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Peroxidase-like catalytic activity of cubic Pt nanocrystals
Colloids and Surfaces A: Physicochem. Eng. Aspects 373 (2011) 6–10
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Peroxidase-like catalytic activity of cubic Pt nanocrystals Ming Ma ∗ , Yu Zhang, Ning Gu State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing 210096, Jiangsu, PR China
a r t i c l e
i n f o
Article history: Received 27 March 2010 Received in revised form 28 July 2010 Accepted 4 August 2010 Available online 12 August 2010 Keywords: Pt nanocrystals Peroxidase-like activity Agglomeration 3,3 ,5,5 -Tetramethylbenzidine
a b s t r a c t Monodispersed cubic platinum (Pt) nanocrystals with an average size of approximately 10 nm were prepared by a reduction method with cetyltrimethylammonium bromide (CTAB) serving as steric stabilizer. The resulting Pt nanocrystals exhibit a peroxidase-like activity and catalyze the H2 O2 -mediated oxidation of 3,3 ,5,5 -tetramethylbenzidine (TMB) to produce two colored products with high catalytic activity. The color-generating activity of this system may be influenced by several factors, and we examined several factors to optimize this colorimetric system including buffer types, pH, and concentrations of both H2 O2 and Pt nanocrystals. The effect of agglomeration of Pt nanocrystals was also investigated, and we find that agglomeration of Pt nanocrystals in aqueous solution distinctly affects Pt nanocrystals catalytic activity. We attribute the catalytic activity of Pt nanocrystals to their acceleration of the electron-transfer process and the consequent facilitation of radical generation. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In enzyme-linked immunosorbent assays (ELISAs), horseradish peroxidase (HRP) and other natural peroxidases have commonly been used as catalysts [1]. Peroxidase catalyzes the conversion of chromogenic substrates (e.g., 3,3 ,5,5 -tetramethylbenzidine TMB, 2,2 -azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt ABTS, o-phenylenediamine dihydrochloride OPD, etc.) into colored molecules using hydrogen peroxide as the oxidizing agent [1–5]. HRP is not the only catalyst possible for catalyzing the reaction of TMB and H2 O2 . In fact, some metal and metal-oxide nanoparticles such as magnetite nanoparticles have recently been found to possess intrinsic enzyme mimetic activity similar to that found in natural peroxidases [6,7]. This fact opens possibility of novel applications of these nanoparticles for biosensors and in immunohistochemistry. Colloidal platinum nanocrystals have recently received considerable interest because of their unique physical and chemical properties [8,9]. In addition to their interesting physical properties, catalysis may be one of their most popular applications because of their high catalytic activities in many chemical reactions, including hydrogenation production [10], fuel-cell technology [11,12], gas sensing [13], fine-chemical synthesis [14], etc. Therefore, colloidal platinum nanoparticles are likely to be effective catalysts because they exhibit peroxidase-like activity just like magnetite nanoparticles.
The catalytic activity of metal nanocrystals is considered to depend strongly on the particle surface properties, its morphology, size, the stabilizing agent, etc. [15]. Exposed particle surfaces can provide active sites for absorbing reactants. If the active sites are blocked, the catalytic activity will be reduced. For instance, the carbonyl group of polyvinylpyrrolidone (PVP) or polyacrylate, which are the most widely used surface-regulating polymers in shapednanoparticle synthesis, interacts strongly with the Pt surface [16] and thus blocks a significant number of active sites. Conversely, compared with the carbonyl group, alkylammonium ions, which have been widely used in synthesizing Au nanoparticles, have much weaker interactions with the Pt surface [17]. Therefore, in the present study, cetyltrimethylammonium bromide (CTAB) is chosen to serve as a surface-stabilizing agent to regulate the shape of nanocrystals while preserving catalytically active sites. Herein, we report a reduction method in which CTAB is used as a surface-stabilizing agent to prepare monodisperse cubic Pt nanocrystals. The resulting Pt nanocrystals can catalyze the H2 O2 -mediated oxidation of TMB with high catalytic activity. To optimize this colorimetric system, we examine several factors including buffer type, pH, and concentrations of both H2 O2 and Pt nanocrystals. In addition, we find that agglomeration of Pt nanocrystals is inevitable in a colloidal solution, and we use this phenomenon to investigate the effect on catalytic activity of reducing the exposed surface. 2. Experimental 2.1. Synthesis of Pt nanocrystals
∗ Corresponding author. E-mail address: [email protected] (M. Ma). 0927-7757/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2010.08.007
In a typical synthesis of Pt nanocrystals, aqueous solutions of 0.0225 mmol K2 PtCl4 (Alfa Aesar, 99.9%) and 2 mmol CTAB (Sun-
M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 373 (2011) 6–10
7
shine, >90%) were mixed in a 20-mL vial at room temperature. The mixture was heated at 50 ◦ C for 5 min until the solution became clear. Ice-cold aqueous solution 0.5 mL 1.35 M NaBH4 (Sinopharm Chemical Reagent Co. Ltd., >96%) was added, and the solution was aged for 7 h in a 50 ◦ C water bath. The product was separated and centrifuged twice at 12,000 rpm for 15 min. The precipitate was collected and redispersed in water. 2.2. Instrumentation Transmission electron microscope (TEM) and high-resolution TEM (HRTEM) images were collected with a JEM-2100 microscope (JEOL). Absorption spectra were recorded with an EL808 Ultra Microplate Reader (Bio-Tek). The hydrodynamic colloid diameters were detected by a submicron particle size analyzer (PCS, N4 Plus, Beckman). 2.3. Testing Pt-catalyzed oxidation of TMB in the presence of H2 O2 The working solution of the substrate for the catalytic reaction was freshly prepared by adding 50 L 4 mg mL−1 TMB (Sigma, >99%) solution (in DMSO) and 60 L 30% (v/v) H2 O2 solution (in H2 O) into 1 mL working buffer (C–P buffer: 0.1 M citrate–0.2 M disodium hydrogen phosphate; C–C buffer: 0.1 M citrate–0.1 M sodium citrate; or A–A buffer: 0.2 M acetate–0.2 M sodium acetate). None of these buffers were sensitive to temperature. The working solution and 50 L 0.03 mM Pt colloid solution were added consecutively to 24-well ELISA plates to start the catalytic reaction. The kinetics of the catalytic reaction was monitored by detecting the absorption at 650 nm as a function of time. To investigate the effect of different buffers and of the pH, the catalytic reactions were performed in working solutions prepared in C–P buffers (pH 7.4, 6.4, 5.4, 4.4, 3.4, and 2.6), C–C buffers (pH 7.4, 6.4, 5.4, 4.4, 3.4, and 2.6), and A–A buffers (pH 6.4, 5.4, 4.4, and 3.4). The absorbance at 650 nm was measured 20 min after the initiation of the reaction. To investigate the effect of the concentration of H2 O2 and Pt, catalytic reactions were performed in working solutions (1 mL C–P buffer, pH 4.4; 50 L 4 mg mL−1 TMB) with different concentrations of H2 O2 or different concentrations of Pt. To investigate the effect of agglomerated-Pt particles, different Pt nanoparticles agglomerates were formed by first adding KCl solution into stock Pt colloid and aging for 1 h. Next, the resulting Pt colloids (0.03 mM, 50 L) were added into a working solution (1 mL C–P buffer, pH 4.4; 50 L 4 mg mL−1 TMB; 60 L 30% H2 O2 ) to initiate the catalytic reactions. The absorbance at 650 nm was measured 20 min later.
Fig. 1. TEM images (a) and HRTEM images (b) of cubic Pt nanoparticle.
the blue-product-formation reaction slowed and became saturated after about 20 min. As a commonly used peroxidase substrate, 3,3 ,5,5 tetramethylbenzidine sulfate (TMB) is colorless and can be oxidized slowly by H2 O2 . Catalysts such as peroxidase can catalyze the oxidation of TMB into two colored products [2]. The first product is a blue charge-transfer complex of the parent diamine. This species exists in rapid equilibrium with the radical cation and its maximal absorption wavelength is 370 and 652 nm. If sufficient acid exists (e.g., excess H2 O2 or a strong acidic condition), the blue product will be further oxidized to a yellow diimine, which is stable in acidic conditions and its maximal absorption wavelength is 450 nm.
3. Results and discussion 3.1. Cubic Pt nanocrystals Fig. 1a shows a TEM image of the resulting Pt nanocrystals. The nanocrystals were generally cubic in shape (84% cubic, 4% tetrahedral, 12% irregular particles), with 10-nm average size. The HRTEM images of a Pt cube along the [1 0 0] zone axis is shown in Fig. 1b. 3.2. Kinetic investigation of Pt-catalyzed oxidation of TMB in the presence of H2 O2 and possible mechanism A kinetic investigation of Pt-catalyzed oxidation of TMB was performed by measuring the absorbance at 652 nm as a function of time after adding Pt colloidal solution (0.03 mM, 50 L) to the working solution (1 mL C–P buffer pH 4.4, 50 L 4 mg mL−1 TMB, 60 L 30% H2 O2 ). The absorbance curves are shown in Fig. 2. Initially, the catalytic reaction proceeded very rapidly. After about 5 min,
Fig. 2. Kinetic curves of Pt-catalyzed oxidation of TMB.
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Scheme 1. Possible mechanism for the TMB–H2 O2 –Pt colloid system.
The use of colloidal platinum nanocrystals as catalysts for electron-transfer reactions has been extensively investigated, and it has been reported that colloidal platinum as a catalyst can transfer electrons to hydrogen peroxide and hydrazine, causing them to decompose [18,19]. Regarding the catalysis for hydrogen peroxide decomposition, many possible mechanisms have been proposed. One such mechanism is the radical chain mechanism, in which the initiation reaction of OH-radical production is catalyzed by a metal or metal ion [20,21]. It is assumed that the oxygen–oxygen bond of H2 O2 is rapidly broken by the catalytic action of platinum nanocrystals to give OH radicals. The resulting OH radicals were stabilized at the surface of the platinum nanocrystals [22,23], and they would react with TMB. This proposed mechanism is summarized in Scheme 1.
Fig. 6 provides the relationship between the concentration of Pt-nanocrystal catalyst and the catalytic activity. We find that the catalytic activity of Pt nanocrystals increases with increasing Pt concentration. When the Pt concentration remains in a relative low range, the relationship between the concentration of Pt-nanocrystal catalyst and catalytic activity is nearly linear. When the Pt concentration increases to over ∼300 ng/mL, the relationship between the concentration of Pt-nanocrystal catalyst and catalytic activity becomes sublinear. With excess reactants, the reaction speed depends on the concentration of Pt-nanocrystal catalyst (over a given range of reaction speed), but when the reaction speed exceeds the given range, the reactants are rapidly depleted, and the reaction speed becomes dependent not only on catalyst concentration but also on reactant concentration.
3.3. Effect of buffers and pH
3.5. Effect of agglomeration of Pt nanocrystals
To investigate whether buffer agents affect the Pt–TMB–H2 O2 catalytic reaction, we performed the catalytic experiments in different buffers and with different pH values. Fig. 3 shows the UV–vis spectra collected 20 min after Pt nanocrystals were added to different working solutions, and Fig. 4 compares the absorbance at 450 and 650 nm of different buffers and different pH values. We find that the catalytic activity of platinum nanocrystals is not significantly affected by the buffer type but is strongly affected by the pH. When the buffer pH value exceeds 6, catalytic activity is very low for all three working buffers. The maximal absorbance at 650 nm appears at pH 3.8 in C–P buffer, at pH 2.6 in A–A buffer, and at pH 4.4 in C–C buffer. The maximal absorbance at 450 nm is apparent at the lowest pH value in all three buffers. Through oxidation, the TMB solution changes color from an achromatic color to blue with absorption at 652 nm, then to green, and finally to yellow with absorption at 450 nm. In addition, excessive acid redounds to produce a yellow diimine as a final product. Thus, it is intelligible that at the lowest pH value, the absorbance at 450 nm is maximal.
The Pt-nanocrystal catalyst used in this work was dispersed in aqueous solution to form a hydrophilic colloid. However, a colloidal dispersion is said to be stable when significant agglomeration does not occur; that is, when the potential barrier is sufficiently high to prevent particles from contacting each other. Whether or not dispersion is stable depends both on the surface electrostatic potential (which depends on the pH of the solution) and the ion concentration of the solution [24]. The size of agglomerates depends greatly on the ionic strength of the solution, and the higher the ionic strength of the liquid in which particles are suspended, the higher the likelihood that agglomeration will occur. Effective control of agglomeration can likely be attained by adjusting the ionic strength. In this work, we obtained different size agglomerates of Pt nanocrystals by adjusting the ionic strength of KCl in the dispersion solution. At first, freshly prepared and cleaned Pt nanocrystals were dispersed in de-ionized water. This Pt colloid was used in all former testing of Pt-catalyzed oxidation of TMB in the presence of H2 O2 . The hydrodynamics diameter of this Pt colloid was measured with PCS (Photon Correlation Spectroscopy, N4 Plus, Beckman). The result was 36 nm which was larger than the result obtained by TEM. This is because that the hydrodynamic diameter is not measuring one single particle, but is effectively measuring the nanoparticle and anything affiliated with its surface that could be a surrounding double-layer, polymers or other capping agents, or nanoparticle aggregates. Then KCl solution at different concentrations was added into this Pt colloid and aging for 1 h. The hydrodynamics diameter of Pt colloid at different ionic strength of KCl was measured immediately after. And the corresponding Pt colloids were added
3.4. Effect of H2 O2 and Pt nanocrystals concentrations As shown in Fig. 5, the catalytic activity of Pt nanocrystals increases with increasing H2 O2 concentration. Furthermore, when the amount of H2 O2 reaches about 1.2 times that of TMB, the catalytic activity of the Pt nanocrystals achieves its highest point. Upon further increasing the concentration of H2 O2 , the catalytic activity of the Pt nanocrystals decreases slowly to a steady level.
M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 373 (2011) 6–10
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Fig. 4. Effect of buffers and pH. (a) Absorbance at 450 nm as a function of pH. (b) Absorbance at 650 nm as a function of pH.
diameter of Pt colloid increases with increasing KCl ionic strength. It means that Pt colloid stability decreased and the aggregation of Pt nanocrystals formed and grew up gradually with increasing KCl ionic strength. In Fig. 7, it shows that the catalytic activity of Pt colloid in the Pt–TMB–H2 O2 system decreases with increasing ionic strength of
Fig. 3. Absorption spectra collected 20 min after Pt nanoparticles were added to working solutions of (a) C–P buffers, (b) C–C buffers, and (c) A–A buffers with different pH values.
into a working solution to initiate TMB–H2 O2 catalytic reactions simultaneously. Various factors control the colloid stability to aggregation. The crucial factors we need to consider include the -potential, the effect of particle size and the effect of ion type and concentration [25]. If any different electrolytic ion were introduced into this colloid, the potential barrier between particles would be impaired so as to not sufficiently prevent particles from contacting one another. And aggregation would form. As shown in Fig. 7, the hydrodynamic
Fig. 5. Effect of H2 O2 concentration. Working buffer is C–P buffer, pH 4.4.
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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30870679 and 30970787) and the National Basic Research Program of China (No. 2006CB933206 and 2006CB705606). References
Fig. 6. Effect of Pt nanoparticles concentration. Working buffer is C–P buffer, pH 4.4.
Fig. 7. Effect of KCl ionic strength on Pt-nanoparticle agglomeration and catalytic activity.
KCl in Pt colloid and with increasing size of Pt-nanocrystal agglomerates. It is known that the rate of heterogeneous catalysis increases with the available active surface area of catalyst. In heterogeneous catalysis, the reactants diffuse to the catalyst surface and adsorb onto it, via the formation of chemical bonds. After reaction, the products desorb from the surface and diffuse away. The total surface area of solid has an important effect on the reaction rate. The smaller the catalyst particle size, the larger the surface area for a given mass of particles [26]. Agglomeration of Pt nanocrystals will cause the available active surface area of Pt-colloid catalyst to decrease, so agglomeration of Pt nanocrystals is likely to decrease the catalytic activity of Pt. 4. Conclusion In this work, we find that Pt nanoparticles are effective as catalyst and possess peroxidase-like activity, and we investigated Pt-nanoparticle-catalyzed H2 O2 -ediated oxidation of TMB. In the present Pt–TMB–H2 O2 catalytic system, platinum nanoparticles may accelerate the electron-transfer process and facilitate radical generation at the surface of Pt nanoparticles in aqueous solution. To facilitate further applications of this catalytic system, several experimental conditions such as pH, buffer type, and H2 O2 and Pt concentrations have been optimized; and we find that the agglomeration of Pt-colloid catalyst in aqueous solution affects its catalytic activity distinctly. These investigations all provide useful information for future applications of Pt nanoparticles for biosensor and in immunohistochemistry.
[1] C.P. Price, D.J. Newman (Eds.), Principles and Practice of Immunoassay, 2nd ed., Stockton, New York, 1997. [2] P.D. Josephy, T.E. Eling, R.P. Mason, The horseradish peroxidase-catalyzed oxidation of 3,5,3 ,5 -tetramethylbenzidine: free radical and charge-transfer complex intermediates, J. Biol. Chem. 257 (1982) 3669–3675. [3] J.P.D. Josephy, T.E. Eling, R.P. Mason, Co-oxidation of benzidine by prostaglandin synthase and comparison with the action of horseradish peroxidase, J. Biol. Chem. 258 (1983) 5561–5569. [4] H.H. Liem, F. Cardenas, M. Tavassoli, M.B. Fitzpatrick, U. Miiller-Eberhard, Quantitative determination of hemoglobin and cytochemical staining for peroxidase using 3,3 ,5,5 -tetramethylbenzidine dihydrochloride, a safe substitute for benzidine, Anal. Biochem. 98 (1979) 388–393. [5] P.E. Thomas, D. Ryan, W. Levin, An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels, Anal. Biochem. 75 (1976) 168–176. [6] L. Gao, J. Zhuang, L. Nie, et al., Peroxidase-like activity of ferromagnetic nanoparticles, Nat. Nanotechnol. 2 (2007) 577–583. [7] Y. Matsuoka, T. Onodera, T. Kojima, Novel enzymatic properties of DNA–Pt complexes, Biomacromolecules 8 (2007) 2684–2688. [8] Z. Wen, S. Ci, J. Li, Pt nanoparticles inserting in carbon nanotube arrays: nanocomposites for glucose biosensors, J. Phys. Chem. C 113 (2009) 13482– 13487. [9] I.J. Plante, S.E. Habas, B.D. Yuhas, et al., Interfacing metal nanoparticles with semiconductor nanowires, Chem. Mater. 21 (2009) 3662–3667. [10] Y. Liu, J. Chen, W. Zhang, et al., Nano-Pt modified aligned carbon nanotube arrays are efficient, robust, high surface area electrocatalysts, Chem. Mater. 20 (2008) 2603–2605. [11] J. Prabhuram, X. Wang, C.L. Hui, I.M. Hsing, Synthesis and characterization of surfactant-stabilized Pt/C nanocatalysts for fuel cell applications, J. Phys. Chem. B 107 (2003) 11057–11064. [12] Z. Chen, X. Qiu, B. Lu, S. Zhang, W. Zhu, L. Chen, Synthesis of hydrous ruthenium oxide supported platinum catalysts for direct methanol fuel cells, Electrochem. Commun. 7 (2005) 593–596. [13] L. Liao, H.X. Mai, Q. Yuan, et al., Single CeO2 nanowire gas sensor supported with Pt nanocrystals: gas sensitivity, surface bond states, and chemical mechanism, J. Phys. Chem. C 112 (2008) 9061–9065. [14] H. Bönnemann, W. Brijoux, A. Schulze Tilling, K. Siepen, Application of heterogeneous colloid catalysts for the preparation of fine chemicals, Top. Catal. 4 (1997) 217–227. [15] R. Narayanan, M. El-Sayed, Catalysis with transition metal nanoparticles in colloidal solution: nanoparticles shape dependence and stability, J. Phys. Chem. B 109 (2005) 12663–12676. [16] Y. Borodko, S.E. Habas, M. Koebe, P. Yang, H. Frei, G.A. Somorjai, Probing the interaction of poly(vinylpyrrolidone) with platinum nanocrystals by UV–Raman and FTIR, J. Phys. Chem. B 110 (2006) 23052–23059. [17] Y. Borodko, L. Jones, H. Lee, H. Frei, G.A. Somorjai, Spectroscopic study of tetradecyltrimethylammonium bromide Pt–C14 TAB nanoparticles: structure and stability, Langmuir 25 (2009) 6665–6671. [18] T. Aiuchi, S. Nakajo, K. Nakaya, Reducing activity of colloidal platinum nanoparticles for hydrogen peroxide, 2,2-diphenyl-1-picrylhydrazyl radical and 2,6-dichlorophenol indophenol, Biol. Pharm. Bull. 27 (2004) 736– 738. ´ [19] A.V. Ananev, M.Y. Boltoeva, G.L. Bykov, B.G. Ershov, Catalytic decomposition of hydrazine in weakly alkaline solutions on platinum nanoparticles, Radiochemistry 46 (2004) 531–535. [20] N. Kitajima, S.I. Fukuzumi, Y. Ono, Formation of superoxide ion during the decomposition of hydrogen peroxide on supported metal oxides, J. Phys. Chem. 82 (1978) 1505–1509. [21] L. Mochida, K. Takshita, Transition metal ions on molecular sieves. II. Catalytic activities of transition metal ions on molecular sieves for the decomposition of hydrogen peroxide, J. Phys. Chem. 78 (1974) 1653–1657. [22] Z. Zhang, A. Berg, H. Levanon, R.W. Fessenden, D. Meisel, On the interactions of free radicals with gold nanoparticles, J. Am. Chem. Soc. 125 (2003) 7959– 7963. [23] A. Hardman, G.R. Millward, P. Neta, M.C. Richoux, J.M. Thomas, Interfacial electron-transfer reactions between platinum colloids and reducing radicals in aqueous solution, J. Phys. Chem. 92 (1988) 1286–1290. [24] P.C. Hiemenz, Principles of Colloid and Surface Chemistry, 2nd ed., Marcel Dekker, New York, 1986. [25] T. Cosgrove, Colloid Science: Principles, Methods and Applications, 2nd ed., John Wiley & Sons Ltd., United Kingdom, 2010. [26] O. Deutschmann, H. Knözinger, K. Kochloefl, T. Turek, Heterogeneous Catalysis and Solid Catalysts, Ullmann’s Encyclopedia of Industrial Chemistry, 7th ed., Wiley-VCH Verlag GmbH & Co. KGaA, 2009.
Contents lists available at ScienceDirect
Colloids and Surfaces A: Physicochemical and Engineering Aspects journal homepage: www.elsevier.com/locate/colsurfa
Peroxidase-like catalytic activity of cubic Pt nanocrystals Ming Ma ∗ , Yu Zhang, Ning Gu State Key Laboratory of Bioelectronics and Jiangsu Key Laboratory for Biomaterials and Devices, Southeast University, Nanjing 210096, Jiangsu, PR China
a r t i c l e
i n f o
Article history: Received 27 March 2010 Received in revised form 28 July 2010 Accepted 4 August 2010 Available online 12 August 2010 Keywords: Pt nanocrystals Peroxidase-like activity Agglomeration 3,3 ,5,5 -Tetramethylbenzidine
a b s t r a c t Monodispersed cubic platinum (Pt) nanocrystals with an average size of approximately 10 nm were prepared by a reduction method with cetyltrimethylammonium bromide (CTAB) serving as steric stabilizer. The resulting Pt nanocrystals exhibit a peroxidase-like activity and catalyze the H2 O2 -mediated oxidation of 3,3 ,5,5 -tetramethylbenzidine (TMB) to produce two colored products with high catalytic activity. The color-generating activity of this system may be influenced by several factors, and we examined several factors to optimize this colorimetric system including buffer types, pH, and concentrations of both H2 O2 and Pt nanocrystals. The effect of agglomeration of Pt nanocrystals was also investigated, and we find that agglomeration of Pt nanocrystals in aqueous solution distinctly affects Pt nanocrystals catalytic activity. We attribute the catalytic activity of Pt nanocrystals to their acceleration of the electron-transfer process and the consequent facilitation of radical generation. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In enzyme-linked immunosorbent assays (ELISAs), horseradish peroxidase (HRP) and other natural peroxidases have commonly been used as catalysts [1]. Peroxidase catalyzes the conversion of chromogenic substrates (e.g., 3,3 ,5,5 -tetramethylbenzidine TMB, 2,2 -azinobis [3-ethylbenzothiazoline-6-sulfonic acid]diammonium salt ABTS, o-phenylenediamine dihydrochloride OPD, etc.) into colored molecules using hydrogen peroxide as the oxidizing agent [1–5]. HRP is not the only catalyst possible for catalyzing the reaction of TMB and H2 O2 . In fact, some metal and metal-oxide nanoparticles such as magnetite nanoparticles have recently been found to possess intrinsic enzyme mimetic activity similar to that found in natural peroxidases [6,7]. This fact opens possibility of novel applications of these nanoparticles for biosensors and in immunohistochemistry. Colloidal platinum nanocrystals have recently received considerable interest because of their unique physical and chemical properties [8,9]. In addition to their interesting physical properties, catalysis may be one of their most popular applications because of their high catalytic activities in many chemical reactions, including hydrogenation production [10], fuel-cell technology [11,12], gas sensing [13], fine-chemical synthesis [14], etc. Therefore, colloidal platinum nanoparticles are likely to be effective catalysts because they exhibit peroxidase-like activity just like magnetite nanoparticles.
The catalytic activity of metal nanocrystals is considered to depend strongly on the particle surface properties, its morphology, size, the stabilizing agent, etc. [15]. Exposed particle surfaces can provide active sites for absorbing reactants. If the active sites are blocked, the catalytic activity will be reduced. For instance, the carbonyl group of polyvinylpyrrolidone (PVP) or polyacrylate, which are the most widely used surface-regulating polymers in shapednanoparticle synthesis, interacts strongly with the Pt surface [16] and thus blocks a significant number of active sites. Conversely, compared with the carbonyl group, alkylammonium ions, which have been widely used in synthesizing Au nanoparticles, have much weaker interactions with the Pt surface [17]. Therefore, in the present study, cetyltrimethylammonium bromide (CTAB) is chosen to serve as a surface-stabilizing agent to regulate the shape of nanocrystals while preserving catalytically active sites. Herein, we report a reduction method in which CTAB is used as a surface-stabilizing agent to prepare monodisperse cubic Pt nanocrystals. The resulting Pt nanocrystals can catalyze the H2 O2 -mediated oxidation of TMB with high catalytic activity. To optimize this colorimetric system, we examine several factors including buffer type, pH, and concentrations of both H2 O2 and Pt nanocrystals. In addition, we find that agglomeration of Pt nanocrystals is inevitable in a colloidal solution, and we use this phenomenon to investigate the effect on catalytic activity of reducing the exposed surface. 2. Experimental 2.1. Synthesis of Pt nanocrystals
∗ Corresponding author. E-mail address: [email protected] (M. Ma). 0927-7757/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2010.08.007
In a typical synthesis of Pt nanocrystals, aqueous solutions of 0.0225 mmol K2 PtCl4 (Alfa Aesar, 99.9%) and 2 mmol CTAB (Sun-
M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 373 (2011) 6–10
7
shine, >90%) were mixed in a 20-mL vial at room temperature. The mixture was heated at 50 ◦ C for 5 min until the solution became clear. Ice-cold aqueous solution 0.5 mL 1.35 M NaBH4 (Sinopharm Chemical Reagent Co. Ltd., >96%) was added, and the solution was aged for 7 h in a 50 ◦ C water bath. The product was separated and centrifuged twice at 12,000 rpm for 15 min. The precipitate was collected and redispersed in water. 2.2. Instrumentation Transmission electron microscope (TEM) and high-resolution TEM (HRTEM) images were collected with a JEM-2100 microscope (JEOL). Absorption spectra were recorded with an EL808 Ultra Microplate Reader (Bio-Tek). The hydrodynamic colloid diameters were detected by a submicron particle size analyzer (PCS, N4 Plus, Beckman). 2.3. Testing Pt-catalyzed oxidation of TMB in the presence of H2 O2 The working solution of the substrate for the catalytic reaction was freshly prepared by adding 50 L 4 mg mL−1 TMB (Sigma, >99%) solution (in DMSO) and 60 L 30% (v/v) H2 O2 solution (in H2 O) into 1 mL working buffer (C–P buffer: 0.1 M citrate–0.2 M disodium hydrogen phosphate; C–C buffer: 0.1 M citrate–0.1 M sodium citrate; or A–A buffer: 0.2 M acetate–0.2 M sodium acetate). None of these buffers were sensitive to temperature. The working solution and 50 L 0.03 mM Pt colloid solution were added consecutively to 24-well ELISA plates to start the catalytic reaction. The kinetics of the catalytic reaction was monitored by detecting the absorption at 650 nm as a function of time. To investigate the effect of different buffers and of the pH, the catalytic reactions were performed in working solutions prepared in C–P buffers (pH 7.4, 6.4, 5.4, 4.4, 3.4, and 2.6), C–C buffers (pH 7.4, 6.4, 5.4, 4.4, 3.4, and 2.6), and A–A buffers (pH 6.4, 5.4, 4.4, and 3.4). The absorbance at 650 nm was measured 20 min after the initiation of the reaction. To investigate the effect of the concentration of H2 O2 and Pt, catalytic reactions were performed in working solutions (1 mL C–P buffer, pH 4.4; 50 L 4 mg mL−1 TMB) with different concentrations of H2 O2 or different concentrations of Pt. To investigate the effect of agglomerated-Pt particles, different Pt nanoparticles agglomerates were formed by first adding KCl solution into stock Pt colloid and aging for 1 h. Next, the resulting Pt colloids (0.03 mM, 50 L) were added into a working solution (1 mL C–P buffer, pH 4.4; 50 L 4 mg mL−1 TMB; 60 L 30% H2 O2 ) to initiate the catalytic reactions. The absorbance at 650 nm was measured 20 min later.
Fig. 1. TEM images (a) and HRTEM images (b) of cubic Pt nanoparticle.
the blue-product-formation reaction slowed and became saturated after about 20 min. As a commonly used peroxidase substrate, 3,3 ,5,5 tetramethylbenzidine sulfate (TMB) is colorless and can be oxidized slowly by H2 O2 . Catalysts such as peroxidase can catalyze the oxidation of TMB into two colored products [2]. The first product is a blue charge-transfer complex of the parent diamine. This species exists in rapid equilibrium with the radical cation and its maximal absorption wavelength is 370 and 652 nm. If sufficient acid exists (e.g., excess H2 O2 or a strong acidic condition), the blue product will be further oxidized to a yellow diimine, which is stable in acidic conditions and its maximal absorption wavelength is 450 nm.
3. Results and discussion 3.1. Cubic Pt nanocrystals Fig. 1a shows a TEM image of the resulting Pt nanocrystals. The nanocrystals were generally cubic in shape (84% cubic, 4% tetrahedral, 12% irregular particles), with 10-nm average size. The HRTEM images of a Pt cube along the [1 0 0] zone axis is shown in Fig. 1b. 3.2. Kinetic investigation of Pt-catalyzed oxidation of TMB in the presence of H2 O2 and possible mechanism A kinetic investigation of Pt-catalyzed oxidation of TMB was performed by measuring the absorbance at 652 nm as a function of time after adding Pt colloidal solution (0.03 mM, 50 L) to the working solution (1 mL C–P buffer pH 4.4, 50 L 4 mg mL−1 TMB, 60 L 30% H2 O2 ). The absorbance curves are shown in Fig. 2. Initially, the catalytic reaction proceeded very rapidly. After about 5 min,
Fig. 2. Kinetic curves of Pt-catalyzed oxidation of TMB.
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Scheme 1. Possible mechanism for the TMB–H2 O2 –Pt colloid system.
The use of colloidal platinum nanocrystals as catalysts for electron-transfer reactions has been extensively investigated, and it has been reported that colloidal platinum as a catalyst can transfer electrons to hydrogen peroxide and hydrazine, causing them to decompose [18,19]. Regarding the catalysis for hydrogen peroxide decomposition, many possible mechanisms have been proposed. One such mechanism is the radical chain mechanism, in which the initiation reaction of OH-radical production is catalyzed by a metal or metal ion [20,21]. It is assumed that the oxygen–oxygen bond of H2 O2 is rapidly broken by the catalytic action of platinum nanocrystals to give OH radicals. The resulting OH radicals were stabilized at the surface of the platinum nanocrystals [22,23], and they would react with TMB. This proposed mechanism is summarized in Scheme 1.
Fig. 6 provides the relationship between the concentration of Pt-nanocrystal catalyst and the catalytic activity. We find that the catalytic activity of Pt nanocrystals increases with increasing Pt concentration. When the Pt concentration remains in a relative low range, the relationship between the concentration of Pt-nanocrystal catalyst and catalytic activity is nearly linear. When the Pt concentration increases to over ∼300 ng/mL, the relationship between the concentration of Pt-nanocrystal catalyst and catalytic activity becomes sublinear. With excess reactants, the reaction speed depends on the concentration of Pt-nanocrystal catalyst (over a given range of reaction speed), but when the reaction speed exceeds the given range, the reactants are rapidly depleted, and the reaction speed becomes dependent not only on catalyst concentration but also on reactant concentration.
3.3. Effect of buffers and pH
3.5. Effect of agglomeration of Pt nanocrystals
To investigate whether buffer agents affect the Pt–TMB–H2 O2 catalytic reaction, we performed the catalytic experiments in different buffers and with different pH values. Fig. 3 shows the UV–vis spectra collected 20 min after Pt nanocrystals were added to different working solutions, and Fig. 4 compares the absorbance at 450 and 650 nm of different buffers and different pH values. We find that the catalytic activity of platinum nanocrystals is not significantly affected by the buffer type but is strongly affected by the pH. When the buffer pH value exceeds 6, catalytic activity is very low for all three working buffers. The maximal absorbance at 650 nm appears at pH 3.8 in C–P buffer, at pH 2.6 in A–A buffer, and at pH 4.4 in C–C buffer. The maximal absorbance at 450 nm is apparent at the lowest pH value in all three buffers. Through oxidation, the TMB solution changes color from an achromatic color to blue with absorption at 652 nm, then to green, and finally to yellow with absorption at 450 nm. In addition, excessive acid redounds to produce a yellow diimine as a final product. Thus, it is intelligible that at the lowest pH value, the absorbance at 450 nm is maximal.
The Pt-nanocrystal catalyst used in this work was dispersed in aqueous solution to form a hydrophilic colloid. However, a colloidal dispersion is said to be stable when significant agglomeration does not occur; that is, when the potential barrier is sufficiently high to prevent particles from contacting each other. Whether or not dispersion is stable depends both on the surface electrostatic potential (which depends on the pH of the solution) and the ion concentration of the solution [24]. The size of agglomerates depends greatly on the ionic strength of the solution, and the higher the ionic strength of the liquid in which particles are suspended, the higher the likelihood that agglomeration will occur. Effective control of agglomeration can likely be attained by adjusting the ionic strength. In this work, we obtained different size agglomerates of Pt nanocrystals by adjusting the ionic strength of KCl in the dispersion solution. At first, freshly prepared and cleaned Pt nanocrystals were dispersed in de-ionized water. This Pt colloid was used in all former testing of Pt-catalyzed oxidation of TMB in the presence of H2 O2 . The hydrodynamics diameter of this Pt colloid was measured with PCS (Photon Correlation Spectroscopy, N4 Plus, Beckman). The result was 36 nm which was larger than the result obtained by TEM. This is because that the hydrodynamic diameter is not measuring one single particle, but is effectively measuring the nanoparticle and anything affiliated with its surface that could be a surrounding double-layer, polymers or other capping agents, or nanoparticle aggregates. Then KCl solution at different concentrations was added into this Pt colloid and aging for 1 h. The hydrodynamics diameter of Pt colloid at different ionic strength of KCl was measured immediately after. And the corresponding Pt colloids were added
3.4. Effect of H2 O2 and Pt nanocrystals concentrations As shown in Fig. 5, the catalytic activity of Pt nanocrystals increases with increasing H2 O2 concentration. Furthermore, when the amount of H2 O2 reaches about 1.2 times that of TMB, the catalytic activity of the Pt nanocrystals achieves its highest point. Upon further increasing the concentration of H2 O2 , the catalytic activity of the Pt nanocrystals decreases slowly to a steady level.
M. Ma et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 373 (2011) 6–10
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Fig. 4. Effect of buffers and pH. (a) Absorbance at 450 nm as a function of pH. (b) Absorbance at 650 nm as a function of pH.
diameter of Pt colloid increases with increasing KCl ionic strength. It means that Pt colloid stability decreased and the aggregation of Pt nanocrystals formed and grew up gradually with increasing KCl ionic strength. In Fig. 7, it shows that the catalytic activity of Pt colloid in the Pt–TMB–H2 O2 system decreases with increasing ionic strength of
Fig. 3. Absorption spectra collected 20 min after Pt nanoparticles were added to working solutions of (a) C–P buffers, (b) C–C buffers, and (c) A–A buffers with different pH values.
into a working solution to initiate TMB–H2 O2 catalytic reactions simultaneously. Various factors control the colloid stability to aggregation. The crucial factors we need to consider include the -potential, the effect of particle size and the effect of ion type and concentration [25]. If any different electrolytic ion were introduced into this colloid, the potential barrier between particles would be impaired so as to not sufficiently prevent particles from contacting one another. And aggregation would form. As shown in Fig. 7, the hydrodynamic
Fig. 5. Effect of H2 O2 concentration. Working buffer is C–P buffer, pH 4.4.
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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30870679 and 30970787) and the National Basic Research Program of China (No. 2006CB933206 and 2006CB705606). References
Fig. 6. Effect of Pt nanoparticles concentration. Working buffer is C–P buffer, pH 4.4.
Fig. 7. Effect of KCl ionic strength on Pt-nanoparticle agglomeration and catalytic activity.
KCl in Pt colloid and with increasing size of Pt-nanocrystal agglomerates. It is known that the rate of heterogeneous catalysis increases with the available active surface area of catalyst. In heterogeneous catalysis, the reactants diffuse to the catalyst surface and adsorb onto it, via the formation of chemical bonds. After reaction, the products desorb from the surface and diffuse away. The total surface area of solid has an important effect on the reaction rate. The smaller the catalyst particle size, the larger the surface area for a given mass of particles [26]. Agglomeration of Pt nanocrystals will cause the available active surface area of Pt-colloid catalyst to decrease, so agglomeration of Pt nanocrystals is likely to decrease the catalytic activity of Pt. 4. Conclusion In this work, we find that Pt nanoparticles are effective as catalyst and possess peroxidase-like activity, and we investigated Pt-nanoparticle-catalyzed H2 O2 -ediated oxidation of TMB. In the present Pt–TMB–H2 O2 catalytic system, platinum nanoparticles may accelerate the electron-transfer process and facilitate radical generation at the surface of Pt nanoparticles in aqueous solution. To facilitate further applications of this catalytic system, several experimental conditions such as pH, buffer type, and H2 O2 and Pt concentrations have been optimized; and we find that the agglomeration of Pt-colloid catalyst in aqueous solution affects its catalytic activity distinctly. These investigations all provide useful information for future applications of Pt nanoparticles for biosensor and in immunohistochemistry.
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