One-Pot Synthesis of Fe3O4 Nanoparticle Loaded 3D Porous

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One-pot synthesis of FeO nanoparticle-loaded 3D porous graphene nanocomposites with enhanced nanozyme activity for glucose detection Qingqing Wang, Xueping Zhang, Liang Huang, Zhiquan Zhang, and Shaojun Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16034 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 27, 2017

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ACS Applied Materials & Interfaces

One-pot synthesis of Fe3O4 nanoparticle-loaded 3D porous graphene nanocomposites with enhanced nanozyme activity for glucose detection Qingqing Wang, †‡ Xueping Zhang, † Liang Huang, † Zhiquan Zhang, ‡* Shaojun Dong †* †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China ‡

College of Chemistry, Jilin University, Changchun, 130012, P. R. China

* Corresponding Author E-mail: [email protected], [email protected].

ACS Paragon Plus Environment

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ABSTRACT

A novel one-pot strategy is proposed to fabricate 3D porous graphene (3D GN) decorated with Fe3O4 nanoparticles (Fe3O4 NPs) by using hemin as iron source. During the process, graphene oxide was simultaneously reduced and self-assembled to form 3D graphene hydrogel while Fe3O4 NPs synthetized from hemin distributed uniformly on 3D GN. The preparation process is simple, facile, economical and green. The obtained freeze-dried product (3D GH-5) exhibits outstanding peroxidase-like activity. Compared to the traditional 2D graphene-based nanocomposites, the introduced 3D porous structure dramatically improved the catalytic activity, as well as the catalysis velocity and its affinity for substrate. The high catalytic activity could be ascribed to the forming Fe3O4 NPs and 3D porous graphene structures. Based on its peroxidaselike activity, 3D GH-5 was used for colorimetric determination of glucose with a low detection limit of 0.8 µM.

KEYWORDS: one-pot, 3D graphene nanocomposites, peroxidase mimics, colorimetry, glucose detection.


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INTRODUCTION Nanomaterials with enzyme-like properties (nanozymes)1-3 have attracted significant research interest owing to their simple preparation, storage, and separation, as well as the low-cost as compared with natural enzymes.1, 4-5 Plenty of nanostructured materials have been reported to mimic the activity of oxidase,6-8 peroxidase,9-14 catalase15-17 and superoxide dismutase18. Among these, peroxidase mimics are the most widely studied, which have shown potential applications in the field of biological detection,19-20 and immunoassay.21-23 Hollow PDA-Au was used for signal amplification to achieve sensitive nonenzymatic colorimetric detection, and a novel colorimetric immunoassay was designed for the sensitive detection of carbohydrate antigen 125.23 In particular, since the first exciting discovery of ferromagnetic nanoparticles (Fe3O4 NPs)24 Wang et al. has developed a sensitive colorimetric method for H2O2 and glucose detection based on their intrinsic peroxide-like activity.25 Besides colorimetric assay, an electrochemical sensor with Fe3O4 NPs was successfully fabricated at the same period.26 An electrochemical immunosensor for sensitive detection of cancer biomarker prostate specific antigen based on NDA-Fe3O4 was also designed.22 Recently, structural effects of Fe3O4 nanocrystals on peroxidase-like activity were investigated systematically27 and various iron based nanomaterials have been demonstrated exhibiting intrinsic peroxidase-like catalytic activities, for instance, FeS,28 MFe2O4,15 CuZnFeS29 and Fe-MOFs.30 Graphene, which possesses remarkable electrical, optical, thermal, and mechanical properties,3132

has been a popular support to interact with various catalysts because of its outstanding 2D

carbon nanostructure.33-36 However, the unavoidable restacking and agglomeration of graphene sheets caused by van der Walls force can lead to a drastic loss of active sites.37-38 The poor intersheet connections between isolated graphene flakes would also break the continuous


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pathway for electron/phonon transports.39 Novel 3D graphene architectures are thus eagerly calling for accomplishment. Up to now, most of 3D graphene nanomaterials, for example 3D graphene bubble network (SG)40 and Ni(OH)2/graphene//porous graphene,41 showed promising application in supercapacitors. You and coworkers successfully prepared graphene aerogel, which exhibited adsorption capacity towards organic dyes and was applied to water purification.42 Inspired by the merits of 3D graphene framework such as large specific surface, high conductivity and good mechanical properties,43-44 we selected 3D porous graphene (3D GN) as a new support for mimic immobilization to improve the enzyme-like activities. Here, we successfully combined 3D GN with Fe3O4 NPs through one-pot strategy in the presence of hemin with proper concentration, which exhibited extremely enhanced peroxidaselike activity, as shown in Scheme 1. The fabrication method is simple, facile, economical and green. More importantly, the as-prepared freeze-dried product exhibits outstanding peroxidaselike activity and can be applied to glucose detection with a wide linear range and low detection limit. Furthermore, a series of experiments were conducted to explore the reason for the high catalytic properties. By comparing with other nanomaterials, the formed Fe3O4 NPs and 3D porous graphene structures are considered playing crucial roles in improving the catalytic activity. EXPERIMENTAL SECTION Materials: Graphene oxide (GO) was synthesized from natural flake graphite by a modified Hummers method.45 L-Cys was obtained from Aladdin. Hemin, 3, 3', 5, 5'-tetramethylbenzidine (TMB) and glucose oxidase (GOD, from Aspergillus niger, 50 KU) were obtained from SigmaAldrich. Ammonia solution (NH3, 25%), glucose, fructose, maltose, lactose and sucrose were


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purchased from Beijing Chemical Reagent Company (Beijing, China). All the chemicals were of analytical grade and used without further purification. Ultrapure water (≥18.2 MΩ cm) was used throughout the study. Apparatus and characterization: UV/Vis absorption measurements were performed on a Cary 500 UV-Vis-NIR spectrometer (Varian). Scanning electron microscopy (SEM) experiments were performed on a PHILIPS XL-30 field-emission scanning electron microscope with an accelerating voltage of 10 kV. High-resolution transmission electron microscopy (HRTEM) measurements were made on a JEM-2100F high-resolution transmission electron microscope operating at 200 kV. Nitrogen adsorption/desorption analysis was performed at 77 K on an AutosorbStation1 (Quantachrome, USA). X-ray diffraction (XRD) patterns were recorded from a D8 ADVANCE (Bruker, Germany) X-ray diffractometer with Cu Kα radiation (λ = 1.5406 Å). X-ray photoelectron spectroscopy (XPS) measurement was performed on an ESCALAB-MKII spectrometer (VG Co., United Kingdom) with Al Kα X-ray radiation as the X-ray source for excitation. Electrochemical measurements were performed using a CHI 832B electrochemical workstation (CH Instruments Inc., China). A conventional three-electrode system was used, which consisted of a Ag/AgCl (saturated KCl) electrode as the reference electrode, a platinum wire as the counter electrode, and the glassy carbon electrode (GCE) as the working electrode. Preparation of Fe3O4 NPs-loaded 3D graphene nanocomposites: The Fe3O4 NPs-loaded 3D graphene nanocomposites were synthesized in a one-pot reaction. Detailedly, 10 mg L-Cys, 40 µL NH3·H2O (NH3, 25%) and different volumes of hemin (2 mg mL-1: 100 µL, 200 µL and 500 µL, respectively) were added into GO aqueous dispersion under ultrasonication. The final volume of the mixture was 2 mL, which contained 2 mg mL-1 GO. Then the mixture was kept in a water bath at 90 °C for 10 h under atmospheric pressure without stirring. Finally, the resulting


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hydrogel was washed thoroughly with double-distilled water and then freeze-dried. The obtained product was named 3D GH-1, 3D GH-2 and 3D GH-5, respectively. When the dosage of hemin was increased to 500 µL or 1 mL (4 mg mL-1), the well-formed 3D graphene hydrogel could not be obtained. The as-prepared material was respectively named GH-10 and GH-20. In the case of 3D GN, a similar procedure was used except for not adding hemin. All the products were dispersed in ethanol for further use. Electrochemistry experiments of 3D GH-5 modified electrode: The glassy carbon electrodes (GCE, 3.0 mm in diameter) were firstly polished successively with 1.0 and 0.3 µm alumina slurry and rinsed with deionized water, followed by sonication in 1:1 nitric acid, acetone and deionized water, respectively. Then, the electrode was dried under blowing nitrogen. 4 µL of 3D GH-5 (1 mg mL-1) was cast on the surface of GCE and dried under an infrared lamp. Steady-State kinetic analysis of 3D GH-5 as peroxidase mimetics: Kinetic experiments were carried out in a reaction volume of 1.0 mL HAc-NaAc buffer solution (0.1 M, pH 4.0) containing 10 µg mL-1 3D GH-5, 5 mM H2O2 and 0.5 mM TMB as substrate, unless otherwise stated. The mixture solutions were incubated at 50 °C for 10 min and then used for absorbance measurement at wavelength 652 nm. The Michaelis-Menten constant was calculated using a Lineweaver-Burk plot: 1/V = Km/Vm (1/[S]+ 1/Km), where V is the initial velocity, Vm is the maximal reaction velocity, [S] corresponds to the substrate concentration, and Km is the Michaelis-Menten constant. Colorimetric detection of glucose based on 3D GH-5 peroxidase mimic: Glucose detection was performed as follows: (1) 50 µL GOD (5 mg mL-1) and 200 µL of different concentrations of glucose in 10 mM Na2HPO4 buffer (pH 7.0) were incubated at 37 °C for 30 min; (2) 10 µL of


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TMB (50 mM), 30 µL of 3D GH-5 (1 mg mL-1) , and 740 µL of 0.1 M HAc-NaAc buffer (pH 4.0) were added into the above 250 µL glucose reaction; (3) the mixed solution was incubated at 50 °C for 10 min and then centrifuged at 8000 rpm for 5 min. Finally, the supernatant was used for absorbance spectroscopy measurement. RESULTS AND DISCUSSION Preparation and Characterization of 3D GH-5 Briefly, a facile one-pot strategy was developed to fabricate the 3D graphene nanocomposites with in situ-formed Fe3O4 NPs. The formation of compact porous structure was attributed to the use of L-Cys. L-Cys could interact with each other to form a polymeric network structure. Besides, L-Cys was easily decomposed into NH3 and H2S at 90 °C, helping to inhibit the restacking and agglomeration of graphene sheets.42 Fe3O4 NPs were formed in situ on the surface of graphene nanosheets in the presence of hemin. And the concentration of hemin (CHemin) has a significant effect on the formation of 3D porous structure. In the absence of or with low concentration of hemin (e.g. 3D GN: no hemin; 3D GH-1: 100 µL hemin; 3D GH-2: 200 µL hemin; 3D GH-5: 500 µL hemin; CHemin: 2 mg mL-1), a relatively well-formed hydrogel was obtained. However, when Chemin was increased (e.g. GH-10: 500 µL hemin; GH-20: 1 mL hemin; CHemin: 4 mg mL-1), the mixture remained its initial condition, and could not form a hydrophobic hydrogel. This may be ascribed to the π-π stacking interactions between excess hemin and graphene, which could reduce the cross-linking of graphene nanosheets, resulting in the failure for the formation of compact 3D architecture, as shown in Fig. S1. So, 3D GH-5 with wellformed porous structure was used for the following experiments.


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Figure 1A shows the photos of 3D GH-5 hydrogel and the free-dried product. From the photographs of front view and top view, we could see that the hybrid hydrogel was stable in water and the water could keep colorless and transparent after the hydrogel was immersed for a long time, indicating that excess free hemin was removed by thoroughly washing. After freezedrying, the shape of 3D GH-5 changed negligibly. Figure 1B and 1C show the SEM images of 3D GH-5. The interconnected 3D porous network of graphene sheets was well-defined, and the pore size ranging from several hundred nanometers to several micrometers. Nitrogen adsorption/desorption experiments were conducted to determine the specific surface area and porous properties of 3D GH-5 (Figure S2). The obtained nanocomposites have an average Brunauer-Emmett-Teller (BET) surface area of 92 m2 g-1 with the Barrett-Joyner-Halenda (BJH) mean pore diameter of ~2.2 nm. These results further confirm the remarkable porous structure of 3D GH-5. Further TEM images (Figure 1D and 1E) reveal that nanoparticles are uniformly distributed on the graphene nanosheets, and their average diameter is about 100 nm. The EDS results (Figure S3) and elemental mappings (Figure S4) both demonstrate the presence of C, N, O, S and Fe components in 3D GH-5. Three elements (C, N and S) coexist on the graphene nanosheets while Fe and O elements mostly exist on the nanoparticles. Combined with the XRD results (Figure S5), the nanoparticles grown on the graphene surfaces are proved to be Fe3O4 NPs. The broad peak located in the range 19°-28° is assigned to (002) plane of the stacked graphene network with 3D porous structure 46. A series of typical peaks at 30.1°, 43.0°, 56.9° and 62.5° correspond to (220), (400), (511) and (440) planes of the standard Fe3O4 (JCPDS no. 19-629). The composition and surface oxidation states of 3G GH-5 were explored with X-ray photoelectron spectroscopy (XPS) measurement. The survey spectra (Figure 2A) indicate the presence of C, N,


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O, S and Fe components in the as-prepared materials once again. To further confirm the reduction of GO, C 1s XPS spectrum of GO samples and 3D GH-5 is compared as shown in Figure 2B and 2C. The C 1s spectra of pure GO display five peaks centered at 284.6, 286.0, 286.8, 287.8 and 289.0 eV, corresponding to C-C, C-OH, C-O-C, C=O and O-C=O groups, respectively.19 After reaction with L-Cys, the oxygen species of C-O-C of GO decreased significantly, indicating that GO was reduced successfully. Three types of nitrogen species are fitted in N 1s spectra (Figure 2D), including pyridinic-N (398.7 eV), pyrrolic-N (400.4 eV) and graphitic-N (401.4 eV).47 And two peaks centered at 163.8 eV and 164.9 eV (Figure 2E) can be assigned to S 2p3/2 and S 2p1/2, respectively. While, the Fe 2p binding energies of 711.4 eV and 724.6 eV (Figure 2F) indicate the oxidation state of Fe3O4,13 further conforming that the nanoparticles grown on the graphene surfaces was Fe3O4 NPs. Peroxidase-like activity of 3D GH-5 To investigate the mimic enzyme catalytic activity of Fe3O4 NPs-loaded 3D graphene nanocomposites, the typical peroxidase substrate 3, 3', 5, 5'-tetramethylbenzidine (TMB) was chosen as the chromogenic substrate. As shown in Figure 3A, 3D GH-5 could catalyze the oxidation of TMB to produce a typical blue color reaction in the presence of H2O2 (curve c). While in the absence of 3D GH-5 or H2O2, control experiments (curve a and b) showed little oxidative reaction, indicating the peroxidase-like activity of 3D GH-5. In addition, the electrocatalytic activity of 3D GH-5 modified glassy carbon electrode (3D GH-5/GCE) towards H2O2 was studied as shown in Figure S6A. In the presence of 1 mM H2O2, the reduction current at 3D GH-5/GCE increased more obviously than that at bare GCE. Figure S6B showed the amperometric response of 3D GH-5/GCE to an aliquot of H2O2 under constant potential of -0.2 V. The response current increased steeply to reach a steady-state with addition of H2O2. These


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results indicated that 3D GH-5 could also transfer electrons between the electrode and H2O2 in the electrochemical process. Continuous cyclic voltammogram measurements were carried out in 0.5 mM H2O2 to evaluate the operational stability. It was found that the reduction current at - 0.4 V retained 90% of its initial values and no obvious potential shifts were observed after 50 scans (Figure S7), which revealed that the modified electrode had good operational stability. In order to explore the reasons for the high catalytic properties of 3D GH-5, a series of experiments were carried out by using different materials as catalysts. Firstly, 3D GN showed very weak catalytic activity as compared with that of 3D GH-5 (Figure 3B). Then, with the dosage of hemin increasing, the catalytic activities of the nanomaterials (3D GH-1, 3D GH-2 and 3D GH-5) clearly increased, and 3D GH-5 exhibited the highest peroxidase-like activity. However, when the dosage of hemin continued increasing, the nanomaterials (GH-10 and GH20) could not form a compact porous structure and they both exhibited lower catalytic activities than 3D GH-5. All these results suggest that three main factors contribute to the high peroxidaselike activity of 3D GH-5. (1) Fe3O4 NPs produced from hemin are demonstrated as an effective peroxidase mimic to catalyze the oxidation reaction. (2) 3D porous graphene structures could provide plenty of active sites for the growth of Fe3O4 NPs. (3) The porous structure of 3D GH-5 could efficiently avoid restacking and agglomeration of graphene sheets and increase the collision probability of the active molecules with Fe3O4, thus improving the nanozyme activity. Similar to HRP, the catalytic activity of 3D GH-5 is depended on the pH, temperature, H2O2 concentration and catalyst dosage (Figure S8). The peroxidase-like activity of 3D GH-5 is much higher in weakly acidic conditions and reaching the highest in pH 4.0, consisting with those reported for HRP.21, 24, 48 In the range of 20-90°C, the optimal temperature is 50°C. Meanwhile, in the wide range 35-65°C, the catalytic activity maintains over 80% (Figure S8B), which would


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certainly expand the field of applications. Therefore, we adopted pH 4.0 and 50 °C for subsequent analysis of 3D GH-5 catalytic activity. The apparent steady-state kinetic parameters for this reaction were determined by changing the concentration of TMB and H2O2 in the system, respectively (Figure S9). The Km and Vm given in Table S1 were obtained by using LineweaverBurk plot (Figure S9B and 9D). It is observed that the oxidation reaction catalyzed by 3D GH-5 follows the typical Michaelis-Menten behavior towards both substrates, TMB and H2O2 (Figure S9A and 9C). Compared with the similar component materials listed in Table S1, 3D GH-5 exhibited favorable catalytic affinity towards H2O2 and TMB. And 3D GH-5 possessed better affinity to TMB than that of nanomaterials based on graphene and Fe3O4 (GO-Fe3O435 and Fe3O4/N-GQDs13), indicating that 3D porous structure notably improves the catalytic affinity. Moreover, the stability of 3D GH-5 after long-term storage was investigated as shown in Figure S10. 86% catalytic activity was maintained even after three months of storage, suggesting the high stability of 3D GH-5. Detection glucose using 3D GH-5 Since the catalytic activity of 3D GH-5 is H2O2 concentration dependent and H2O2 is the main product of glucose oxidase (GOD)-catalyzed reaction, a simple and sensitive colorimetric glucose biosensor was developed by using the TMB system. As presented in Figure 4, the absorption spectra of TMB was increased as the concentration of glucose increasing and the absorbance was linearly correlated to glucose concentration from 0.005 mM to 0.5 mM with a detection limit of 0.8 µM, which was comparative to the conventional methods.49-55 The photo in Figure 4A showed the color changing with different concentration of glucose, indicating the obtained glucose biosensor exhibited the advantages of visual determination. The 3D GH-5 is also superior to other nanomaterials with peroxidase-like acivity in lower detection limit for


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colorimetric determination of glucose as shown in Table S2. Furthermore, the specificity of the biosensor was tested using fructose, maltose, lactose and sucrose. As shown in Figure S11, the naked eye can distinguish the color difference even the concentration of control samples was 10 times larger than glucose, suggesting an excellent selectivity of the developed glucose biosensor. To demonstrate the feasibility of this biosensor for practical applications, the analysis of glucose was carried out in 20-fold dilution human serum samples and the results are listed in Table S3. It can be seen that the recoveries of glucose fall in the range of 99.0–102.7% using the standard addition method, indicating that the GOD-based colorimetric method is applicable to determining glucose in real samples.

CONCLUSIONS In summary, we have prepared Fe3O4 NPs loaded 3D porous N-doped graphene nanocomposites through one-pot strategy by using hemin as iron source. The as-prepared 3D GH-5 exhibits outstanding peroxidase-like activity and high stability during long time storage. Compared with different catalysts (3D GN, 3D GH-1, 3D GH-2, 3D GH-5, GH-10, GH-20), the enhanced nanozyme activity of 3D GH-5 is ascribed to the intrinsic catalytic activity of Fe3O4 NPs, as well as the plenty of active sites and huge accessible surface area forming in 3D porous nanostructure. Then, a novel, highly sensitive and selective biosensor for the colorimetric detection of glucose was successfully developed based on the peroxidase-like activity of 3D GH-5. This work provides a new method to fabricate 3D porous graphene nanocomposites and will facilitate the study of the nanomaterials as peroxidase mimics.


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FIGURES.

Scheme 1. Schematic presentation for Fe3O4 NPs-loaded 3D graphene nanocomposites with peroxidase-like activity.

Figure 1. Photographs of 3D GH-5 and the freeze-dried product (A). SEM images of 3D GH-5 with low (B) and high (C) magnifications. TEM images of 3D GH-5 with low (D) and high (E) magnifications.


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Figure 2. XPS spectra of survey scan of 3D GH-5 (A); High-resolution C 1s XPS spectra of GO (B). High-resolution C 1s (C), N 1s (D), S 2p (E) and Fe 2p (F) XPS spectra of 3D GH-5.

Figure 3. The absorption spectra and digital photos of different colorimetric reaction systems: (a) TMB+H2O2, (b) TMB+3D GH-5, and (c) TMB+H2O2+3D GH-5. Experimental conditions: 0.1 M HAc-NaAc buffer solution, pH 4.0; TMB, 0.5 mM; H2O2, 5 mM; 3D GH-5, 3 µg mL-1; and different systems were incubated at 50 °C for 10 min (A). Time-dependent absorbance of TMB at 652 nm varied with different catalysts used. Experimental conditions: 0.1 M HAc-NaAc buffer


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solution, pH 4.0; TMB, 0.5 mM; H2O2, 5 mM; catalysts, 10 µg mL-1; and measured at room temperature (B).

Figure 4. The absorption spectra of TMB with different glucose concentrations from a to j: 0, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.5, 0.7 and 1.0 mM (A). Dose-response curve for glucose detection at 652 nm, the error bars represent the standard deviation of three measurements (B).

ASSOCIATED CONTENT Supporting Information Additional

information

about

characterization:

TEM,

SEM,

EDS,

typical

nitrogen

adsorption/desorption isotherm, dark-field STEM and XRD patterns; the electrocatalytic activity of 3D GH-5 modified glassy carbon electrode towards H2O2; the effect of pH and temperature on the peroxidase-like activity of 3D GH-5; kinetic studies on the peroxidase-like activity of 3D GH-5; the stability of 3D GH-5 after long-term storage; selectivity study for glucose detection. This material is available free of charge on the ACS Publications website at DOI. AUTHOR INFORMATION


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Corresponding Author *Shaojun Dong, E-mail: [email protected]. * Zhiquan Zhang, E-mail: [email protected]. Notes The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 21375123 and 21675151) and the Ministry of Science and Technology of China (Nos. 2013YQ170585 and 2016YFA0203201).

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One-Pot Synthesis of Fe3O4 Nanoparticle Loaded 3D Porous

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