Biosensors and Bioelectronics 64 (2015) 165–170
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Peroxidase-like activity of apoferritin paired gold clusters for glucose detection Xin Jiang a, Cuiji Sun b, Yi Guo a,n, Guangjun Nie b, Li Xu a,c a
Key laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Collage of Life Science, Jilin University, Changchun 130012, PR China CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology of China, Beijing 100190, PR China c National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun 130012, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 June 2014 Received in revised form 15 August 2014 Accepted 28 August 2014 Available online 3 September 2014
The discovery and application of noble metal nanoclusters have received considerable attention. In this paper, we reported that apoferritin paired gold clusters (Au–Ft) could efficiently catalyze oxidation of 3.3′,5.5′-tetramethylbenzidine (TMB) by H2O2 to produce a blue color reaction. Compared with natural enzyme, Au–Ft exhibited higher activity near acidic pH and could be used over a wide range of temperatures. Apoferritin nanocage enhanced the reaction activity of substrate TMB by H2O2. The reaction catalyzed by Au–Ft was found to follow a typical Michaelis–Menten kinetics. The kinetic parameters exhibited a lower Km value (0.097 mM) and a higher Kcat value (5.8 104 s 1) for TMB than that of horse radish peroxidase (HRP). Base on these findings, Au–Ft, acting as a peroxidase mimetic, performed enzymatic spectrophotometric analysis of glucose. This system exhibited acceptable reproducibility and high selectivity in biosening, suggesting that it could have promising applications in the future. & 2014 Elsevier B.V. All rights reserved.
Keywords: Apoferritin Gold clusters Peroxidase-like activity Kinetic study Glucose
1. Introduction In recent years, enzyme-based biosensing has attracted considerable attention due to its low cost, simplicity, and practicality (Li et al., 2013). It arises from the unique catalytic properties of enzymes with highly efficient and selective catalysis under mild conditions (Mireia et al., 2004; Wolfenden and Snider, 2001). Therefore, there has for thousands of years existed a great interest in utilizing enzymes in food processing, agriculture, the chemical industry and medicine. Peroxidases, especially horse radish peroxidase (HRP), can activate hydrogen peroxide to perform a variety of chemical reactions such as oxidation, which has been extensively investigated and applied in many different areas (Veitch, 2004). However, the most practical applications of natural enzymes are limited by the preparation techniques, reaction conditions and storage requirements (Breslow, 1995; Shoji and Freund, 2001). To minimize these limitations, researchers have devoted great efforts to construct highly stable and low-cost alternatives to enzymes (Kotov, 2010; Murakami et al., 1996). Recently, advances in nanotechnology and progress in designing functional nanomaterials, provide exciting new opportunities for catalysis. The n
Corresponding author. Tel.: þ 86 431 85155246; fax: þ 86 431 85155226. E-mail address: [email protected] (Y. Guo).
http://dx.doi.org/10.1016/j.bios.2014.08.078 0956-5663/& 2014 Elsevier B.V. All rights reserved.
concept of “nanozyme” was initially proposed by Scrimin, Pasquato, and co-workers to represent their thiol monolayer protected gold clusters with ribonuclease-like activity (Manea et al., 2004). Some nanozymes possess intrinsic peroxidase-like activity, such as ceria nanoparticles (NPs) (Jiao et al., 2012), carbon NPs (Dong et al., 2012; Li et al., 2013), Pt NPs (Fan et al., 2011; Gao et al., 2013), and Fe3O4 magnetic NPs (Dong et al., 2012; Gao et al., 2007; Wu et al., 2011). Gold NPs have also been considered as one of the most important nanozymes because of their unique properties, such as high chemical stability, feasible surface modifications, and excellent biocompatibility (Burda et al., 2005). In addition, they possess intrinsic peroxidase-like activity and provide a new route for colorimetric detection of H2O2, Hg2 þ , xanthine, etc. (Jv et al., 2010; Wang et al., 2011; Long et al., 2011). Glucose, as a source of energy for the living cells and metabolic intermediate, is very important for the public health. Maintaining blood glucose levels between 3.0 mM and 8.0 mM (Xu et al., 2007) is critical as diabetes mellitus may result in heart disease, kidney failure, blindness, etc. (Si et al., 2011; Lu et al., 2011). This necessitates the development of a fast, reliable, sensitive, and selective method for glucose determination. Many approaches including surface plasmon resonance (Al-Ogaidi et al., 2014), fluorescence (Deng et al., 2014), electrochemiluminescence (Xiao et al., 2014), and colorimetry (Tabrizi and Varkani, 2014) have been performed for glucose detection. These assays can be based on
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enzymatic activity of new nanomaterials or nanostructures within the sensor. Nanozyme is becoming a new tool for glucose detection due to its low cost, simplicity, and practicality. Moreover, the catalytic ability of nanozyme can be tuned by many important factors (Daniel and Astruc, 2004; Wang et al., 2012). According to previous studies, gold nanozymes can be divided into two major categories based on the structural features of nano-gold. One category utilizes monolayer protected gold particles as catalysts and biomimetic catalytic activities, which derive from the functional groups of the modification shell (Bonomi et al., 2008; Kisailus et al., 2005). Another category involves small and stable gold NPs with various surface states and the intrinsic activities, which are attributable to the nanomaterial core (Luo et al., 2010). For example, the charge properties of the coating, which affect the interaction between nanoparticles and substrates, plays an important role in their activity (Jv et al., 2010). Additionally, ultrasmall metal clusters exhibit superior catalytic activity due to their high surface energy that makes surface atoms fairly active (Biswas et al., 2012; Wang et al., 2014). Apoferritin, is a 24-subunit spherical protein complex (450 kDa) with a nanoscale hollow interior and good biocompatiblity (Arosio et al., 2009). It is a physiological protein that impacts iron metabolism and oxidative stress regulation, and also shows unexpected enzymatic activities (Friedman et al., 2011). Recently, apoferritin has been used as a size-constrained reaction vessel to direct nanostructure synthesis. We have already successfully synthesized apoferritin paired gold clusters (Au–Ft) according to the “points of control” synthesis strategy (Sun et al., 2011). Within the apoferritin nanocage, the ferroxidase center is composed of six amino acid residues: histidine (His), aspartic acid (Asp), glutamine (Gln), and three glutamic acids (Glu). Gold clusters can bind strongly with the His residues at ferroxidase center of H-ferritin (Ueno et al., 2009). This can produce an enzyme active center that simulates natural enzyme micro-environment, thereby facilitating substrate molecular binding ability and stabilizing the enzyme–substrate complex. Therefore, this model could be a great mimicry system of enzymes. Protein– inorganic nanozymes designed by utilizing nature's strategy could insure biocompatibility and enable controllable catalysis. This gold nanozyme with paired ultrasmall size could be a novel platform for peroxidase-like activity. In this work, we discovered that apoferritin paired gold clusters (Au–Ft) can act as enzymatic mimics possessing intrinsic peroxidase-like activity. The clusters can catalyze the reaction of peroxidase substrate 3.3.5.5-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a color reaction. Compared to larger colloidal gold nanoparticles, it was found that Au–Ft exhibit highest catalytic activity. Moreover, kinetic studies demonstrated that the catalytic reaction followed a ping-pong mechanism and Au–Ft had even higher catalytic activity to TMB than natural HRP. Combining the catalytic reaction by glucose oxidase (GOx) and Au– Ft, this novel mimicry enzyme was used successfully to detect glucose detection (as shown in Fig. 1). This method exhibits a relatively high selective response to glucose detection and could also have potential applications in the fields of environmental chemistry and biomedicine.
2. Materials and methods 2.1. Reagents and materials Horseradish peroxidase (HRP) and glucose oxidase were purchased from Roche. 3.3′,5.5′-tetramethylbenzidine dihydrochloridewere (TMB) was obtained from Boston Biomedical Inc. (USA). The BCA protein assay kit, horse spleen apoFt, and HAuCl4 were
Fig. 1. Schematic illustration of colorimetric determination of glucose using glucose oxidase (GOx) and Au–Ft catalyzed reactions.
purchased from Sigma-Aldrich. All other chemicals, such as H2O2 solution (30 wt% aqueous), NaOH, HOAc, NaOAc, and HCl were obtained from Beijing Chemicals Reagent Company (Beijing, China). All reagents were of analytical grade and used without further purification. All solutions were prepared with ultrapure water purified by a Millipore water purification system (Z18.2 MΩ, Milli-Q, Millipore). 2.2. Synthesis and characterization of apoferritin paired gold clusters The preparation of Au–Ft was carried out in an aqueous solution as previously described (Sun et al., 2011). 300 μL of 5 mM HAuCl4 was added to 300 μL of horse spleen apoFt (49 mg/mL). To avoid apoFt subunit dissociation under acidic conditions, HAuCl4 was first adjusted to pH 7 before mixing with apoFt. After the mixture was mixed for 2 min, 20 μL of 1 M NaOH was added to the solution, followed by incubation for 12 h at 37 °C. The reaction solution was ultrafiltered with a centrifugal filter device (Amicon Ultra-15; 30,000 molecular weight cut off) and washed three times with 3 mL of ultrapure water. The concentration of Au–Ft was measured by the bicinchoninic acid method (BCA). An aliquot of 4 μL of Au–Ft was applied to a glow-discharged grid coated with a layer of amorphous carbon film, and excess fluid was gently blotted off with filter paper. The HRTEM images were recorded on an FEI Tecnai F20 U-TWIN electron microscope. TEM analysis was performed on a JEOL JEM-1200EX model transmission electron microscope. For negative staining, a dispersion of Au–Ft clusters was dropped onto a carbon-coated copper grid, dried inair at room temperature, and stained with 2% uranyl acetate. 2.3. Mimetic peroxidase activity assays HRP-like activity was examined using TMB as a chromogenic substrate. Experiments were carried out using 0.58 μg Au–Ft or 0.50 ng HRP in a reaction volume of 1 mL, in 0.2 M HOAc–NaOAc buffer with 300 mM TMB. The concentration was 300 mM for Au– Ft and 4 mM for HRP. The pH was 4.0 for both Au–Ft clusters and HRP. Before the reaction, the mixture was incubated at 37 °C for HRP and 45 °C for Au–Ft clusters. After 3 min, the color formation was monitored at 652 nm using Shimadzu UV-2550 spectrophotometer after adding H2O2 to the reaction. To compare the influence of the reaction buffer pH on the relative activity of Au– Ft and HRP, 0.2 M NaOAc buffer solutions from pH 2.0 to 12.0 were investigated at 45 °C and 37 °C, respectively. To examine the influence of incubation temperature on the relative activity of Au–Ft and HRP, catalytic reactions incubated in water baths from 4 to 90 °C were investigated at pH 4.0. 2.4. Reaction mechanism and kinetic analysis The reaction kinetics for the catalytic oxidation of TMB was carried out by recording the absorption spectra at 652 nm with a
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1 min interval in scanning kinetics mode using a Shimadzu UV2550 spectrophotometer. Unless otherwise stated, the reaction was carried out in the presence of varied concentrations of TMB and H2O2. The apparent kinetic parameters were calculated based on the Michaelis equation: v ¼Vmax 1/(Km þ[S]), where V is the initial velocity, Vmax is the maximal reaction velocity, [S] is the concentration of substrate and Km is the Michaelis constant. Km and Vmax were obtained by the Lineweavere–Burk plot method. To further investigate the mechanism, assays were carried out under standard reaction conditions as described above by varying concentrations of TMB at a fixed concentration of H2O2 or vice versa. 2.5. Glucose detection Glucose detection was performed as follows: (a) 20 uL of 20 mg/mL GOx and 200 uL of glucose at different concentrations in 10 mM phosphate buffered saline (PBS, pH 7.0) were incubated at 37 °C for 40 min; (b) 40 uL of 30 mM TMB, 10 uL of the Au–Ft stock solution, and 800 uL of 0.2 HOAc–NaOAc buffer (pH 4.0) were added into the 220 uL glucose reaction solution; (c) the mixed solution was incubated in a 45 °C water bath for 10 min, and 900 uL of the final reaction solution was used to perform the adsorption spectroscopy measurement at 652 nm. In control
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experiments, 5 mM maltose, 5 mM lactose, and 5 mM fructose were used instead of glucose, under conditions identical to those reported above.
3. Results and discussion 3.1. Characterization of Au-Ft The aqueous solution of the Au cluster–apoferritin nanostructures showed orange in color and exhibited light pink fluorescence under ultraviolet light. As shown in Fig. S1(a), the emission spectra of the prepared Au–Ft clusters was about 504 nm upon excitation at 455 nm. The features of the spectra were similar to those obtained in our previous study (Sun et al., 2011). The paired Au clusters with a diameter of less than 2 nm could be observed in HRTEM (Fig. S1(b)). Negatively stained TEM of Au–Ft clusters show that apoferritin cages are monodisperse (Fig. S1(c)). As reported in our previous study (Sun et al., 2011), this nanostructure was formed by reduction of Au ions in a apoferritin shell. The gold clusters that were formed in the apoferritin solution could have been stabilized by the strong binding of Au3 þ to the imidazole ring of the His residues in the ferroxidase center of H-ferritin. 3.2. Au-Ft catalyses the oxidation of peroxidase substrates To investigate the catalytic activity of Au–Ft, the catalytic oxidation process of the peroxidase substrate TMB in the presence of H2O2 was tested. As shown in Fig. 2, Au–Ft can catalyze the oxidation of TMB in the presence of H2O2 to produce a blue color reaction. The absorbance peak at 652 nm appeared with higher peak value, which originated from the oxidation product of TMB (Marquez and Dunford, 1997). Notably, the experimental conditions without Au–Ft or H2O2 showed negligible color variations. This result indicates that both components are required for catalysis. We also examined the size effect of gold nanoparticles on peroxidase-like activity. Compared with 6 nm or 20 nm gold NPs, Au–Ft showed markedly higher catalytic activity toward TMB (Table S1). 3.3. Catalytic relative activity of Au–Ft clusters at varying pH and temperature
Fig. 2. Typical absorption spectra of aqueous suspensions of the corresponding reactions and the images of TMB reaction solutions incubated at 45 °C with 0.6 mM TMB in 0.2 M HOAc–NaOAc buffer (pH 4.0), and (a) 0 mM H2O2 with Au–Ft, (b) 10 mM H2O2 without Au–Ft, and (c) 10 mM H2O2 with Au–Ft. (For interpretation of the references to color in this figure the reader is referred to the web version of this article.)
Similarly to natural HRP, the catalytic activity of the Au–Ft nanozyme systems is also dependent on pH and temperature. We examined the peroxidase-like activity of the Au–Ft while varying the pH from 2 to 12 and the temperature from 4 °C to 85 °C. The results were then compared to the results found with HRP over the
Fig. 3. The catalytic relative activity of HRP and Au–Ft is pH (a) and temperature (b) dependent. The error bars represent the standard deviation for three measurements.
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same range (Fig. 3(a) and (b)). The catalytic oxidation activity of TMB with H2O2 using Au–Ft was much higher in acidic solution than in neutral or basic solutions, as was the case for the natural enzyme. The optimum pH was around 4. However, the optimal temperature of 45 °C was considerably higher than that of native HRP (37 °C). The peroxidase mimicry system displayed higher thermal stability than HRP. Based on these results, a pH of 4.0 and a temperature of 45 °C was used as optimal reaction conditions for subsequent analysis of Au–Ft. According to a previous study, the subunits of apoferritin can disassemble at low pH (2.0) (Aime et al., 2002). Therefore, the composition of Au– Ft at pH 2.0 is naked gold clusters, causing a marked decrease in activity. This observation indicates that the apoferritin protein shell dramatically improves the catalytic activity of gold clusters.
3.4. Kinetic study and mechanism of Au–Ft catalytic reactivity To investigate the reaction mechanism of Au–Ft as a mimetic enzyme, the steady-state kinetic parameters for the reaction were measured. The slopes of the initial absorbance curves were calculated with the Lambert–Beer Law using a molar absorption coefficient for TMB derived oxidation products over time. Typical Michaelis–Menten curves were obtained for Au–Ft with TMB or H2O2 as substrates (Fig. 4(a) and (b)). An obvious dependence relationship was found between the initial reaction rate and the substrate concentration. Next, the Lineweaver–Burk plot method was used to investigate whether the catalytic reaction of Au–Ft followed the Michaelis–Menten behavior. As shown in Fig. 4 (c) and (d), the reciprocal of the initial rate was directly proportional to the reciprocal of the substrate concentration. The double
Fig. 4. Steady-state kinetic assay and catalytic mechanism of Au–Ft (a–f): The velocity (v) of the reaction was measured using 0.58 μg mL 1 Au–Ft in 1 mL NaOAc buffer at pH 3.0 and 45 °C. (a and c) The concentration of H2O2 was 100 mM for Au–Ft and the TMB concentration varied; (b and d) The concentration of TMB was 0.3 mM and the H2O2 concentration varied; and (e and f) double reciprocal plots of activity of Au–Ft with the concentration of one substrate (H2O2 or TMB) fixed and the other varied. The error bars represent the standard deviation for three measurements.
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reciprocal of the Michaelis–Menten equation was applied to obtain the catalytic parameters, as shown in Table S2. The apparent Km value of Au–Ft was significantly higher than that for HRP towards H2O2 (Table S2), indicating that Au–Ft had a lower affinity than HRP with H2O2 as a substrate. That result was corresponding to the observation that a higher concentration of H2O2 was required to obtain a maximal reaction velocity for Au–Ft. Meanwhile, the apparent Km value of Au–Ft was more than four times lower than that of HRP towards TMB, indicating that Au–Ft had a higher affinity for TMB than HRP. In addition, the Kcat value of Au–Ft showed a significantly higher level of activity than HRP at the same molar concentration. In comparison with other reported nanoparticles with peroxidase-like activities such as FeS (Km ¼0.13) (Dai et al., 2009), Prussian blue-Fe2O3 (Km ¼0.307) (Zhang et al., 2010), and ZnFe2O4 (Km ¼0.85) (Su et al., 2012) (Table S2), Au–Ft had the smallest Km value, which may be due to its small size and unique cage structure. To further explore the catalytic mechanism of Au–Ft, the catalytic efficiency of Au–Ft was measured under standard reaction conditions with varying concentrations of H2O2 and a fixed concentration of TMB or vice versa. Fig. 4(e) and (f) showed double reciprocal plots of initial velocity against the concentration of one substrate in a range of concentrations of the second substrate. Several parallel slopes obtained from the data lines, were the characteristic of a ping-pong mechanism, as has been observed for HPR (Gao et al., 2007). These results indicate that Au–Ft reacts with the first substrate and then releases the first product before reacting with the second substrate. As previous reports have pointed out, this product should be a hydroxyl radical (HO∙) which originated from the decomposition of H2O2 during the catalytic reaction of noble metal nanoparicles (Gao et al., 2007; Song et al., 2010). In fact, the O-O bond of H2O2 may be broken into double HO∙ by AuNPs (Murakami et al., 1996). Recently, Jv et al. have proposed that the stabilization of hydroxide free radicals may take place through partial electron exchange with AuNPs, which could aid the catalytic ability of AuNPs (Jv et al., 2010). Consequently, the generated HO∙ radical would become more stable as a result of two gold clusters within the apoferritin cavity, which could increase the catalytic activity of Au–Ft. This phenomenon could explain the decrease in catalytic activity upon disassembly of apoferritin nanostructures. 3.5. Comparison of rubustness of Au–Ft and native enzymes The stability of Au–Ft was evaluated and compared to that of HRP (shown in Fig. S2). HRP and Au–Ft were incubated in solutions
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with various pH (0–12) and temperatures (4–90 °C) for 2 h, after which their activities were measured under optimal reaction conditions. The activity of Au–Ft was maintained at different pH values (above 80% activity). However, the native HRP progressively lost most of its activity above pH 6. Au–Ft was stable at all temperatures, except above 70 °C, while the native HRP showed significant enzymatic activity only between 4 °C (storage temperature) and 50 °C. The robustness of Au–Ft makes it a suitable tool for a broad range of applications.
3.6. Glucose detection Based on the intrinsic peroxidase catalytic activity of Au–Ft, a colorimetric detection system for glucose detection was evaluted by combining the mimetic enzyme and glucose oxidase. As demonstrated above, the color variation of TMB oxidation catalyzed by Au–Ft was H2O2 concentration dependent, which indicated that the change of the absorbance at 652 nm can be used for H2O2 detection. When coupled with the glucose catalytic reaction by glucose oxidase, we established a colorimetric sensor for glucose detection. Fig. 5(a) shows the calibration curve for glucose detection obtained under conventional conditions. The linear response (R¼0.997) of the absorbance (652 nm) versus glucose concentration is in the range from 2.0 mM to 10.0 mM. According to a previous report (Xu et al., 2007), the blood glucose range in a healthy persons is 3.0–8.0 mM. Higher glucose values will damage the body and can lead to multiple health problems associated with diabetes. Therefore, glucose concentration range from 2.0 mM to 10.0 mM, has practical relevance from a physiological perspective. The coefficient of variation (CV) of the different standard samples is listed in Table S3. To examine the selectivity of detection of glucose, the control experiments were taken under the same conditions using maltose, fructose, and lactose. As shown in Fig. 5(b), there was no detectable signal obtained from the control samples with other saccharides at concentrations of 5.0 mM. Thus, other saccharides will not interfere with the detection of glucose. The value of coefficient of variation in three repeated measurements of 5.0 mM was 4.35% (Table S3). In essence, the developed method shows acceptable reproducibility and high selectivity towards glucose at a normal physiological range. Thus, the Au–Ft glucose sensor system could be a new future candidate for glucose detection.
Fig. 5. Glucose detection based on peroxidase-like Au–Ft. (a): The linear calibration plot for glucose. (b): Specificity analysis of glucose detection. The difference in absorbance between glucose and other sugars (the concentration is 5 mM). The error bars represent the standard deviation for three measurements.
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4. Conclusion In conclusion, our results demonstrate that Au–Ft possesses intrinsic peroxidase-like activity and performs catalysis over a wide range of pH values and temperatures comparable with natural enzymes. Intriguingly, the paired gold clusters in the apoferritin cavity possess high catalytic activity to the substrates. Catalysis by Au–Ft shows a typical Michaelis–Menten kinetics and exhibits a ping-pong mechanism, which is similar to that of the natural enzyme. Based on these findings, a simple and selective assay for glucose detection was developed. This work will not only promote the development of noble metal nanomaterials as peroxidase mimics but also provide a wide range of potential applications for Au mimetic enzyme systems in bio-detection, catalysis, and clinical diagnostics.
Acknowledgment The work was supported by the National Natural Science Foundation of China (No. 81271697), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Nos. 20100061120077, 20120061110021), the Social Development Project of Science and Technology Department of Jilin Province, China (Nos. 20106031, 20120967, YYZX201264, 20130206069GX), the Fundamental Research Funds for the Central Universities, and “Significant New Drug Creation” Science and Technology Major Program (No. 2012ZX09503001-003).
Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.078.
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Peroxidase-like activity of apoferritin paired gold clusters for glucose detection Xin Jiang a, Cuiji Sun b, Yi Guo a,n, Guangjun Nie b, Li Xu a,c a
Key laboratory for Molecular Enzymology and Engineering, the Ministry of Education, Collage of Life Science, Jilin University, Changchun 130012, PR China CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology of China, Beijing 100190, PR China c National Engineering Laboratory for AIDS Vaccine, Jilin University, Changchun 130012, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 June 2014 Received in revised form 15 August 2014 Accepted 28 August 2014 Available online 3 September 2014
The discovery and application of noble metal nanoclusters have received considerable attention. In this paper, we reported that apoferritin paired gold clusters (Au–Ft) could efficiently catalyze oxidation of 3.3′,5.5′-tetramethylbenzidine (TMB) by H2O2 to produce a blue color reaction. Compared with natural enzyme, Au–Ft exhibited higher activity near acidic pH and could be used over a wide range of temperatures. Apoferritin nanocage enhanced the reaction activity of substrate TMB by H2O2. The reaction catalyzed by Au–Ft was found to follow a typical Michaelis–Menten kinetics. The kinetic parameters exhibited a lower Km value (0.097 mM) and a higher Kcat value (5.8 104 s 1) for TMB than that of horse radish peroxidase (HRP). Base on these findings, Au–Ft, acting as a peroxidase mimetic, performed enzymatic spectrophotometric analysis of glucose. This system exhibited acceptable reproducibility and high selectivity in biosening, suggesting that it could have promising applications in the future. & 2014 Elsevier B.V. All rights reserved.
Keywords: Apoferritin Gold clusters Peroxidase-like activity Kinetic study Glucose
1. Introduction In recent years, enzyme-based biosensing has attracted considerable attention due to its low cost, simplicity, and practicality (Li et al., 2013). It arises from the unique catalytic properties of enzymes with highly efficient and selective catalysis under mild conditions (Mireia et al., 2004; Wolfenden and Snider, 2001). Therefore, there has for thousands of years existed a great interest in utilizing enzymes in food processing, agriculture, the chemical industry and medicine. Peroxidases, especially horse radish peroxidase (HRP), can activate hydrogen peroxide to perform a variety of chemical reactions such as oxidation, which has been extensively investigated and applied in many different areas (Veitch, 2004). However, the most practical applications of natural enzymes are limited by the preparation techniques, reaction conditions and storage requirements (Breslow, 1995; Shoji and Freund, 2001). To minimize these limitations, researchers have devoted great efforts to construct highly stable and low-cost alternatives to enzymes (Kotov, 2010; Murakami et al., 1996). Recently, advances in nanotechnology and progress in designing functional nanomaterials, provide exciting new opportunities for catalysis. The n
Corresponding author. Tel.: þ 86 431 85155246; fax: þ 86 431 85155226. E-mail address: [email protected] (Y. Guo).
http://dx.doi.org/10.1016/j.bios.2014.08.078 0956-5663/& 2014 Elsevier B.V. All rights reserved.
concept of “nanozyme” was initially proposed by Scrimin, Pasquato, and co-workers to represent their thiol monolayer protected gold clusters with ribonuclease-like activity (Manea et al., 2004). Some nanozymes possess intrinsic peroxidase-like activity, such as ceria nanoparticles (NPs) (Jiao et al., 2012), carbon NPs (Dong et al., 2012; Li et al., 2013), Pt NPs (Fan et al., 2011; Gao et al., 2013), and Fe3O4 magnetic NPs (Dong et al., 2012; Gao et al., 2007; Wu et al., 2011). Gold NPs have also been considered as one of the most important nanozymes because of their unique properties, such as high chemical stability, feasible surface modifications, and excellent biocompatibility (Burda et al., 2005). In addition, they possess intrinsic peroxidase-like activity and provide a new route for colorimetric detection of H2O2, Hg2 þ , xanthine, etc. (Jv et al., 2010; Wang et al., 2011; Long et al., 2011). Glucose, as a source of energy for the living cells and metabolic intermediate, is very important for the public health. Maintaining blood glucose levels between 3.0 mM and 8.0 mM (Xu et al., 2007) is critical as diabetes mellitus may result in heart disease, kidney failure, blindness, etc. (Si et al., 2011; Lu et al., 2011). This necessitates the development of a fast, reliable, sensitive, and selective method for glucose determination. Many approaches including surface plasmon resonance (Al-Ogaidi et al., 2014), fluorescence (Deng et al., 2014), electrochemiluminescence (Xiao et al., 2014), and colorimetry (Tabrizi and Varkani, 2014) have been performed for glucose detection. These assays can be based on
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enzymatic activity of new nanomaterials or nanostructures within the sensor. Nanozyme is becoming a new tool for glucose detection due to its low cost, simplicity, and practicality. Moreover, the catalytic ability of nanozyme can be tuned by many important factors (Daniel and Astruc, 2004; Wang et al., 2012). According to previous studies, gold nanozymes can be divided into two major categories based on the structural features of nano-gold. One category utilizes monolayer protected gold particles as catalysts and biomimetic catalytic activities, which derive from the functional groups of the modification shell (Bonomi et al., 2008; Kisailus et al., 2005). Another category involves small and stable gold NPs with various surface states and the intrinsic activities, which are attributable to the nanomaterial core (Luo et al., 2010). For example, the charge properties of the coating, which affect the interaction between nanoparticles and substrates, plays an important role in their activity (Jv et al., 2010). Additionally, ultrasmall metal clusters exhibit superior catalytic activity due to their high surface energy that makes surface atoms fairly active (Biswas et al., 2012; Wang et al., 2014). Apoferritin, is a 24-subunit spherical protein complex (450 kDa) with a nanoscale hollow interior and good biocompatiblity (Arosio et al., 2009). It is a physiological protein that impacts iron metabolism and oxidative stress regulation, and also shows unexpected enzymatic activities (Friedman et al., 2011). Recently, apoferritin has been used as a size-constrained reaction vessel to direct nanostructure synthesis. We have already successfully synthesized apoferritin paired gold clusters (Au–Ft) according to the “points of control” synthesis strategy (Sun et al., 2011). Within the apoferritin nanocage, the ferroxidase center is composed of six amino acid residues: histidine (His), aspartic acid (Asp), glutamine (Gln), and three glutamic acids (Glu). Gold clusters can bind strongly with the His residues at ferroxidase center of H-ferritin (Ueno et al., 2009). This can produce an enzyme active center that simulates natural enzyme micro-environment, thereby facilitating substrate molecular binding ability and stabilizing the enzyme–substrate complex. Therefore, this model could be a great mimicry system of enzymes. Protein– inorganic nanozymes designed by utilizing nature's strategy could insure biocompatibility and enable controllable catalysis. This gold nanozyme with paired ultrasmall size could be a novel platform for peroxidase-like activity. In this work, we discovered that apoferritin paired gold clusters (Au–Ft) can act as enzymatic mimics possessing intrinsic peroxidase-like activity. The clusters can catalyze the reaction of peroxidase substrate 3.3.5.5-tetramethylbenzidine (TMB) in the presence of H2O2 to produce a color reaction. Compared to larger colloidal gold nanoparticles, it was found that Au–Ft exhibit highest catalytic activity. Moreover, kinetic studies demonstrated that the catalytic reaction followed a ping-pong mechanism and Au–Ft had even higher catalytic activity to TMB than natural HRP. Combining the catalytic reaction by glucose oxidase (GOx) and Au– Ft, this novel mimicry enzyme was used successfully to detect glucose detection (as shown in Fig. 1). This method exhibits a relatively high selective response to glucose detection and could also have potential applications in the fields of environmental chemistry and biomedicine.
2. Materials and methods 2.1. Reagents and materials Horseradish peroxidase (HRP) and glucose oxidase were purchased from Roche. 3.3′,5.5′-tetramethylbenzidine dihydrochloridewere (TMB) was obtained from Boston Biomedical Inc. (USA). The BCA protein assay kit, horse spleen apoFt, and HAuCl4 were
Fig. 1. Schematic illustration of colorimetric determination of glucose using glucose oxidase (GOx) and Au–Ft catalyzed reactions.
purchased from Sigma-Aldrich. All other chemicals, such as H2O2 solution (30 wt% aqueous), NaOH, HOAc, NaOAc, and HCl were obtained from Beijing Chemicals Reagent Company (Beijing, China). All reagents were of analytical grade and used without further purification. All solutions were prepared with ultrapure water purified by a Millipore water purification system (Z18.2 MΩ, Milli-Q, Millipore). 2.2. Synthesis and characterization of apoferritin paired gold clusters The preparation of Au–Ft was carried out in an aqueous solution as previously described (Sun et al., 2011). 300 μL of 5 mM HAuCl4 was added to 300 μL of horse spleen apoFt (49 mg/mL). To avoid apoFt subunit dissociation under acidic conditions, HAuCl4 was first adjusted to pH 7 before mixing with apoFt. After the mixture was mixed for 2 min, 20 μL of 1 M NaOH was added to the solution, followed by incubation for 12 h at 37 °C. The reaction solution was ultrafiltered with a centrifugal filter device (Amicon Ultra-15; 30,000 molecular weight cut off) and washed three times with 3 mL of ultrapure water. The concentration of Au–Ft was measured by the bicinchoninic acid method (BCA). An aliquot of 4 μL of Au–Ft was applied to a glow-discharged grid coated with a layer of amorphous carbon film, and excess fluid was gently blotted off with filter paper. The HRTEM images were recorded on an FEI Tecnai F20 U-TWIN electron microscope. TEM analysis was performed on a JEOL JEM-1200EX model transmission electron microscope. For negative staining, a dispersion of Au–Ft clusters was dropped onto a carbon-coated copper grid, dried inair at room temperature, and stained with 2% uranyl acetate. 2.3. Mimetic peroxidase activity assays HRP-like activity was examined using TMB as a chromogenic substrate. Experiments were carried out using 0.58 μg Au–Ft or 0.50 ng HRP in a reaction volume of 1 mL, in 0.2 M HOAc–NaOAc buffer with 300 mM TMB. The concentration was 300 mM for Au– Ft and 4 mM for HRP. The pH was 4.0 for both Au–Ft clusters and HRP. Before the reaction, the mixture was incubated at 37 °C for HRP and 45 °C for Au–Ft clusters. After 3 min, the color formation was monitored at 652 nm using Shimadzu UV-2550 spectrophotometer after adding H2O2 to the reaction. To compare the influence of the reaction buffer pH on the relative activity of Au– Ft and HRP, 0.2 M NaOAc buffer solutions from pH 2.0 to 12.0 were investigated at 45 °C and 37 °C, respectively. To examine the influence of incubation temperature on the relative activity of Au–Ft and HRP, catalytic reactions incubated in water baths from 4 to 90 °C were investigated at pH 4.0. 2.4. Reaction mechanism and kinetic analysis The reaction kinetics for the catalytic oxidation of TMB was carried out by recording the absorption spectra at 652 nm with a
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1 min interval in scanning kinetics mode using a Shimadzu UV2550 spectrophotometer. Unless otherwise stated, the reaction was carried out in the presence of varied concentrations of TMB and H2O2. The apparent kinetic parameters were calculated based on the Michaelis equation: v ¼Vmax 1/(Km þ[S]), where V is the initial velocity, Vmax is the maximal reaction velocity, [S] is the concentration of substrate and Km is the Michaelis constant. Km and Vmax were obtained by the Lineweavere–Burk plot method. To further investigate the mechanism, assays were carried out under standard reaction conditions as described above by varying concentrations of TMB at a fixed concentration of H2O2 or vice versa. 2.5. Glucose detection Glucose detection was performed as follows: (a) 20 uL of 20 mg/mL GOx and 200 uL of glucose at different concentrations in 10 mM phosphate buffered saline (PBS, pH 7.0) were incubated at 37 °C for 40 min; (b) 40 uL of 30 mM TMB, 10 uL of the Au–Ft stock solution, and 800 uL of 0.2 HOAc–NaOAc buffer (pH 4.0) were added into the 220 uL glucose reaction solution; (c) the mixed solution was incubated in a 45 °C water bath for 10 min, and 900 uL of the final reaction solution was used to perform the adsorption spectroscopy measurement at 652 nm. In control
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experiments, 5 mM maltose, 5 mM lactose, and 5 mM fructose were used instead of glucose, under conditions identical to those reported above.
3. Results and discussion 3.1. Characterization of Au-Ft The aqueous solution of the Au cluster–apoferritin nanostructures showed orange in color and exhibited light pink fluorescence under ultraviolet light. As shown in Fig. S1(a), the emission spectra of the prepared Au–Ft clusters was about 504 nm upon excitation at 455 nm. The features of the spectra were similar to those obtained in our previous study (Sun et al., 2011). The paired Au clusters with a diameter of less than 2 nm could be observed in HRTEM (Fig. S1(b)). Negatively stained TEM of Au–Ft clusters show that apoferritin cages are monodisperse (Fig. S1(c)). As reported in our previous study (Sun et al., 2011), this nanostructure was formed by reduction of Au ions in a apoferritin shell. The gold clusters that were formed in the apoferritin solution could have been stabilized by the strong binding of Au3 þ to the imidazole ring of the His residues in the ferroxidase center of H-ferritin. 3.2. Au-Ft catalyses the oxidation of peroxidase substrates To investigate the catalytic activity of Au–Ft, the catalytic oxidation process of the peroxidase substrate TMB in the presence of H2O2 was tested. As shown in Fig. 2, Au–Ft can catalyze the oxidation of TMB in the presence of H2O2 to produce a blue color reaction. The absorbance peak at 652 nm appeared with higher peak value, which originated from the oxidation product of TMB (Marquez and Dunford, 1997). Notably, the experimental conditions without Au–Ft or H2O2 showed negligible color variations. This result indicates that both components are required for catalysis. We also examined the size effect of gold nanoparticles on peroxidase-like activity. Compared with 6 nm or 20 nm gold NPs, Au–Ft showed markedly higher catalytic activity toward TMB (Table S1). 3.3. Catalytic relative activity of Au–Ft clusters at varying pH and temperature
Fig. 2. Typical absorption spectra of aqueous suspensions of the corresponding reactions and the images of TMB reaction solutions incubated at 45 °C with 0.6 mM TMB in 0.2 M HOAc–NaOAc buffer (pH 4.0), and (a) 0 mM H2O2 with Au–Ft, (b) 10 mM H2O2 without Au–Ft, and (c) 10 mM H2O2 with Au–Ft. (For interpretation of the references to color in this figure the reader is referred to the web version of this article.)
Similarly to natural HRP, the catalytic activity of the Au–Ft nanozyme systems is also dependent on pH and temperature. We examined the peroxidase-like activity of the Au–Ft while varying the pH from 2 to 12 and the temperature from 4 °C to 85 °C. The results were then compared to the results found with HRP over the
Fig. 3. The catalytic relative activity of HRP and Au–Ft is pH (a) and temperature (b) dependent. The error bars represent the standard deviation for three measurements.
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same range (Fig. 3(a) and (b)). The catalytic oxidation activity of TMB with H2O2 using Au–Ft was much higher in acidic solution than in neutral or basic solutions, as was the case for the natural enzyme. The optimum pH was around 4. However, the optimal temperature of 45 °C was considerably higher than that of native HRP (37 °C). The peroxidase mimicry system displayed higher thermal stability than HRP. Based on these results, a pH of 4.0 and a temperature of 45 °C was used as optimal reaction conditions for subsequent analysis of Au–Ft. According to a previous study, the subunits of apoferritin can disassemble at low pH (2.0) (Aime et al., 2002). Therefore, the composition of Au– Ft at pH 2.0 is naked gold clusters, causing a marked decrease in activity. This observation indicates that the apoferritin protein shell dramatically improves the catalytic activity of gold clusters.
3.4. Kinetic study and mechanism of Au–Ft catalytic reactivity To investigate the reaction mechanism of Au–Ft as a mimetic enzyme, the steady-state kinetic parameters for the reaction were measured. The slopes of the initial absorbance curves were calculated with the Lambert–Beer Law using a molar absorption coefficient for TMB derived oxidation products over time. Typical Michaelis–Menten curves were obtained for Au–Ft with TMB or H2O2 as substrates (Fig. 4(a) and (b)). An obvious dependence relationship was found between the initial reaction rate and the substrate concentration. Next, the Lineweaver–Burk plot method was used to investigate whether the catalytic reaction of Au–Ft followed the Michaelis–Menten behavior. As shown in Fig. 4 (c) and (d), the reciprocal of the initial rate was directly proportional to the reciprocal of the substrate concentration. The double
Fig. 4. Steady-state kinetic assay and catalytic mechanism of Au–Ft (a–f): The velocity (v) of the reaction was measured using 0.58 μg mL 1 Au–Ft in 1 mL NaOAc buffer at pH 3.0 and 45 °C. (a and c) The concentration of H2O2 was 100 mM for Au–Ft and the TMB concentration varied; (b and d) The concentration of TMB was 0.3 mM and the H2O2 concentration varied; and (e and f) double reciprocal plots of activity of Au–Ft with the concentration of one substrate (H2O2 or TMB) fixed and the other varied. The error bars represent the standard deviation for three measurements.
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reciprocal of the Michaelis–Menten equation was applied to obtain the catalytic parameters, as shown in Table S2. The apparent Km value of Au–Ft was significantly higher than that for HRP towards H2O2 (Table S2), indicating that Au–Ft had a lower affinity than HRP with H2O2 as a substrate. That result was corresponding to the observation that a higher concentration of H2O2 was required to obtain a maximal reaction velocity for Au–Ft. Meanwhile, the apparent Km value of Au–Ft was more than four times lower than that of HRP towards TMB, indicating that Au–Ft had a higher affinity for TMB than HRP. In addition, the Kcat value of Au–Ft showed a significantly higher level of activity than HRP at the same molar concentration. In comparison with other reported nanoparticles with peroxidase-like activities such as FeS (Km ¼0.13) (Dai et al., 2009), Prussian blue-Fe2O3 (Km ¼0.307) (Zhang et al., 2010), and ZnFe2O4 (Km ¼0.85) (Su et al., 2012) (Table S2), Au–Ft had the smallest Km value, which may be due to its small size and unique cage structure. To further explore the catalytic mechanism of Au–Ft, the catalytic efficiency of Au–Ft was measured under standard reaction conditions with varying concentrations of H2O2 and a fixed concentration of TMB or vice versa. Fig. 4(e) and (f) showed double reciprocal plots of initial velocity against the concentration of one substrate in a range of concentrations of the second substrate. Several parallel slopes obtained from the data lines, were the characteristic of a ping-pong mechanism, as has been observed for HPR (Gao et al., 2007). These results indicate that Au–Ft reacts with the first substrate and then releases the first product before reacting with the second substrate. As previous reports have pointed out, this product should be a hydroxyl radical (HO∙) which originated from the decomposition of H2O2 during the catalytic reaction of noble metal nanoparicles (Gao et al., 2007; Song et al., 2010). In fact, the O-O bond of H2O2 may be broken into double HO∙ by AuNPs (Murakami et al., 1996). Recently, Jv et al. have proposed that the stabilization of hydroxide free radicals may take place through partial electron exchange with AuNPs, which could aid the catalytic ability of AuNPs (Jv et al., 2010). Consequently, the generated HO∙ radical would become more stable as a result of two gold clusters within the apoferritin cavity, which could increase the catalytic activity of Au–Ft. This phenomenon could explain the decrease in catalytic activity upon disassembly of apoferritin nanostructures. 3.5. Comparison of rubustness of Au–Ft and native enzymes The stability of Au–Ft was evaluated and compared to that of HRP (shown in Fig. S2). HRP and Au–Ft were incubated in solutions
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with various pH (0–12) and temperatures (4–90 °C) for 2 h, after which their activities were measured under optimal reaction conditions. The activity of Au–Ft was maintained at different pH values (above 80% activity). However, the native HRP progressively lost most of its activity above pH 6. Au–Ft was stable at all temperatures, except above 70 °C, while the native HRP showed significant enzymatic activity only between 4 °C (storage temperature) and 50 °C. The robustness of Au–Ft makes it a suitable tool for a broad range of applications.
3.6. Glucose detection Based on the intrinsic peroxidase catalytic activity of Au–Ft, a colorimetric detection system for glucose detection was evaluted by combining the mimetic enzyme and glucose oxidase. As demonstrated above, the color variation of TMB oxidation catalyzed by Au–Ft was H2O2 concentration dependent, which indicated that the change of the absorbance at 652 nm can be used for H2O2 detection. When coupled with the glucose catalytic reaction by glucose oxidase, we established a colorimetric sensor for glucose detection. Fig. 5(a) shows the calibration curve for glucose detection obtained under conventional conditions. The linear response (R¼0.997) of the absorbance (652 nm) versus glucose concentration is in the range from 2.0 mM to 10.0 mM. According to a previous report (Xu et al., 2007), the blood glucose range in a healthy persons is 3.0–8.0 mM. Higher glucose values will damage the body and can lead to multiple health problems associated with diabetes. Therefore, glucose concentration range from 2.0 mM to 10.0 mM, has practical relevance from a physiological perspective. The coefficient of variation (CV) of the different standard samples is listed in Table S3. To examine the selectivity of detection of glucose, the control experiments were taken under the same conditions using maltose, fructose, and lactose. As shown in Fig. 5(b), there was no detectable signal obtained from the control samples with other saccharides at concentrations of 5.0 mM. Thus, other saccharides will not interfere with the detection of glucose. The value of coefficient of variation in three repeated measurements of 5.0 mM was 4.35% (Table S3). In essence, the developed method shows acceptable reproducibility and high selectivity towards glucose at a normal physiological range. Thus, the Au–Ft glucose sensor system could be a new future candidate for glucose detection.
Fig. 5. Glucose detection based on peroxidase-like Au–Ft. (a): The linear calibration plot for glucose. (b): Specificity analysis of glucose detection. The difference in absorbance between glucose and other sugars (the concentration is 5 mM). The error bars represent the standard deviation for three measurements.
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4. Conclusion In conclusion, our results demonstrate that Au–Ft possesses intrinsic peroxidase-like activity and performs catalysis over a wide range of pH values and temperatures comparable with natural enzymes. Intriguingly, the paired gold clusters in the apoferritin cavity possess high catalytic activity to the substrates. Catalysis by Au–Ft shows a typical Michaelis–Menten kinetics and exhibits a ping-pong mechanism, which is similar to that of the natural enzyme. Based on these findings, a simple and selective assay for glucose detection was developed. This work will not only promote the development of noble metal nanomaterials as peroxidase mimics but also provide a wide range of potential applications for Au mimetic enzyme systems in bio-detection, catalysis, and clinical diagnostics.
Acknowledgment The work was supported by the National Natural Science Foundation of China (No. 81271697), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Nos. 20100061120077, 20120061110021), the Social Development Project of Science and Technology Department of Jilin Province, China (Nos. 20106031, 20120967, YYZX201264, 20130206069GX), the Fundamental Research Funds for the Central Universities, and “Significant New Drug Creation” Science and Technology Major Program (No. 2012ZX09503001-003).
Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.078.
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