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Positively-charged gold nanoparticles as peroxidiase mimic and their application in hydrogen peroxide and glucose detectionw Yun Jv, Baoxin Li* and Rui Cao Received 21st July 2010, Accepted 13th September 2010 DOI: 10.1039/c0cc02698k Positively-charged gold nanoparticles possess intrinsic peroxidaselike activity, and can catalyze oxidation of the peroxidase substrate 3,3,5,5-tetramethylbenzidine (TMB) by H2O2 to develop a blue color in aqueous solution, thus providing a simple approach to colorimetric detection of H2O2 and glucose. Artificial enzyme mimics are a current research interest1,2 because natural enzymes bear some serious disadvantages, such as their catalytic activity which can be easily inhibited.3 Furthermore, the preparation, purification and storage of natural enzymes are usually time-consuming and expensive.4 Peroxidase enzymes activate H2O2 to perform a myriad of oxidations in nature and have long been targeted by biomimetic chemists.5 Many peroxidase mimics including hemin,6 porphyrin,7 molecularly imprinted hydrogels8 and DNAzyme9 have been applied in different fields. The emergence and recent advance of nanoscience and nanotechnology open new opportunities for the application of nanomaterials in catalysis.10,11 Because of their large surface-to-volume ratio, nanomaterials are attractive to use as high-efficiency catalysts,12 and some nanomaterials have been discovered with peroxidase-like activity. Recently, Yan and co-workers found that Fe3O4 magnetic nanoparticles possessed intrinsic enzyme mimetic activity similar to that found in natural peroxidases, though Fe3O4 magnetic nanoparticles are thought to be biologic and chemical inert.13 Fe3O4 magnetic nanoparticles are used as peroxidase mimic to detection H2O214 and thrombin.15 Subsequently, sheet-like FeS nanostructure,16 single-wall carbon nanotube,17 polymercoated CeO2 nanoparticles,18 BiFeO3 nanoparticles19 and bimetallic alloy nanoparticles20 were also found with peroxidase- or oxidase-like activity. The new functions make them potentially useful in the environmental chemistry and biomedicine fields. Because of easy preparation, excellent biocompatibility, and their unique optoelectronic properties, gold nanoparticles (AuNPs) have attracted increasing attention in many fields.21–23 Historically, gold has been regarded as being catalytically inert, but in recent times it has been shown that it becomes active when stabilized in the form of nanoparticles on metal oxide supports (such as Co3O4, Fe2O3, or TiO2).22 Remarkably, these supported AuNPs can catalyze some gas-phase redox reactions, such as CO and H2 oxidation,24,25
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Materials Science, Shaanxi Normal University, Xi’an 710062, P.R. China. E-mail:
[email protected]; Fax: (+86)-29-85307774 w Electronic supplementary information (ESI) available: Experimental details. See DOI: 10.1039/c0cc02698k
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NO reduction,26 CO2 hydrogenation,27 and synthesis of H2O2 from H2 and O2.28 Many attempts have been made to utilize AuNPs supported on various matrices as the catalyst in selective oxidation of organic compounds.29 So far, there are few reports on unsupported AuNPs (i.e., gold colloids) directly used as the catalysts of liquid-phase chemical reactions.30 In addition, conjugation chemistry for protein and DNA binding at the surface of AuNPs has been well established, which paves the way for using biomolecule– AuNPs conjugated in biosensing applications.31 These methods mainly rely on the brilliant red color of AuNPs arising from their strong surface plasmas resonance absorption and high extinction coefficient in the visible region. In addition, AuNPs are observed to enhance the activities of glucose oxidase (GOD)32 and horseradish peroxidase.33 In this work, we made the surprising discovery that the positively-charged AuNPs ((+)AuNPs) possess intrinsic peroxidase-like activity. The unsupported (+)AuNPs could catalyze the oxidation of the peroxidase substrate 3,3,5,5tetramethylbenzidine (TMB) by H2O2 to develop a blue color in aqueous solution, which provided colorimetric detection of H2O2. The accurate and rapid determination of H2O2 is of practical importance due to its application in food, pharmaceutical, clinical, industrial, and environmental analysis.34,35 By coupling the oxidation of glucose catalyzed by GOD, the colorimetric method was further developed for quantitative analysis of glucose. The (+)AuNPs were synthesized by sodium borohydride reduction of hydrogen tetrachloroaurate(III) in the presence of cysteamine36 (see ESIw). The average size of the as-prepared AuNPs was about 34 nm (Fig. S1 and S2w), and the zeta potential of the as-prepared AuNPs was positive (+11.3 mV) due to the adsorption of the positively charged –NH3+ group of cysteamine. To investigate the peroxidase-like activity of the as-prepared (+)AuNPs, the catalytic oxidation of peroxidase substrate TMB in the presence of H2O2 was tested. As shown in Fig. 1, the (+)AuNPs can catalyze the oxidation of TMB by H2O2 to produce a blue color. The resulting solution shows a maximum absorbance at 655 nm (Fig. 2), which originates from the oxidation of TMB. Furthermore, we prepared the citrate-capped AuNPs (ca. 13 nm) and the citrate-capped silver nanoparticles (AgNPs, ca. 20 nm) for a comparison study, and the zeta potential experiments showed that the two kinds of nanoparticles were negatively charged. As shown in Fig. 3, the citrate-capped AgNPs cannot catalyze the TMB–H2O2 reaction, and the catalytic activity of the citrate-capped AuNPs is lower than that of (+)AuNPs. H2O2 can be adsorbed on the surface of AuNPs, and the O–O bond of H2O2 might be broken up into double HO radical.30 The generated HO radical might be stabilized by AuNPs via the Chem. Commun., 2010, 46, 8017–8019
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Fig. 1 Typical images of 100 mL of 5 mM TMB reaction solution catalytically oxidized by the (+)AuNPs in the presence of H2O2 incubated at 45 1C in 100 mL of 0.5 M pH 4.0 acetate buffer (from left to right: 0 mM H2O2 with (+)AuNPs, 10 mM H2O2 without (+)AuNPs, 10 mM H2O2 with (+)AuNPs).
Fig. 2 Typical absorption spectra of the TMB–H2O2 mixed solution in the absence (black line) and in the presence (red line) of 70 mL of 1.62 nM (+)AuNPs incubated at 45 1C in 100 mL of 0.5 M pH 4.0 acetate buffer (100 mL of 5 mM TMB, 4 mM H2O2).
activity of the mercaptoacetic acid-capped AuNPs (ca. 36 nm) was also lower than that of (+)AuNPs (Fig. S3w). On the other hand, the as-prepared (+)AuNPs solution was centrifuged at 13 000 rpm at 4 1C, and then the precipitate was again re-dispersed using ultrapure water. The resulting (+)AuNPs solution, not including any unreacted species (such as AuCl4 ), showed almost the same catalytic activity as the original (+)AuNPs solution. These results further confirmed that the catalytic activity originated from the (+)AuNPs themselves. The catalytic efficiency of the (+)AuNPs was strongly dependent on pH and temperature. The 0.2 M acetate buffer was used as the reaction media, and we examined the influence of pH in the range 2.0–12.0. The results showed that the catalytic oxidation of TMB with H2O2 in the presence of (+)AuNPs was much faster in acidic solutions than in neutral or basic solutions. The possible reason is that the cysteaminecapped AuNPs have enough positive charges only in acidic solution, ensuring stability of the as-prepared AuNPs. However, compared to (+)AuNPs, the citrate-capped AuNPs always exhibited low catalytic behavior in acidic or basic solution. The maximum catalytic activity occurred at approximately pH 4.0. In the range 20 to 60 1C, the absorbance of the TMB–H2O2 system in the absence and presence of the (+)AuNPs increased with increasing temperature (Fig. S4w). The DA, where DA = A(AuNPs) A(blank), reached the maximum at 45 1C. Thus, we chose 45 1C as the reaction temperature. As the properties of nanoscale materials are often dependent on size, we also studied the catalysis of the (+)AuNPs at different sizes (34 and 48 nm). The experimental results showed that the activity of the 34 nm (+)AuNPs was higher than that of the 48 nm (+)AuNPs. This phenomenon may be due to the smaller nanoparticles having a greater surface-to-volume ratio to interact with substrates. The control experiments showed that 13 nm citrate-capped AuNPs exhibited higher catalytic cativity than 38 nm citrate-capped AuNPs in this system. Because the catalytic activity of the (+)AuNPs is H2O2 concentration dependent, the system discussed above could be used to detect H2O2. Under the optimized conditions, the developed method was used for H2O2 detection. As shown in
Fig. 3 Images of the TMB–H2O2 mixed solution (1) in the absence of nanoparticles, (2) in the presence of 20 mL 20 nm citrate-capped AgNPs, (3) in the presence of 20 mL 13 nm citrate-capped AuNPs, (4) in the presence of 20 mL 34 nm (+)AuNPs. Experimental conditions: 200 mL of 5 mM TMB, 200 mL of 10 mM H2O2, 5 M acetate buffer (pH 4.0).
partial electron exchange interaction,37,38 which may contribute to the catalytic ability of AuNPs. The surface property of AuNPs would influence the absorption of H2O2 and the particle-mediated electron transfer processes.38 The citratecapped AuNPs are negatively charged, whereas the (+)AuNPs (cysteamine-capped AuNPs) are positively charged. So, the (+)AuNPs and the citrate-capped AuNPs exhibited different catalytic behavior. In addition, the results showed that the 8018
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Fig. 4 Linear calibration plot between the absorbance at 655 nm and concentration of H2O2. The insert shows the dependence of the absorbance at 655 nm on the concentration of H2O2 in the range 2 mM to 1.6 mM.
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This study was supported by the National Natural Science Foundation of China (No. 21075080) and the Fundamental Research Funds for the Central Universities.
Notes and references
Fig. 5 Linear calibration plot between the absorbance at 655 nm and glucose concentration in the range 1.8 10 5–1.1 10 3 M.
Fig. 4, the absorbance of this system increased with increasing H2O2 concentration. The calibration graph of the absorbance at 655 nm to H2O2 concentration was linear in the range 2.0 10 6 – 2.0 10 4 M with a detection limit of 5 10 7 M. In addition, (+)AuNPs as peroxidiase mimics performed with good specificity (Fig. S5w). When the catalytic reaction is coupled with the glucose catalytic reaction by GOD, the colorimetric glucose detection could be readily realized. Because the activity of GOD is not optimal at pH 4.0 and 45 1C, glucose detection was performed in two separated steps: (1) glucose and GOD were reacted in PBS (10 mM, pH 7.4) at 37 1C for 15 min; (2) the produced H2O2 was detected using the TMB–(+)AuNPs system at pH 4.0 and 45 1C. Fig. 5 displays the linear response (R = 0.9993) of the absorbance (655 nm) versus glucose concentration in the range 1.8 10 5 1.1 10 3 M. The detection limit of this assay for glucose was 4 10 6 M, which is lower than that obtained using Fe3O4 magnetic nanoparticles as peroxidase mimetic.14 Furthermore, we tested the specificity of the proposed method by conducting several control experiments using fructose, lactose and maltose. As high as 5 mM control samples were investigated and no detectable signals were obtained (Fig. S6w). Thus, the colorimetric method developed here showed high selectivity toward glucose detection. In conclusion, we report that (+)AuNPs possess unique peroxidase-like activity. The catalytic oxidation of peroxidase substrate TMB with H2O2 using the (+)AuNPs was realized. The (+)AuNPs as a peroxidase mimic provide a colorimetric assay for H2O2. The colorimetric method showed good response toward H2O2. Furthermore, the colorimetric method for glucose was also fabricated using (+)AuNPs and GOD. These results demonstrate that the (+)AuNPs act as an effective peroxidase mimic. Because the (+)AuNPs can rival natural enzymes due to their easy preparation, robustness, and stability under harsh conditions, our findings open up a wide range of new potential applications for AuNPs in biotechnology, environmental chemistry and medicine.
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