Simultaneous Voltammetric Determination of Acetaminophen and Isoniazid (Hepatotoxicity-Related Drugs) Utilizing Bismuth Oxide Nanorod Modified Screen-Printed Electrochemical Sensing Platforms

Article abstract of DOI:10.1021/acs.analchem.6b05130

To overcome the recent outbreaks of hepatotoxicity-related drugs, a new analytical tool for the continuously determination of these drugs in human fluids is required. Electrochemical-based analytical methods offer an effective, rapid, and simple tool for on-site determination of various organic and inorganic species. However, the design of a sensitive, selective, stable, and reproducible sensor is still a major challenge. In the present manuscript, a facile, one-pot hydrothermal synthesis of bismuth oxide (Bi₂O_2.33) nanostructures (nanorods) was developed. These BiO nanorods were cast onto mass disposable graphite screen-printed electrodes (BiO-SPEs), allowing the ultrasensitive determination of acetaminophen (APAP) in the presence of its common interference isoniazid (INH), which are both found in drug samples. The simultaneous electroanalytical sensing using BiO-SPEs exhibited strong electrocatalytic activity toward the sensing of APAP and INH with an enhanced analytical signal (voltammetric peak) over that achievable at unmodified (bare) SPEs. The electroanalytical sensing of APAP and INH are possible with accessible linear ranges from 0.5 to 1250 μM and 5 to 1760 μM with limits of detection (3σ) of 30 nM and 1.85 μM, respectively. The stability, reproducibility, and repeatability of BiO-SPE were also investigated. The BiO-SPEs were evaluated toward the sensing of APAP and INH in human serum, urine, saliva, and tablet samples. The results presented in this paper demonstrate that BiO-SPEs sensing platforms provide a potential candidate for the accurate determination of APAP and INH within human fluids and pharmaceutical formulations.

Full text of DOI:10.1021/acs.analchem.6b05130

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Article
Simultaneous Voltammetric Determination of Acetaminophen and
Isoniazid (Hepatotoxicity-related Drugs) utilizing Bismuth Oxide
Nanorod Modified Screen-printed Electrochemical Sensing Platforms
Bahaa G. Mahmoud, Mohamed Khairy, Farouk A Rashwan, and Craig E Banks
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05130 • Publication Date (Web): 06 Jan 2017
Downloaded from http://pubs.acs.org on January 8, 2017
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Simultaneous Voltammetric Determination of Acetaminophen and
Isoniazid (Hepatotoxicityꢀrelated Drugs) utilizing Bismuth Oxide
Nanorod Modified Screenꢀprinted Electrochemical Sensing Platꢀ
forms
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†
†
†
‡
Bahaa G. Mahmoud, Mohamed Khairy, * Farouk A. Rashwan and Craig E. Banks *
† Chemistry Department, Faculty of Science, Sohag University, 82524.
‡ Faculty of Science and Engineering, Manchester Metropolitan University, Chester Street, Manchester
M1 5GD, UK.
ABSTRACT: To overcome the recent outbreaks of hepatotoxicity relatedꢀdrugs, a new analytical tool for the continuously deterꢀ
mination of these drugs in human fluids is required. Electrochemical based analytical methods offer an effective, rapid and simple
tool for onꢀsite determination of various organic and inorganic species. However, the design of a sensitive, selective, stable and
reproducible sensor is still a major challenge. In the present manuscript, a facile, oneꢀpot hydrothermal synthesis of bismuth oxide
(Bi2O2.33) nanostructures (nanorods) was developed. These BiO nanorods were cast onto mass disposable graphite screenꢀprinted
electrodes (BiOꢀSPEs) allowing the ultrasensitive determination of acetaminophen (APAP) in the presence of it’s the common inꢀ
terference isoniazid (INH) which are both found in drug samples. The simultaneous electroanalytical sensing using BiOꢀSPEs exꢀ
hibited strong electrocatalytic activity towards the sensing of APAP and INH with an enhanced analytical signal (voltammetric
peak) over that achievable at unmodified (bare) SPEs. The electroanalytical sensing of APAP and INH are possible with accessible
linear ranges from 0.5 to 1250 µM and 5 to 1760 µM with limits of detection (3σ) of 30 nM and 1.85 µM, respectively. The stabilꢀ
ity, reproducibility and repeatability of BiOꢀSPE were also investigated. The BiOꢀSPEs were evaluated towards the sensing of
APAP and INH in human serum, urine, saliva and tablet samples. The results presented in this paper demonstrate that BiO–SPEs
sensing platforms provide a potential candidate for the accurate determination of APAP and INH within human fluids and pharmaꢀ
ceutical formulations.
Hepatotoxicity implies chemicalꢀdriven liver damage and
the liver acts as central organ for metabolism of virtually every
foreign substance (i.e. drugs).1 More than 900 drugs and herbs
have been reported to cause severe liver disease and drugs
account for 20–40% of all instances of fulminant hepatic failꢀ
ure. Approximately 75% of the idiosyncratic drug reactions
result in liver transplantation or death.2 Drugꢀinduced liver
disease is responsible for 5% of all hospital admissions and
50% of all acute liver failures in western countries.3 Therefore,
physicians must be vigilant in identifying drugꢀrelated hepatic
injury because early detection can reduce the severity of inꢀ
duced hepatotoxicity. Knowledge of the commonly implicated
drugs is essential in clinical diagnosis. In the United States,
approximately 2000 cases of acute liver failure occur annually
and nearly 60% of these are caused by acetaminophen or idioꢀ
syncratic drug reactions.4 Development of an analytical tool
for accurate determination of acetaminophen (APAP) and its
interferences has a significant importance not only in clinical
diagnosis but also for quantity control of their pharmaceutical
formulations.
and effective painkiller associated with headache, backache,
arthritis and postoperative pain. It has been also used for reꢀ
duction of cough, cold and fevers symptoms.5 The analgesic–
antipyretic effect of APAP is similar to aspirin, but APAP is
normally preferred especially for the patients who are sensitive
to acetylsalicylic acid.6 The mechanism of action of APAP to
relieve pain is linked to inhibition of prostaglandin synthesis
in the central nervous system and relieves fever by sedating
hypothalamic heatꢀregulating centre.7 Single doses of APAP
shows analgesic activity in a variety of acute pain syndromes
without any side effects; however, overdoses (4.0 g/daily) are
now the most common cause of acute liver failure, which may
progress to death. Therapeutic concentration levels in plasma
is between 10ꢀ25 µg/mL, however, its toxicity concentration
level is higher than 200 µg/mL (≈ 1.33mM). It is therefore
important to develop an analytical technique to determine acꢀ
etaminophen in pharmaceutical dosages and human fluids
accurately. A variety of analytical methods such as titrimetry,8
UV/Vis spectrophotometry,9 flowꢀinjection,10 high perforꢀ
mance liquid chromatography (HPLC),11 chemiluminesꢀ
cence,12 capillary electrophoresis (CE),13 and electrochemical
techniques14ꢀ16 have been recently reported. While, improving
the analytical method in terms of sensitivity, stability, repeataꢀ
bility and selectivity are essential for human health and securiꢀ
ty.17
Acetaminophen, APAP (Nꢀacetylꢀpꢀaminophenol) is
known as Paracetamol or Tylenol and is one of the most wideꢀ
ly antipyretic and analgesic drugs these days. It is an extremeꢀ
ly weak acid (pKa = 9.7) and is, therefore, essentially unionꢀ
ized at physiological pH values. It is commonly used as a safe
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Isoniazid (isonicotinoyl hydrazine, INH) is an antibacterial
ing 0.05 M of each boric acid, phosphoric acid and acetic acid
and 0.1 M sodium hydroxide was added to reach the required
pH value of the solution.
drug available as more than 300 mg tablets for oral administraꢀ
tion. It is one of effective tuberculostatic drugs widely used for
prevention and treatment of tuberculosis due to restriction of
mycobacterium strains18,19 and is an essential and indispensaꢀ
ble basic remedy for tuberculosis. However, it is associated
with hepatotoxicity and peripheral neuropathy while slow
acetylators may be at increased risk of toxicity. The toxicity
can occur with the acute ingestion of as little as 1.5 g (10.9
mM). Moreover, hydrazine was produced during the isoniazid
metabolism not only inducing hepatotoxicity with inflammaꢀ
tion but also might lead to death.20 The Public Health Service
Surveillance Study examined 13,838 patients treated with INH
in USA; there were eight deaths among 174 cases of hepatitis.
Furthermore, some antipyretic drugs like APAP should be
combined with INH to control fever during the course of disꢀ
ease. This combination enhances drugꢀdrug interactions, ultiꢀ
mately affecting their bioavailability and pharmacokinetic
behaviors. Such interaction might also increase the extent of
bioactive drug resulting in its toxic effects or decrease it to
subꢀtherapeutic levels in blood that may lead to antimicrobial
resistance. The preꢀtreatment with INH could increase the
APAPꢀinduced hepatic necrosis score by 85%.21 Therefore, the
people taking INH and APAP are at risk of APAPꢀ toxicity.22
The patients treated with INH should be carefully monitored
and interviewed periodically every month intervals. HPLC,23
spectrophotometry,24 chemiluminescence,25 CE,26 and electroꢀ
chemical technique27,28 have been reported recently for deterꢀ
mination of INH. Although electrochemical methods offer fast
response, easy handling and low operating cost, there is only
one literature report detailing the simultaneous determination
of APAP and INH using thionine immobilized multiꢀwalled
carbon nanotube modified carbon paste electrode.29 However,
the cost effective, portability and difficulty to use such elecꢀ
trodes leads us to explore new generation of screenꢀprinted
electrodes in a form of a pointꢀofꢀcare sensor.
Synthesis of bismuth oxide nanostructures: The hydroꢀ
thermal synthesis of bismuth oxide nanorods has been develꢀ
oped using bismuth nitrate Bi(NO3)3 and ammonium hydroxꢀ
ide (NH4OH, 28%) as starting precursors without using any
further additives or templateꢀdirecting agents. In our synthesis
approach, 2.42 g of Bi(NO3)3 was dissolved in 50.0 mL acidiꢀ
fied deionized water. The transparent solution of Bi(NO3)3 was
introduced into a 100.0 mL Teflonꢀlined stainless steel autoꢀ
clave. Then, NH4OH solution was introduced until a white
precipitate was formed. The solution mixture has a pH value
of 2.2. The autoclave was sealed and placed in an oven at 160
°C for 12 hours for hydrothermal treatment after which was
then allowed to cool at room temperature. After the hydroꢀ
thermal process, a white precipitate was collected and rinsed
several times with ethanol/water mixture to remove the reꢀ
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maining agents before being dried at 50 C. Finally, the large
yield of bismuth oxide nanorods was formed by calcination at
300 °C for 3 hours.
Fabrication of bismuth oxide modified screenꢀprinted
electrode: The SPEs were fabricated inꢀhouse with appropriꢀ
ate stencil using a DEK 248 screenꢀprinting machine (DEK,
Weymouth, U.K.).These electrodes have been used extensiveꢀ
ly in previous studies For their fabrication, first, a carbonꢀ
graphite ink formulation (product code C2000802P2; Gwent
Electronic Materials Ltd., U.K.) was screenꢀprinted onto a
polyester (Autostat, 250 ꢁm thickness) flexible film (denoted
throughout as standardꢀSPEs); these electrodes have been used
extensively in other work.17, 30ꢀ37 This layer was cured in a fan
oven at 60 °C for 30 minutes. Next, a silver/silver chloride
reference electrode was included by screenꢀprinting Ag/AgCl
paste (product code C2040308D2; Gwent Electronic Materials
Ltd., U.K.) onto the polyester substrates and a second curing
step was undertaken where the electrodes were cured at 60 °C
for 30 minutes. Finally, a dielectric paste (product code
D2070423D5; Gwent Electronic Materials Ltd., U.K.) was
then printed onto the polyester substrate to cover the connecꢀ
tions. After a final curing at 60 °C for 30 minutes these SPEs
are ready to be used. These SPEs have been reported previousꢀ
ly and shown to exhibit a heterogeneous electron transfer
(HET) rate constant, ko, of ca. 10–3 cm s–1, as measured using
the [Ru(NH3)6]3+/2+ redox probe.
Herein, we explored the simultaneous voltammetric deꢀ
termination of APAP and its common interference of INH
using BiO nanostructured modified screenꢀprinted electrodes
(SPEs). Oneꢀpot hydrothermal synthesis of BiO nanostructures
has been developed for the first time. The dispersed BiO nanoꢀ
rods are simply dropꢀcasted onto SPE surfaces after which the
sensors are ready to be utilized. The BiOꢀSPEs are found to
exhibit greater electrocatalytic activity, stability, and reproducꢀ
ibility for the simultaneous determination of APAP and INH
in aqueous pH 2 buffer solution over unmodified (bare) SPEs.
The suggested BiOꢀSPE electrochemical sensing platform was
successfully employed to detect the concentration of APAP
and INH in human fluids and pharmaceutical formulations.
The SPEs were modified with BiO nanostructures by a
simple dropꢀcasting process. The BiO suspension was preꢀ
pared by dispersing 5.0 mg of BiO powder in 5.0 mL deionꢀ
ized water and then sonicated for 10 min. 5.0 µL of the BiO
suspension was dropped onto the SPE surface and left it to dry
in oven at 50 oC for 30 minutes.
EXPERIMENTAL SECTION
All chemicals were used without any further purification.
Bismuth nitrate (Bi(NO3)3, 99.9%) was obtained from Sigmaꢀ
Aldrich Company Ltd. Acetaminophen was kindly supplied by
Glopal Napi Pharmaceutical Co. (6th of October, Egypt) and
Isoniazid purchased from BDH Co., Ltd. All experiments were
performed in Britton Robinson (BR) buffer solutions containꢀ
Real sample analysis: Fresh human blood (5 mL) was
collected from healthy volunteers in the hospital of Sohag
University, with the aliquot of the sample collected in a test
tube. The collected blood sample was kept at room temperaꢀ
ture for 30 minutes and then centrifuged for 5 minutes at 3500
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rpm. Finally, the supernatant serum sample was collected in a
new test tube and stored at 4 C in a refrigerator when not in
a CCD camera. The BiO sample was dispersed in ethanol soꢀ
o
lution by using an ultrasonic bath, and then dropped on a copꢀ
per grid. Prior to inserting the samples into the TEM column,
the grid was vacuum dried for 20 min.
use. The APAP and INH in low, medium and high concentraꢀ
tion levels have been spiked into blood serum sample. Then,
the recovery test was carried out by spiking of APAP and INH
in serum sample into10 mL electrochemical cell containing pH
2.0 B.R buffer and differential pulse voltammetry (DPV) was
recorded. A standard addition method was used to determine
the APAP and INH concentrations.
Wideꢀangle powder Xꢀray diffraction (XRD) was perꢀ
formed by Xꢀray diffractometer (Model FW 1700 series,
Philips, Netherlands) using with monochromatic CuKα radiaꢀ
tion (λ = 1.54 Å), employing a scanning rate of 0.06°/min and
2θ ranges from 20° to 80°. The diffraction data were analysed
using PDF software Released in1996.
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The applicability of BiOꢀSPEs was also tested in several
real samples (human blood, urine, saliva and pharmaceutical
tablets). APAP and INH were injected to the real samples and
left for 1.0 h, stored at 4 oC in a refrigerator. For human blood,
the sample was centrifuged for 5 minutes at 3500 rpm. The
supernatant serum sample containing APAP and INH was
collected in a new test tube and the pH was adjusted to pH 2
using hydrochloric acid. Then, A 100 µL of serum samples
was dropped onto three electrode configuration cell of BiOꢀ
SPEs working electrode. DPV was utilised and the standard
addition method was used to determine the APAP and INH
concentration.
The textural surface properties and pore size distribution
was determined by N2 adsorption/desorption isotherms at 77 K
with a BELSORP36 analyzer (JP. BEL Co., Ltd.). The specific
surface area (SBET) was calculated using the Brunauer–
Emmett–Teller (BET) method with multipoint adsorption data
from the linear segment of the N2 adsorption isotherm. The
pore size distribution was determined from the analysis of
desorption branch of isotherm using BarrettꢀJoynerꢀHalenda
(BJH) method.
The voltammetric measurements were carried out using
Autolab 302N potentiostat/galvanostat workstation and data
was controlled by NOVA software version 1.11.2 for Winꢀ
dows7. Experiments were performed using 3 mm bismuth
oxide modified graphite screenꢀprinted working electrode,
platinum counter electrode and saturated calomel electrode
(SCE) reference electrode.
For urine and saliva samples, 300 µL of saliva or urine
samples was also injected with APAP and INH and stored at 4
oC in refrigerator for 1.0 h. Then, the sample solution was
adjust to pH 2 using HCl. A 100 µL of adjusted saliva or
urine samples was dropped onto the working electrode of the
BiOꢀSPEs and DPV was utilised.
Ten tablets of APAP (Paracetamol, 500 mg) and INH (Isoꢀ
cid forte, 200 mg) pharmaceutical formulations were accurateꢀ
ly weighed and finely grinded in a mortar. A chosen amount of
these powders were transferred into a 100 mL volumetric
flask. About 20 mL of ethanol was added, swirled and sonicatꢀ
ed for 5 min. The volume of the sample was completed to the
mark and finally filtered using fine filter paper. The first porꢀ
tion of the filtrate was rejected. A specific volume of the stock
solution of the drugs was diluted with deionized water to obꢀ
tain the suitable concentration. A 100 µL of a particular conꢀ
centration of APAP and INH solutions were diluted by B.R.
buffer pH 2 and dropped onto the BiOꢀSPEs and compared
with the standard concentrations of APAP and INH drugs. The
concentrations of APAP and INH were measured by DPV
method.
RESULTS AND DISCUSSION
Physicochemical characterization of the bismuth oxide
nanostructures: Bismuth oxide is an important metal oxide
semiconductor which has been intensively investigated due to
excellent optical and electrical properties such as wide bandꢀ
gap, high refractive index, high dielectric permittivity and
good photoconductivity.38,39 Owing to these unique features, it
has been widely used in many applications such as schottky
barrier solar cells, sensor technology, varistors, optical coatꢀ
ings, photovoltaic cells, microwave integrated circuits and
transparent ceramic glass manufacturing.38,39 Control over the
morphology, size, crystallinity and surface composition of
bismuth oxide nanostructures can be effectively alter its physiꢀ
cochemical characteristics. Herein, a simple, oneꢀpot hydroꢀ
thermal synthesis of BiO nanostructures has been carried out
by adjusting the pH of acidic solution of bismuth nitrate by
ammonium hydroxide solution until a white precipitate was
formed. After which, calcination of obtained white powder at
300 oC for 3.0 hours was performed.
Characterization of bismuth oxide nanostructures: The
morphology of the bismuth oxide sample was investigated
using field emission scanning electron microscopy (FEꢀSEM,
JEOL model 6500). The bismuth oxide powder was ground
and fixed onto a specimen stub using doubleꢀsided carbon
tape. To obtain highꢀresolution micrographs, a 10 nm Pt film
was coated on the bismuth oxide using anion sputtering (Hitaꢀ
chi Eꢀ1030) at room temperature. The SEM was operated at 15
KeV to obtain highꢀresolution SEM images. Further, Transꢀ
mission electron microscopy (TEM) of BiO sample was perꢀ
formed using a JEOL JEM microscope model 2100. TEM was
conducted at an acceleration voltage of 200 kV to obtain a
lattice resolution of 0.1 nm. TEM images were recorded using
Although the particle morphology, composition and size
are crucial factors for metal oxide nanostructures applicabilꢀ
ity,30, 30ꢀ42 very few literatures have been reported to control the
size and morphology of bismuth oxide (Bi2O3)
nanostrucures.38, 39, 43 Figure 1A shows the SEM micrograph of
bismuth oxide sample synthesized by a facile oneꢀpot hydroꢀ
thermal method without any additives. The SEM image exhibꢀ
its highly dispersed and nanoꢀsized bismuth oxide with rodꢀ
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like morphology. The average diameter of bismuth oxide naꢀ
of the bismuth oxide powder was characterized using XRD
norods is about 50 nm with a length of 400 nm. Further, TEM
image (Figure 1B) shows dispersed BiO nanorods with an
average diameter of 50 nm and a length of about 300ꢀ400 nm.
Significantly, our synthesis conditions provide a facile and
templateꢀfree method for fabrication of highly crystalline bisꢀ
muthꢀrich bismuth oxide nanostructures without any agglomꢀ
eration.
and is shown in Figure 2(B). Different diffraction peaks have
been indexed with corresponding (hkl) values using JCPDS
data file (27ꢀ0051) indicating the nanoꢀcrystalline structure of
the bismuth oxide. The XRD patterns also confirmed preferenꢀ
tial growth of tetragonal phases of Bi2O2.33. Interestingly, the
XRD shows stoichiometric changes due to oxygen loss. The
slight excess of Bi atoms over oxygen atoms in the tetragonal
crystal might offer enhanced electron conductivity for better
electrochemical performance.
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Electrochemical characterization of bismuth oxide
nanostructures: To explore the electrochemical performance
of (BiOꢀSPE), the redox probe of K3Fe(CN)6 has been studied.
Figure 3 and Figure S1 show the cyclic voltammetric curves of
BiO modified and bare/unmodified screen printedꢀelectrode in
2×10ꢀ4 mol.Lꢀ1 K3[Fe(CN)6] / 0.1 mol.Lꢀ1 KCl recorded over a
range of voltammetric scan rates. In both cases, the peak
heights were increased with increasing the potential scan rate
as well as they are linearly correlated with the square root of
the scan rates in the range of 10–200 mV/s ( as shown in Figꢀ
ure 3B) suggesting a diffusion controlled process of reactants
at the electrode surface. Comparing the CV curves at 10 mV/s,
it was found that the peak current ratio (Ipc/Ipa) is equal to 1.1
for the unmodified SPEs while the BiOꢀSPEs exhibit a Ipc/Ipa
equal to 1.37. Therefore, the BiOꢀSPEs may be claimed to
promote electron transfer reaction better than the unmodified
SPE does. Moreover, the peak separation was slightly inꢀ
creased to 179 mV at the BiOꢀSPEs compared to 170 mV for
unmodified SPEs, which is due to the electrostatic repulsion
between [Fe(CN)6]3ꢀ and BiO nanorod surface. This behaviour
suggests that the BiO surface is more negatively charged, in
agreement with the behaviour observed for Titanate nanoꢀ
tubes.40 The result show in Figure 3 that the BiO nanorods
have enhanced the electrochemical signal / performance of the
graphite screenꢀprinted electrode by an order of ~2 times,
which might be attributed to the free bismuth atoms on the
surface of BiO nanoparticles that facilitated the electron transꢀ
fer process and/or an increase in the electrochemical surface
area (i.e. the BiO kinetics dominate).30
Figure 1 (A) FEꢀSEM and (B) TEM images of BiO nanostrucꢀ
tures prepared via a simple hydrothermal treatment.
Figure 2 (A) Nitrogen adsorption /desorption isotherm and its
corresponding BJH pore distribution curve, (B) Typical Xꢀray
powder diffraction pattern of the BiO nanostructures
The specific surface area and porosity of the BiO nanorods
were investigated by using N2 adsorption/desorption isoꢀ
therms. Figure 2, shows
a representative N2 adsorpꢀ
20
tion/desorption isotherm measured at 77 K and distribution of
pore diameter for the synthesized BiO sample. The N2 isoꢀ
therm can be categorized as type IV with a distinct H3 hysteꢀ
resis loop was observed in the range of 0.5–1.0 P/P0, which
was characteristic of macroꢀ/ mesoꢀporous materials. The SBET
specific surface area was calculated to be 7.9 m2 gꢀ1. Further,
the BiO nanorods contained small mesopores with average
diameter about 5, 34 and 55 nm, determined by using the Barꢀ
ret–Joyner–Halenda (BJH) method (Figure 2B). The formation
of the lower pore size was probably related to the pores preꢀ
sent inside the nanorods, which were formed between primary
crystallites. In turn, the higher pore size may be related to the
voids between the nanorod particles. Such bimodal porous
texture of BiO nanorods is very important in electrochemical
sensors because it provides enhanced surface area with macroꢀ
/ mesopores. These significant features are the key factors for
controlling the diffusion pathway of analytes and alter the
solution/electrode interface. Furthermore, the crystal structure
ꢀ20
ꢀ15
ꢀ10
ꢀ5
SPE
Bi2OꢀSPE
(A)
(B)
3
10
0
SPEs
BiOꢀSPE
0
ꢀ10
ꢀ20
5
1.0 0.8 0.6 0.4 0.2 0.0 ꢀ0.2
0.1
0.2
0.3
0.4
ꢀ1 1/2
(Scanrate)1/2 (Vs )
Potential (V)
Figure 3 (A) Cyclic voltammetric curves of SPE and BiOꢀ
SPEs in 0.1 M KCl containing 2×10ꢀ4 M Potassium ferricyaꢀ
nide. (B) Cyclic voltammetric peak current analysis at various
scan rates.
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were observed (For APAP; E/V = 0.043 Ep (V) /pH + 0.55 V,
bg
25
25
75
75
140
140
225
225
300
300
12
APAP
INH
R2= 0.95; For INH; E/V = 0.064 Ep (V) /pH + 0.83 V, R2=
0.98). These values are close to that of Nernstian slope (59
mV/pH) which related to transfer an equal number of protons
and electrons and likely to be in agreement with the literaꢀ
ture.5,29,50ꢀ59 Therefore, we supposed that the oxidation of
APAP and INH at BiO–SPEs is likely involves 2eꢀ/2H+ transꢀ
fer processes as shown in Scheme 1. In APAP, it was observed
that, the peak current of APAP was decreased in alkaline soluꢀ
tions. The pKa value APAP in solution is about 9.7, above this
value the APAP was existed in deprotonated form which exꢀ
hibit a net negative charge. Such negative charge will be reꢀ
pulsed with the negative charge of the BiOꢀSPE surface.
Moreover, the deprotonated APAP form is likely to be more
soluble in aqueous solution than its neutral species; hence the
amount of diffused APAP to BiOꢀSPEs surface is much lower
than its fraction in the solution. Therefore, the acidic medium
is highly desirable for oxidation of APAP on BiOꢀSPEs. The
oxidation of APAP includes transfer of 2eꢀ/2H+ to form quiꢀ
noneꢀlike structure followed by hydrolysis of the acetamide
functional group to produce pꢀaminophenol (Scheme 1). Furꢀ
ther, the pKa value of INH is about 4.84 and therefore, protoꢀ
nated form of isoniazid will be also very important in the elecꢀ
trostatic attraction with negatively charged BiO nanorods. In
alkaline medium, the oxidation peak was splitted to oneꢀ
electron coupled with oneꢀproton reaction process for each of
these two peaks. The oxidation of INH on BiOꢀSPEs is underꢀ
going transfer of 2eꢀ/2H+ and subsequently hydrolyses to Isonꢀ
icotinic acid. The optimal voltammetric response in Figure
S4C suggested that B. R. buffer pH 2 is a suitable supporting
electrolyte for determination of APAP and INH in real samꢀ
ples.
µ
µ
µ
µ
µ
µ
µ
µ
MAPAP
MAPAP+25µMINH
MAPAP+25µMINH
MAPAP+75 MINH
MAPAP+75 MINH
MAPAP+140 MINH
MAPAP+140 MINH
MAPAP+225 MINH
MAPAP+225 MINH
MAPAP+300 MINH
µ
µ
9
6
3
0
µ
µ
µ
µ
µ
µ
µ
(A)
(B)
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55
56
57
58
59
60
0
50 100 150 200 250 300
Concentration(µM)
0.0
1.2
P0o.t4ential (0V.8)
Figure 4 (A) Cyclic voltammetric curves observed at BiO
nanoꢀrods modified SPEs for successive additions of APAP
and INH into B.R. buffer pH 2, (B) Corresponding cyclic voltꢀ
ammetric peak current analysis of APAP and INH additions at
scan rate 50 mVsꢀ1.
Electrochemical behaviour of APAP and INH: The
voltammetric behaviour of APAP and INH were investigated
on BiOꢀSPEs because the isoniazid was considered as an imꢀ
portant drug interferes with APAP in pharmaceuticals. Figure
S2 illustrates the cyclic voltammograms of 75 µmol.Lꢀ1 APAP
and 75 µmol.Lꢀ1 INH obtained in B.R. Buffer solution (pH
2.0) on unmodified and BiO modified SPEs. BiO nanorods
showed an efficient electrocatalytic activity for the oxidation
of APAP and INH molecules compared to bare SPEs. In order
to explore the efficiency of BiO nanorods, different metal oxꢀ
ide nanostructure materials and bismuthꢀbased materials have
been synthesised and used for determination of APAP and
INH drugs. NiO,40 CuO,30 CdO,44 MgO,45 , Bi2S3,46 Bi12NiO19
47
and bulky Bi2O3 have been investigated (as shown in Figure
S3). Interestingly, BiO nanorodsꢀSPEs show high sensitivity
and stability as well as good peak potential separation in the
determination of APAP and INH molecules. Figure 4A shows
cyclic voltammetry of successive additions individually of
APAP and INH in BR buffer pH 2.0 at scan rate of 50 mVsꢀ1
on BiOꢀSPEs. As it can be seen, APAP and INH exhibit wellꢀ
defined oxidation peaks around 0.64 and 1.02V, respectively.
The peak potentials difference of about 0.38 V (vs. SCE) beꢀ
tween both oxidation peaks clearly allows the simultaneous
determination of APAP and INH without any interference. Of
interest is the voltammetric peak current response of APAP
and INH on the BiOꢀSPE linearly correlates with their concenꢀ
trations up to 300 µmol.Lꢀ1 as shown in Figure 4B.
6
4
4
3
2
0
2
ꢀ2
(A)
(B)
0.5
1
ꢀ0.2 0.0 0.2 0.4 0.6 0.8 1.0
0.1
0.2
0.3
0.4
0.6
(scanrate)1/2 (V/s)1/2
Potential (V)
9
6
3
7
6
5
4
3
2
1
0
Next attention was turned to explore the effect of pH upon
the electrochemical signals of APAP and INH, since the selecꢀ
tion of suitable medium is very important for simultaneous
electrochemical responses of APAP and INH. Figure S4
shows the cyclic voltammetric curves of 100µM of APAP and
100µM INH onto BiOꢀSPEs in different pH values in different
pH values. Moreover, effect of solution pHs on the CV curves
for 50 µmol.Lꢀ1 APAP and 50 µmol.Lꢀ1 INH onto BiOꢀSPEs
also presented. With increasing the pH value, the oxidation
potentials of APAP and INH were shifted to less positive poꢀ
tential values and the oxidation peak of INH interferes with
oxidation peak of APAP. This result indicates that, the pH 2 is
the suitable medium for simultaneous determination of APAP
and INH. A plot of peak potential (E) vs. pH, as shown in Figꢀ
ure S4D, was constructed where a linear range with gradient
of 43 mV/pH and 64 mV/pH for APAP and INH, respectively
0
(C)
(D)
ꢀ3
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
0.0 0.3 0.6
1.2 1.5
Potential0(.V9 )
(scanrate)1/2 (V/s)1/2
Figure 5. Effect of scan rate on cyclic voltammetric curves of
100 µM of APAP (A, and B) and 100 µM of INH (C and D)
observed at BiO nanorods modified SPEs in B.R. buffer pH 2.
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Next, the effect of scan rates upon the electrochemical oxiꢀ
linear fitting equations of APAP and INH, the limits of detecꢀ
dation behaviours of APAP and INH was explored in B.R.
buffer pH 2.0. Figure 5 shows the cyclic voltammetric profiles
of 100 µmol.Lꢀ1 APAP and 100 µmol.Lꢀ1 INH in pH 2 in difꢀ
ferent scan rate in the range of 10 –300 mVsꢀ1. No reduction
peak has been observed in the cyclic voltammograms of INH.
However, the intensity of APAP reduction peak becomes sigꢀ
nificant at higher scan rates. Meanwhile, there was a positive
shift of peak potential with the increase of scan rates. The
analysis of peak height against the square root of scan rate was
found to be linear indicating a diffusional controlled processes
(For APAP; Ipa/µA = 5.74 µA (Vsꢀ1)ꢀ1/2 + 0.68 µA, R2 = 0.992,
For INH; Ipa/µA = 8.67 µA (Vsꢀ1)ꢀ1/2 + 0.23 µA, R2 = 0.997).
Moreover, the linear relationship of log (Ipa) and log υ can be
expressed as following; For APAP: log Ipa = 0.33 log υ +
0.73, For INH: log Ipa/µA = 0.45 log υ + 0.925. The slop valꢀ
ues are very close to the theoretical value of 0.5, which clearly
indicates a diffusion controlled electrode processes (rather than
a thin layer effect). Form the above results the electroꢀ
oxidation of APAP and INH can be described as follows;
tion were calculated to be 30 nmol.Lꢀ1 and 1.85 µmol.Lꢀ1, reꢀ
spectively. The superior performance of the BiOꢀSPEs sugꢀ
gests a promising platform for the electrochemical determinaꢀ
tion of APAP and INH in real samples.
7
6
5
4
3
2
1
0
6
5
4
3
2
1
0
bg
0.5
2.49
9.9
19.6
29.3
58
140
320
460
650
µ
µ
M
M
1.4
4.98
14.8
µM
µM
µM
(A)
(B)
µ
M
10
11
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
µ
µ
M
M
24
39
µM
µM
µ
M
97
µM
µ
µ
µ
µ
M
M
M
M
220 µM
890
1220
µ
M
µ
M
0.2
0.4
0.6
0.8
1.0
0
500
1000
1500
Potenial (V)
[APAP] (µM)
5
9
14
24
39
94
180
200
250
319
469 µM
669
916
µ
M
7µM
2.5
2.0
1.5
1.0
0.5
0.0
2.8
2.4
2.0
1.6
1.2
0.8
0.4
µ
M
12
µM
(C)
(D)
µ
M
19
µM
µM
29
µM
µM
59
µM
µ
M
µM
144µM
µ
M
µ
M
µM
µM
µM
O
1260
1760
µM
M
O
µ
O
N
H
H
N+
N
H
N
0
500
1000
1500
0.4 0.6 0.8 1.0 1.2 1.4 1.6
[INH] (µM)
-
Potenial (V)
+H+
ꢀ2e
H O
2
ꢀ2H+
Figure 6. DPV responses from successive additions of APAP
(A) and INH (C) into B.R. buffer pH 2 at BiO nanostructures
modified SPEs, (B and D) Typical calibration plots correꢀ
sponding to APAP and INH additions up to 1800 µM. DPV
parameters: step potential 0.01 V, modulation potential 0.025
V, modulation time 0.05s, interval time 0.2s, scan rate 50
mVsꢀ1, deposition time 5s and equilibrium time 5s.
O
H
O
O
O
APAP
O
O
NH2
NH
OH
-
NH
ꢀ2e
N
O
N
H O
2
N
+
ꢀ 2H
N
INH
As mentioned previously, simultaneous determination of
APAP and INH was studied in different pH values, and B.R.
buffer pH 2.0 was selected as a suitable supporting electrolyte
solution. DPV experiments for simultaneous determination of
APAP and INH are carried out in pH 2.0 as represented in
Figure 7. DPV of successive additions of APAP in presence of
50 µmol.Lꢀ1 INH revealed two wellꢀseparated and distinct
oxidation peaks of APAP and INH were observed (Figure 7A).
Interestingly, there was no significant change of the voltamꢀ
metric response of INH with consequence additions of APAP.
The calibration curve of APAP was shown in Figure 7B with
the linear relationship from 10 to 210 µmol.Lꢀ1 with a linear
equation; Ip(µA) = 0.02 µA µMꢀ1 + 0.083 µA (R2 = 0.997).
While, Figure 7C shows DPV of successive additions of INH
in presence of 50 µmol.Lꢀ1 APAP. As can see from the voltꢀ
ammograms, relatively the current response of APAP does not
effect by addition of INH even at high concentrations. A linear
calibration curve for INH additions was presented in Figure
7D; Ip (µA) = 0.007 µA µMꢀ1 + 0.937 µA (R2 = 0.97). The
limits of detection of APAP and INH were calculated based on
these linear fitting equations to be 37 nmol.Lꢀ1 and 1.61
µmol.Lꢀ1, respectively. These results synergistically proved the
feasibility of simultaneous determination and distinguished
electrochemical sensing performance of BiOꢀSPEs to APAP
and INH drugs as shown in Table 1.
Scheme 1. Oxidation mechanisms of APAP and INH at BiOꢀ
SPEs.
Electrochemical determination of APAP and INH:
Next, DPV was employed to investigate the sensitivity of BiOꢀ
SPEs in terms of linear ranges and the detection limits for the
APAP and INH drugs. The DPV experiments were recorded
using the following parameters; step potential 0.01 V, moduꢀ
lation potential 0.025 V, modulation time 0.05s, interval time
0.2s, scan rate 50 mVsꢀ1, deposition time 5s and equilibrium
time 5s. Figure 6 displays the DPV responses of the electroꢀ
chemical oxidation of APAP and INH using BiOꢀSPEs in pH
2.0 of BR buffer under the optimized working conditions. The
peak height was increased with continuous additions of APAP
and INH solutions. For APAP, two linear relationships were
established (as shown in Figure 6B) in the range of 0.5 to 97
µmol.Lꢀ1 and 140 to 1250 µmol.Lꢀ1 as Ip (µA) = 0.027 µAµMꢀ1
+ 0.06 µA (R2 = 0.997) and Ip (µA) = 0.0034 µAµMꢀ1 + 1.64
µA (R2= 0.98), respectively. As well as, two linear segments
of INH were also presented (as shown in Figure 6D) in the
range of 5 to 100 µmol.Lꢀ1 and 144 to 1760 µmol.Lꢀ1 as Ip (µA)
= 0.008 µAµMꢀ1 – 0.009 µA (R2= 0.99) and Ip (µA) = 0.0012
µA µMꢀ1 + 0.49 µA (R2= 0.98), respectively. Based on the first
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2.5
Repeatability of BiOꢀSPEs for APAP and INH deterꢀ
bg
50
15
25
35
40
110
130
170
2.0
1.5
1.0
0.5
0.0
(A)
(B)
µ
µ
µ
µ
µ
µ
µ
MINH
MAPAP
MAPAP
MAPAP
MAPAP
MAPAP
MAPAP
mination: Screenꢀprinted electrode is a favourable approach
in electroanalytical chemistry because it provides a low cost,
singleꢀshot disposable yet highly reproducible and reliable
platform for electrochemical measurement of the target anaꢀ
lyte. However, the sensitivity and selectivity are major chalꢀ
lenge. Therefore, the modification of SPEs with active nanoꢀ
materials or bioꢀrecognition events has been developed. The
reproducibility of BiOꢀSPEs was investigated by using the
same BiOꢀSPEs for five repetitive measurements of 50
µmol.Lꢀ1 APAP and 100 µmol.Lꢀ1 INH under the optimum
conditions. The relative standard deviation was calculated to
be 0.27 % and 0.08 % for APAP and INH respectively. Furꢀ
thermore, BiOꢀSPEs show not only reproducible signal on the
same electrode but also with separately employed SPEs. Three
electrodes were examined in presence of 50 µmol.Lꢀ1 APAP
and 100 µmol.Lꢀ1 INH under the same conditions. The relative
standard deviation was calculated to be 1.7 % and 2.1 % for
APAP and INH drugs, respectively. These results revealed that
BiO nanorods are promising materials in electrochemical deꢀ
termination of APAP and INH in real samples.
2.0
1.5
1.0
0.5
0.0
µMAPAP
190
210
µMAPAP
µ
MAPAP
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58
59
60
0.0 0.2 0.4 0.6 0.8 1.0 1.2
0
50
100
150
200
Potential (V)
[APAP] (µM)
1.8
1.6
1.4
1.2
1.0
0.8
0.6
bg
1.8
1.6
1.4
1.2
1.0
50 µMAPAP
50 µMINH
75 µMINH
100µMINH
125µMINH
150µMINH
175µMINH
200µMINH
225µMINH
250µMINH
275µMINH
(C)
(D)
0.3
0.6
0.9
1.2
50 100 150 200 250 300
Potential (V)
[INH](µM)
Figure 7. (A) DPV curves from successive addition of APAP
in presence 50 µM INH and (B) Corresponding peak current
as function of APAP concentrations. (C) DPV curves for sucꢀ
cessive addition of INH in presence 50 µM APAP and (D)
Corresponding peak current as function of INH concentration.
DPV parameters : step potential 0.01 V, modulation potential
0.025 V, modulation time 0.05s, interval time 0.2s, scan rate
50 mVsꢀ1 , deposition time 5s and equilibrium time 5s.
Analysis of real samples: To verify the applicability of
BiOꢀSPE sensor, the recovery experiment for determination
APAP and INH in a human serum environment was performed
as in section 2.4. Before DPV determination, the APAP and
INH were spiked into human serum and left it for 30 minutes
in fridge at 4 oC. Then, these spiked samples were added to 10
mL electrochemical cell containing B. R. buffer pH 2.0. The
concentrations of APAP and INH were determined by standꢀ
ard addition protocol. The results were summarized and listed
in Table 2. The recovery experiment for human serum samples
demonstrated that the BiOꢀSPE electrochemical sensing platꢀ
forms held great promise for reliable and sensitive application
in the clinical analysis.
Table 1. Comparisons of the proposed bismuth oxideꢀSPEs
with previous reported electrochemical methods for APAP and
INH determinations.
Electrode
Target
Molecule
Linear
Range
(µM)
0.1 ꢀ100
1 ꢀ100
LOD
( µM)
Ref.
[29]
Table 2. Determination of APAP and INH added in human
serum samples using DPV (each sample was measured three
times n = 3).
Thionine immobilized
MWCNT/CPE
APAP
INH
0.05
0.8
EGR,ZnO/GCE
Tyrosine/GCE
APAP
INH
0.02 – 10
25ꢀ125
0.1–100
0.2 – 10
0.1ꢀ110
0.1ꢀ370
2ꢀ70
33
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[5]
6.93
Blood serum
Added (µM)
Detected (µM)
Recovery (%) ± SDa
Fe3O4/PDDA/GR/GCE
AuNP–MWCNT/GCE
MIPRu–AuNP–MCNT/GCE
OMC/GC
APAP
INH
0.037
0.0003
0.08
APAP
10
INH
40
APAP
9.8
INH
37.2
62.4
39.2
55.2
APAP
INH
Sample 1
98 ± 1.94
102 ± 2.04
102 ± 1.49
102.7 ± 1.75
93 ± 3.15
104 ± 4.17
98 ± 2.44
92 ± 4.34
INH
15
60
15.3
10.2
15.4
INH
0.084
0.17
Sample 2
10
40
ERGO/GCE
INH
15
60
RGO–Au/GCE
(MWCNT–chit/GCE)
GR/GCE
INH
10ꢀ1000
10ꢀ100
0.01
a Relative standard deviation.
INH
0.055
0.032
APAP
APAP
APAP
APAP
Moreover, to explore the applicability of BiOꢀSPEs in
simultaneous and sensitive determination of APAP and INH,
The recovery experiments were performed in human blood,
urine and saliva samples. As explained in experimental secꢀ
tion, the APAP and INH were injected to the human fluid
Au and glutamic Acid /CPE
GCE
0.05ꢀ70
4ꢀ100
0.369
0.21
TiO2–GR/GCE
BiOꢀSPEs
1ꢀ100
o
samples and stored at 4 C in a refrigerator. After 1.0 h, the
APAP
INH
0.5 – 97
5 ꢀ 100
0.03
1.85
This
work
human blood was centrifuged for 5 min at 3500 rpm. The suꢀ
pernatant serum sample containing APAP and INH was colꢀ
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The Supporting Information is available free of charge on the
ACS Publications website.
lected in a new test tube and the pH was adjusted to pH 2 by
HCl. A 100 µL of treated serum samples was dropped onto
BiOꢀSPEs cell, DPV was utilised and the standard addition
method was used to determine the APAP and INH concentraꢀ
tions. On other hand, 300µL of urine or saliva samples was
AUTHOR INFORMATION
Corresponding Author
o
injected by APAP and INH and stored at 4 C in refrigerator
Email: mohamed.khairy@science.sohag.edu.eg, Tel:
+(02)01092099116
for 1.0 h. The sample solution was adjusted to pH 2 using HCl
and dropped onto BiOꢀSPEs and DPV was utilised. Further,
the APAP and INH were determined in pharmaceutical dosꢀ
age. The concentrations or “recoveries” of APAP and INH
were obtained. As shown in Figure S5 and Table 3, BiOꢀSPEs
are promising candidates in determination of APAP and INH
in different matrix samples.
10
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34
35
36
37
38
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Email: c.banks@mmu.ac.uk
ACKNOWLEDGMENT
The authors acknowledge funding from a British Council Instituꢀ
tional Link grant (No. 172726574, ID: 18435) for the support of
this research.
Table 3. Recovery of APAP and INH in different real samples
measured by DPV using 3ꢀelectrode BiOꢀSPEs configuration
cell (each sample was measured three times n = 3).
REFERENCES
(1) Russmann, S.; KullakꢀUblick, G. A.; Grattagliano, I.
Curr Med Chem., 2009, 16, 3041–3053.
(2) Friedman, S. E.; Grendell, J. H.; McQuaid, K. R.
Current diagnosis & treatment in gastroenterology.
New York: Lang Medical Books/McGrawꢀHill.,
2003, 664–679.
(3) Darvas, F.; Guttman, A.; Dormán, G. Chemical Geꢀ
nomics and Proteomics, second addition, CRC Press,
Taylor & Francis group, LLC, New York, 2013, 138.
(4) Lee, W. M. Clin Liver Dis., 2013, 17, 575ꢀ586.
(5) Fan, Y.; Liu, J.ꢀH.; Lu, H.ꢀT.; Zhang, Q. Colloids
Surface B, 2011, 85, 289–292.
ample
Added/mM
Detected/mM
Recovery (%) ±
SDa
APAP
INH
0.50
APAP
0.213
INH
APAP
INH
Human blood
Urine samples
0.25
0.475
85 ±
4.04
111 ±
3.09
109±
4.34
95 ±
2.10
98 ±
1.04
99 ±
2.50
2.0
2.0
2.2
1.96
1.0
4.0
1.09
0.43
2.08
1.08
0.245
0.103
3.96
0.40
2.0
1.50
2.0
1.45
108 ±
3.75
97 ±
2.30
102 ±
2.01
Saliva Samples
2.04
104 ±
3.2
1.0
3.0
3.15
108 ±
2.1
105 ±
1.02
101.2
± 3.2
99 ±
3.6
(6) Prabakar, S.J.R.; Narayanan, S.S. Talanta, 2007, 72
,
1818.
Pharmaceutical
Tablets
0.25
0.1
0.25
0.1
0.253
0.099
98 ±
2.08
103 ±
3.05
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D.; Lewis, S. E.; Andersson, A.; Bucher, B.; Galzi, Jꢀ
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screenꢀprinted electrode mutually facilitate the electron transꢀ
fer and mass transport as well as electrocatalytic activity toꢀ
ward oxidation of APAP and INH drugs. The unique properꢀ
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