- Email: [email protected]
MWCNT nanocomposite
Accepted Manuscript Title: Nonenzymatic H2 O2 sensing based on silver nanoparticles capped polyterthiophene/MWCNT nanocomposite Author: Adel A. Abdelwahab Yoon-Bo Shim PII: DOI: Reference:
S0925-4005(14)00523-1 http://dx.doi.org/doi:10.1016/j.snb.2014.05.004 SNB 16878
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-3-2014 22-4-2014 1-5-2014
Please cite this article as: A.A. Abdelwahab, Y.-B. Shim, Nonenzymatic H2 O2 sensing based on silver nanoparticles capped polyterthiophene/MWCNT nanocomposite, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
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► AgNPs modified Ox-pTTBA/MWCNT nanocomposite film for highly sensitive and selective H2O2
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sensor. ► Fast amperometric response with lowest detection limit was obtained for H2O2. ► The
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reliability of the proposed sensor was evaluated using real samples analysis.
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Nonenzymatic H2O2 sensing based on silver nanoparticles
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capped polyterthiophene/MWCNT nanocomposite
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Adel A. Abdelwahab a*, Yoon-Bo Shim b**
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Department of Chemistry, Faculty of Science, Al-Azhar University, Assiut 71524, Egypt b
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Department of Chemistry and Institute of BioPhysio Sensor Technology, Pusan National University, Busan 609-735, South Korea
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* Corresponding author. Tel.: (+20) 88-231-2193; Fax: (+20) 88-232-5436.
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E-mail address: [email protected] (A.A. Abdelwahab).
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** Corresponding author. Tel.: (+82) 51-510-2244; Fax: (+82) 51-514-2430.
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E-mail address: [email protected] (Y.-B. Shim).
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Abstract
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A novel method for highly sensitive H2O2 sensor is proposed using silver nanoparticles
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(AgNPs) modified oxidized poly-2,2′:5′,2′′-terthiophene-3-p-benzoic acid/multi wall
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carbon nanotube (Ox-pTTBA/MWCNT). The Ox-pTTBA/MWCNT nanocomposite
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film was prepared via electropolymerization of a TTBA monomer and MWCNT
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mixture solution, followed by in situ electrooxidation of the pTTBA/MWCNT film.
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Then, AgNPs were formed on the Ox-pTTBA/MWCNT layer through immersing the
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freshly
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characterization of sensor probe and experimental parameters affecting its activity were
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investigated employing UV-visible spectroscopy, transition electronic microscopy
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(TEM), scanning electron microscopy (SEM), electrochemical impedance spectroscopy
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(EIS), X-ray photoelectron spectroscopy (XPS), and cyclic voltammetry (CV). The
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AgNPs/Ox-pTTBA/MWCNT nanocomposite showed an excellent electrocatalytic
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activity to H2O2 by significantly increasing the reduction peak current and completely
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inhibiting the effect of other interfering species. The sensor probe displays a fast
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response time less than 5 s with a linear range from 10 – 260 µM and detection limit of
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0.24 µM. The sensitive, stable and specific response to H2O2 demonstrates that the
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present sensor is potentially suitable for monitoring H2O2 concentrations in biological
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system. The application was conducted for the determination of H2O2 in human urine
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real samples.
Ox-pTTBA/MWCNT
electrode
in
AgNPs
solution.
The
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Keywords: Nonenzymatic sensor, Hydrogen peroxide, Silver nanoparticles, Carbon
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nanotubes, Conducting polymer, Nanocomposite.
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Introduction
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Hydrogen peroxide (H2O2) is an essential intermediate that plays an important role in
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both environmental and biological systems [1]. Numerous analytical techniques, such as
4
spectrophotometry [2], fluorescence [3], and chemiluminescence [4] have been
5
employed for the determination of H2O2. Because of the redox behavior of H2O2, the
6
electrochemical techniques have received a significant interest in the determination of
7
H2O2 over other methods due to its simplicity, selectivity, and high sensitivity.
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Generally, H2O2 reduced to water via two-electron transfer process in an aqueous
9
solution. However, the direct electrochemical reduction of H2O2 with the conventional
10
electrodes is not effective for analytical application due to slow electrode kinetics.
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Therefore, modification of the electrode surface is of practically important to enhance
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the rate of electron transfer and hence minimize the overpotential of redox reactions.
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Metals nanoparticles have received considerable interest due to their conducting nature
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and biocompatibility that makes these materials excellent to develop a wide variety of
15
sensors and biosensors. Electrochemical reduction of H2O2 have been studied using
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different metals and metal oxides nanoparticles, such as gold [5], platinum [6],
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palladium [7], copper oxide [8], ruthenium oxide [9], and iron oxide [10]. Among them,
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silver nanoparticles were also used to investigate the electrochemical behavior and
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kinetics of H2O2 reduction [11-13]. Although, some studies have shown success in this
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direction, there is still much effort to develop more sensitive and selective H2O2 sensor
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is needed for monitoring H2O2 release in biological systems.
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Conducting polymers have received considerable interest due to their conducting nature
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and high stability that makes them extremely attractive for immobilizing nanomaterials
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and biomolecules to develop a wide variety of sensors and biosensors [14-18]. Of these,
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poly-2,2′:5′,2′′-terthiophene-3-p-benzoic acid (pTTBA), has been recently employed for
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electrode modification and successfully showed a great ability for biosensors
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applications [19,20]. The purpose of using conducting polymer pTTBA herein, is to
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introduce negative charge in the polymer film via electrooxidation of pTTBA. The
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anionic polymeric film can offer exchange sites and serve as a charge selective
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compound, which can be used for strong interaction with Ag nanoparticles. On the other
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hand, carbon nanotube (CNT) has received much attention over the past decade as
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suitable materials for electrode modification and biosensor applications [21-23]. Due to
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their unique characteristics, such as large active surface area, high electrical
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conductivity, chemical stability and biocompatibility, CNT has been used to improve
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electrocatalytic performance [24-26]. The nanocomposites of CNT with conducting
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polymers have recently been employed to enhance the electrical conductivity of the
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polymer film by increasing the active surface area and hence facilitating the rate of
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electron transfer reactions between target analyte and electrode surface [14,16,23].
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In the present study, an electrochemical sensor for direct analytical detection of H2O2 is
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proposed. The sensor was successfully fabricated by immobilizing AgNPs on the
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oxidized pTTBA/MWCNT nanocomposite layer. The electrochemical oxidation of
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pTTBA into Ox-pTTBA play an important role in the present sensor fabrication. Since
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this process introduces active sites in the Ox-pTTBA surface through the formation of
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negatively charged carboxylate groups and conjugated π electron rich-polymer
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backbone after the oxidation of pTTBA. Thus, the Ox-pTTBA film becomes cationic
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permselective and more conductive which may therefore act as templates for strong
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adsorption of AgNPs. Additionally, MWCNT was used in the nanocomposite to increase
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sensor conductivity and then amplified electron transfer process on the electrode surface.
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The mechanism of nanocomposite sensor formation and experimental parameters
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affecting its electrochemical activity are investigated and discussed in details.
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2. Materials and Methods
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2.1. Materials
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H2O2, ascorbic acid (AA), dopamine (DA), uric acid (UA), glutamic acid (GA), glucose
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(Glu), acetaminophen (AP), AgNO3, sodium citrate and NaBH4 were purchased from
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Sigma and Aldrich (USA). Multi wall carbon nanotubes (MWCNT) was purchased
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from Iljin Nanotech (South Korea). Tetrabutylammonium perchlorate (TBAP) was
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received from Fluka (USA). A 2,2′:5′,2′′-terthiophene-3-p-benzoic acid monomer
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(TTBA) was recently synthesized as a reported method [27]. All other chemicals were
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of extra pure analytical grade and used without further purification. Nitrogen gas
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(99.99%) was used for maintain deoxygenating in the measurement cell solution before
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and during the experiments.
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2.2. Instruments
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Cyclic voltammograms (CV) and chronoamperograms were recorded using a
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Potentiostat/Galvanostat, Kosentech model PT-1 (Busan, S. Korea). A UV-visible
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spectrum was obtained using a UV-3101PC (Shimadzu). A JEOL JEM-2010 electron
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microscope (Jeol High-Tech. Co.) was used to obtain transition electronic microscopy
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(TEM) image. Scanning electron microscopy (SEM) images were obtained using a
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Cambridge Stereoscan 240 (KBSI at Busan). Electrochemical impedance spectroscopy
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(EIS) was recorded with the EG&G PAR 273A Potentiostat/Galvanostat and a lock-in
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amplifier (PAR EG&G, Model 5210) linked to a personal computer. The frequency was
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scanned from 100 kHz to 10 Hz at the open circuit voltage, acquiring five points per
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decade. The amplitude of sinusoidal voltage of 10 mV was used. XPS experiments were
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performed using a VG scientific ESCA lab 250 XPS spectrometer coupled with a
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monochromated Al Kα source having charge compensation. The AgNPs/Ox-
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pTTBA/MWCNT/GCE, Ox-pTTBA/MWCNT/GCE and bare GCE, with an electrode
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area of 0.07 cm2, were used as working electrodes. Reference and counter electrodes
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were Ag/AgCl and platinum wire, respectively.
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2.3. Preparation of colloidal AgNPs
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AgNPs were prepared by borohydride reduction of AgNO3 as reported previously [28].
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Briefly, a 10 mL of 0.25 mM AgNO3 was mixed with 10 mL of 0.25 mM trisodium
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citrate. Next, 0.6 mL of ice-cold 0.1 M NaBH4 solution was added to the mixture
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solution while stirring. Upon this addition, the solution turned to yellow, indicating the
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formation of AgNPs. An adsorption band in the UV-visible spectrum of AgNPs solution
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was observed at 389 nm (Fig. 1(i)) and TEM images confirmed that the nanoparticles
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size in the solution was about 3.5 nm (Fig. 1(ii)).
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2.4. Functionalization of MWCNT
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The MWCNT was functionalized by acid treatment according to the previous procedure
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[14]. Firstly, 50 mL of a mixture solution of concentrated HNO3 and H2SO4 (1:3)
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containing 50 mg of MWCNT was sonicated for about 8 h. Thereafter, the mixture was
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washed with distilled water and filtrated for several times using the filter membrane (0.2
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μm) until the pH of the filtrates became neutral. Finally, the carboxylated MWCNT was
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dried in the oven at 80 °C for about 12 h and stored at room temperature until use.
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2.5. Sensor fabrication
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For the preparation of AgNPs/Ox-PTTBA/MWCNT sensor, a 1.0 mM TTBA monomer
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was mixed with 1.0 mg/ml carboxylated MWCNT in a 0.1 M TBAP/CH2Cl2 solution
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and sonicated for 30 min. The Ox-pTTBA/MWCNT nanocomposite film were formed
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on the electrode surface by cycling the potential from 0.0 to +1.6 V in the above
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mixture solution for five cycles at the scan rate of 0.1 V/s [14]. After that, an
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electrochemical oxidation of pTTBA/MWCNT film to produce Ox-pTTBA/MWCNT
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was occurred in PBS pH 7.0 from 0.0 to +1.8 V for three cycles at 0.1 V/s. This process
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introduces active sites on the Ox-pTTBA/MWCNT film, and may therefore act as
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templates for strong adsorption of AgNPs from the solution. This is due to the cationic
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selectivity of the negatively charged carboxylate groups of the Ox-pTTBA/MWCNT
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nanocomposite [14,29]. In order to incorporate AgNPs onto the Ox-pTTBA/MWCNT
10
film, the freshly prepared Ox-pTTBA/MWCNT electrode was immersed at open circuit
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in a well stirred colloidal AgNPs solution for 2 h. Then, the sensor was rinsed
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thoroughly with distilled water and stored until use. Scheme 1 shows the schematic of
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the AgNPs/Ox-pTTBA/MWCNT nanocomposite sensor fabrication.
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3. Results and discussion
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3.1. Characterization of AgNPs/Ox-pTTBA/MWCNT nanocomposite
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Fig. 1 shows the SEM images of stepwise of the sensor fabrication: (iii) GC, (iv) Ox-
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pTTBA/MWCNT/GC,
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morphology of the Ox-pTTBA/MWCNT layer shows a homogeneous nanocomposite
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film. This might be due to the incorporation of MWCNT into pTTBA during the
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electropolymerization process [14]. The diameter of MWCNT in the nanocomposite
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film was determined to be about 10 nm. In addition, the SEM image of the AgNPs/Ox-
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pTTBA/MWCNT surface shows the formation of AgNPs on the Ox-pTTBA/MWCNT
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film, indicating successful fabrication of the nanocomposite sensor probe.
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(v)
AgNPs/Ox-pTTBA/MWCNT/GC.
As
shown,
the
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EIS was carried out to study the conductivity of the electrode surfaces after
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modification. Fig. 1vi shows the Nyquist plots recorded for bare and AgNPs/Ox-
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pTTBA/MWCNT electrodes in a 5.0 mM [Fe(CN)6]3-/4- solution. A Randle circuit was
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employed to analyze the obtained impedance results (Inset of Fig. 1vi). Where, Rs is the
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solution resistance, Rp1, Rp2 are the polarization resistances, W is the Warburg element,
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and CPE1, CPE2 are the constant phase elements. The parameter values were obtained
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by fitting the results to the equivalent circuit using Zview 2 impedance software. A plot
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of bare electrode shows the charge transfer resistance in Rp1 and Rp2 to be 1020 and
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22920 Ω, respectively. While, these values decreased with the AgNPs/Ox-
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pTTBA/MWCNT electrode to 450 and 10060 Ω for Rp1 and Rp2, respectively. This
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result indicates that, the AgNPs/Ox-pTTBA/MWCNT nanocomposite layer improves
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the conductivity by facilitating the electron transfer process on the electrode surface.
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The characterization of AgNPs/Ox-pTTBA/MWCNT nanocomposite was further
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studied using XPS. All XPS spectra were taken after Ar ion gas etching for 50 s and
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corrected using a C1s peak at 284.6 eV as an internal standard. Figure 1vii shows the
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S2p spectra observed for (a) bare GC and (b) AgNPs/Ox-pTTBA/MWCNT/GC surfaces.
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As
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pTTBA/MWCNT/GC surface shows a peak at 163.5 eV, which due to the S−C bond in
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the polymer structure supporting the formation of nanocomposite layer on the GC
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surface. In addition, Fig. 1viii (b) shows the appearances of the two peaks of Ag 3d5/2
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and Ag3d3/2 at 368.8 and 374.7, respectively for the AgNPs/Ox-pTTBA/MWCNT/GC
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nanocomposite. While, these peaks was not observed with a bare GC as shown in Fig.
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1viii (a), indicating that the AgNPs have been successfully immobilized onto the Ox-
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pTTBA/MWCNT film.
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shown,
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3.2. Electrochemical characterization and performance
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The electrochemical properties of the Ox-pTTBA/MWCNT nanocomposite layer was
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investigated employing CV. Fig. 2A shows the CVs recorded for the Ox-
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pTTBA/MWCNT (solid line) and bare (dashed line) electrodes in 1.0 mM
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[Ru(NH3)6]Cl3 solution. As shown, the enhanced redox peaks appeared for the Ox-
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pTTBA/MWCNT nanocomposite as compared with the bare electrode. However, when
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the Ox-pTTBA/MWCNT electrode was exposed to a solution containing 1.0 mM
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K3[Fe(CN)6] (Fig. 2B), a well-defined redox peak of the [Fe(CN)6]4−/[Fe(CN)6]3−
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couple was observed with the bare electrode (dotted line), while no peak was observed
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for the CV recorded for the Ox-pTTBA/MWCNT (solid line). This result indicates the
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formation of a negatively charged carboxylate groups of the polymeric nanocomposite
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film which facilitates the electron transfer process of [Ru(NH3)6]3+ ions due to the
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electrostatic interaction. While, the electron transfer process of [Fe(CN)6]3− ions
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completely inhibited and hence the redox peak current was negligible [30,31]. In order
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to characterize the positively charged AgNPs, the CVs of the AgNPs/Ox-
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pTTBA/MWCNT electrode in [Ru(NH3)6]Cl3 and K3[Fe(CN)6] solutions were recorded
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in Fig 2A and 2B (dotted lines), respectively. As shown, well-defined redox peaks of
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both [Ru(NH3)6]4+/[Ru(NH3)6]3+ and [Fe(CN)6]4−/[Fe(CN)6]3− couples were observed.
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This clearly indicated that, the AgNPs interact with the Ox-pTTBA/MWCNT
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electrostatically and covered wholly and uniformly the electrode surface and hence the
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effect of the negatively charged of the Ox-pTTBA/MWCNT nanocomposite was
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completely prevented. In addition, to investigate the formation of AgNPs onto the Ox-
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pTTBA/MWCNT nanocomposite film, the CV of the AgNPs/Ox-pTTBA/MWCNT
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electrode in 0.1 M H2SO4 solution was recorded (Fig. 2C). A well-defined oxidation
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peak of the Ag/Ag+ at 0.45 V is obtained with the AgNPs/Ox-pTTBA/MWCNT (solid
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line), and no signal was observed for the Ox-pTTBA/MWCNT (dotted line). This result
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conformed the successful fabrication of nanostructured AgNPs on the Ox-
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pTTBA/MWCNT nanocomposite layer and hence it can be applied for further analytical
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detection of H2O2.
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3.3. Electrochemical detection of H2O2
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Metal nanoparticles-based sensor electrodes often gave the enhanced current response
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and higher sensitivity and selectivity for H2O2 detection [32,33]. Thus, the AgNPs/Ox-
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pTTBA/MWCNT electrode was employed for electrochemical detection of H2O2. Fig.
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3A shows the CVs of a (a) bare, (b) Ox-pTTBA/MWCNT and (c) AgNPs/Ox-
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pTTBA/MWCNT electrodes in PBS containing 100 μM H2O2. In comparison, a large
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enhanced current response can be seen with the AgNPs/Ox-pTTBA/MWCNT electrode,
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while no significant signals were observed with either bare nor Ox-pTTBA/MWCNT
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electrodes. This might be attributed to the hybrid nanomaterials of AgNPs/Ox-
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pTTBA/MWCNT nanocomposite integrating the advantage properties of nanometal
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AgNPs and may therefore significanetly enlarged the catalytic peak current of H2O2
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reduction (Scheme 1).
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In order to investigate the suitability of this method for the determination of H2O2, the
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catalytic activities of the AgNPs/Ox-pTTBA/MWCNT electrode towards H2O2 were
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examined at various concentrations (Fig. 3B). It was found that, the catalytic peak
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current gradually increased with the increasing concentration of H2O2 in the solution.
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This indicates that the proposed sensor is potentially suitable for the electrochemical
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detection of H2O2, which would probably behave well in the amperometric experiments.
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Furthermore, the scan rate dependency of the CV peaks of the sensor probe was also
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studied in PBS containing 100 μM H2O2 as shown in Fig. 3C. The cathodic peak current
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increased as the scan rate increased from 10 to 500 mV/s. Inset of Fig. 3C shows a
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linear relationship of cathodic peak currents versus the square root of scan rates,
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indicating the process is diffusion-controlled [34].
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3.4. Optimization of H2O2 detection
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The effect of pH on the response of the sensor probe to H2O2 was studied in the range
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from pH 5.0 to 9.0 (Fig. 4A). The results indicated that, the current increased with the
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increasing pH of the solution until it reached 7.0. However, further increase over pH 7,
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the current response decreased. The maximum peak current was obtained at pH 7.0,
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indicating the catalytic activity of the sensor probe was more effective at pH 7.0 and
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hence a higher peak current for H2O2 reduction was observed. Therefore, the optimum
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pH for H2O2 sensor was selected to be pH 7.0.
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The effect of adsorption time of AgNPs on the Ox-pTTBA/MWCNT nanocomposite
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was investigated (Fig. 4B). The catalytic peak current of H2O2 increased as the
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adsorption time increased from 0.5 to 2.0 h. Thereafter, the current response did not
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significantly increase when the adsorption time increased further up to 4.0 h. This might
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be attributed to that, AgNPs has been capped entirely the surface of Ox-
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pTTBA/MWCNT electrode after 2 h. Hence, 2.0 h was used as the optimum adsorption
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time of AgNPs.
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The effect of applied potential on the amperometric current response was also studied
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(Fig. 4C). The current response increased as the applied potential shifted from –0.1 V to
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a more negative value, where the maximum current response was observed at –0.6 V.
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Applying more negative potential up to –0.9 V, the current response did not
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significantly increase. Hence, –0.6 V was chosen as the optimum applied potential for
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H2O2 detection.
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3.5. Amperometric response, selectivity and stability of H2O2 sensor
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Fig. 5A shows the typical current-time response of the sensor probe upon successive
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addition of varying concentrations of H2O2. The sensor showed a fast amperometric
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response time less than 5 s after each addition of H2O2 with a linear relationship from
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10 – 260 µM. The linear regression equation was expressed as: Ip (μA) = 2.42 (±0.42) +
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0.58 (±0.01) [H2O2] (µM), with the correlation coefficient of 0.998. The relative
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standard deviation (RSD) was determined to be 4.8 % and the detection limit was
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estimated to be 0.24±0.04 µM. The lower detection limit was obtained as compared
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with other recently reported nonenzymatic H2O2 sensors based nanometals (Table 1),
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suggesting that the proposed sensor might be effective for the detection of H2O2 in
12
practical applications.
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To investigate the selectivity of the sensor probe, the amperometric responses to 5 µM
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H2O2 and 0.1 mM of different biological interfering species was recorded (Fig. 5B). As
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shown, these compounds did not interfere with H2O2 detection, indicating that the
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proposed sensor completely prevented the diffusion of interfering species. Moreover,
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the stability of the sensor towards H2O2 was examined with respect to the storage time.
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The sensor retained about 95% of its initial response to H2O2 over a period of one
19
month. The repeatability of the sensor-to-sensor variation has been studied for five
20
electrodes prepared under the same condition. The relative standard deviation (R.S.D.)
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of these electrodes was 3.8% for the current response to H2O2 indicating a good
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repeatability of the nanocomposite sensor. In addition, the stability of the sensor to
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multiple uses was also studied. The sensor lost only 2.5% from its initial response to
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H2O2 after 20 continuous measurements of H2O2 concentration. Although the
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AgNPs/Ox-pTTBA/MWCNT sensor probe is simple and easy to fabricate, it has also a
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long time stability without significant change of its response to H2O2, indicates that the
3
present method is an excellent for H2O2 detection.
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3.6. Real samples application
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To study the reliability of the AgNPs/Ox-pTTBA/MWCNT sensor for practical
6
applications, the determination of H2O2 in human urine real samples was investigated.
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The samples was collected and diluted 100 times with 0.1 M PBS (pH 7.0). Table 2
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shows the analytical values obtained for three urine samples. The recovery of spiked
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urine samples were in the range from 98.2 to 104.6%, which indicates the appreciable
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4. Conclusion
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We developed and characterized a novel sensor for analytical detection of H2O2 based
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on the incorporation of AgNPs onto the Ox-pTTBA/MWCNT nanocomposite film. The
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AgNPs/Ox-pTTBA/MWCNT probe exhibited high sensitivity and selectivity to H2O2
16
by significantly enhancing the reduction peak current and the effect of other interfering
17
compounds was completely eliminated. The present sensor provides a novel route for
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synthesizing hybrid nanomaterials which integrating the advantages of nanometals
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AgNPs for highly sensitive H2O2 sensor. The stable Ox-pTTBA/MWCNT
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nanocomposite polymer was used as a matrix for strong capturing of AgNPs. The higher
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catalytic response of the sensor to H2O2 might be attributed to the close compaction
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between AgNPs and electrode for fast electron transfer. The advantages of the proposed
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sensor are less expensive, easy to fabricate, highly stable, very sensitive, specific
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response to H2O2, and more convenient as compared with other reports demonstrating
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that the present method is an ideally H2O2 sensor.
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Acknowledgement
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This study was financially supported by the NRF grant funded by the MEST, Korea
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(No. 20100029128).
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References [1] M.R. Guascito, E. Filippo, C. Malitesta,D. Manno, A. Serra, A. Turco, A new
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amperometric nanostructured sensor for the analytical determination of hydrogen
4
peroxi, Biosens. Bioelectron. 24 (2008) 1057–1063.
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[5] A. Liu, W. Dong, E. Liu, W. Tang, J. Zhu, J. Han, Non-enzymatic hydrogen
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tetrahedral amorphous carbon electrodes, Electrochim. Acta 55 (2010) 1971–1977.
[6] S. Hrapovic, Y. Liu, K.B. Male, J.H.T. Luong, Electrochemical biosensing platforms using platinum nanoparticles and carbon nanotubes, Anal. Chem. 76 (2004) 1083–1088.
[7] J. Huang, D. Wang, H. Hou, T. You, Electrospun palladium nanoparticle-loaded
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carbon nanofibers and their electrocatalytic activities towards hydrogen peroxide
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and NADH, Adv. Funct. Mater. 18 (2008) 441–448.
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2
electrode by a novel film plating/potential cycling method and its characterization,
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Sens. Actuators B: Chem. 141 (2009) 147–153.
ip t
1
[9] R.C.Peña, V.O.Silva, F.H. Quina, M. Bertotti, Hydrogen peroxide monitoring in
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Fenton reaction by using a ruthenium oxide hexacyanoferrate/multiwalled carbon
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[10] J. Wang, H. Gao, F. Sun, Q. Hao, C. Xu, Highly sensitive detection of hydrogen
8
peroxide based on nanoporous Fe2O3/CoO composites, Biosens. Bioelectron. 42
9
(2013) 550–555.
an
7
[11] X. He, C. Hu, H. Liu, G. Du, Y. Xi, Y. Jiang, Building Ag nanoparticle 3D catalyst
11
via Na2Ti3O7 nanowires for the detection of hydrogen peroxide, Sens. Actuators
12
B: Chem. 144 (2010) 289–294.
d
M
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[12] F.W. Campbell, S.R. Belding, R. Raron, L. Xiao, R.G. Compton, Hydrogen
14
peroxide electroreduction at a silver-nanoparticle array: Investigating nanoparticle
15
size and coverage effects, J. Phys. Chem. C 113 (2009) 9053–9062.
Ac ce p
te
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16
[13] L. Zhong, S. Gan, X. Fu, F. Li, D. Han, L. Guo, L. Niu, Electrochemically
17
controlled growth of silver nanocrystals on graphene thin film and applications for
18
efficient nonenzymatic H2O2 biosensor, Electrochim. Acta 89 (2013) 222–228.
19
[14] A.A. Abdelwahab, W.C.A. Koh, H.-B. Noh, Y.-B. Shim, A selective nitric oxide
20
nanocomposite biosensor based on direct electron transfer of microperoxidase:
21
Removal of interferences by co-immobilized enzymes, Biosens. Bioelectron. 26
22
(2010) 1080–1086.
17
Page 17 of 31
[15] A.A. Abdelwahab, M.-S. Won, Y.-B. Shim, Direct electrochemistry of cholesterol
2
oxidase immobilized on a conducting polymer: Application for a cholesterol
3
biosensor, Electroanalysis 22 (2010) 21–25. [16] Y. Zhu, W.C.A. Koh, Y.-B. Shim, An amperometric immunosensor for IgG based
5
on
6
Electroanalysis 22 (2010) 2908–2914.
polymer
and
carbon
nanotube-linked
hydrazine
label,
us
conducting
cr
4
ip t
1
[17] T.-Y. Lee, Y.-B. Shim, Direct DNA hybridization detection based on the
8
oligonucleotide-functionalized conductive polymer, Anal. Chem. 73 (2001), 5629–
9
5632.
11
[18] M.A. Rahman, P. Kumar, D.S. Park, Y.-B. Shim, Electrochemical sensors based on organic conjugated polymers, Sensors, 8 (2008) 118–141.
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10
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[19] W. C. A. Koh, P. Chandra, D.-M. Kim, Y.-B. Shim, Electropolymerized self-
13
assembled layer on gold nanoparticles: Detection of inducible nitric oxide
14
synthase in neuronal cell culture, Anal. Chem., 83 (2011) 6177–6183.
16 17
te
[20] P. Chandra, H.-B. Noh, M.-S. Won, Y.-B. Shim, Detection of daunomycin using
Ac ce p
15
d
12
phosphatidylserine and aptamer co-immobilized on Au nanoparticles deposited conducting polymer, Biosens. Bioelectron., 26 (2011) 4442–4429.
18
[21] J. Wang, M. Musameh, Y. Lin, Solubilization of carbon nanotubes by nafion
19
toward the preparation of amperometric biosensors, J. Am. Chem. Soc. 125 (2003)
20
2408–2409.
21
[22] Y. Xu, P.E. Pehrsson, L. Chen, R. Zhang, W. Zhao, Double stranded DNA-single
22
walled carbon nanotube hybrids for optical hydrogen peroxide and glucose
23
sensing, Phys. Chem. C 111 (2007) 8638–8643.
18
Page 18 of 31
[23] Y. Zhu, J. I. Son, Y.-B. Shim, Amplification strategy based on gold nanoparticle-
2
decorated carbon nanotubes for neomycin immunosensors, Biosens. Bioelectron,
3
26 (2010) 1002–1008.
5
[24] B. Wu, Y. Kuang, X. Zhang, J. Chen, Noble metal nanoparticles/carbon nanotubes nanohybrids: Synthesis and applications, Nano Today 6 (2011) 75–90.
cr
4
ip t
1
[25] J.-J. Zhang, M.-M. Gu, T.-T. Zheng, J.-J. Zhu, Synthesis of gelatin-stabilized gold
7
nanoparticles and assembly of carboxylic single-walled carbon nanotubes/Au
8
composites for cytosensing and drug uptake, Anal. Chem. 81 (2009) 6641–6648.
an
us
6
[26] B.-S. Kong, D.-H. Jung, S.-K. Oh, C.-S. Han, H.-T. Jung, Single-walled carbon
10
nanotube gold nanohybrids: application in highly effective transparent and
11
conductive films, Phys. Chem. C 111 (2007) 8377–8382.
M
9
[27] D.-M. Kim, J.-H. Yoon, M.-S. Won, Y.-B. Shim, Electrochemical characterization
13
of newly synthesized polyterthiophene benzoate and its applications to an
14
electrochromic device and a photovoltaic cell, Electrochim. Acta 67 (2012)
15
201–207.
17
te
Ac ce p
16
d
12
[28] R.C. Doty, T.R. Tshikhudo, M. Brust, D.G. Fernig, Extremely stable water-soluble Ag nanoparticles, Chem. Mater. 17 (2005) 4630–4635.
18
[29] A.A. Abdelwahab, O.-S. Jung, Y.-B. Shim, Enhanced electrocatalytic reduction of
19
oxygen with a molecule having multi-quinone moieties adsorbed in the nanofiber
20
film, J. Electroanal. Chem. 632 (2009) 102–108.
21
[30] A.A. Abdelwahab, H.-M. Lee, Y.-B. Shim, Selective determination of dopamine
22
with a cibacron blue/poly-1,5-diaminonaphthalene composite film, Anal. Chim.
23
Acta 650 (2009) 247–253.
24
[31] A.A. Abdelwahab, D.-M. Kim, N.M. Halappa, Y.-B. Shim, A selective catalytic
19
Page 19 of 31
1
oxidation of ascorbic acid at the aminopyrimidyl functionalized-conductive
2
polymer electrode, Electroanalysis 25 (2013) 1178–1184.
4
platinum-coated
5
Bioelectron. 41 (2013) 576–581.
gold
nanoparticles
with
core@shell
ip t
[32] Y. Li, Q. Lu, S. Wu, L. Wang, X. Shi, Hydrogen peroxide sensing using ultrathin structure,
Biosens.
cr
3
[33] J.M. You, Y.N. Jeong, S.K. Kim, H.C. Choi, S. Jeon, Reductive determination of
7
hydrogen peroxide with MWCNTs-Pd nanoparticles on a modified glassy carbon
8
electrode, Biosens Bioelectron. 26 (2011) 2287-2291.
an
10
[34] R.L. McCreery, in: A.J. Bard (Ed), Electroanalytical Chemistry, vol. 17, Marcel Dekker, New York, 1991.
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Page 20 of 31
Biographies
2
Adel A. Abdelwahab received his Ph.D. in analytical chemistry from Department of
3
Chemistry at Pusan National University, South Korea, in 2010. He is working as a
4
lecturer at Department of Chemistry, Al-Azhar University. His current research interests
5
are the development of electrochemical sensors and biosensors, modified electrodes,
6
study of electron transfer reaction of enzymes and proteins, and characterization of
7
conducting polymers and their applications.
8
Yoon-Bo Shim received his Ph.D. in Department of Chemistry at Pusan National
9
University, South Korea, in 1985. He is working as a professor at Department of
10
Chemistry and a director at Institute of BioPhysio Sensor Technology (IBST), Pusan
11
National University. His current research interests are the development of bio-(protein,
12
DNA, enzyme, etc.)/chemical-sensors, electroanalytical method of trace biological,
13
organic species with modified electrodes, electron transfer of organic compounds and
14
proteins on the biomembranes, and characterization of conducting polymers and their
15
applications.
17 18 19 20
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Page 21 of 31
Figure Captions
2
Scheme 1. Schematic representation of the fabrication and overall detection of H2O2 by
3
AgNPs/Ox-pTTBA/MWCNT nanocomposite.
4
Fig. 1. (i) UV-visible spectra and (ii) TEM image of AgNPs solution. SEM images of
5
(iii) bare GC, (iv) Ox-pTTBA/MWCNT/GC, and (v) AgNPs/Ox-pTTBA/MWCNT/GC.
6
(vi) Nyquist plots of bare GC (blue dotted line) and AgNPs/Ox-pTTBA/MWCNT/GC
7
(red dotted line) in a 5.0 mM [Fe(CN)6]3-/4- solution. XPS spectra of (vii) S2p peak and
8
(viii) Ag3d peaks for (a) bare GC and (b) AgNPs/Ox-pTTBA/MWCNT/GC surfaces.
9
Fig. 2. CVs recorded for the AgNPs/Ox-pTTBA/MWCNT (dotted lines) Ox-
10
pTTBA/MWCNT (solid lines) and bare (dashed lines) electrodes in (A) 1.0 mM
11
[Ru(NH3)6]Cl3 and (B) 1.0 mM K3[Fe(CN)6] solutions. (C) CVs of the AgNPs/Ox-
12
pTTBA/MWCNT (solid line) and Ox-pTTBA/MWCNT (dashed line) electrodes in 0.1
13
M H2SO4.
14
Fig. 3. (A) CVs recorded for (a) bare, (b) Ox-pTTBA/MWCNT and (c) AgNPs/Ox-
15
pTTBA/MWCNT electrodes in PBS containing 100 μM H2O2. (B) CVs recorded for
16
AgNPs/Ox-pTTBA/MWCNT electrode in PBS contains various concentrations of H2O2
17
(a) 0 (b) 50, (c) 100 and (d) 150 μM. (C) CVs recorded for AgNPs/Ox-TTBA/MWCNT
18
electrode in PBS contains 100 μM H2O2 at various scan rates. Inset shows a plot of
19
cathodic peak currents vs. the square root of scan rates.
20
Fig. 4. Optimization of H2O2 detection using the AgNPs/Ox-pTTBA/MWCNT probe:
21
(A) pH and (B) adsorption time of AgNPs, (C) applied potential.
22
Fig. 5. Amperometric response obtained using the sensor probe in PBS after multiple
23
additions of H2O2. Inset shows the corresponding calibration plot. (B) Amperogram of
24
the addition of 5 μM H2O2 and 0.1 mM of other interfering species.
Ac ce p
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Page 22 of 31
1
Table 1. Comparison of recently reported nonenzymatic H2O2 sensors based nanometals
2
composite Linear range (µM)
Detection limit (µM) 4.7
Fe2O3/CoO
50 – 485
0.1
Ag nanocrystals/graphene
20 – 1000
PdNPs/MWCNTs
1 – 1000
AgNPs/Ox-pTTBA/MWCNT
10 – 260
us
1 – 450
3
an
Pt/AuNPs
3
[10] [13]
1.18
[32]
0.3
[33]
0.24
[this work]
M
4
[9]
cr
100 – 1000
RuOHCF/MWCNT
References
ip t
Nanocomposite
Ac ce p
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Page 23 of 31
1 2 3
Table 2 Determination of H2O2 in human urine samples at the AgNPs/OxpTTBA/MWCNT nanocomposite (n = 5)
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Sample
Spiked (μM)
Found (μM)
RSD
1
10
10.46
4.1 %
2
10
10.25
3.8 %
102.5
3
10
9.82
3.2 %
98.2
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cr
104.6
an
5
Recovery
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Page 24 of 31
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cr
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Scheme 1
S
+ MWCNT
S S
TTBA
COOH
COOH
H2 O
COO
COO
COO
Ag
Ag
COO
COO
Ag
AgNPs GCE
GCE
OpTTBA-MWCNT
AgNPs/OpTTBA-MWCNT
Ac
pTTBA-MWCNT
pH 7.0
Ag
COO
COO
Oxidation
GCE
H2 O2
Ag
COO
ce pt
COOH COOH
ed
Electropolymerization
M an
COOH
Scheme 1. Page 25 of 31
ip t
R-Figure 1
30000
(ii)
(i)
1.5
cr
max = 389 nm
CPE2
(vi)
W
Rp1
Rp2W
0.5
400 500 600 700 Wavelength/nm
800
22 00 nn m m
Bare AgNPs/Ox-pTTBA/MWCNT
0
(iv)
20000
Figure 1.
160 165 170 Binding Energy/eV
60000
Ag3d5/2
S2p
Intensity/a.u.
Ag3d3/2
(vii)
(viii)
(b) (a)
(b) (a)
155
40000 Zre/
(v)
ce pt Ac
Intensity/a.u.
10000
0
ed
(iii)
Zim/
us
1.0
0.0 300
CPE1
20000
M an
Absorbance
2.0
Rs
175
365
370 375 Binding Energy/eV
380 Page 26 of 31
20
(A)
15
(B)
cr
30
ip t
R-Figure 2
20
us
0
-10
-400
-200 E/mV
0
ed
-20 -600
200
I/A
10
M an
I/A
10 5 0
-5 -10 -15 -400
-200
0
200 400 E/mV
600
800
ce pt
20
(C)
10
I/A
Ac
0
-10 -20 -30 -40 -100
Figure 2.
100
300
500 E/mV
700
900
1100 Page 27 of 31
80
150
(A) c
100 d
us I/A
20 a
-20 -900
-700
ed
0
-500 -300 E/mV
-100
ce pt
a
0
100
-900
250 (C)
-700
-500 -300 E/mV
-100
100
250 200
I/A
200
I/A
c
50 b
M an
b
Ac
I/A
60 40
(B)
cr
100
ip t
Figure 3
150
150 100 50
100
0
5
10
15
20
25
1/2/(mV/s)1/2
50 0
Figure 3.
-900
-700
-500 -300 E/mV
-100
100 Page 28 of 31
120
110
(B)
cr
(A) 100
us
100
I/A
90
M an
I/A
ip t
R-Figure 4
80 70 60 6
7 pH
8
9
ed
5
60 40
10
0
1
2 3 Time/min
4
5
ce pt
4
80
50
(C)
I/A
Ac
40 30 20
Figure 4.
10 0.0
-0.2
-0.4 -0.6 E/V
-0.8
-1.0 Page 29 of 31
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Figure 5
200
cr
(A)
us 200
100
150
I/A
M an
I/A
150
0
200
ce pt
0
ed
50
100 50
400 Time/s
0 0
100 200 H2O2/M
600
300
800
15
AP
Glu
GA
UA
DA
H2O2
I/A
Ac
10
AA
(B)
H2O2
5
0
Figure 5.
0
100
200 300 Time/s
400
500 Page 30 of 31
M an
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cr
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Graphical Abstract (for review)
100
H2 O
H2 O2
Ag
Ag
COO
COO
COO
Detection
GCE
Ac
AgNPs/OpTTBA-MWCNT
H2 O2
I/A
COO
60
ed
Ag
80
ce pt
Ag
Bare GC OpTTBA-MWCNT AgNPs/OpTTBA-MWCNT
40 20 0 -20 -900
-700
-500
-300 E/mV
-100
100
Graphic abstract Page 31 of 31
S0925-4005(14)00523-1 http://dx.doi.org/doi:10.1016/j.snb.2014.05.004 SNB 16878
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-3-2014 22-4-2014 1-5-2014
Please cite this article as: A.A. Abdelwahab, Y.-B. Shim, Nonenzymatic H2 O2 sensing based on silver nanoparticles capped polyterthiophene/MWCNT nanocomposite, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights
2
► AgNPs modified Ox-pTTBA/MWCNT nanocomposite film for highly sensitive and selective H2O2
3
sensor. ► Fast amperometric response with lowest detection limit was obtained for H2O2. ► The
4
reliability of the proposed sensor was evaluated using real samples analysis.
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Page 1 of 31
Nonenzymatic H2O2 sensing based on silver nanoparticles
2
capped polyterthiophene/MWCNT nanocomposite
3
cr
Adel A. Abdelwahab a*, Yoon-Bo Shim b**
4
7 8
Department of Chemistry, Faculty of Science, Al-Azhar University, Assiut 71524, Egypt b
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6
a
Department of Chemistry and Institute of BioPhysio Sensor Technology, Pusan National University, Busan 609-735, South Korea
an
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* Corresponding author. Tel.: (+20) 88-231-2193; Fax: (+20) 88-232-5436.
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E-mail address: [email protected] (A.A. Abdelwahab).
20
** Corresponding author. Tel.: (+82) 51-510-2244; Fax: (+82) 51-514-2430.
21
E-mail address: [email protected] (Y.-B. Shim).
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2
Page 2 of 31
Abstract
2
A novel method for highly sensitive H2O2 sensor is proposed using silver nanoparticles
3
(AgNPs) modified oxidized poly-2,2′:5′,2′′-terthiophene-3-p-benzoic acid/multi wall
4
carbon nanotube (Ox-pTTBA/MWCNT). The Ox-pTTBA/MWCNT nanocomposite
5
film was prepared via electropolymerization of a TTBA monomer and MWCNT
6
mixture solution, followed by in situ electrooxidation of the pTTBA/MWCNT film.
7
Then, AgNPs were formed on the Ox-pTTBA/MWCNT layer through immersing the
8
freshly
9
characterization of sensor probe and experimental parameters affecting its activity were
10
investigated employing UV-visible spectroscopy, transition electronic microscopy
11
(TEM), scanning electron microscopy (SEM), electrochemical impedance spectroscopy
12
(EIS), X-ray photoelectron spectroscopy (XPS), and cyclic voltammetry (CV). The
13
AgNPs/Ox-pTTBA/MWCNT nanocomposite showed an excellent electrocatalytic
14
activity to H2O2 by significantly increasing the reduction peak current and completely
15
inhibiting the effect of other interfering species. The sensor probe displays a fast
16
response time less than 5 s with a linear range from 10 – 260 µM and detection limit of
17
0.24 µM. The sensitive, stable and specific response to H2O2 demonstrates that the
18
present sensor is potentially suitable for monitoring H2O2 concentrations in biological
19
system. The application was conducted for the determination of H2O2 in human urine
20
real samples.
Ox-pTTBA/MWCNT
electrode
in
AgNPs
solution.
The
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Keywords: Nonenzymatic sensor, Hydrogen peroxide, Silver nanoparticles, Carbon
23
nanotubes, Conducting polymer, Nanocomposite.
24
3
Page 3 of 31
Introduction
2
Hydrogen peroxide (H2O2) is an essential intermediate that plays an important role in
3
both environmental and biological systems [1]. Numerous analytical techniques, such as
4
spectrophotometry [2], fluorescence [3], and chemiluminescence [4] have been
5
employed for the determination of H2O2. Because of the redox behavior of H2O2, the
6
electrochemical techniques have received a significant interest in the determination of
7
H2O2 over other methods due to its simplicity, selectivity, and high sensitivity.
8
Generally, H2O2 reduced to water via two-electron transfer process in an aqueous
9
solution. However, the direct electrochemical reduction of H2O2 with the conventional
10
electrodes is not effective for analytical application due to slow electrode kinetics.
11
Therefore, modification of the electrode surface is of practically important to enhance
12
the rate of electron transfer and hence minimize the overpotential of redox reactions.
13
Metals nanoparticles have received considerable interest due to their conducting nature
14
and biocompatibility that makes these materials excellent to develop a wide variety of
15
sensors and biosensors. Electrochemical reduction of H2O2 have been studied using
16
different metals and metal oxides nanoparticles, such as gold [5], platinum [6],
17
palladium [7], copper oxide [8], ruthenium oxide [9], and iron oxide [10]. Among them,
18
silver nanoparticles were also used to investigate the electrochemical behavior and
19
kinetics of H2O2 reduction [11-13]. Although, some studies have shown success in this
20
direction, there is still much effort to develop more sensitive and selective H2O2 sensor
21
is needed for monitoring H2O2 release in biological systems.
22
Conducting polymers have received considerable interest due to their conducting nature
23
and high stability that makes them extremely attractive for immobilizing nanomaterials
24
and biomolecules to develop a wide variety of sensors and biosensors [14-18]. Of these,
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Page 4 of 31
poly-2,2′:5′,2′′-terthiophene-3-p-benzoic acid (pTTBA), has been recently employed for
2
electrode modification and successfully showed a great ability for biosensors
3
applications [19,20]. The purpose of using conducting polymer pTTBA herein, is to
4
introduce negative charge in the polymer film via electrooxidation of pTTBA. The
5
anionic polymeric film can offer exchange sites and serve as a charge selective
6
compound, which can be used for strong interaction with Ag nanoparticles. On the other
7
hand, carbon nanotube (CNT) has received much attention over the past decade as
8
suitable materials for electrode modification and biosensor applications [21-23]. Due to
9
their unique characteristics, such as large active surface area, high electrical
10
conductivity, chemical stability and biocompatibility, CNT has been used to improve
11
electrocatalytic performance [24-26]. The nanocomposites of CNT with conducting
12
polymers have recently been employed to enhance the electrical conductivity of the
13
polymer film by increasing the active surface area and hence facilitating the rate of
14
electron transfer reactions between target analyte and electrode surface [14,16,23].
15
In the present study, an electrochemical sensor for direct analytical detection of H2O2 is
16
proposed. The sensor was successfully fabricated by immobilizing AgNPs on the
17
oxidized pTTBA/MWCNT nanocomposite layer. The electrochemical oxidation of
18
pTTBA into Ox-pTTBA play an important role in the present sensor fabrication. Since
19
this process introduces active sites in the Ox-pTTBA surface through the formation of
20
negatively charged carboxylate groups and conjugated π electron rich-polymer
21
backbone after the oxidation of pTTBA. Thus, the Ox-pTTBA film becomes cationic
22
permselective and more conductive which may therefore act as templates for strong
23
adsorption of AgNPs. Additionally, MWCNT was used in the nanocomposite to increase
24
sensor conductivity and then amplified electron transfer process on the electrode surface.
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Page 5 of 31
1
The mechanism of nanocomposite sensor formation and experimental parameters
2
affecting its electrochemical activity are investigated and discussed in details.
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2. Materials and Methods
5
2.1. Materials
6
H2O2, ascorbic acid (AA), dopamine (DA), uric acid (UA), glutamic acid (GA), glucose
7
(Glu), acetaminophen (AP), AgNO3, sodium citrate and NaBH4 were purchased from
8
Sigma and Aldrich (USA). Multi wall carbon nanotubes (MWCNT) was purchased
9
from Iljin Nanotech (South Korea). Tetrabutylammonium perchlorate (TBAP) was
10
received from Fluka (USA). A 2,2′:5′,2′′-terthiophene-3-p-benzoic acid monomer
11
(TTBA) was recently synthesized as a reported method [27]. All other chemicals were
12
of extra pure analytical grade and used without further purification. Nitrogen gas
13
(99.99%) was used for maintain deoxygenating in the measurement cell solution before
14
and during the experiments.
15
2.2. Instruments
16
Cyclic voltammograms (CV) and chronoamperograms were recorded using a
17
Potentiostat/Galvanostat, Kosentech model PT-1 (Busan, S. Korea). A UV-visible
18
spectrum was obtained using a UV-3101PC (Shimadzu). A JEOL JEM-2010 electron
19
microscope (Jeol High-Tech. Co.) was used to obtain transition electronic microscopy
20
(TEM) image. Scanning electron microscopy (SEM) images were obtained using a
21
Cambridge Stereoscan 240 (KBSI at Busan). Electrochemical impedance spectroscopy
22
(EIS) was recorded with the EG&G PAR 273A Potentiostat/Galvanostat and a lock-in
23
amplifier (PAR EG&G, Model 5210) linked to a personal computer. The frequency was
24
scanned from 100 kHz to 10 Hz at the open circuit voltage, acquiring five points per
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decade. The amplitude of sinusoidal voltage of 10 mV was used. XPS experiments were
2
performed using a VG scientific ESCA lab 250 XPS spectrometer coupled with a
3
monochromated Al Kα source having charge compensation. The AgNPs/Ox-
4
pTTBA/MWCNT/GCE, Ox-pTTBA/MWCNT/GCE and bare GCE, with an electrode
5
area of 0.07 cm2, were used as working electrodes. Reference and counter electrodes
6
were Ag/AgCl and platinum wire, respectively.
7
2.3. Preparation of colloidal AgNPs
8
AgNPs were prepared by borohydride reduction of AgNO3 as reported previously [28].
9
Briefly, a 10 mL of 0.25 mM AgNO3 was mixed with 10 mL of 0.25 mM trisodium
10
citrate. Next, 0.6 mL of ice-cold 0.1 M NaBH4 solution was added to the mixture
11
solution while stirring. Upon this addition, the solution turned to yellow, indicating the
12
formation of AgNPs. An adsorption band in the UV-visible spectrum of AgNPs solution
13
was observed at 389 nm (Fig. 1(i)) and TEM images confirmed that the nanoparticles
14
size in the solution was about 3.5 nm (Fig. 1(ii)).
15
2.4. Functionalization of MWCNT
16
The MWCNT was functionalized by acid treatment according to the previous procedure
17
[14]. Firstly, 50 mL of a mixture solution of concentrated HNO3 and H2SO4 (1:3)
18
containing 50 mg of MWCNT was sonicated for about 8 h. Thereafter, the mixture was
19
washed with distilled water and filtrated for several times using the filter membrane (0.2
20
μm) until the pH of the filtrates became neutral. Finally, the carboxylated MWCNT was
21
dried in the oven at 80 °C for about 12 h and stored at room temperature until use.
22
2.5. Sensor fabrication
23
For the preparation of AgNPs/Ox-PTTBA/MWCNT sensor, a 1.0 mM TTBA monomer
24
was mixed with 1.0 mg/ml carboxylated MWCNT in a 0.1 M TBAP/CH2Cl2 solution
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Page 7 of 31
and sonicated for 30 min. The Ox-pTTBA/MWCNT nanocomposite film were formed
2
on the electrode surface by cycling the potential from 0.0 to +1.6 V in the above
3
mixture solution for five cycles at the scan rate of 0.1 V/s [14]. After that, an
4
electrochemical oxidation of pTTBA/MWCNT film to produce Ox-pTTBA/MWCNT
5
was occurred in PBS pH 7.0 from 0.0 to +1.8 V for three cycles at 0.1 V/s. This process
6
introduces active sites on the Ox-pTTBA/MWCNT film, and may therefore act as
7
templates for strong adsorption of AgNPs from the solution. This is due to the cationic
8
selectivity of the negatively charged carboxylate groups of the Ox-pTTBA/MWCNT
9
nanocomposite [14,29]. In order to incorporate AgNPs onto the Ox-pTTBA/MWCNT
10
film, the freshly prepared Ox-pTTBA/MWCNT electrode was immersed at open circuit
11
in a well stirred colloidal AgNPs solution for 2 h. Then, the sensor was rinsed
12
thoroughly with distilled water and stored until use. Scheme 1 shows the schematic of
13
the AgNPs/Ox-pTTBA/MWCNT nanocomposite sensor fabrication.
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3. Results and discussion
16
3.1. Characterization of AgNPs/Ox-pTTBA/MWCNT nanocomposite
17
Fig. 1 shows the SEM images of stepwise of the sensor fabrication: (iii) GC, (iv) Ox-
18
pTTBA/MWCNT/GC,
19
morphology of the Ox-pTTBA/MWCNT layer shows a homogeneous nanocomposite
20
film. This might be due to the incorporation of MWCNT into pTTBA during the
21
electropolymerization process [14]. The diameter of MWCNT in the nanocomposite
22
film was determined to be about 10 nm. In addition, the SEM image of the AgNPs/Ox-
23
pTTBA/MWCNT surface shows the formation of AgNPs on the Ox-pTTBA/MWCNT
24
film, indicating successful fabrication of the nanocomposite sensor probe.
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(v)
AgNPs/Ox-pTTBA/MWCNT/GC.
As
shown,
the
8
Page 8 of 31
EIS was carried out to study the conductivity of the electrode surfaces after
2
modification. Fig. 1vi shows the Nyquist plots recorded for bare and AgNPs/Ox-
3
pTTBA/MWCNT electrodes in a 5.0 mM [Fe(CN)6]3-/4- solution. A Randle circuit was
4
employed to analyze the obtained impedance results (Inset of Fig. 1vi). Where, Rs is the
5
solution resistance, Rp1, Rp2 are the polarization resistances, W is the Warburg element,
6
and CPE1, CPE2 are the constant phase elements. The parameter values were obtained
7
by fitting the results to the equivalent circuit using Zview 2 impedance software. A plot
8
of bare electrode shows the charge transfer resistance in Rp1 and Rp2 to be 1020 and
9
22920 Ω, respectively. While, these values decreased with the AgNPs/Ox-
10
pTTBA/MWCNT electrode to 450 and 10060 Ω for Rp1 and Rp2, respectively. This
11
result indicates that, the AgNPs/Ox-pTTBA/MWCNT nanocomposite layer improves
12
the conductivity by facilitating the electron transfer process on the electrode surface.
13
The characterization of AgNPs/Ox-pTTBA/MWCNT nanocomposite was further
14
studied using XPS. All XPS spectra were taken after Ar ion gas etching for 50 s and
15
corrected using a C1s peak at 284.6 eV as an internal standard. Figure 1vii shows the
16
S2p spectra observed for (a) bare GC and (b) AgNPs/Ox-pTTBA/MWCNT/GC surfaces.
17
As
18
pTTBA/MWCNT/GC surface shows a peak at 163.5 eV, which due to the S−C bond in
19
the polymer structure supporting the formation of nanocomposite layer on the GC
20
surface. In addition, Fig. 1viii (b) shows the appearances of the two peaks of Ag 3d5/2
21
and Ag3d3/2 at 368.8 and 374.7, respectively for the AgNPs/Ox-pTTBA/MWCNT/GC
22
nanocomposite. While, these peaks was not observed with a bare GC as shown in Fig.
23
1viii (a), indicating that the AgNPs have been successfully immobilized onto the Ox-
24
pTTBA/MWCNT film.
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shown,
no
peak
appeared
for
a
bare
GC,
where
the
AgNPs/Ox-
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Page 9 of 31
3.2. Electrochemical characterization and performance
2
The electrochemical properties of the Ox-pTTBA/MWCNT nanocomposite layer was
3
investigated employing CV. Fig. 2A shows the CVs recorded for the Ox-
4
pTTBA/MWCNT (solid line) and bare (dashed line) electrodes in 1.0 mM
5
[Ru(NH3)6]Cl3 solution. As shown, the enhanced redox peaks appeared for the Ox-
6
pTTBA/MWCNT nanocomposite as compared with the bare electrode. However, when
7
the Ox-pTTBA/MWCNT electrode was exposed to a solution containing 1.0 mM
8
K3[Fe(CN)6] (Fig. 2B), a well-defined redox peak of the [Fe(CN)6]4−/[Fe(CN)6]3−
9
couple was observed with the bare electrode (dotted line), while no peak was observed
10
for the CV recorded for the Ox-pTTBA/MWCNT (solid line). This result indicates the
11
formation of a negatively charged carboxylate groups of the polymeric nanocomposite
12
film which facilitates the electron transfer process of [Ru(NH3)6]3+ ions due to the
13
electrostatic interaction. While, the electron transfer process of [Fe(CN)6]3− ions
14
completely inhibited and hence the redox peak current was negligible [30,31]. In order
15
to characterize the positively charged AgNPs, the CVs of the AgNPs/Ox-
16
pTTBA/MWCNT electrode in [Ru(NH3)6]Cl3 and K3[Fe(CN)6] solutions were recorded
17
in Fig 2A and 2B (dotted lines), respectively. As shown, well-defined redox peaks of
18
both [Ru(NH3)6]4+/[Ru(NH3)6]3+ and [Fe(CN)6]4−/[Fe(CN)6]3− couples were observed.
19
This clearly indicated that, the AgNPs interact with the Ox-pTTBA/MWCNT
20
electrostatically and covered wholly and uniformly the electrode surface and hence the
21
effect of the negatively charged of the Ox-pTTBA/MWCNT nanocomposite was
22
completely prevented. In addition, to investigate the formation of AgNPs onto the Ox-
23
pTTBA/MWCNT nanocomposite film, the CV of the AgNPs/Ox-pTTBA/MWCNT
24
electrode in 0.1 M H2SO4 solution was recorded (Fig. 2C). A well-defined oxidation
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Page 10 of 31
peak of the Ag/Ag+ at 0.45 V is obtained with the AgNPs/Ox-pTTBA/MWCNT (solid
2
line), and no signal was observed for the Ox-pTTBA/MWCNT (dotted line). This result
3
conformed the successful fabrication of nanostructured AgNPs on the Ox-
4
pTTBA/MWCNT nanocomposite layer and hence it can be applied for further analytical
5
detection of H2O2.
6
3.3. Electrochemical detection of H2O2
7
Metal nanoparticles-based sensor electrodes often gave the enhanced current response
8
and higher sensitivity and selectivity for H2O2 detection [32,33]. Thus, the AgNPs/Ox-
9
pTTBA/MWCNT electrode was employed for electrochemical detection of H2O2. Fig.
10
3A shows the CVs of a (a) bare, (b) Ox-pTTBA/MWCNT and (c) AgNPs/Ox-
11
pTTBA/MWCNT electrodes in PBS containing 100 μM H2O2. In comparison, a large
12
enhanced current response can be seen with the AgNPs/Ox-pTTBA/MWCNT electrode,
13
while no significant signals were observed with either bare nor Ox-pTTBA/MWCNT
14
electrodes. This might be attributed to the hybrid nanomaterials of AgNPs/Ox-
15
pTTBA/MWCNT nanocomposite integrating the advantage properties of nanometal
16
AgNPs and may therefore significanetly enlarged the catalytic peak current of H2O2
17
reduction (Scheme 1).
18
In order to investigate the suitability of this method for the determination of H2O2, the
19
catalytic activities of the AgNPs/Ox-pTTBA/MWCNT electrode towards H2O2 were
20
examined at various concentrations (Fig. 3B). It was found that, the catalytic peak
21
current gradually increased with the increasing concentration of H2O2 in the solution.
22
This indicates that the proposed sensor is potentially suitable for the electrochemical
23
detection of H2O2, which would probably behave well in the amperometric experiments.
24
Furthermore, the scan rate dependency of the CV peaks of the sensor probe was also
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Page 11 of 31
studied in PBS containing 100 μM H2O2 as shown in Fig. 3C. The cathodic peak current
2
increased as the scan rate increased from 10 to 500 mV/s. Inset of Fig. 3C shows a
3
linear relationship of cathodic peak currents versus the square root of scan rates,
4
indicating the process is diffusion-controlled [34].
5
3.4. Optimization of H2O2 detection
6
The effect of pH on the response of the sensor probe to H2O2 was studied in the range
7
from pH 5.0 to 9.0 (Fig. 4A). The results indicated that, the current increased with the
8
increasing pH of the solution until it reached 7.0. However, further increase over pH 7,
9
the current response decreased. The maximum peak current was obtained at pH 7.0,
10
indicating the catalytic activity of the sensor probe was more effective at pH 7.0 and
11
hence a higher peak current for H2O2 reduction was observed. Therefore, the optimum
12
pH for H2O2 sensor was selected to be pH 7.0.
13
The effect of adsorption time of AgNPs on the Ox-pTTBA/MWCNT nanocomposite
14
was investigated (Fig. 4B). The catalytic peak current of H2O2 increased as the
15
adsorption time increased from 0.5 to 2.0 h. Thereafter, the current response did not
16
significantly increase when the adsorption time increased further up to 4.0 h. This might
17
be attributed to that, AgNPs has been capped entirely the surface of Ox-
18
pTTBA/MWCNT electrode after 2 h. Hence, 2.0 h was used as the optimum adsorption
19
time of AgNPs.
20
The effect of applied potential on the amperometric current response was also studied
21
(Fig. 4C). The current response increased as the applied potential shifted from –0.1 V to
22
a more negative value, where the maximum current response was observed at –0.6 V.
23
Applying more negative potential up to –0.9 V, the current response did not
24
significantly increase. Hence, –0.6 V was chosen as the optimum applied potential for
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Page 12 of 31
H2O2 detection.
2
3.5. Amperometric response, selectivity and stability of H2O2 sensor
3
Fig. 5A shows the typical current-time response of the sensor probe upon successive
4
addition of varying concentrations of H2O2. The sensor showed a fast amperometric
5
response time less than 5 s after each addition of H2O2 with a linear relationship from
6
10 – 260 µM. The linear regression equation was expressed as: Ip (μA) = 2.42 (±0.42) +
7
0.58 (±0.01) [H2O2] (µM), with the correlation coefficient of 0.998. The relative
8
standard deviation (RSD) was determined to be 4.8 % and the detection limit was
9
estimated to be 0.24±0.04 µM. The lower detection limit was obtained as compared
10
with other recently reported nonenzymatic H2O2 sensors based nanometals (Table 1),
11
suggesting that the proposed sensor might be effective for the detection of H2O2 in
12
practical applications.
13
To investigate the selectivity of the sensor probe, the amperometric responses to 5 µM
14
H2O2 and 0.1 mM of different biological interfering species was recorded (Fig. 5B). As
15
shown, these compounds did not interfere with H2O2 detection, indicating that the
16
proposed sensor completely prevented the diffusion of interfering species. Moreover,
17
the stability of the sensor towards H2O2 was examined with respect to the storage time.
18
The sensor retained about 95% of its initial response to H2O2 over a period of one
19
month. The repeatability of the sensor-to-sensor variation has been studied for five
20
electrodes prepared under the same condition. The relative standard deviation (R.S.D.)
21
of these electrodes was 3.8% for the current response to H2O2 indicating a good
22
repeatability of the nanocomposite sensor. In addition, the stability of the sensor to
23
multiple uses was also studied. The sensor lost only 2.5% from its initial response to
24
H2O2 after 20 continuous measurements of H2O2 concentration. Although the
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Page 13 of 31
AgNPs/Ox-pTTBA/MWCNT sensor probe is simple and easy to fabricate, it has also a
2
long time stability without significant change of its response to H2O2, indicates that the
3
present method is an excellent for H2O2 detection.
4
3.6. Real samples application
5
To study the reliability of the AgNPs/Ox-pTTBA/MWCNT sensor for practical
6
applications, the determination of H2O2 in human urine real samples was investigated.
7
The samples was collected and diluted 100 times with 0.1 M PBS (pH 7.0). Table 2
8
shows the analytical values obtained for three urine samples. The recovery of spiked
9
urine samples were in the range from 98.2 to 104.6%, which indicates the appreciable
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4. Conclusion
13
We developed and characterized a novel sensor for analytical detection of H2O2 based
14
on the incorporation of AgNPs onto the Ox-pTTBA/MWCNT nanocomposite film. The
15
AgNPs/Ox-pTTBA/MWCNT probe exhibited high sensitivity and selectivity to H2O2
16
by significantly enhancing the reduction peak current and the effect of other interfering
17
compounds was completely eliminated. The present sensor provides a novel route for
18
synthesizing hybrid nanomaterials which integrating the advantages of nanometals
19
AgNPs for highly sensitive H2O2 sensor. The stable Ox-pTTBA/MWCNT
20
nanocomposite polymer was used as a matrix for strong capturing of AgNPs. The higher
21
catalytic response of the sensor to H2O2 might be attributed to the close compaction
22
between AgNPs and electrode for fast electron transfer. The advantages of the proposed
23
sensor are less expensive, easy to fabricate, highly stable, very sensitive, specific
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Page 14 of 31
1
response to H2O2, and more convenient as compared with other reports demonstrating
2
that the present method is an ideally H2O2 sensor.
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Acknowledgement
5
This study was financially supported by the NRF grant funded by the MEST, Korea
6
(No. 20100029128).
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1
oxidation of ascorbic acid at the aminopyrimidyl functionalized-conductive
2
polymer electrode, Electroanalysis 25 (2013) 1178–1184.
4
platinum-coated
5
Bioelectron. 41 (2013) 576–581.
gold
nanoparticles
with
core@shell
ip t
[32] Y. Li, Q. Lu, S. Wu, L. Wang, X. Shi, Hydrogen peroxide sensing using ultrathin structure,
Biosens.
cr
3
[33] J.M. You, Y.N. Jeong, S.K. Kim, H.C. Choi, S. Jeon, Reductive determination of
7
hydrogen peroxide with MWCNTs-Pd nanoparticles on a modified glassy carbon
8
electrode, Biosens Bioelectron. 26 (2011) 2287-2291.
an
10
[34] R.L. McCreery, in: A.J. Bard (Ed), Electroanalytical Chemistry, vol. 17, Marcel Dekker, New York, 1991.
M
9
us
6
11
15 16 17 18 19 20
te
14
Ac ce p
13
d
12
21 22 23 24
20
Page 20 of 31
Biographies
2
Adel A. Abdelwahab received his Ph.D. in analytical chemistry from Department of
3
Chemistry at Pusan National University, South Korea, in 2010. He is working as a
4
lecturer at Department of Chemistry, Al-Azhar University. His current research interests
5
are the development of electrochemical sensors and biosensors, modified electrodes,
6
study of electron transfer reaction of enzymes and proteins, and characterization of
7
conducting polymers and their applications.
8
Yoon-Bo Shim received his Ph.D. in Department of Chemistry at Pusan National
9
University, South Korea, in 1985. He is working as a professor at Department of
10
Chemistry and a director at Institute of BioPhysio Sensor Technology (IBST), Pusan
11
National University. His current research interests are the development of bio-(protein,
12
DNA, enzyme, etc.)/chemical-sensors, electroanalytical method of trace biological,
13
organic species with modified electrodes, electron transfer of organic compounds and
14
proteins on the biomembranes, and characterization of conducting polymers and their
15
applications.
17 18 19 20
cr
us
an
M
d
te
Ac ce p
16
ip t
1
21 22 23 24
21
Page 21 of 31
Figure Captions
2
Scheme 1. Schematic representation of the fabrication and overall detection of H2O2 by
3
AgNPs/Ox-pTTBA/MWCNT nanocomposite.
4
Fig. 1. (i) UV-visible spectra and (ii) TEM image of AgNPs solution. SEM images of
5
(iii) bare GC, (iv) Ox-pTTBA/MWCNT/GC, and (v) AgNPs/Ox-pTTBA/MWCNT/GC.
6
(vi) Nyquist plots of bare GC (blue dotted line) and AgNPs/Ox-pTTBA/MWCNT/GC
7
(red dotted line) in a 5.0 mM [Fe(CN)6]3-/4- solution. XPS spectra of (vii) S2p peak and
8
(viii) Ag3d peaks for (a) bare GC and (b) AgNPs/Ox-pTTBA/MWCNT/GC surfaces.
9
Fig. 2. CVs recorded for the AgNPs/Ox-pTTBA/MWCNT (dotted lines) Ox-
10
pTTBA/MWCNT (solid lines) and bare (dashed lines) electrodes in (A) 1.0 mM
11
[Ru(NH3)6]Cl3 and (B) 1.0 mM K3[Fe(CN)6] solutions. (C) CVs of the AgNPs/Ox-
12
pTTBA/MWCNT (solid line) and Ox-pTTBA/MWCNT (dashed line) electrodes in 0.1
13
M H2SO4.
14
Fig. 3. (A) CVs recorded for (a) bare, (b) Ox-pTTBA/MWCNT and (c) AgNPs/Ox-
15
pTTBA/MWCNT electrodes in PBS containing 100 μM H2O2. (B) CVs recorded for
16
AgNPs/Ox-pTTBA/MWCNT electrode in PBS contains various concentrations of H2O2
17
(a) 0 (b) 50, (c) 100 and (d) 150 μM. (C) CVs recorded for AgNPs/Ox-TTBA/MWCNT
18
electrode in PBS contains 100 μM H2O2 at various scan rates. Inset shows a plot of
19
cathodic peak currents vs. the square root of scan rates.
20
Fig. 4. Optimization of H2O2 detection using the AgNPs/Ox-pTTBA/MWCNT probe:
21
(A) pH and (B) adsorption time of AgNPs, (C) applied potential.
22
Fig. 5. Amperometric response obtained using the sensor probe in PBS after multiple
23
additions of H2O2. Inset shows the corresponding calibration plot. (B) Amperogram of
24
the addition of 5 μM H2O2 and 0.1 mM of other interfering species.
Ac ce p
te
d
M
an
us
cr
ip t
1
25 22
Page 22 of 31
1
Table 1. Comparison of recently reported nonenzymatic H2O2 sensors based nanometals
2
composite Linear range (µM)
Detection limit (µM) 4.7
Fe2O3/CoO
50 – 485
0.1
Ag nanocrystals/graphene
20 – 1000
PdNPs/MWCNTs
1 – 1000
AgNPs/Ox-pTTBA/MWCNT
10 – 260
us
1 – 450
3
an
Pt/AuNPs
3
[10] [13]
1.18
[32]
0.3
[33]
0.24
[this work]
M
4
[9]
cr
100 – 1000
RuOHCF/MWCNT
References
ip t
Nanocomposite
Ac ce p
te
d
5
23
Page 23 of 31
1 2 3
Table 2 Determination of H2O2 in human urine samples at the AgNPs/OxpTTBA/MWCNT nanocomposite (n = 5)
ip t
4
Sample
Spiked (μM)
Found (μM)
RSD
1
10
10.46
4.1 %
2
10
10.25
3.8 %
102.5
3
10
9.82
3.2 %
98.2
us
cr
104.6
an
5
Recovery
Ac ce p
te
d
M
6
24
Page 24 of 31
us
cr
ip t
Scheme 1
S
+ MWCNT
S S
TTBA
COOH
COOH
H2 O
COO
COO
COO
Ag
Ag
COO
COO
Ag
AgNPs GCE
GCE
OpTTBA-MWCNT
AgNPs/OpTTBA-MWCNT
Ac
pTTBA-MWCNT
pH 7.0
Ag
COO
COO
Oxidation
GCE
H2 O2
Ag
COO
ce pt
COOH COOH
ed
Electropolymerization
M an
COOH
Scheme 1. Page 25 of 31
ip t
R-Figure 1
30000
(ii)
(i)
1.5
cr
max = 389 nm
CPE2
(vi)
W
Rp1
Rp2W
0.5
400 500 600 700 Wavelength/nm
800
22 00 nn m m
Bare AgNPs/Ox-pTTBA/MWCNT
0
(iv)
20000
Figure 1.
160 165 170 Binding Energy/eV
60000
Ag3d5/2
S2p
Intensity/a.u.
Ag3d3/2
(vii)
(viii)
(b) (a)
(b) (a)
155
40000 Zre/
(v)
ce pt Ac
Intensity/a.u.
10000
0
ed
(iii)
Zim/
us
1.0
0.0 300
CPE1
20000
M an
Absorbance
2.0
Rs
175
365
370 375 Binding Energy/eV
380 Page 26 of 31
20
(A)
15
(B)
cr
30
ip t
R-Figure 2
20
us
0
-10
-400
-200 E/mV
0
ed
-20 -600
200
I/A
10
M an
I/A
10 5 0
-5 -10 -15 -400
-200
0
200 400 E/mV
600
800
ce pt
20
(C)
10
I/A
Ac
0
-10 -20 -30 -40 -100
Figure 2.
100
300
500 E/mV
700
900
1100 Page 27 of 31
80
150
(A) c
100 d
us I/A
20 a
-20 -900
-700
ed
0
-500 -300 E/mV
-100
ce pt
a
0
100
-900
250 (C)
-700
-500 -300 E/mV
-100
100
250 200
I/A
200
I/A
c
50 b
M an
b
Ac
I/A
60 40
(B)
cr
100
ip t
Figure 3
150
150 100 50
100
0
5
10
15
20
25
1/2/(mV/s)1/2
50 0
Figure 3.
-900
-700
-500 -300 E/mV
-100
100 Page 28 of 31
120
110
(B)
cr
(A) 100
us
100
I/A
90
M an
I/A
ip t
R-Figure 4
80 70 60 6
7 pH
8
9
ed
5
60 40
10
0
1
2 3 Time/min
4
5
ce pt
4
80
50
(C)
I/A
Ac
40 30 20
Figure 4.
10 0.0
-0.2
-0.4 -0.6 E/V
-0.8
-1.0 Page 29 of 31
ip t
Figure 5
200
cr
(A)
us 200
100
150
I/A
M an
I/A
150
0
200
ce pt
0
ed
50
100 50
400 Time/s
0 0
100 200 H2O2/M
600
300
800
15
AP
Glu
GA
UA
DA
H2O2
I/A
Ac
10
AA
(B)
H2O2
5
0
Figure 5.
0
100
200 300 Time/s
400
500 Page 30 of 31
M an
us
cr
ip t
Graphical Abstract (for review)
100
H2 O
H2 O2
Ag
Ag
COO
COO
COO
Detection
GCE
Ac
AgNPs/OpTTBA-MWCNT
H2 O2
I/A
COO
60
ed
Ag
80
ce pt
Ag
Bare GC OpTTBA-MWCNT AgNPs/OpTTBA-MWCNT
40 20 0 -20 -900
-700
-500
-300 E/mV
-100
100
Graphic abstract Page 31 of 31