Development of Methodology Based on the Formation Process of Gold

Anal. Chem. 2009, 81, 8916–8922

Development of Methodology Based on the Formation Process of Gold Nanoshells for Detecting Hydrogen Peroxide Scavenging Activity Hui Li, Xiaoyuan Ma, Jian Dong, and Weiping Qian* State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, P. R. China In the present work, we have developed a novel nanocomposite-based method for estimating antioxidant activity. The assay implements a new enzyme-free optical nanoprobe for assessing hydrogen peroxide (H2O2) scavenging activity based on the formation process of gold nanoshells (GNSs). H2O2 could enlarge the gold nanoparticles (GNPs) on the surface of GNSs precursor nanocomposites (SiO2/GNPs), and the preadsorbed GNPs served as nucleation sites for Au deposition. As the concentration of H2O2 increases, more GNPs on the SiO2 cores are enlarged until continuous GNSs are formed. During the growth procedure, the spectra changes correlate well with H2O2 concentrations which indicate that this nanocomposite is a good nanoprobe for detecting H2O2. H2O2 scavenging activities of several antioxidants were determined by restraining the H2O2-mediated formation of GNSs from SiO2/ GNPs, and the changes of the corresponding plasmon absorption bands correlated well with H2O2 scavenging activity of antioxidants. The spectra were monitored by a UV-vis-near-infrared (NIR) spectrophotometer, and the wavelength changes were adopted as detection signal. The results obtained expressed the difference of H2O2 scavenging activity between various tested compounds, and the relationship between function and structure of antioxidants was also discussed in this article. The new method based on the formation process of GNSs is simple, rapid, and sensitive and, additionally, can be used in visual analysis to a certian extent for antioxidant functional evaluation. Oxidative stress has been implicated in the pathogenesis of several human diseases and conditions including aging, diabetes, chronic inflammation, and cancer. It occurs when excessive quantities of reactive oxygen species (ROS), such as superoxide radical anion (O2•-), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), overwhelm host antioxidant defenses, resulting in cellular damage, protein damage, lipid peroxidation, DNA alteration, and enzyme inactivation.1-5 ROS can be scavenged by antioxidants, which are defined as any substances that when * To whom correspondence should be addressed. Phone: +8625-83795719. Fax: +862583795719. E-mail: [email protected]. (1) Magalhaes, L. M.; Segundo, M. A.; Reis, S.; Lima, J. L. F. C. Anal. Chim. Acta 2008, 613, 1–19.

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present at low concentration compared to those of an oxidizable substrate significantly delay or prevent oxidation of that substrate. Antioxidants are not only used in biomedicine and clinical medicine but also have extensive applications in food industry and food nutriology. ROS scavenging activity is an important aspect of antioxidant activity. H2O2 is a key part of ROS and generated in vivo under physiological conditions by peroxisomes, several oxidative enzymes including glucose oxidase and D-amino acid oxidase, and by dismutation of superoxide radical catalyzed by superoxide dismutase; it together with O2•- can damage many cellular components and furthermore convert into more ROS such as •OH. Thus, the determination of H2O2 scavenging activity is a very important part for evaluation of reactive oxygen scavenging activity.1,2 Because of the importance of the measurement of antioxidant activity, much research has gone into the study of antioxidant activity assessment and a considerable amount of relevant articles have been published. In view of many different antioxidants having various scavenging activities against O2•-, H2O2, or •OH, many methods for measuring these properties have been developed with different mechanisms, respectively, such as chromatography,6 chemiluminescence (CL),7 electrochemiluminescence (ECL),8 spectrophotometry,1-5 cyclic voltammetry (CV),1 automatic flow injection based methodologies,9 electron spin resonance (ESR),1 etc. Recently, several nanomaterials have been utilized in the measurement of antioxidant activity, such as metal nanoparticles,10,11 quantum dots,8 polymer spheres,12 and so on. (2) Pazdzioch- Czochra, M.; Widenska, A. Anal. Chim. Acta 2002, 452, 177– 184. (3) Mansouri, A.; Makris, D. P.; Kefalas, P. J. Pharm. Biomed. Anal. 2005, 39, 22–26. (4) Wood, L. G.; Gibson, P. G.; Garg, M. L. J. Sci. Food Agric. 2006, 86, 2057– 2066. (5) Arnous, A.; Petrakis, C.; Makris, D. P.; Kefalas, P. J. Pharmacol. Toxicol. Methods 2002, 48, 171–177. (6) Winston, G. W.; Regoli, F.; Dugas, A. J.; Fong, J. H.; Blanchard, K. A. Free Radical Biol. Med. 1998, 24, 480–493. (7) Magalhaes, L. M.; Segundo, M. A.; Reis, S.; Lima, J. L. F. C.; Estela, J. M.; Cerda, V. Anal. Chem. 2007, 79, 3933–3939. (8) Jiang, H.; Ju, H. X. Anal. Chem. 2007, 79, 6690–6696. (9) Magalhaes, L. M.; Lucio, M.; Segundo, M. A.; Reis, S.; Lima, J. L. F. C. Talanta 2009, 78, 1219–1226. (10) Lee, H. K.; Lee, K.; Kim, I.-K.; Park, T. G. Adv. Funct. Mater. 2009, 19, 1–7. (11) Wang, J.; Zhou, N. D.; Zhu, Z. Q.; Huang, J. Y.; Li, G. X. Anal. Bioanal. Chem. 2007, 388, 1199–1205. (12) Kim, S. H.; Kim, B.; Yadavalli, V. K.; Pishko, M. V. Anal. Chem. 2005, 77, 6828–6833. 10.1021/ac901534b CCC: $40.75  2009 American Chemical Society Published on Web 10/13/2009

Compared with conventional methods, these approaches with high sensitivity, rapid determination, relative ease of measurement, inexpensive, and simple apparatus have attracted a widespread attention. As a new metal and semiconductor composite nanomaterial, gold nanoshells (GNSs) consisting of a silica core with a gold shell have attracted considerable attentions owing to their unique chemical, physical properties, and good biocompatibility, especially highly tunable plasmon resonance. The localized surface plasmon resonance (LSPR) of GNSs has strongly depended on the core radius-shell thickness ratio and can be placed from visible to near-infrared (NIR) region. Compared with gold nanoparticles (GNPs), the utility of GNSs is more extensive because their plasmon resonance can be changed in a controlled manner easily during a relatively broad wavelength range. For example, the LSPR of GNPs with a diameter range from 8 to 99 nm in the spectra region is only from 518 to 575 nm. While, the LSPR of GNSs spans a range of ∼300 nm in the wavelength with the core radius-shell thickness ratio varied between 3 and 12.13 The unique optical properties of GNSs that lead to this new nanomaterial are both useful and sensitive, accordingly GNSs have prompted considerable attention for a variety of applications in biomedicine, material science, diagnoses and therapy of disease, and chemical and biological sensors.13-17 Among various GNSs synthesis methods, the seed-mediated growth technique has been widely exploited because it is readily adaptable to produce uniform gold layers on the surfaces of silica spheres compared with the direct synthesis procedure. This technique has also been used to prepare other core/shell nanostructures such as nanorice and nanorod, etc.18,19 As the intermediate in the synthesis of GNSs adopting the seed-mediated growth method, GNSs precursor nanocomposites (SiO2/GNPs) consist of a SiO2 core and attached GNPs; the preadsorbed GNPs act as nucleation sites for further reduction of gold onto the silica core by reduction of AuCl4- solution in the presence of reducing agent.20 Our previous work has shown that13,16 as more gold is reduced, the surface coverage increases until coalescence into complete GNSs. During the process, the LSPR spans at least 200 nm, and this highly optical tunability offers this nanocomposite extensive application perspectives. In general, the detection methods of H2O2 scavenging activity are dependent on peroxidase originally.1,2 The peroxidase-based approaches with a common disadvantage are that the peroxidase may interfere with the determination. Thus, a variety of enzymefree luminescent methods coupled with numerous techniques have been presented for assessing the H2O2 scavenging activity in recent years.1,3,5,9 However, these techniques require complicated sample preparation processes or expensive and sophisticated (13) Wang, Y.; Qian, W. P.; Tan, Y.; Ding, S. H. Biosens. Bioelectron. 2008, 23, 1166–1170. (14) Stewart, M. E.; Anderton, C. R.; Thompson, L. B.; Maria, J.; Gray, S. K.; Rogers, J. A.; Nuzzo, R. G. Chem. Rev. 2008, 108, 494–521. (15) Scampicchio, M.; Wang, J.; Blasco, A. J.; Arribas, A. S.; Mannino, S.; Escarpa, A. Anal. Chim. Acta 2006, 78, 2060–2063. (16) Ding, S. H.; Qian, W. P.; Tan, Y.; Wang, Y. Langmuir 2006, 22, 7105– 7108. (17) Jain, P. K.; El-Sayed, M. A. Nano Lett. 2007, 7, 2854–2858. (18) Skrabalak, S. E.; Xia, Y. N. ACS Nano 2009, 3, 10–15. (19) Wang, H.; Brandl, D. W.; Le, F.; Nordlander, P.; Halas, N. J. Nano Lett. 2006, 6, 827–832. (20) Hirsch, L. R.; Gobin, A. M.; Lowery, A. R.; Tam, F.; Drezek, R. A.; Halas, N. J.; West, J. L. Ann. Biomed. Eng. 2006, 34, 15–22.

instrumentation, which are quite time-consuming. Therefore, the development of new and effective methods for the detection of H2O2 scavenging activity still remains a great challenge. Herein, the purpose of this investigation is to apply SiO2/GNPs as a nanoprobe for estimating antioxidant activity and implement a new reliable, enzyme-free spectrophotometric methodology for assessing H2O2 scavenging activity in vitro based on the formation process of GNSs. EXPERIMENTAL SECTION Chemicals and Reagents. Silica colloidal spheres (∼110 nm) and 3-(aminopropyl)-triethoxysilane (APTES, 98%) were obtained from Nissian Chemical Ind., Ltd., Japan, and Sigma, respectively. Sodium borohydride (NaBH4), potassium carbonate (K2CO3), chloroauric acid terahydrate (HAuCl4 · 4H2O), hydrogen peroxide (H2O2, 30%), and anhydrous ethanol were all purchased from Nanjing Sunshine Biotechnology Ltd., China. Tannic acid, ferulic acid, L-apple acid, tartaric acid, salicylic acid, and citric acid were received from Shanghai Aladdin Chemical Ltd., China, and used without further purification. All of the chemical reagents were analytical grade. K2CO3/HAuCl4 solution was prepared as follows: 100 mL of aqueous K2CO3 solution (0.25 g/L) was mixed with 1.5 mL of HAuCl4 (1%) stock solution under continuous stirring for 20 min and aging in the dark at 4 °C for 24 h. The final concentration of H2O2 used in the system is 200 µM. Aqueous solutions used in the experiments were prepared using Milli-Q water from Milli-Q system (resistivity >18 MΩ). Synthesis of GNPs. GNPs (∼5 nm) were prepared by the reduction of HAuCl4 with NaBH4 according to our previous method:13,16 3 mL of HAuCl4 (1%) was mixed with 200 mL of water under vigorous stirring, followed by the addition of 1 mL of K2CO3 (0.2 M). After that, 9 mL of freshly prepared NaBH4 (0.5 mg/mL) was quickly added to the mixture. The mixture tuned to wine red rapidly, which indicated the generation of GNPs. The obtained solution was stored at 4 °C until use. Synthesis of GNSs Precursor Nanocomposites (SiO2/ GNPs). The SiO2/GNPs were prepared as reported in our previous work13 with some modifications. Silica colloidal spheres were purified by centrifuging and redispersing five times in ethanol. Then 39 mL of monodisperse silica colloidal spheres (0.013 g/mL in ethanol) were amino-functionalized by mixing with 60 µL of APTES under vigorous magnetic stirring at 70 °C for 3 h to present positively charged amino groups. After centrifugation and redispersion in ethanol, aiming to remove excess reactants, the pure amino-functionalized silica spheres (593 µL) were redispersed in ethanol (20 mL) and added dropwise to 200 mL of GNPs with vigorous stirring, resulting in GNPs adsorption on the surface of silica nanoparticles and the formation of SiO2/GNPs. After centrifugation and removal of the supernatant three times, pure SiO2/GNPs were redispersed in 150 mL of water. The absorbance spectrum of obtained SiO2/GNPs stock solution was 0.5 at ∼533 nm. The SiO2/GNPs nanocomposites can be dispersed homogeneously in solvents such as water and ethanol to form colloidal suspensions. In this case, they can be kept at least 1 month at 4 °C. Analytical Chemistry, Vol. 81, No. 21, November 1, 2009

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Scheme 1. Schematic Illustration of the Formation For H2O2-Mediated Gold Nanoshells

H2O2 Scavenging Activity Detection Assay. The testing procedures were as follows: 3 mL of water was mixed with 200 µL of sample and 100 µL of H2O2 (13.6 mM), after incubation for 2 min, 0.5 mL of SiO2/GNPs solution and 3 mL of K2CO3/ HAuCl4 were immediately added and mixed for 5 min under vigorous stirring at room temperature, finally the spectra were performed on a Shimadzu UV3150 UV-vis-NIR spectrophotometer in transmission mode. A JEM2100EX transmission electron microscope (TEM) was used to image the morphology of SiO2/GNPs at different conditions. Control was prepared by mixing 0.5 mL of SiO2/GNPs, 3 mL of K2CO3/HAuCl4, and 3.3 mL of water. Reference samples were tested by adding 200 µL of water instead of an antioxidant solution. All measurements were performed at least in triplicate and values were averaged. Results were given as means ± the standard deviation (SD). RESULTS AND DISCUSSION H2O2-Mediated Growth of SiO2/GNPs. As we know that many chemicals could induce the formation of GNSs, such as formaldehyde, hydroxylamine-hydrochloride, sodium borohydride, and carbon monoxide gas.21 Our work observes that H2O2 could reduce AuCl4- to Au which deposit on the surface of SiO2/ GNPs and enlarge GNPs. As more gold is reduced, the GNPs grow larger and ultimately merge, resulting in the formation of a continuous shell layer. During this procedure the preadsorbed GNPs serve as nucleation sites for Au deposition. As the concentration of H2O2 increases, more AuCl4- can be reduced and more GNPs on cores are enlarged; if the concentration of H2O2 reaches to some level, continuous nanoshells are formed as Scheme 1 illustrates. The concentration of H2O2 could affect the growth of SiO2/ GNPs and the corresponding spectra. Figure 1A shows the evolution of the spectra in the presence of variable concentrations of H2O2. Also, the corresponding derived calibration curve is shown in Figure 1B. The TEM images indicate the morphology variation of SiO2/GNPs upon reaction with the increasing concentration of H2O2 (Figure 2). As the concentration of H2O2 increases, the wavelength is constantly red-shifted, and the (21) Brinson, B. E.; Lassiter, J. B.; Levin, C. S.; Bardhan, R.; Mirin, N.; Halas, N. J. Langmuir 2008, 24, 14166–14171.

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absorbance is intensified. When the final concentration of H2O2 reaches to 200 µM, the wavelength maximum occurs which reveals the formation of continuous nanoshells (core diameter is ∼110 nm, shell thickness is ∼20 nm). The reaction process completes in 5 min with color changes from faint pink to blue, and the spectra transfers obviously from ∼530 to ∼750 nm. Thus, the 200 µM H2O2 is the optimal concentration for the formation of GNSs in the reaction system. The K2CO3/HAuCl4 aged time is a key parameter for the GNSs formation. When the K2CO3/HAuCl4 aged time reaches to 24 h, the wavelength maximum occurs which reveals that continuous nanoshells are formed. Less than or exceeding 24 h, the obtained wavelengths are all lower than this value. While the K2CO3/HAuCl4 aged time exceeds 72 h, the SiO2/GNPs stop growing and the spectra do not change. Therefore, 24 h is the optimal aged time of the K2CO3/HAuCl4 solution. Detection of H2O2 Scavenging Activity. During the past years, there have been some optical studies reporting on methods for assessing the H2O2 scavenging activity, involving UV spectrophotometry and fluorescence spectrophotometry. The most common UV spectrophotometric determination method is a direct way based on the intrinsic absorption of H2O2 in the UV region at 230 nm, yet this UV enzyme-free assay has a problem that samples may also absorb at this wavelength, which makes it difficult to distinguish a small change when the background absorption is large.1 Some fluorimetric methods are based on the oxidation of scopoletin or homovanillic acid to their nonfluorescent product or fluorescent biphenyl dimer in the presence of H2O2 and peroxidase. However, these peroxidase-based approaches have a disadvantage that antioxidants may be a substrate for the peroxidase enzyme, resulting in error determination.1,2 There are also some enzyme-free luminescent methods that rely on peroxyoxalate chemiluminescence (POCL) using 9,10diphenylanthracene, luminol, or lucigenin as a fluorophore (probe). POCL involves H2O2 imidazole-catalyzed oxidation of a substance to a high-energy intermediate and transfers its energy to the fluorophore. The transition of the excited state of the fluorophore to its ground state causes the emission of light. Accordingly, any compound with H2O2

Figure 1. (A) Absorbance spectra for the growth of SiO2/GNPs in the presence of variable concentrations of H2O2 from 0 to 300 µM. (B) The calibration curve corresponding to the peak absorbance and wavelength of reaction system with different concentrations of H2O2. All systems include 3.2 mL of water, 3 mL of K2CO3/HAuCl4, 0.5 mL of SiO2/GNPs solution, and 100 µL of H2O2 or water. Spectra were recorded after the mixture reacted for 5 min under vigorous stirring.

Figure 2. TEM images of (a) SiO2/GNPs upon reaction with variable concentrations of H2O2 of (b) 25, (c) 100, and (d) 200 µM.

scavenging capacity promotes CL inhibition. The above POCL methods are proposed to evaluate mainly lipophilic antioxidants because of the nonpolar environment employed.1,3,5 This article reports on the H2O2-mediated growth of SiO2/ GNPs to form GNSs and apply this process to develop a method for estimating H2O2 scavenging activity of antioxidants. The presence of substances with H2O2 scavenging activity prevents SiO2/GNPs from forming complete GNSs and causes a variation in spectra. Thus, both the shifts of wavelength and absorbance can be used as an optical signature for the change of SiO2/GNPs morphology. Some conventional spectrophotometry for measuring antioxidant capacity mainly use the absorbance change at a fixed wavelength as an optical signature, such as the ABTS•+ assay, DPPH• assay, ferric reducing antioxidant power (FRAP assay), folion-ciocalten reducing capacity (FC assay),

etc.1 Compared with the variation of the absorption value, the variation of wavelength is more direct for visual discrimination, which has more potential application in visual colorimetric detection. In addition, to estimate the antioxidant activity of a pure compound, it is also essential to assess the antioxidant ability of relevant food and beverage. However, some tested samples such as tea, fruit juice, and red wine also absorb in the measured wavelength range and the spectra are not horizontal. The instinct color of these natural antioxidant substances may interfere with determination and lead to error estimation. Therefore, the wavelength shift is chosen as the detection signal to avoid this type of determination error. Besides, if there is an interfering agent that may influence our detection, the SiO2/GNPs nanocomposite can be separated through centrifugation (∼3000 rpm) and ultrasonic redispersion from the supernatant conveniently. According to our experiments, the wavelength of the nanocomposite is unchanged through centrifuging and redispersing, which does not affect the detection results. As the concentration of antioxidant increases, the residual H2O2 decreases and the growth of SiO2/GNPs is more inhibited, so the H2O2 scavenging activity of samples was measured in different concentration ranges. For comparison of the H2O2 scavenging activity of antioxidants, the obtained results are presented as the percentage wavelength inhibition (Iw) using the following formula: Iw ) ∆w /∆w0 (%), where ∆w0 is the wavelength difference of the reaction mixture with and without H2O2 in the absence of sample, ∆w is the reaction mixture wavelength difference without and with sample in the presence of H2O2. The amount of each antioxidant to provoke a 50% wavelength inhibition ratio is called IC50, while hydrogen peroxide scavenging activity (SAHP) is defined as SAHP ) 1/IC50 (Figure 3). A relationship between the concentration of antioxidant and the percentage Analytical Chemistry, Vol. 81, No. 21, November 1, 2009

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Figure 3. Schematic diagram of the method for expressing H2O2 scavenging activity: (A) The percentage wavelength inhibition (Iw) and (B) the hydrogen peroxide scavenging activity (SAHP).

Figure 4. The percentage wavelength inhibition of antioxidants including tannic acid, ferulic acid, citric acid, L-apple acid, tartaric acid, and salicylic acid.

wavelength inhibition are obtained (Figure 4). According to those results, IC50 values can be read. The concentration range of tested antioxidants with their IC50 values and the corresponding SAHP values are shown in Table 1. On the grounds that the spectral change resulting from the addition of antioxidants with their H2O2 scavenging activity is in the visible range from ∼530 to ∼750 nm, the change in color corresponding to the spectra can be observed directly without a spectrophotometer, which makes the visual colo8920

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rimetric detection of antioxidant activity feasible in qualitative or semiquantitative analysis. So compared with traditional methods for the determination of antioxidant capacity, such as CL and chromatography, this means has more potential practical application in rapid screening analysis of antioxidants. Figure 5 shows the spectra changes and the relevant color changes of the solution in the presence of variable concentrations of tannic acid. The color change from faint pink to blue could be directly observed by the naked eye with

Table 1. Concentration Ranges, IC50, and SAHP Values of Samples sample

range (µM)

IC50 (µM)

SAHP (× 10-2 µM-1)

salicylic acid tartaric acid L-apple acid citric acid ferulic acid tannic acid

100-800 50-1200 5-400 5-400 3-100 5-200

470 273 162 106 8 7

0.21 0.36 0.62 0.94 12.5 14.28

decreasing concentrations of tannic acid, and this color alteration is consistent with the H2O2-mediated growth of the SiO2/GNPs assay as the H2O2 concentration increases. The TEM images indicate that increasing the concentration of tannic acid indeed decreases the amount of H2O2 and inhibits the growth of SiO2/GNPs (Figure 6). Relationship between Activity and Structure of Antioxidants. The results indicate that tannic acid and ferulic acid express superior H2O2 scavenging activity than other tested antioxidants, salicylic acid shows the weakest activity, three organic acids (L-apple acid, tartaric acid, citric acid) represent the median activity. These results with phenolic compounds are in agreement with many other researchers that tannic acid was more potent in radical scavenging activity than ferulic acid.22-24 The H2O2 scavenging activity may have a relationship with the chemical structures and in particular the number of hydroxyl groups.5,15 The structure of phenolic compounds, which comprise an aromatic ring with one or more hydroxyl substituents that range from simple phenolic molecules to highly polymerized compounds, is an important determinant of their radical scavenging activity, and the antioxidant activity of phenolic acids increases with an increasing degree of hydroxylation and extent of conjugation.23,25 As is well-known, monophenols are less efficient as antioxidants than polyphenols and tannic acid as largemolecular weight polyphenols are strong radical scavengers, which may be related to the formation of stable radicals; ferulic acid and salicylic acid are monophenols and show lower antioxidant activity than tannic acid. As to the monophenols, the inductive effect of the hydroxyl group is an important factor that enhances antioxidant ability and methoxyl substitution is another factor. For this reason, salicylic acid with one hydroxyl is less efficient than ferulic acid with one hydroxyl and methoxyl in the aromatic ring.22 Besides, three organic acids also express H2O2 scavenging activity due to their hydroxyl structures although are not phenolic acids. In conclusion, the degree of hydroxylation and extent of conjugation seem to be the key factor for the radical scavenging activity. The method reported here is comparing the H2O2 scavenging activity of plant-based antioxidants, and this is a portion function of these multifunctional compounds. According to many researches, fruits and vegetables are excellent sources of exogenously obtained antioxidants. Ingestion of these com(22) Sanchez-Moreno, C.; Larrauri, J. A.; Saura-Calixto, F. Food Res. Int. 1999, 32, 407–412. (23) Pulido, R.; Bravo, L.; Saura-Calixto, F. J. Agric. Food Chem. 2000, 48, 3396– 3402. (24) Kumar, G. S.; Nayaka, H.; Dharmesh, S. M.; Salimath, P. V. J. Food Compos. Anal. 2006, 19, 446–452. (25) Scherer, R.; Godoy, H. T. Food Chem. 2009, 112, 654–658.

Figure 5. Color images and the corresponding UV-vis spectra for H2O2-mediated growth of (a) SiO2/GNPs in the presence of a fixed concentration of 200 µM H2O2 and different concentrations of tannic acid from (b) 200 to (h) 0 µM.

Figure 6. TEM images of (a) SiO2/GNPs upon reaction with 200 µM H2O2 and variable concentrations of tannic acid for (b) 200, (c) 25, and (d) 0 µM.

pounds from dietary sources may reinforce antioxidants in the body, keep the balance between antioxidants and oxidants in living organisms, and help prevent cardiovascular disease and cancer.26 The tested antioxidants were chosen because they are all naturally occurring antioxidants that come from plants and have superior antioxidant ability, meanwhile, many of them exist in daily foodstuff universally. For example, tannic acid is the most important component of various tea beverages, and tannic acid, tartaric acid, and apple acid are major ingredients (26) Prior, R. L.; Wu, X. L.; Schaich, K. J. Agric. Food Chem. 2005, 53, 4290– 4302.

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of red wine, while many fruits and vegetables contain citric acid, ferulic acid, and apple acid. Therefore, the relevant plantbased foods and drinks should have the same function as pure compounds. It is widely accepted that different antioxidants represent variable behavior in tests performed under different conditions, and no single assay will accurately reflect all of the radical sources and estimate all the multifunctional antioxidants in a complex system.27 Therefore, more than one method is required to provide adequate information for in vitro antioxidant characteristics evaluation. In this article, the method presented here may serve as an additional means for estimating the activities of multifunctional antioxidants. CONCLUSIONS Here, we have developed a new nanocomposite-based method for estimating the H2O2 scavenging activity that relies on the H2O2-mediated growth of SiO2/GNPs. The corresponding plasmon absorption bands change correlate well with H2O2 (27) Wolfe, K. L.; Liu, R. H. J. Agric. Food Chem. 2007, 55, 8896–8907.

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scavenging activity of antioxidants. This methodology is simple to design, easy to operate, and furthermore, could be used as visual colorimetric detection without a spectrophotometer in qualitative or semiquantitative analysis. It is a convenient tool for the screening analysis of H2O2 scavenging activity. Studies are under way to optimize the new method and evaluate more antioxidants, not only pure compounds but also relevant beverage and food with their functional estimation. ACKNOWLEDGMENT H.L. and X.M. contributed equally to this work. We gratefully acknowledge support from the National Natural Science Foundation of China (Grants 90923010 and 20475009) and the Ministry of Science & Technology of China (Grant 2007AA022007).

Received for review July 10, 2009. Accepted September 29, 2009. AC901534B