Monitoring analgesic drug using sensing method based on nanocomposite

Article abstract of DOI:10.1039/c4ra11255e

This paper reports a rapid, reliable and sensitive electrochemical method for the determination of acetaminophen, a safe analgesic drug. Most methods currently used for therapeutic drug monitoring require a pre-treatment of the sample. Biosensors avoid this kind of drawbacks. A horseradish peroxidase (HRP) was immobilized using core-shell ZrO@Fe₃O₄ nanoparticles on chitosan hybrid film electrodeposited on the surface of an Au electrode. The surface functionalization of core-shell ZrO@Fe₃O₄NPs on a chitosan hybrid film was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). The experimental variables that can affect the acetaminophen amperometric response, such as the pH, temperature and applied potential, have been optimized to perform a selective determination of acetaminophen. An average limit of detection of 0.01 μM (S/N = 3) was obtained. The biosensor was finally applied to the determination of acetaminophen in complex matrices, such as pharmaceutical drugs.

Full text of DOI:10.1039/c4ra11255e

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PAPER
Monitoring analgesic drug using sensing method
based on nanocomposite
Cite this: RSC Adv., 2015, 5, 2396
Jagriti Narang, a Nitesh Malhotra,b Sandeep Singh,c Gajendra Singhd and C. S. Pundirc
*
This paper reports a rapid, reliable and sensitive electrochemical method for the determination of
acetaminophen, a safe analgesic drug. Most methods currently used for therapeutic drug monitoring
require a pre-treatment of the sample. Biosensors avoid this kind of drawbacks. A horseradish peroxidase
(HRP) was immobilized using core–shell ZrO@Fe3O4 nanoparticles on chitosan hybrid film
electrodeposited on the surface of an Au electrode. The surface functionalization of core–shell
ZrO@Fe3O4NPs on a chitosan hybrid film was characterized by cyclic voltammetry (CV), scanning
electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). The experimental
variables that can affect the acetaminophen amperometric response, such as the pH, temperature and
applied potential, have been optimized to perform a selective determination of acetaminophen. An
average limit of detection of 0.01 mM (S/N ¼ 3) was obtained. The biosensor was finally applied to the
determination of acetaminophen in complex matrices, such as pharmaceutical drugs.
Received 26th September 2014
Accepted 7th November 2014
DOI: 10.1039/c4ra11255e
www.rsc.org/advances
pretreatment.11 Hence, the amperometric biosensors based on
direct enzyme immobilization on a transducer surface are the
main analytical strategies used for acetaminophen analysis.12
Nanoparticles exhibit very unique electrical and magnetic
properties that are distinct from their bulk counterparts.
Among these, magnetic nanoparticles are biocompatible and
potentially non-toxic for biosensor applications.13–15 The
immobilization of enzymes on magnetic nanoparticles has the
advantage of distinctive characters like enhancing their activity,
mediating rapid contact between the enzyme and substrate, and
reducing mass-transfer limitations.16,17
Magnetic nanoparticles provide a large surface area and
biocompatible micro-environment to the immobilized enzyme
that helps in providing close proximity to the analyte and
sensing element, and have been proven to be the best sensing
interface for the fabrication of a biosensor. Magnetic nano-
particles have a large surface area that can be oxidized easily to
form aggregates but this can change their original structure and
unique properties. To overcome this difficulty, the surface of
NPs has been coated with a protective layer of various mate-
rials18,19 Here, ZrO2 (zirconium oxide) has become a favored
coating material owing to its good insulating property, simple
synthetic procedure and chemical functionality, chemical
inertness, and wear resistance.20 It is believed that magnetic
nanoparticles can avoid being oxidized and maintain their
magnetic properties (such as coercivity or blocking tempera-
ture) by a ZrO2 coating. Therefore, the magnetic cores can be
protected from oxidation and corrosion.21
1. Introduction
APAP (N-acetyl-p-aminophenol or paracetamol) is an analgesic
and one of the most commonly used drugs.1 It is an effective
mild analgesic and antipyretic agent used extensively at thera-
peutic dosage for the safe relief of mild to moderate pain2–4 but
acute overdoses can cause serious hepatic damage, which may
result in death.5 Overdoses of APAP causes 2600 hospitaliza-
tions, >56 000 emergency room visits, and approximately 458
acute liver failures each year in the United States.6 Therefore,
extensive efforts should be made to detect the cause of its
toxicity and its mitigation but a determination of its exact level
would require an accurate method. Various methods are avail-
able for the determination of acetaminophen, like Raman
spectrometry,7
liquid
chromatography
(LC),8
chem-
iluminescence,9 and spectrometric methods.10 These methods,
however, have some drawbacks that make them unsuitable for
routine analyses, such as high cost, time consuming, need to
pretreat the sample, skilled people to operate, and even low
sensitivity and selectivity in some cases. Therefore, there should
be a reliable and precise method for measuring APAP to
maintain a constant concentration, thereby optimizing the
individual dosage regimen. The use of biosensors is a method
that overcomes these problems due to their intrinsic specicity,
low cost, fast analyses and minimal requirements for sample
aAmity Institute of Nanotechnology, AMITY University, Noida, UP, India. E-mail:
jags_biotech@yahoo.co.in; Tel: +91 9811792572
bAmity Institute of Physiotherapy, AMITY University, Noida, UP, India
cDepartment of Biochemistry, M. D. University, Rohtak-124 001, Haryana, India
dFaculty of Pharmaceutical Science PGIMS, Rohtak, India
Chitosan (CHIT) is an extensive biopolymer for the immo-
bilization of biomolecules because of its excellent lm-forming
ability, high permeability, mechanical strength, non-toxicity,
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biocompatibility, low cost, and easy availability.22 It was chosen centrifuged at 10 000 rpm for 5 min. The encapsulated particles
as the orientation directing matrix because large quantities of were removed and washed several times with ethanol. This
amino and hydroxyl groups are present on the CHIT units to zirconia coated iron oxide nanoparticles (ZrO@Fe3O4NP) were
amplify the binding ability to enzymes.23–27
kept at 40 C for drying. The zirconia-coated iron oxide nano-
ꢀ
We describe the therapeutic drug monitoring of acetamin- particles were characterized by UV/visible spectroscopy, X-ray
ophen using biosensor based on core–shell ZrO@Fe3O4 nano- diffraction and transmission electron microscopy.
particles on chitosan hybrid lm.
2.5. Construction of ZrO@Fe3O4NP/CHIT hybrid lm onto
Au electrode
2. Materials and methods
The surface of the Au electrode was polished with an alumina
slurry and dipped into sodium phosphate buffer (0.1 M, pH 7.0)
containing chitosan (500 ml) and ZrO@Fe3O4NP suspension
(500 ml) and subjected to 10 successive deposition cycles at ꢂ0.1
V to 0.1 V using a potentiostat–galvanostat (Fig. 1). The modi-
ed Au electrode was washed thoroughly with DW to remove the
unbound matter.
2.1. Materials
Acetaminophen (paracetamol) was purchased from Sigma (St.
Louis, MO). Iron(III) chloride hexahydrate (98%), ferrous chlo-
ride tetrahydrate (FeCl2$4H2O) sodium borohydride powder
(98%) and zirconium(IV) tert-butoxide were obtained from Sisco
Research Laboratory Pvt. Ltd., Mumbai, India. All other chem-
icals were of analytic reagent grade. Double distilled water (DW)
was used throughout the experiments.
2.6. Preparation of the enzyme electrode for the
electrochemical sensing of analgesic drug
2.2. Apparatus and methods
The puried HRP enzyme was immobilized on the surface of
ZrO@Fe3O4NP/CHIT hybrid lm. First, Au electrode was dipped
into glutaraldehyde (2.5%) at room temperature and washed
thoroughly with DW. This modied electrode was dipped into
10 ml of an enzyme solution (40 mg mlꢂ1 protein) and kept
Cyclic voltammetry (CV) and electrochemical impedance spec-
troscopy (EIS) were performed on a potentiostat/galvanostat
(Autolab, Eco Chemie, the Netherlands. Model: AUT83785)
with a three electrode system consisting of a Pt wire as an
auxiliary electrode, an Ag/AgCl electrode as the reference elec-
trode and modied Au wire as the working electrode. All elec-
trochemical experiments were performed at ambient
temperature (25 ꢀC). Fourier transform infrared (FTIR) spec-
troscopy was performed on a FTIR spectrometer (Make: iS10,
Thermoelectron, USA). Scanning electron microscopy (SEM)
was carried out at the Department of Chemistry, M. D. Univer-
sity, Rohtak. Transmission electron microscopy (TEM) was
performed at Punjab University, Chandigarh. Ultrasonication
was performed on Misonix Ultrasonic Liquid Processors (mode
XL-2000 series). X-ray diffraction (XRD) was conducted on a
X-ray diffractometer (Make: Rigaku, D/Max2550, Tokyo, Japan)
at the Department of Physics, G. J. University, Hisar, collecting
data from 20ꢀ to 70ꢀ 2q. The database of the Joint Committee on
Powder Diffraction Standards (JCPDS) was used for phase
identication.
ꢀ
undisturbed for approximately 12 h at 4 C. The electrode was
nally washed with 0.1 M Tris HCl buffer (pH 8.5) to remove the
unbound enzyme. The resulting HRP/ZrO@Fe3O4NP/CHIT/Au
ꢀ
electrode was used as the working electrode and stored at 4 C,
when not in use. The working electrode was characterized by
SEM at different stages of its construction (Scheme 1).
2.7. Electrochemical characterization of HRP/
ZrO@Fe3O4NP/CHIT/Au electrode
Cyclic voltammetry was carried out using a three electrode
system composed of HRP/ZrO@Fe3O4NP/CHIT/Au electrode as
the working electrode, Ag/AgCl as the reference electrode and Pt
wire as the auxiliary electrode. Cyclic voltammograms of the
2.3. Synthesis of Fe3O4NP
Fe3O4NP were prepared according to the method reported by
Predoi.28 0.5 M ferrous chloride tetrahydrate (FeCl2$4H2O) in 2
M HCl and 0.5 M ferric chloride hexahydrate (FeCl3$6H2O) in
DW were mixed at room temperature. The mixture was added to
200 ml of a 1.5 M NaOH solution with vigorous stirring for
about 30 min. The resulting precipitates were isolated by
ꢀ
centrifugation at 8000ꢁ g and dried at 40 C.
2.4. Preparation of ZrO@Fe3O4NP
ZrO@Fe3O4NPs were prepared by the hydrolysis and conden-
sation method reported by Chaubey.20 Fe nanoparticles (3 g)
and zirconium(IV) tert-butoxide (10 ml) were taken in a 50 ml
beaker in inert atmosphere. To this mixture, 50 ml of ethanol
Fig. 1 Linear response of concentrations of acetaminophen (substrate
was added and stirred mechanically for 2 h. The mixture was concentration/mM) vs. current (I/mA).
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Scheme 1 Graphical representations of the stepwise amperometric sensor fabrication process.
bare Au electrode, ZrO@Fe3O4NP/CHIT/Au electrode, and HRP/ with a phosphate buffer solution (0.1 M; pH 7.0). Finally,
ZrO@Fe3O4NP/CHIT/Au electrode were recorded in sodium each pharmaceutical product (400 ml) was added to the
phosphate buffer (0.1 M, pH 7.0, containing 0.1 mM H2O2) in
potential ranging between ꢂ0.1 V sꢂ1 to +1 V sꢂ1 at a scan rate of
50 mV sꢂ1
.
2.8. Preparation of analgesic drug solution
Paracetamol (acetaminophen) was prepared in a phosphate
buffer solution (pH 7.0). Solutions of different concentrations of
paracetamol (acetaminophen) ranging from 0.01 mM to 10 000
mM were preparꢀed in 0.1 M sodium phosphate buffer (pH 7.0)
and stored at 4 C until needed.
2.9. Optimization of analgesic drug biosensor
To optimize the working conditions of the biosensor, the effects
of pH, incubation temperature, time, and substrate concentra-
tion on the biosensor response were studied. To determine the
optimal pH, the pH of the reaction buffer, sodium phosphate
buffers was varied from 5.0 to 8.0, each at a nal concentration
of 0.1 M. To determine the optimal temperature, the reaction
mixture was incubated at different temperatures (20 ꢀC to 50 ꢀC)
at intervals of 5 ꢀC. The effect of the substrate concentration on
the biosensor response was determined by varying the
concentration of acetaminophen in the range 0.01 mM to 10 000
mM. To optimize the applied potential for acetaminophen
determination, the effect of the applied potential on the
response current was investigated over the range, ꢂ0.1 V to +0.1
V vs. Ag/AgCl. The optimal current was measured at ꢂ0.75 V vs.
Ag/AgCl. Hence, subsequent electrochemical studies were
carried at ꢂ0.07 V vs. Ag/AgCl.
2.10. Amperometric determination of analgesic drug
Fig. 2 (A) X-ray diffraction (XRD) pattern of ZrO@Fe3O4NPs. (B)
Transmission electron microscope (TEM) image of ZrO@Fe3O4NP.
Each pharmaceutical product (1.0 ml) was stirred until
complete dissolution and then diluted to 10, 20 and 50 ml
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cell containing 10 ml of phosphate buffer solution (0.1 M; pH
7.0). The measurements were performed aer successive
additions of the pharmaceutical product. Aer each addition,
cyclic voltammograms was recorded by cycling the potential
3. Results and discussions
3.1. Characterization of ZrO@Fe3O4NP
Fig. 2A shows the XRD pattern of the ZrO@Fe3O4NP hybrid lm.
The peaks observed were matched up to only the bcc Fe struc-
ture and showed no peak for Fe oxide or crystalline ZrO2. TEM
image of ZrO@Fe3O4NP (Fig. 2B) showed the occurrence of
spherical particles tending to form chains, indicating ferro-
magnetic interactions. Iron nanoparticles aggregation led to the
trapping of a zirconia shell during the coating process (30 nm).
These observations conrm the formation of ZrO@Fe3O4NP.
between ꢂ0.1 V and +0.1 V at a scan rate of 100 mV sꢂ1
.
The acetaminophen content in the pharmaceutical product
was determined by the present biosensor and recording
the current (mA) under the optimal working conditions
(Fig. 1).
2.11. Storage stability of HRP/ZrO@Fe3O4NP/CHIT/Au
electrode
3.2. Scanning electron microscopy (SEM), electrochemical
impedance studies (EIS) and Fourier transform infrared
(FTIR) spectroscopy of the modied Au electrode
The long-term storage and stability of the working electrode and
its amperometric current response to 100 mM ꢀof paracetamol To conrm the enzyme immobilization on the ZrO@Fe3O4NP
was investigated over a period of 1 month at 4 C.
lm, the surface morphology of the bare electrode (a),
Fig. 3 (A) SEM image of bare Au electrode (top), ZrO@Fe3O4NP/CHIT hybrid film modified Au electrode (middle) and HRP/ZrO@Fe3O4NP/CHIT
hybrid film modified Au electrode (bottom). (B) EIS of ZrO@Fe3O4NP/CHIT hybrid film (a), HRP/ZrO@Fe3O4NP/CHIT hybrid film modified Au
electrode (b) and bare Au electrode (c) in a solution containing 1 mM Fe(CN)63ꢂ/4ꢂ with 0.1 M KCl at 0.20 mV sꢂ1 (frequency range of 0.01 Hz to 10
kHz). (C) FTIR spectra of ZrO@Fe3O4NP/CHIT/Au electrode (upper curve) and HRP/ZrO@Fe3O4NP/CHIT/Au electrode (lower curve).
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ZrO@Fe3O4NP/CHIT/Au electrode (b) and HRP/ZrO@Fe3O4NP/ enzyme, the Rct value of the HRP/ZrO@Fe3O4NP/CHIT/Au elec-
CHIT/Au bioelectrode (c) were investigated by SEM (Fig. 3). trode increased. This is because most biological molecules,
The bare electrode showed a smooth surface (top image). A including enzymes, are poor electrical conductors, which
roughness granular morphology observed on the ZrO@Fe3O4- impede electron transfer.
NP/CHIT/Au electrode conrmed the coating of nanoparticles
Fig. 3C shows the FTIR spectra of ZrO@Fe3O4NP/CHIT/Au
on chitosan (middle image). Some ake-like structures were electrode (upper curve) and HRP/ZrO@Fe3O4NP/CHIT/Au elec-
observed, which conrms the immobilization of the enzyme on trode (lower curve). The CHIT exhibited the characteristic
the electrode (bottom image).
absorption bands of amino saccharide at 3421 cmꢂ1 (due to
Fig. 3B shows the electrochemical impedance spectra (EIS) of overlap of the OH and NH2 stretching bands), 2811 cmꢂ1 (due to
CHIT/Au electrode (a), ZrO@Fe3O4NP/CHIT/Au electrode (b) –CH2 stretching band) and 1647 cmꢂ1 (due to C–O stretching
and HRP/ZrO@Fe3O4NP/CHIT/Au electrode (c). The Rct values band), while the 634 cmꢂ1 in ZrO@Fe3O4NP are the characteristic
for the CHIT/Au electrode, the ZrO@Fe3O4NP/CHIT/Au elec- bands for a Zr–O lm (curve i). The enzyme mixture was immo-
trode and HRP/ZrO@Fe3O4NP/CHIT/Au electrode were 800 U, bilized onto chitosan through covalent binding with glutaralde-
380 U and 600 U, respectively. Upon immobilization of the hyde. One CHO group of glutaraldehyde was linked to a NH2
Fig. 4 Cyclic voltammograms of (i) ZrO@Fe3O4NP/CHIT hybrid film, (ii) HRP/ZrO@Fe3O4NP/CHIT hybrid film and (iii) bare Au electrode modified
Au electrode in a 2.5 mM K3Fe(CN)6/K4Fe(CN)6 solution and sodium phosphate buffer 0.05 M (pH 7.2) at a scan rate of 50 mV sꢂ1
.
Scheme 2 Electrochemical reaction at the HRP/ZrO@Fe3O4NP/CHIT hybrid film modified Au electrode.
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group on the surface of the enzymes, while another CHO group modied electrode for 0.1 M pH 7.0 PBS. Fe3O4 NPs not only act as
was bound to the NH2 group of chitosan on the CHIT/ electron-transfer mediators, but also play an important role in the
ZrO@Fe3O4NP composite lm, which provided a physically more preparation of immobilized enzymes because of their desirable
stable complex. The FTIR spectra of the HRP/CHIT/ZrO@Fe3O4- characteristics: large pore size and volume, and good electron
NP/Au electrode showed a broadening of the peak at 3264 cmꢂ1 conductivity (CV curve i).18 Fe3O4 NPs also create a suitable
and 1653 cmꢂ1 due to the addition of carbonyl and amino groups microenvironment that benets the exposition of the active center,
conrming the binding of enzyme with the CHIT/ZrO@Fe3O4NP and increases the activity of the enzyme.19 In contrast, a decrease in
matrix (curve ii). This change suggests that the enzyme was the peaks was observed at the enzyme modied electrode as
attached to the ZrO@Fe3O4NP/CHIT/Au composite lm.
protein might impede electron transfer (CV curve ii). No peak was
observed on the voltammogram of the unmodied electrode (CV
curve iii). The results were matched with the EIS study.
3.3. Construction of HRP/ZrO@Fe3O4NP/CHIT modied Au
electrode and cyclic voltammetry measurements
3.4. The principle of ZrO@Fe3O4NP/CHIT hybrid lm
modied Au electrode for electrochemical sensing of
analgesic drug
To conrm the electron transfer regime, the CV technique was
employed at the electrode surface. Unmodied electrode is unable
to take redox reactions at the electrode surface. Modication of the
electrode with ZrO@Fe3O4NP/CHIT resulted in fast electron An electrocatalytic mechanism initiated by HRP is likely
transfer reactivity. As shown in Fig. 4, a pair of well dened, quasi- to catalyze the oxidation of paracetamol to N-acetyl-p-ben-
reversible redox peaks can be obtained with a ZrO@Fe3O4NP/CHIT zoquinoneimine (Scheme 2). The resulting current is
Fig. 5 Cyclic voltammograms of the HRP/ZrO@Fe3O4NP/CHIT/Au electrode in PBS (pH 7.0) in presence of substrate (i) and in the absence of
substrate (ii), at a scan rate of 5 mV sꢂ1
.
Fig. 6 Cyclic voltammograms of (i) HRP/Au (ii) HRP/ZrO@Fe3O4NP/CHIT hybrid film modified Au electrode in a 2.5 mM K3Fe(CN)6/K4Fe(CN)6
solution and sodium phosphate buffer 0.05 M (pH 7.2) at a scan rate of 50 mV sꢂ1
.
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proportional to the concentration of phenolic compounds in
solution. This is expected because of the participation of
proton(s) in the oxidation reaction of acetaminophen to N-
acetyl-p-benzoquinoneimine, and vice versa within a quasi-
reversible two-electron process.29 Therefore, the signi-
cantly increased redox peak currents greatly increased the
electron transfer rate of APAP at the ZrO@Fe3O4NP/CHIT/
AuE. As shown in Fig. 5, oxidation peak signal signicantly
increases to 750 mA in the presence of substrate. Fig. 6
presents the CV curve showing the electrode modied with
HRP only and with HRP/ZrO@Fe3O4NP/CHIT/AuE, oxidation
peak signal signicantly increases to 1000 mA when the nano-
composite is decorated on the Au electrode. These results show
that the electrochemical reactivity of APAP is improved
remarkably on the ZrO@Fe3O4NP/CHIT/AuE.
3.5. Optimization of the biosensor
To improve the performance of the biosensor, the effect of the
determination conditions, such as the working potential, pH,
response time, and temperature on the response of the
ZrO@Fe3O4NP/CHIT/Au electrode was investigated.
The effect of the working potential on the response
current of the ZrO@Fe3O4NP/CHIT/Au electrode was
studied. When the applied potential was changed from ꢂ0.1
V to +0.1 V, the response current increased signicantly. The
maximum response current was achieved at around ꢂ0.07 V.
When the applied potential became more negative, there
might have been interfering reactions from other electro-
active species in the solution. Therefore, an applied poten-
tial of ꢂ0.07 V was selected to give a high detection
sensitivity and good signal/noise ratio. The effect of the pH
value on the response current of the ZrO@Fe3O4NP/CHIT/Au
electrode was studied between 5.0 and 8.0 in 0.05 M PBS.
The response current increased from 5.0 to 7.0 and
decreased from 7.0 to 8.0, and the maximum current
response was observed at pH 7.0 (Fig. 7a). Therefore, the pH
7.0 was suitable for the maximum activity of immobilized
HRP, which is in agreement with that reported for soluble
HRP.Effect of temperature on biosensor was also studied in
order to ensure the optimization. The current response
ꢀ
reaches a maximum at approximately 50 C, and then goes
down as the temperature turn higher. In contrast, the
modied electrode without ZrO@Fe3O4NP shows that the
response declines when temperature is higher than 40 ꢀC.
The result indicates that enzyme bioconjugated with
ZrO@Fe3O4NP has good thermodynamic stability and life
span. In order to keep consistent with the temperature of
ꢀ
human body, 35 C was selected for this work (Fig. 7b). The
Fig. 7 Effects of pH (a), temperature (b) and response time (c) on the
electrochemical response of the fabricated acetaminophen biosensor
based on HRP/ZrO@Fe3O4NP/CHIT in 0.1 M sodium phosphate buffer.
response time was less than 4 s, which shows a rapid
response and the immobilized HRP could well catalyze the
reduction of H2O2 (Fig. 7c). The faster response was mainly
ascribed to the fact that the ZrO@Fe3O4NPs provide a
favorable orientation and conductive pathway to transfer
electrons. In addition, through the ZrO@Fe3O4NP exposed
3.6. Voltammetric determination of analgesic drug
surface, H2O2 molecules can freely diffuse to the HRP Cyclic voltammetry (CV) was used to determine the acetamin-
molecules.
ophen concentration in an array to obtain higher sensitivity.
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enzyme electrode. The relative standard deviation (R.S.D.) was
1.2% for ten successive assays.
3.8. Selectivity and real sample analysis
The effect of the substances that might interfere with the
response of the biosensor was studied. The selectivity of the
biosensor was examined in the presence of acetaminophen (0.2
mM). The addition of the same concentration of citric acid,
sodium benzoate, stearic acid, sodium metabisulphite, and
saccharin did not cause any observable interference. Only
stearic acid decreased the response by 10% and showed a
signicant interference. The proposed procedure was applied to
determine the paracetamol concentrations in pharmaceutical
formulations. Table 1 presents the results obtained for four
commercial samples by replacing acetaminophen with
samples. To study the accuracy of the present method, the
acetaminophen level in the samples were determined by both
the pharmacopoeia method (x) and the present method (y). The
values obtained by both methods matched each other with a
good correlation (r ¼ 0.95).
Fig. 8 Cyclic voltammograms of the HRP/ZrO@Fe3O4NP/CHIT at
various concentrations of acetaminophen.
The CV curves of different concentrations of acetaminophen at
ZrO@Fe3O4NP/CHIT/AuE modied electrode were obtained
(Fig. 8). The peak current increased linearly with increasing
acetaminophen concentration. The limit of detection was
0.01 mM.
3.9. Stability of the enzyme electrode
The stability of the biosensor was investigated and the
current response of the biosensor retained about 90% of its
original response aer 40 repetitions of uninterrupted
detection. In addition, the long-term stability was also tested
aer a month. The results showed that the current response
of the sensor maintained 84% of the initial current response.
This means that ZrO@Fe3O4NP ensure good stability of the
3.7. Reproducibility
The repeatability of the biosensor was valued at an acetamino-
phen concentration of 0.1 mM in PBS (0.1 M) with the same
Table 1 Determination of acetaminophen using present sensor based biosensor.
on the HRP/ZrO@Fe3O4NP/CHIT/Au electrode and standard enzy-
matic method
A comparison of the present biosensor with other biosensing
methods is given in Table 2.
Label value
(mg)
Pharmacopoeia
method (mg)
Present method
(mg)
S. no.
Sample
4. Conclusions
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
A
B
C
D
E
F
G
H
I
500
250
100
50
100
450
350
550
150
500
500
250
100
50
100
440
360
540
145
490
499
255
98
A novel strategy for developing a composite electrode consisting
HRP/ZrO@Fe3O4NP/CHIT/AuE, which showed relatively rapid
response, high sensitivity, broad linear range, low detection
limit, good reproducibility, and long term stability, was repor-
ted. This biosensor almost eliminated the interference. There-
fore, this novel biosensor can be readily extended to the
detection of other clinically important antigens using
ZrO@Fe3O4NPs to develop other simple and practical
biosensors.
49
102
450
350
552
152
500
J
Table 2 Comparison of the present method with other biosensing methods
Response
Detection limit
Matrix/method
Enzyme
time
(mM)
Linearity (mM)
Stability
Reference
C–Ni/GCE/DPV
—
—
—
—
—
7.8–110
0.1–20
0.1–100
2.5–1000
0.2–1500
0.01–10 000
—
—
—
—
30
31
32
33
34
Present
Chronoamperometry MWCNT-lm coated electrode
Carbon nanoparticles (CNPs)/GCE/voltammetry
Cobalt hydroxyl nanoparticles/cyclic voltammetry
Nanogold/ITOE/cyclic voltammetry
HRP/ZrO@Fe3O4NP/CHIT/Au
0.04
0.05
10
0.18
0.01
—
—
HRP
—
—
1 s
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16 L. M. Rossi, A. D. Quach and Z. Rosenzweig, Anal. Bioanal.
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Acknowledgements
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The present work was supported to one of the author (Jagriti
Narang) by SERB, Department of Science and Technology (DST),
India. Thanks to all scientists referenced throughout the paper
whose valuable work has guided the way through to this
research.
20 G. S. Chaubey and J. Kim, Bull. Korean Chem. Soc., 2007, 28,
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