Biosensors and Bioelectronics - University of Florida · 2019-08-14 · Biosensors and...

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Hollow graphitic nanocapsules as efcient electrode materials for sensitive Hydrogen peroxide detection Wei-Na Liu, Ding Ding, Zhi-Ling Song, Xia Bian, Xiang-Kun Nie, Xiao-Bing Zhang, Zhuo Chen n , Weihong Tan n Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082, PR China article info Article history: Received 18 July 2013 Received in revised form 11 August 2013 Accepted 13 August 2013 Available online 24 August 2013 Keywords: Hollow graphitic nanocapsule Electrode materials Hydrogen peroxide Methylene blue Amperometric sensors abstract Carbon nanomaterials are typically used in electrochemical biosensing applications for their unique properties. We report a hollow graphitic nanocapsule (HGN) utilized as an efcient electrode material for sensitive hydrogen peroxide detection. Methylene blue (MB) molecules could be efciently adsorbed on the HGN surfaces, and this adsorption capability remained very stable under different pH regimes. HGNs were used as three-dimensional matrices for coimmobilization of MB electron mediators and horse- radish peroxidase (HRP) to build an HGNHRPMB reagentless amperometric sensing platform to detect hydrogen peroxide. This simple HGNHRPMB complex demonstrated very sensitive and selective hydrogen peroxide detection capability, as well as high reproducibility and stability. The HGNs could also be utilized as matrices for immobilization of other enzymes, proteins or small molecules and for different biomedical applications. & 2013 Elsevier B.V. All rights reserved. 1. Introduction Carbon nanomaterials have attracted wide attention based on their unique physical and chemical properties. Different kinds of carbon-based nanomaterials have been developed, such as fullerene, carbon nanotubes (CNTs), graphene, carbon dots, and carbon cages (Smalley, 1992; Tans et al., 1998; Chen et al., 2008; Novoselov et al., 2004; Chen et al., 2010; Sun et al., 2006; Sheng and Wang, 2008; Yang et al., 2010). Among these materials, graphitic nanocapsules, as a type of carbon cage, have recently been explored for such applications as lithium ion batteries (Wang et al., 2005), catalysts (Wu et al., 2007; Wu et al., 2008; Schaefer et al., 2010), super- capacitors (Bushueva et al., 2008; Xie et al., 2012), immunoassays (Cui et al., 2008; Ho et al., 2009), and drug delivery (Uo et al., 2005). Unlike other amorphous carbon nanocapsules, graphitic nanocap- sules have properties similar to those of graphene and carbon nanotubes, such as unique structures, very high conductivity, and good mechanical and biocompatible properties (Hwang, 2010). Their high surface area, good conductivity and three-dimensional structure could make graphitic nanocapsules a good electrode material for electrochemical biosensing. Graphitic nanocapsules also have high chemical stability and excellent dispersion characteristics which are important both for optimizing synergistic nanoparticle-support interactions and maximizing the mass activity of expensive precious enzymes. However, many applications and properties unique to graphitic nanocapsules remain to be explored. For example, increas- ing interest has been shown in controllable synthesis of graphitic nanocapsules and constructing bio-hybrid nanocomposites and structures for broader biomedical applications. In particular, hydrogen peroxide is a universal molecule and has signicant functions as a signaling molecule in the regulation of a variety of biological processes, including aging and carcinogenesis (Veal et al., 2007; Giorgio et al., 2007; López-Lázaro, 2007). As such, the accurate determination of hydrogen peroxide is essential in the biological, environmental and clinical elds. Accordingly, many methods have been developed for the highly sensitive detection of hydrogen peroxide. The electrochemical method is a widely used approach by its simplicity and high sensitivity, different strategies and electrode systems have been explored, such as HRP-peptide on gold electrode (Zhao et al., 2013), HRPAuchitosanclay (Zhao et al., 2008), HRPgold nanowire (Xu et al., 2010), HRPattapulgite on glassy carbon (GC) (Wu et al., 2011), HRPCNTchitosansolgel (Kang et al., 2009), HRPCNTmethylene blue (Xu et al., 2003; Zhang et al., 2010), Pt on glassy carbon (O'Neil et al., 2004), PtMnOgraphene (Xiao et al., 2012), TiO 2 cytochrome c (Luo et al., 2009), and PtPdFe 3 O 4 (Sun et al., 2012). While these methods have all demonstrated their utility, it is worthwhile exploring new electrode materials and developing a simpler and more effective approach to fabricate reagentless amperometric biosensors for hydrogen peroxide. One such candidate is the graphitic nanocapsule. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.08.023 n Corresponding authors. Tel.: þ86 731 88821834; fax: þ86 731 88821894. E-mail addresses: [email protected] (Z. Chen), [email protected].edu (W. Tan). Biosensors and Bioelectronics 52 (2014) 438444

Transcript of Biosensors and Bioelectronics - University of Florida · 2019-08-14 · Biosensors and...

Page 1: Biosensors and Bioelectronics - University of Florida · 2019-08-14 · Biosensors and Bioelectronics 52 (2014) 438–444. Herein, we developed a method of synthesizing hollow gra-phitic

Hollow graphitic nanocapsules as efficient electrode materialsfor sensitive Hydrogen peroxide detection

Wei-Na Liu, Ding Ding, Zhi-Ling Song, Xia Bian, Xiang-Kun Nie, Xiao-Bing Zhang,Zhuo Chen n, Weihong Tan n

Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and ChemicalEngineering, College of Biology, Hunan University, Changsha 410082, PR China

a r t i c l e i n f o

Article history:Received 18 July 2013Received in revised form11 August 2013Accepted 13 August 2013Available online 24 August 2013

Keywords:Hollow graphitic nanocapsuleElectrode materialsHydrogen peroxideMethylene blueAmperometric sensors

a b s t r a c t

Carbon nanomaterials are typically used in electrochemical biosensing applications for their uniqueproperties. We report a hollow graphitic nanocapsule (HGN) utilized as an efficient electrode material forsensitive hydrogen peroxide detection. Methylene blue (MB) molecules could be efficiently adsorbed onthe HGN surfaces, and this adsorption capability remained very stable under different pH regimes. HGNswere used as three-dimensional matrices for coimmobilization of MB electron mediators and horse-radish peroxidase (HRP) to build an HGN–HRP–MB reagentless amperometric sensing platform to detecthydrogen peroxide. This simple HGN–HRP–MB complex demonstrated very sensitive and selectivehydrogen peroxide detection capability, as well as high reproducibility and stability. The HGNs could alsobe utilized as matrices for immobilization of other enzymes, proteins or small molecules and for differentbiomedical applications.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Carbon nanomaterials have attracted wide attention based ontheir unique physical and chemical properties. Different kinds ofcarbon-based nanomaterials have been developed, such as fullerene,carbon nanotubes (CNTs), graphene, carbon dots, and carbon cages(Smalley, 1992; Tans et al., 1998; Chen et al., 2008; Novoselov et al.,2004; Chen et al., 2010; Sun et al., 2006; Sheng and Wang, 2008;Yang et al., 2010). Among these materials, graphitic nanocapsules, asa type of carbon cage, have recently been explored for suchapplications as lithium ion batteries (Wang et al., 2005), catalysts(Wu et al., 2007; Wu et al., 2008; Schaefer et al., 2010), super-capacitors (Bushueva et al., 2008; Xie et al., 2012), immunoassays(Cui et al., 2008; Ho et al., 2009), and drug delivery (Uo et al., 2005).Unlike other amorphous carbon nanocapsules, graphitic nanocap-sules have properties similar to those of graphene and carbonnanotubes, such as unique structures, very high conductivity, andgood mechanical and biocompatible properties (Hwang, 2010). Theirhigh surface area, good conductivity and three-dimensional structurecould make graphitic nanocapsules a good electrode material forelectrochemical biosensing. Graphitic nanocapsules also have highchemical stability and excellent dispersion characteristics which areimportant both for optimizing synergistic nanoparticle-support

interactions and maximizing the mass activity of expensive preciousenzymes. However, many applications and properties unique tographitic nanocapsules remain to be explored. For example, increas-ing interest has been shown in controllable synthesis of graphiticnanocapsules and constructing bio-hybrid nanocomposites andstructures for broader biomedical applications.

In particular, hydrogen peroxide is a universal molecule and hassignificant functions as a signaling molecule in the regulation of avariety of biological processes, including aging and carcinogenesis(Veal et al., 2007; Giorgio et al., 2007; López-Lázaro, 2007). As such,the accurate determination of hydrogen peroxide is essential in thebiological, environmental and clinical fields. Accordingly, manymethods have been developed for the highly sensitive detection ofhydrogen peroxide. The electrochemical method is a widely usedapproach by its simplicity and high sensitivity, different strategiesand electrode systems have been explored, such as HRP-peptide ongold electrode (Zhao et al., 2013), HRP–Au–chitosan–clay (Zhao et al.,2008), HRP–gold nanowire (Xu et al., 2010), HRP–attapulgite onglassy carbon (GC) (Wu et al., 2011), HRP–CNT–chitosan–sol–gel(Kang et al., 2009), HRP–CNT–methylene blue (Xu et al., 2003;Zhang et al., 2010), Pt on glassy carbon (O'Neil et al., 2004),Pt–MnO–graphene (Xiao et al., 2012), TiO2–cytochrome c (Luoet al., 2009), and PtPd–Fe3O4 (Sun et al., 2012). While these methodshave all demonstrated their utility, it is worthwhile exploring newelectrode materials and developing a simpler and more effectiveapproach to fabricate reagentless amperometric biosensors forhydrogen peroxide. One such candidate is the graphitic nanocapsule.

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/bios

Biosensors and Bioelectronics

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bios.2013.08.023

n Corresponding authors. Tel.: þ86 731 88821834; fax: þ86 731 88821894.E-mail addresses: [email protected] (Z. Chen), [email protected] (W. Tan).

Biosensors and Bioelectronics 52 (2014) 438–444

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Herein, we developed a method of synthesizing hollow gra-phitic nanocapsules (HGN) and using them as an electrodematerial for hydrogen peroxide detection. These HGNs were usedas three-dimensional matrices to effectively immobilize enzymes,proteins and small molecules. Specifically, a reagentless ampero-metric biosensor was created by coimmobilization of methyleneblue (MB) electron mediators and horseradish peroxidase (HRP)enzyme on the HGN-coated electrode. The positively charged HRPand MB molecules were anchored on the HGN surface throughelectrostatic adsorption. As a consequence of the extra strong π–πinteraction, HGNs exhibited a high loading capacity for the MBelectrochemical mediator-molecules. The constructed HGN–HRP–MB platform ably facilitated electron shuttling between the activecenter of HRP enzyme and the surface of the electrode. Thus, byusing HGN as the electrode matrix, together with a simple MB andHRP assembly strategy, the sensing platform demonstrated theability to detect hydrogen peroxide with high sensitivity, selectiv-ity, reproducibility and stability. Indeed, hydrogen peroxide mole-cules could be detected less than 1 μM.

2. Experimental

2.1. Chemicals

Methylene blue and horseradish peroxidase (300 U/mg) wereobtained from Shanghai Reagent Company (Shanghai, China). Allother chemicals of analytical reagent grade or higher were obtainedfrom Changsha Chemical Reagents Company (Changsha, China) andused as received without further purification. All aqueous solutionswere prepared using ultrapure water (Milli-Q, Millipore). The bufferused in this work was 10 mM phosphate buffer solution (PBS) and0.15 M NaCl (pH 7.4).

2.2. Preparation of HGN

Hollow graphitic nanocapsules were obtained from a core-shellmagnetic graphitic nanomaterial (MG). MG was synthesized witha chemical vapor deposition (CVD) system as reported previously(Chen et al., 2012; Song et al., 2013). Briefly, we first impregnatedfumed silica (1.00 g, Aladdin) with Co(NO3)2 �6H2O (2.06 g) inmethanol. Then the methanol was removed, the mixture dried,and the powder ground. Typically, 0.50 g of the powder was usedfor methane CVD growth in a tube furnace at 800 1C for 5 min.After growth, the sample was etched with 10% HF in H2O (80%)and ethanol (10%) to dissolve the silica. We collected the MG solidproduct through centrifugation and washed thoroughly. To obtainthe HGN, a solution of sulfuric and nitric acid was utilized to polishthe as-received MG for 4 h and solubilize it in water. The excessMGs were removed by an external magnet. The HGNs werecollected through centrifugation and washed thoroughly withultrapure water.

2.3. Adsorption measurement of methylene blue on HGN

Adsorption measurements were performed with simple mixing ofthe MB and HGN solutions. Typically, the MB solution at a concen-tration of 2 mg/L was used for the preparation of MB–HGN nano-composites through a premixing procedure with different amountsof HGNs. The mixture was shaken for 30 min at room temperatureand then centrifugated at 7000 rpm for 5 min. The resulting super-natant was collected, and UV–vis spectra were recorded using aShimadzu UV-2550 spectrophotometer (Shimadzu InternationalTrading Co., Ltd., Shanghai, China).

2.4. Preparation of HGN–HRP–MB-modified electrode

Prior to modification, the bare glassy carbon electrode wasthoroughly polished with emery paper and alumina slurry in theorder of 1.0, 0.5, and 0.03 μm, followed by ultrasonication in water.Next, the electrode was immersed in a freshly prepared piranhasolution (30% H2O2 and 98% H2SO4, 1/3, v/v) for 40 min. After this,the electrode was rinsed with ultrapure water and electrochemi-cally pretreated with cyclic potential scanning to obtain a cleanglassy carbon electrode. Then, 5 μL, 1.75 mg/mL HGN was cast onthe electrode surface and air-dried at room temperature. Follow-ing this step, the electrode was soaked in 10 mg/mL HRP solutionand incubated overnight. Meanwhile, a homogeneous solution(I) composed of 1 mg/mL HRP and 0.33 mg/mL MB was preparedin PBS (pH 7.4). The HGN–HRP–MB electrode was obtained bydipping the HRP-coated HGN glassy carbon electrode into solutionI (PBS, pH 7.4) for 12 h at 4 1C, followed by careful rinsing withultrapure water. Finally, 5 μL of 1% Nafion ethanolic solution wascast on the electrode surface and air-dried at room temperature toready the instrument for electrochemical measurements.

2.5. Characterization and electrochemical measurements

Raman spectroscopy was performed on a Horiba Jobin YvonLabRAM-010 Raman microscope with 632 nm He–Ne laser excita-tion. The hydrodynamic diameters of the HGNs under investiga-tion were measured using a Zetasizer Nano ZS90 Dynamic LightScattering (DLS) system equipped with a red (633 nm) laser and anAvalanche photodiode detector (APD) (quantum efficiency450%at 633 nm) (Malvern Instruments Ltd., Worcestershire, England).Zeta potential measurements were performed in water. Themeasurements were carried out at room temperature on theZetaSizer Nano ZS90 equipped with MPT-2 Autotitrator and4 mW He�Ne laser (λ0¼633 nm) using the Laser Doppler Elec-trophoresis technique. Electrochemical measurements were car-ried out with a CHI 760 electrochemical analyzer (CH InstrumentCompany, Shanghai, China). A conventional three-electrode cellwas used. A bare or modified glassy carbon electrode (GCE) wasused as a working electrode, and a platinum disk was used as anauxiliary electrode, with a saturated calomel electrode (SCE) asreference.

3. Results and discussion

3.1. Characterization of hollow graphitic nanocapsules

The prepared HGNs were analyzed by scanning electron micro-scopy (SEM), as shown in the images pictured in Fig. 1A and B.These HGNs showed uniform size distribution. Transmissionelectron microscopy (TEM), which was utilized for the structuralcharacterization of HGNs, showed a hollow structure with somewrinkles on the surface (Fig. 1C). In the high-resolution TEM imageof the HGN (Fig. 1D), the graphitic shell structure was clearlyobserved, and the intralayer distance of the shell was around0.34 nm, which is consistent with that of graphite. The digitalcamera image shows the graphitic nanocapsule in water solutionbefore and after the hollow structure was prepared (Fig. 1E). Afterthe acid treatment, the HGNs form a black suspension which couldremain stable for months.

The HGNs were further characterized with selected area electrondiffraction (SAED) and Raman spectroscopy, and both techniquesindicated the graphitic structure of the nanocapsules. As demonstratedin Fig. 2A, the SAED diffraction patterns can be assigned to the (002),(100), (004) and (112) facets of the hexagonal crystalline graphite,respectively. The graphitic shell was also proved through Raman

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spectroscopy (Fig. 2B, Fig. S1, Supporting information), showing agraphitic carbon (G) peak at �1590 cm�1 and a disordered (D) peakat �1300 cm�1. The high Raman D peak reflects the high strain of thegraphite shells caused by the small size of the MG and defectspresented during the acid treatments. Analyses of the hydrodynamicdiameters and surface ζ-potential of the HGNs were performed inwater, and the size distribution of the suspended HGNs was investi-gated with dynamic light scattering (Fig. 2C). The average size wasaround 60 nm, which agreed with the size measured from TEM.Fig. 2D shows the ζ-potential curves of the HGN water solution. HGNswere negatively charged, and the ζ-potential was around 36mV. Themixed-acid treatments produced massive suspended hydroxyl andcarboxyl groups at the HGN surface, which were believed to be theorigin of the negative charge observed in the ζ-potential curves.

3.2. Isothermal adsorption of hollow graphitic nanocapsules

HGNs have high surface areas resulting from their hollow andthree-dimensional graphitic structure. Methylene blue was used asthe model molecule to explore the isothermal adsorption propertiesof HGNs (Fig. 3). HGNs exhibited high loading capacity for MBmolecules, and the positively charged MB molecules were anchoredon the HGN surface through strong electrostatic adsorption and π–πinteractions. Fig. 3A shows the UV–vis spectra of the 2 mg/L MBmolecules (a) mixed with 0.175 mg/mL HGNs after 0 min (b), 30 min(c), 1 h (d) and 5 h (e) incubation and separation, respectively. Twoabsorption peaks around 613 and 664 nm, respectively, can beobserved in the UV–vis spectra. These peaks belong to the MBmolecules whose intensities are proportional to the concentration ofMB in solution. As determined from the spectra, most MB moleculeswere adsorbed by the HGNs and reached isothermal equilibriumafter 30 min. The inset of Fig. 3A shows the digital camera images of

the original MB solution (a) and following the addition of HGNs (b).The HGNs demonstrated good MB adsorbing capability, whichremained very stable under different pH regimes (Fig. 3B). Morespecifically, HGNs can stably adsorb the MB dyes, ranging from veryacidic pH 3 to alkaline pH 11 conditions, suggesting that HGNs couldbe a potentially efficient adsorbent for the removal of organicenvironmental contaminants. We further investigated MB adsorptionas a function of HGN concentration. Accordingly, UV–vis spectra werecollected from MB solution after different concentrations of HGNwere incubated and separated (Fig. 3C). By adding certain amounts ofHGNs into the MB solution, the peaks around 613 and 664 nm nearlydisappeared. The inset shows the digital camera images of the MBsolution with different concentrations of HGN added. The curve ofadsorption efficiency as a function of the added HGN concentration isshown in Fig. 3D, which could be well fit into an equilibriumisotherm adsorption equation. With around 0.08 mg/mL HGN addedinto the 2 mg/L MB solution, the adsorption reached saturation, andmost of the MB molecules were adsorbed on the HGN surfaces.

3.3. Hydrogen peroxide detection strategies

Having determined the efficient surface adsorption capability andexcellent conductivity of the HGNs, we designed an HGN electro-chemical sensing platform for hydrogen peroxide detection. Hydrogenperoxide, the simplest perioxide, is a strong oxidizer and considered ahighly reactive oxygen species. Therefore, its accurate determination isessential in biological, environmental and clinical fields. Fig. 4 showsthe assembly of the HGN–HRP–MB electrochemical sensing platform.Briefly, HGNs were used as three-dimensional matrices for coimmo-bilization of methylene blue (MB) electron mediators and horseradishperoxidase (HRP) (Fig. S2, Supporting information). First, HGNs werecoated on the glassy carbon electrode (GCE) surface through physical

Fig. 1. Electron microscopy characterization of hollow graphitic nanocapsules. (A) and (B) were SEM images of the HGNs. (C) TEM image of HGNs. (D) High-resolution TEM ofthe HGN shell, which was consistent with the graphitic structure. (E) Suspensions of HGN aqueous solution before (left) and after (right) mixed-acid treatments.

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absorption (Fig. S3 for electrochemical impedance characterization,Supporting information). Next, the electrode was soaked in the HRPsolution for the preassembly of HRP enzymes. Following that, the HRP-coated electrode was immersed in the HRP–MB solution for furtherassembly (Fig. 4A). Fig. 4B displays the interface view of the activeHGN–HRP–MB electrode structure and the hydrogen peroxide detec-tion processes. The high conductivity of the HGN graphitic shellsclearly enhances the electrochemical performance of the HGN elec-trode. The HGNs were also a high surface area supporter, and both theHRP and MB molecules could be easily adsorbed, while maintainingtheir activity as a direct result of the three-dimensional structure ofthe HGN. The adsorbed MB molecules on the HGN–HRP particlesurface could act as an efficient electrochemical mediator to improveelectron transport. While the HRP-coated electrode loses its enzymaticbioactivity and shows poor electron transport, the HGN platform hashigher hydrogen peroxide sensing activity and better electron trans-port efficiency.

3.4. Electrochemical properties of HGN–HRP–MB-modified electrode

HGNs, as an efficient electrode material, have unique and stableelectrochemical properties, as characterized in the HGN–HRP–MBdetection platform shown in Fig. 5. Typical cyclic voltammograms ofHGN–HRP–MB in PBS solution (Fig. S4, Supporting information) atdifferent scanning rates in the potential range from 0.7 V to �0.7 Vdemonstrate the symmetrical and good redox behavior of MB incontributing to the efficiency of HGNs (Fig. 5B). As the scanning rateincreased, the redox current correspondingly increased. The peakcurrents of HGN–HRP–MB complexes are proportional to the scan-ning rate up to 700 mV/s, indicating the redox reaction of HGN–HRP–MB complexes on the electrode surfaces. The redox peak currents

increased linearly (linear correlation coefficient R240.999) with thescanning rate between 50 and 700 mV/s, as expected, furtherindicating a surface-confined process of the HGN–HRP–MB redoxreaction (Fig. 5B).

3.5. Electrochemical detection of H2O2 by HGN–HRP–MB electrode

We next investigated the hydrogen peroxide detection capabilityof the HGN–HRP–MB electrochemical sensing platform (Fig. 6).In particular, electrochemical activity is compared among the bareglassy carbon (a), HRP only (b) and HGN–HRP–MB (c) electrode in100 μM of hydrogen peroxide, respectively (Fig. 6A). Similar to thepeak current of the bare GCE, the HRP-only electrode also shows arelatively low peak current, possibly caused by the denaturation ofthe HRP enzyme molecules and the low electron transport efficiencybetween the electrode and HRP molecules. However, for the HGN–HRP–MB matrix-modified electrode, the redox peak current issignificantly increased. The high conductivity of HGN was believedto improve the electron transport, and the 3D hollow structure of theHGN also helps to maintain HRP enzyme activity during the electro-chemical measurements. MB, as an electrochemical reaction med-iator, displayed excellent mediating ability in the electrocatalyticreactions and also contributed to the increase of peak current. TheHGN–HRP–MB platform was further applied to detect hydrogenperoxide, and Fig. 6B shows the results of redox current vs. hydrogenperoxide concentration. The HGN–HRP–MB sensing platform hasgood stability and can be stored for more than one week at 4 1Cwhile still remained more than 95% of its initial current value for thehydrogen peroxide detection. The response of a same HGN–HRP–MBelectrode was further investigated under optimum conditions andthe sensing platform demonstrated very good operational stability.

Fig. 2. Advanced structural analysis of HGNs. (A) Based on selected area electron diffraction measurement of the HGNs, the diffraction patterns can be assigned to the (002),(100), (004) and (112) facets of the hexagonal crystalline graphitic structure. (B) A Raman spectrum (excitation 632 nm) of the HGNs with the G and D bands of graphiticcarbon. (C) and (D) were the size and ζ-potential distribution of the HGNs, respectively, as measured by the DLS system equipped with a red (633 nm) laser at roomtemperature. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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The HGN–HRP–MB platform also demonstrated high detectionsensitivity and good reproducibility for the determination of hydro-gen peroxide in its linear range. Hydrogen peroxide could be detected

down to 1 μM. The cyclic voltammograms of PBS solution without(a) and with (b) the addition of 1 μM hydrogen peroxide clearlydemonstrate the sensing capability of the HGN–HRP–MB platform

Fig. 3. The isothermal adsorption properties of the HGNs. (A) UV–vis spectra of 2 mg/L MB molecules (a) mixed with 0.175 mg/mL HGNs after 0 min (b), 30 min (c), 1 h(d) and 5 h (e) incubation and separation, respectively (see inset for digital camera images of solution a and b before separation). (B) UV–vis absorption of MB moleculesadsorbed on HGNs under different pH regimes (A, A0 were the intensity of the solution with and without HGN adsorption, respectively). (C) UV–vis spectra of 2 mg/L MBsolution after different amounts of HGNs were added (see inset for the digital camera images of the different solutions). (D) Adsorption efficiency of 2 mg/L MB as a functionof the added HGN concentration.

Fig. 4. The strategy of using HGNs for hydrogen peroxide detection. (A) Procedure for preparing the HGN–HRP–MB matrix on the glassy carbon electrode (GCE). (B) Interfaceview of the active electrode structure and the hydrogen peroxide detection process.

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(Fig. 6B). Sensing response of five different platforms was alsoinvestigated with fixing the hydrogen peroxide concentration underoptimum conditions, and the HGN–HRP–MB platform demonstratedgood fabrication reproducibility. Moreover, HGN as electrode mate-rial can also be utilized for different electrochemical detection and itdemonstrated good generality and reproducibility (Supporting infor-mation, Figs. S5, S6 and S7).

3.6. Amperometric and selectivity measurements of hydrogenperoxide detection

The amperometric response of the HGN–HRP–MB platform wasattempted under the optimized operating electrode potential of�0.4 V with successive injections of hydrogen peroxide in PBSsolution. Fig. 7A shows a typical amperometric response by varying

Fig. 5. Electrochemical characterization of HGN–HRP–MB-modified GCE electrode. (A) Cyclic voltammograms of HGN–HRP–MB in PBS solution at different scanning rates inthe potential range from 0.7 V to�0.7 V. (B) Calibration plots of redox peak currents vs. scanning rate.

Fig. 6. Hydrogen peroxide detection with HGN–HRP–MB-modified GCE electrode. (A) Electrochemical activity compared among the bare glassy carbon (a), HRP only (b) andHGN–HRP–MB (c) electrodes in 100 μM hydrogen peroxide solution. (B) Plot of redox current (Irel, the relative current between the solution with and without hydrogenperoxide) vs. hydrogen peroxide concentration (see inset for the cyclic voltammograms of PBS solution without (a) and with (b) addition of 1 μM hydrogen peroxide).

Fig. 7. Amperometric and selectivity measurements of HGN–HRP–MB-modified GCE electrode. (A) amperometric response with successive additions of (a) 500 nM, (b) 5 μM,(c) 50 μM and (d) 500 μM of hydrogen peroxide. (B) Redox current changes of the HGN–HRP–MB sensing platform for 50 μM of H2O2, L-Cys, NO3

� and glycine, respectively.

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H2O2 concentration from 500 nM to 500 μM, respectively. Ampero-metric response down to 500 nM H2O2 indicates the good sensitivityof the HGN–HRP–MB sensing platform. Selectivity of the HGN–HRP–MB platform was also investigated. Hydrogen peroxide, L-Cys, NO3

and glycine were compared with the platform, as demonstratedunder votammetry tests (Fig. 7B). The 50 μM H2O2 solution showedaround three times higher positive signal than the 50 μM L-Cys, NO3

or glycine solution, indicating good selectivity of the HGN–HRP–MBsensing platform. For maximum efficiency and selectivity, more effortshould be directed toward optimizing HGN size and graphitic shellthickness, designing multilayer HRP and MB coating structures, orfurther amplifying the sensing with other strategies.

4. Conclusion

In summary, we prepared and utilized hollow graphitic nanocap-sules as efficient electrode materials for the sensitive detection ofhydrogen peroxide. The HGNs were synthesized through a CVDsystem and thenwith mixed-acid treatments. HGNs have large surfaceareas, high conductivity, as well as good mechanical and biocompa-tible properties, based on their hollow and three-dimensional graphi-tic structure. Methylene blue molecules can be efficiently adsorbed onthe HGN surfaces, and this adsorption capability remained very stableunder different pH regimes. The positively charged MB moleculeswere anchored on the HGN surface through strong electrostaticadsorption and π–π interactions. Using HGNs as three-dimensionalmatrices for coimmobilization of methylene blue electron mediatorsand horseradish peroxidase, an HGN reagentless amperometric sen-sing platform was designed to detect hydrogen peroxide. The simpleHGN–HRP–MB electrochemical sensing platform was able to facilitateelectron shuttling between the active center of HRP enzyme and thesurface of the electrode. Benefiting from efficient surface adsorptioncapability and excellent conductivity of the HGN, the sensing platformdemonstrated very sensitive hydrogen peroxide detection capability,and less than 1 μM hydrogen peroxide could be detected. The HGN–HRP–MB electrochemical sensing platform also demonstrated goodhydrogen peroxide detection selectivity and high reproducibility andstability. Considering that no noble metal electrode is used, thisdetection capability of HGN platform is excellent. The HGNs couldalso be utilized as matrices for immobilization of other enzymes,proteins or small molecules and for different biomedical applications.

Acknowledgments

This work was financially supported by the National Key BasicResearch Program of China (No. 2013CB932702), the Research Fund forthe Program on National Key Scientific Instruments and EquipmentDevelopment (No. 2011YQ0301241402), and the National NaturalScience Foundation of China (No. 21105025).

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.bios.2013.08.023.

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