with AuNPs(0.5%)ꢀNanoꢀZSMꢀ5 displayed an
almost straight line indicating negligible electron transfer resistance. Improved charge transfer
rate at the electrode surface and diffusion controlled behavior observed in the EIS measurement
with AuNPs(0.5%)ꢀNanoꢀZSMꢀ5 modified electrode is important for the individual and
simultaneous determination of analytes.
Voltammetric studies of cysteine and glutathione
The electrochemical behavior of CySH and GSH was investigated in detail at
AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE due to its high electrocatalytic activity. In order to mimic the
physiological environment, pH 7.4 was chosen as the optimum pH value for all the experiments.
Fig. 6 shows a comparison of CVs for CySH and GSH oxidation, individually, at AuNPs(0.5%)ꢀ
NanoꢀZSMꢀ5/GCE and bare GCE in 0.1 M PBS (pH 7.4) at a scan rate of 50 mV/s. CV result
shows that AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE exhibited well defined and sharp oxidation peaks
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for CySH and GSH with high current response (Fig. 6). However, bare GCE did not show any
distinguishable oxidation peak for CySH and GSH. The poor voltammetric response at bare GCE
may be attributed to the sluggish electron transfer kinetics. These results clearly show that the
modification of GCE with AuNPs(0.5%)ꢀNanoꢀZSMꢀ5 nanocomposite effectively accelerated
the rate of electron transfer between the analytes and electrode. Therefore, the electrochemical
behavior of CySH and GSH is considerably improved at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE.
Based on the results obtained, it can be concluded that AuNPs(0.5%)ꢀNanoꢀZSMꢀ5 have the
ability to promote the electron transfer rate for CySH and GSH oxidation and have high
electrocatalytic activity.
The effect of scan rate on the electrochemical oxidation of CySH and GSH was
investigated using CV. Fig. S7aꢀb, Supporting Information shows the CVs at AuNPs(0.5%)ꢀ
NanoꢀZSMꢀ5/GCE containing CySH and GSH in 0.1 M PBS (pH 7.4) at various scan rates (10ꢀ
600 mV/s). The CV results for CySH and GSH show only an oxidation peak and no reduction
peak was observed in the reverse scan which confirms that oxidation of CySH and GSH is an
irreversible electrode process. Further, with increase in the scan rate, the oxidation peak current
was increased for both CySH and GSH. The plot of the oxidation peak current against the square
root of scan rate showed a linear relationship indicating a diffusion controlled process. The
Semerano coefficient can be calculated from the logarithmic dependence of the electroꢀoxidation
peak current on the scan rate (tan α = ꢁlog I /ꢁlog ν, where I is the oxidation peak current and ν
p
p
2
3
is the scan rate). The plot of logarithm of anodic peak current vs. logarithm of scan rate
2
provided a straight line with the linear regression equation log I = 0.452 log v + 0.434 (R =
p
2
0
.987) and log I = 0.527 log v + 0.019 (R = 0.996), for CySH and GSH, respectively (Fig. S7cꢀ
p
d, Supporting Information). Slopes obtained from the linear behavior between the logarithm of
anodic peak current vs. logarithm of scan rate for CySH and GSH were found to be 0.45 and
0.53, which are close to the theoretical value of 0.5 for a diffusion controlled process.
Chronoamperometry was used to calculate the kinetic parameters such as diffusion
coefficient (D) and rate constant (k) for the electroꢀcatalytic oxidation of CySH and GSH at
AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE (Fig. S8ꢀS9, Supporting Information includes experimental
−
5
details along with a brief discussion). The diffusion coefficients were found to be 3.1 × 10 and
−
5
2
1
.4 × 10 cm /s for CySH and GSH, respectively. The rate constant values for electroꢀcatalytic
3
3
oxidation of CySH and GSH were found as 17.0 × 10 and 3.2 × 10 1/s M, respectively. This
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chronoamperometric data indicates that the electrochemical reaction rate for CySH and GSH at
AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE is fast.
The total number of electrons (n) involved in the electrochemical oxidation of CySH and
GSH at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE can be calculated from the plot of oxidation peak
currents against the square root of scan rate according to the following equation for a totally
2
9ꢀ31
irreversible diffusionꢀcontrolled process.
.8 and 2.1 for CySH and GSH, respectively. Hence, a total of 2 electrons were involved in the
electrochemical oxidation of CySH and GSH.
The total number of electrons was calculated to be
1
5
1/2
1/2 1/2
I = 3.01 x 10 n[n (1ꢀα)] ACD ν
p
α
Where I is the peak current (A), n is the number of electrons in rate determining step, α is
p
α
2
charge transfer coefficient, A is electrode surface area (0.07 cm ), C is the bulk concentration of
3
−5
2
analyte (mol/cm ), D is the diffusion coefficient of analyte (3.1 × 10 cm /s for CySH and 1.4 ×
−
5
2
1
0 cm /s for GSH; obtained from the chronoamperometric study), and ν is the scan rate (V/s).
The charge transfer coefficient between the analyte (CySH and GSH) and AuNPs(0.5%)ꢀNanoꢀ
ZSMꢀ5/GCE was obtained from Tafel plot using the rising part of the currentꢀvoltage plot (Fig.
ꢀ1
ꢀ1
S10, Supporting Information). A slope of 8.31 V and 8.12 V was obtained for CySH and
GSH, respectively. Assuming one electron transfer in the rateꢀdetermining step (n = 1), charge
α
transfer coefficient (α) was calculated as 0.51 and 0.53 for CySH and GSH, respectively,
according to the equation b = n (1ꢀ α) F/2.3 RT, where b is slope of tafel plot. Based on the
α
above results and the literature reports, the mechanism for the electrochemical oxidation of
1
2, 15, 32
CySH and GSH at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE is proposed (Scheme 2).
Individual electro-catalytic oxidation of CySH and GSH
The individual electrocatalytic oxidation of CySH and GSH at AuNPs(0.5%)ꢀNanoꢀ
ZSMꢀ5/GCE was carried out using DPV in 0.1 M PBS (pH 7.4) at a scan rate 20 mV/s (Fig. 7).
The DPV results show that both the analytes were oxidized with wellꢀdefined and
distinguishable sharp oxidation peaks with peak potentials at 334 and 573 mV for CySH and
GSH, respectively, at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE (Fig. 7). The peak currents were found
to increase linearly with the increase in the concentration of CySH and GSH in the
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2

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electrochemical cell. A linear dynamic range from 1 nM to 1000 µM with a calibration equation
2
of ICySH(µA) = 9.788 + 0.083 CCySH(µM) (R = 0.997) was obtained for CySH (Fig. 7a, inset). A
linear calibration for GSH was found to be in the range of 2 nM to 1000 µM, with a calibration
2
equation of IGSH(µA) = 8.309 + 0.061 CGSH (µM) (R = 0.995) (Fig. 7b, inset). The limit of
detection (S/N = 3) was found to be 0.2 nM and 0.5 nM for CySH and GSH, respectively.
Simultaneous electrochemical determination of CySH and GSH
DPV was employed for the simultaneous determination of CySH and GSH at
AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE in 0.1 M PBS (pH 7.4) at a scan rate 20 mV/s (Fig. 8). Fig. 8
shows that two distinguished and sharp anodic peaks at potentials 334 and 573 mV
corresponding to the oxidation of CySH and GSH were obtained in the simultaneous
determination. These peak potentials matched well with their individual anodic peak potentials
as discussed in the above section. The voltammograms for the binary mixture were well
separated from each other with a potential difference of ꢁECySH–GSH = 239 mV. This peak
potential difference is large enough for the simultaneous determination of CySH and GSH from
their binary solution. A significant increase in the oxidation peak current was observed for both
the analytes with the increase in concentration of analytes in the electrochemical cell. The anodic
peak current obtained was found to be linearly dependent on the concentration of analytes in the
2
2
range of 2 nM to 800 µM (R = 0.998) for CySH and 3 nM to 800 µM (R = 0.998) for GSH with
2
the sensitivity of 1.4 and 1.1 µA/ µM cm and the limit of detection (S/N = 3) of 0.3 nM and 0.6
nM for CySH and GSH, respectively, (Fig. 8, inset). These results demonstrate that
AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE exhibited excellent electroꢀcatalytic activity toward the
simultaneous determination of CySH and GSH.
A comparison for the electrochemical oxidation of CySH and GSH at gold nanoparticles
decorated NanoꢀZSMꢀ5 modified GCE with different weight ratios is provided in Fig. S11,
Supporting Information. Fig. S11, Supporting Information shows that with increase in the
amount of AuNPs on NanoꢀZSMꢀ5 support, the electrochemical activity was increased. This
shows that higher loading of AuNPs in nanocomposite material is better for the high
electrocatalytic activity. However, with further increase in AuNPs content (after AuNPs(0.5%)),
the electrocatalytic activity was decreased. These results clearly show that the highly dispersed
AuNPs on the surface of NanoꢀZSMꢀ5 enhanced the accessibility of analytes to the active sites
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and improved the electrocatalytic activity of material. With further increase in the Au content,
less dispersed AuNPs with larger size were formed that resulted in the lower current response.
These results show that optimum loading and high dispersion of AuNPs on NanoꢀZSMꢀ5 surface
is required to achieve high electrocatalytic activity. Therefore, AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE
was chosen for the determination of CySH and GSH. A comparison of AuNPs(0.5%)ꢀNanoꢀ
ZSMꢀ5/GCE, NanoꢀZSMꢀ5/GCE, and bare GCE in the simultaneous determination of CySH and
GSH is shown in (Fig. 9 and Fig. S12, Supporting Information). Bare GCE did not exhibit any
oxidation peak in the simultaneous determination of CySH and GSH (Fig. S12, Supporting
Information). NanoꢀZSMꢀ5/GCE exhibited oxidation peaks for CySH and GSH but with very
low current response when compared to AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE (Fig. S12, Supporting
Information). This further confirms that Au nanoparticles play an important role in the
electrochemical oxidation of CySH and GSH and have electrocatalytic effect in the oxidation of
CySH and GSH. The remarkable high activity of AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE can be
attributed to the synergistic contribution provided by highly dispersed AuNPs and high surface
area of NanoꢀZSMꢀ5. The interꢀcrystalline mesoporosity in NanoꢀZSMꢀ5 provides an efficient
transport path for reactant/product molecules because of the short diffusion length. The
comparison of results shown in this paper with literature reports are provided in Table S1,
Supporting Information. It is clear from Table S1, Supporting Information, that the proposed
sensor is able to detect CySH and GSH simultaneously with a wide linear range and remarkably
low limit of detection when compared to literature reports.
Reproducibility, stability, and anti-interference property of the sensor
The reproducibility, repeatability, and long term stability of the developed sensor were
evaluated in the sensing studies. Five, AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE were constructed and
their current response to 1 µM concentration of CySH and GSH was investigated (Fig. S13a,
Supporting Information). The relative standard deviation (RSD) was found to be 1.3 % and 1.5
%
for CySH and GSH, respectively, confirming that the fabrication method was highly
reproducible. The stability of AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE was examined by recording
repetitive CVs for 50 scans in PBS (pH 7.4) at a scan rate 50 mV/s (Fig. S14 and Fig. S15,
Supporting Information). No obvious change in the peak current was observed after 50 cycles,
which confirms that AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE is highly stable. The long term stability of
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the sensor was evaluated by measuring its sensitivity toward 1 µM concentration of CySH and
GSH for 30 days (Fig. S13b, Supporting Information). The sensor was stored in refrigerator at
278 K and its sensitivity was tested at the interval of 5 days. Prior to each measurement,
electrode was washed with 0.1 M PBS and repetitive CV were run in blank 0.1 M PBS (pH 7.4).
The results confirmed that the DPV response of the electrode to the same concentration of CySH
and GSH remained almost same with RSD 2.1 % and 2.8 % for CySH and GSH respectively,
indicating that the modified electrode has excellent stability. The selectivity of AuNPs(0.5%)ꢀ
NanoꢀZSMꢀ5/GCE toward simultaneous determination of CySH and GSH was investigated in
the presence of various interfering agents. The DPV responses are shown in Fig. S16, Supporting
Information. No obvious deviation upon the addition of very high concentration of interferents
(500 times excess) was observed. The results showed that other Lꢀamino acids such as alanine,
glycine, arginine, histidine, phenylalanine, proline, tyrosine, valine, tryptophan, glutamic acid,
serine, threonine, and methionine had no effect on the peak currents for the oxidation of CySH
and GSH. The presence of other common interferents such as glucose, ascorbic acid, dopamine,
and uric acid did not show any change in the peak current response confirming that no
interference for these common species occurred. These results confirmed that AuNPs(0.5%)ꢀ
NanoꢀZSMꢀ5/GCE is highly selective for the determination of CySH and GSH.
Determination of CySH and GSH in pharmaceutical preparations
In order to show the analytical application of the developed sensor; experiments were
performed to determine the concentration of CySH and GSH in pharmaceutical preparations and
the results are listed in Table 2. The standard addition method was used by spiking with standard
solution of CySH and GSH. The values of recovery were in the range from 99.4 to 100.6 %,
suggesting the accuracy of AuNPs(0.5%)ꢀNanoꢀZSMꢀ5 based sensor. These results confirm that
proposed sensor is reliable and sensitive enough for the determination of CySH and GSH in real
pharmaceutical samples even in the presence of other amino acids (see the details in the
experimental section).
Conclusions
In summary, gold nanoparticles decorated nanocrystalline zeolite was synthesized.
Transmission electron microscopy investigations confirmed that highly dispersed gold
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nanoparticles (particle size 3ꢀ5 nm) were supported on nanocrystalline zeolite NanoꢀZSMꢀ5. An
electrochemical sensor based on gold nanoparticles supported NanoꢀZSMꢀ5 modified glassy
carbon electrode was fabricated for the simultaneous determination of thiol containing bioꢀ
molecules cysteine and glutathione. The results demonstrate that the developed sensor exhibited
high electroꢀcatalytic activity, sensitivity, selectivity, and stability in the detection of cysteine
and glutathione. The high activity of AuNPs(0.5%)ꢀNanoꢀZSMꢀ5 can be attributed to the
synergistic contribution provided by highly dispersed Au nanoparticles, large surface area and
mesopores of NanoꢀZSMꢀ5 support. The analytical performance of the developed sensor was
extended in the determination of these biomolecules in pharmaceutical preparations with
satisfactory results. The high sensitivity and selectivity together with very low limit of detection
of the proposed sensor makes it a promising candidate for the determination of very low amount
of cysteine and glutathione in pharmaceutical preparations. The proposed methodology is simple,
rapid and provides a potentially new analytical platform for the sensitive and selective analysis
of thiols.
Acknowledgements
Authors thank Department of Science and Technology, New Delhi for financial
assistance (DST grant SB/S1/PCꢀ91/2012). BK is grateful to CSIR, New Delhi for SRF
fellowship. Authors are also thankful to Director IIT Ropar for his constant encouragement.
Supporting Information
The supporting information contains additional diffusion coefficient and rate constant calculation
and results and discussion as noted in text.
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Figures, Tables and Scheme captions
Figures
Fig. 1
(a) XRD patterns of NanoꢀZSMꢀ5, AuNPs(0.5%)ꢀNanoꢀZSMꢀ5, and AuNPs(3%)ꢀ
NanoꢀZSMꢀ5; and (b) N ꢀadsorption isotherms of NanoꢀZSMꢀ5 and NanoꢀZSMꢀ5ꢀPrꢀ
2
NH . Inset shows the pore size distribution.
2
Fig. 2
(a) Low magnification TEM image, (b) High magnification TEM image, (c) High
magnification TEM image showing gold nanoparticles decorated on NanoꢀZSMꢀ5
surface, (d) HRTEM image showing single crystal structure of one AuNP shown in (c).
(a) STEMꢀHAADF image of gold nanoparticles decorated on NanoꢀZSMꢀ5, (b) EDX
spectrum from a rectangular region in (a).
Fig. 3
Fig. 4
CV response of AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE in 0.1 M PBS (pH 7.4) at a scan rate
of 50 mV/s. Inset shows the CV response at bare GCE in 0.1 M PBS (pH 7.4) at a scan
rate of 50 mV/s.
Fig. 5
Fig. 6
Fig. 7
Nyquist plots of impedance profiles at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE, NanoꢀZSMꢀ
3
ꢀ/4ꢀ
5
/GCE, and bare GCE in 0.1 M KNO solution containing 10 mM [Fe(CN) ]
over
3
6
5
the frequency range from 0.1 Hz to 10 Hz at an applied potential of 0.3 V.
Comparison of CVs in a 0.1 M PBS (pH 7.4) containing (a) CySH (10 µM) and (b)
GSH (10 µM) at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE and bare GCE at a scan rate of 50
mV/s.
DPVs at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE in 0.1 M PBS (pH 7.4) by varying the
concentrations of (a) CySH and (b) GSH. DPV parameters were selected as: pulse
amplitude: 50 mV, pulse width: 50 ms, scan rate: 20 mV/s. Inset shows the calibration
plot.
Fig. 8
Fig. 9
DPVs of the binary mixture containing varying concentrations of CySH and GSH at
AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE in 0.1 M PBS (pH 7.4). DPV parameters were
selected as: pulse amplitude: 50 mV, pulse width: 50 ms, scan rate: 20 mV/s. Inset
shows the calibration plot for CySH and GSH.
Comparison of the sensitivity for CySH and GSH electroꢀoxidation at AuNPs(0.5%)ꢀ
NanoꢀZSMꢀ5/GCE and NanoꢀZSMꢀ5/GCE .
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Table
Table 1 Physicoꢀchemical characteristics of zeolite materials investigated in this study.
Table 2 Determination of CySH and GSH in pharmaceutical preparations at AuNPs(0.5%)ꢀNanoꢀ
ZSMꢀ5/GCE.
Scheme
Scheme 1 Schematic representation for the fabrication of AuNPs decorated NanoꢀZSMꢀ5 based
electrochemical sensor.
Scheme 2 Electrochemical oxidation of CySH and GSH at AuNPs(0.5%)ꢀNanoꢀZSMꢀ5/GCE.
2
0

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Table 1
S.No.
Sample
Total surface
area
External
Total pore
volume
surface area
2
2
3
SBET (m /g)
(m /g)
(cm /g)
1
.
.
NanoꢀZSMꢀ5
548
335
365
164
0.58
0.33
2
NanoꢀZSMꢀ5ꢀPrꢀNH2
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Table 2
S. No.
a
Sample
Spiking
Original
(nM)
Detected RSD (%)
Recovery
(%)
(nM)
(nM)
1.
2.
3.
4.
5.
6.
7.
8.
CySH Syrup
CySH Syrup
CySH Syrup
CySH Syrup
GSH Injection
GSH Injection
GSH Injection
GSH Injection
ꢀ
500
500
600
700
500
500
600
700
502
599
703
801
497
603
702
804
2.4
2.6
3.2
1.9
2.7
2.9
1.8
2.6
100.4
99.8
100
100
100
ꢀ
100.5
100.1
99.4
100
100
100
100.6
100.3
100.6
a
Average value of five determinations.
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6
5
4
3
00
00
00
00
AuNPs(3%)-Nano-ZSM-5
1.2
(
a)
(111)
(b)
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
2
0
0
40
60
80
0
10 20 30 40 50
Pore size (nm)
AuNPs(0.5%)-Nano-ZSM-5
2
40
60
80
80
(051)
200
100
Nano-ZSM-5
Nano-ZSM-5
Nano-ZSM-5-Pr-NH2
0
.2
0.4
0.6
0.8
1.0
10
20
30
40
50
60
70
Relative pressure (P/P0)
2
θ
(degree)
Fig. 1

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Fig. 2

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Fig. 3

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1
2
8
4
0
4
8
8
4
0
4
8
Bare GCE
-
-
-
1.0
-0.5
0.0
0.5
1.0
Potential (V)
-
-
-1.0
-0.5
0.0
0.5
1.0
Potential (V)
Fig. 4

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Bare GCE
Nano-ZSM-5/GCE
AuNPs(0.5%)-Nano-ZSM-5/GCE
1
500
000
1
500
0
0
500
1000
1500
2000
2500
Z' (Ohm)
Fig. 5

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30
20
10
0
Bare GCE
AuNPs(0.5%)-Nano-ZSM-5/GCE
-10
(
a)
-
0.2
0.0
0.2
0.4
0.6
0.8
Potential (V)
2
0
0
0
(
b)
1
-10
Bare GCE
AuNPs(0.5%)-Nano-ZSM-5/GCE
0.2
0.4
0.6
0.8
1.0
1.2
Potential (V)
Fig. 6

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100
1
00
(
a)
CySH
8
0
80
60
40
20
0
60
4
2
0
0
0
1
mM
nM
0
200 400 600 800 1000
Conc. (ꢀM)
1
0.0
0.2
0.4
0.6
0.8
1.0
Potential (V)
80
60
40
20
0
8
0
(b)
GSH
6
4
2
0
0
0
0
1 mM
0
200 400 600 800 1000
Conc. (ꢀM)
2
nM
0.2
0.4
0.6
0.8
Potential (V)
Fig. 7