Horseradish Peroxidase Functionalized Fluorescent Gold

Jan 24, 2011 - ... range of 100 nM to 100 μM with high sensitivity (LOD = 30 nM, S/N = 3). ...... Devi , S. Salini , A.H. Anulekshmi , G.L. Praveen ,...
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Horseradish Peroxidase Functionalized Fluorescent Gold Nanoclusters for Hydrogen Peroxide Sensing Fang Wen, Yanhua Dong, Lu Feng, Song Wang, Sichun Zhang, and Xinrong Zhang* Key Laboratory for Atomic and Molecular Nanosciences of the Education Ministry, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China

bS Supporting Information ABSTRACT: The fluorescence of metal nanoclusters provides an amusing optic feature to be applied in various fields. However, rational design of dual functional fluorescent metal nanoclusters directed by active enzyme with targeted application remains little explored. In this work, we report a new strategy to construct enzyme functionalized fluorescent gold nanoclusters via a biomineralization process for the detection of hydrogen peroxide. Horseradish peroxidase (HRP) was used as a model functional template to direct the synthesis of fluorescent gold nanoclusters (Au NCs) at physiological conditions to form HRP-Au NCs bioconjugates. We found that the fluorescence of HRP-Au NCs can be quenched quantitatively by adding H2O2, indicating that HRP enzyme remains active and enables catalytic reaction of HRP-Au NCs and H2O2. Upon the addition of H2O2 under optimal conditions, the fluorescence intensity quenched linearly over the range of 100 nM to 100 μM with high sensitivity (LOD = 30 nM, S/N = 3). This study would be potentially extended to other functional proteins to generate dual functional nanoclusters and applied to real time monitoring of biologically important targets in living cells.

he fluorescence of metal nanoclusters has drawn continuous research interest in the fields of chemistry, biology, and materials.1-4 There has been a great deal of research work on the fluorescence of metal nanoclusters, especially gold and silver.5-7 Owing to their ultrasmall size, biocompatibility, and highly fluorescent properties, applications of these fluorescent gold nanoclusters (Au NCs) would be an attractive field to study. Up to now, the applications of fluorescent Au NCs in analysis are mainly based on the fluorescence quenching effect5,8-12 of Au NCs through the interaction between gold and the limited analytes like Hg2þ,8,9,13 Cu2þ,11 and CN-.10 As we know, the fluorescence of Au NCs correlates not only with the metal quantization effect but also with the surface ligands or scaffolds.1,14 How to rational design fluorescent Au NCs with functional ligands or scaffolds to make them with broad applications remains to be explored. Biomolecules like bovine serum albumin (BSA) used in most studies merely serve as stabilizer and reducer to form fluorescent Au NCs.15 Buildup of bioinorganic hybrid Au NCs directed by active enzymes would integrate the catalysis function of the enzyme shell and the fluorescence of the core in a single nanocluster. In this work, we demonstrated that enzyme functionalized fluorescent nanoclusters can be obtained through a green biomineralization process and applied for biosensing using the enzyme activity. We chose the commonly used horseradish peroxidase (HRP)-hydrogen peroxide system to verify the concept. Our results are (i) HRP functionalized fluorescent gold nanoclusters (HRP-Au NCs) were successfully synthesized via a simple biomineralization process; (ii) HRP remains active and

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Scheme 1. Schematic of the Formation and the H2O2 Directed Quenching of HRP-Au NCs

possesses its intrinsic catalytic activity, thus HRP-Au NCs have dual functions including the fluorescence of the gold core and catalytic ability of the enzyme shell; (iii) taking advantage of the dual funcions, HRP-Au NCs have been successfully applied to hydrogen peroxide detection. HRP functionalized fluorescent Au NCs that possess dual functions including catalysis ability and fluorescence have been designed for hydrogen peroxide detection (Scheme 1). Fluorescent HRP-Au NCs have been synthesized at first (see Supporting Information for the details). The blackish green Received: November 29, 2010 Accepted: January 14, 2011 Published: January 24, 2011 1193

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Figure 1. Fluorescence spectra of HRP (black line), HRP-Au NCs in the absence (red line) and presence of 100 mM H2O2 (blue line). Inset: photograph displaying the fluorescence of HRP-Au NPs in the absence (A) and presence (B) of H2O2 upon excitation at 365 nm under a handheld UV lamp.

solution of the as-prepared HRP-Au NCs emits intense red fluorescence (λem max = 650 nm), while being accompanied with a weak peak at 450 nm under UV light excitation (365 nm). In contrast, the control HRP solution was reddish brown and emits no fluorescence (Figure 1). The proof-of-concept experiment was first conducted by measuring the fluorescence of HRP-Au NCs (7.8 μM, the concentration of HRP) in 50 mM glycine buffer (pH 9.0) in the presence of H2O2 (100 mM). Notably, the fluorescence at 450 nm significantly increased, while the peak at 650 nm completely disappeared in the presence of H2O2. Compared with the absorption spectrum of HRP and HRP-Au NCs (Figure S1, Supporting Information), the disappearance of absorption region between 300 and 450 nm indicated that H2O2 might change the conformation/structure of HRP and HRP-Au NCs. Thus, the change of the microenvironment around the Au NCs may be one of the reasons for fluorescence quenching. To prove that the catalytic activity of HRP plays a major role in H2O2 induced quenching, we conduct a comparison experiment by employing BSA-Au NCs as control. The addition of 100 μM H2O2 induced a significant decrease of the two peaks (650 and 450 nm) ratio (denoted as I650/I450) for HRP-Au NCs. On the contrary, the existence of 100 μM H2O2 caused a neglectable variation of the peak ratio (I670/I450) in the case of BSA-Au NCs. This result demonstrated that HRP remains active in the HRP-Au NCs and enables catalytic reduction/oxidation of H2 O2 . To test the possibility of the use of our HRP-Au NCs for the detection of H2O2, we investigated the effects of temperature and buffer solution pH on the fluorescence intensity. We examined the fluorescence of HRP-Au NCs over the temperature range from 25 to 60 °C (Figure S2, Supporting Information). The results reveal that the sensor performs best at 25 °C. Through monitoring the relative I650/I450 ratio during the reaction, we found that the H2O2induced fluorescence quenching of HRP-Au NCs reached completion within 10 min (Figure S3, Supporting Information). Next, we tested the fluorescence intensities of HRP-Au NCs in the absence and presence of H2O2 (1 mM) at various values of pH. The results show the sensor provided the optimal sensitivity at pH 9.0 (Figure S4, Supporting Information). Under the optimal conditions, HRP-Au NCs (7.8 μM) in 50 mM glycine buffer (pH 9.0) at 25 °C, we used our probe to

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Figure 2. Fluorescence responses of HRP-Au NCs after the addition of H2O2 (0-100 μM). Inset: Plot of the fluorescence ratio [I650/I450] of HRP-Au NCs versus the log concentration of H2O2.

Figure 3. Relative fluorescence intensity ratio (I650/I450) in the absence and presence of 100 μM ROS including H2O2, O2-, TBPH, OCl-, and •OH. (O2-, generated by ionization of KO2 in aqueous medium; TBPH, from commercially available tert-butyl hydroperoxide solution, t-BuOOH; •OH, generated from Fenton reaction (Fe2þ þ H2O2); OCl-, generated from ionization of NaOCl solution) and 100 μM interferences (GSH, Vc, glucose, Naþ, Ca2þ, and Kþ).

detect various concentrations of H2O2 in solution. As shown in Figure 3, the peak ratio I650/I450 decreased linearly (R2 = 0.97) upon increasing the concentration of H2O2 over the range of 100 nM to 100 μM, (Figure 2). The limit of detection (LOD) for H2O2 was 30 nM (S/N = 3). Interestingly, the fluorescence intensities at 450 nm increased significantly, while the peaks at 650 nm kept on decreasing when further increasing the concentrations of H2O2 up from 100 μM to 100 mM (Figure S5, Supporting Information). Next, we tested the specificity of HRP-Au NCs by conducting several contol experiments using Naþ, Kþ, Ca2þ, glucose, ascorbic acid (Vc), and glutathione (GSH) as interferences. No obvious changes have been observed as shown in Figure 3. Meanwhile, we tested the possibility of the use of HRP-Au NCs for sensing reactive oxygen species (ROS), which are ubiquitous in life and death processes of cells.16,17 The addition of 100 μM ROS including H2O2, O2-, tert-butyl hydroperoxide (TBPH), OCl-, and •OH induced a significant decrease of the two peaks ratio I650/I450 for HRP-Au NCs. Our results suggest that 1194

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surface of the Au core in the absence of H2O2, while 14.5% and 5.0% exist in the presence of 50 μM and 100 mM H2O2, respectively. In conclusion, we have demonstrated that dual functional fluorescent Au NCs can be formed in situ through HRP as scaffold. HRP remains active and enables catalytic reaction of HRP-Au NCs itself and H2O2, resulting in the quenching of its fluorescence that can be applied to hydrogen peroxide detection. This finding represents an important advance over previous Au NC synthesis in which the proteins are merely used as stabilizers and reducers whereas their biological functions are neglected. This study can be extended to other functional proteins to generate dual functional nanoclusters by integrating the functions of biomolecules and nanoclusters via a simple one-step synthesis.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel: þ86-10-62787678. Fax: þ86-10-6278-2485.

Figure 4. High resolution transmission electron microscopy (HRTEM) images and size distributions analysis of HRP-Au NCs in the absence (a, d, 2.7 ( 0.6 nm) and presence of 50 μM H2O2 (b, e, 3.0 ( 0.5 nm) and 100 mM H2O2 (c, f, 3.6 ( 0.7 nm); 180 particles are measured to get the size distribution.

this probe has potential for use in determining ROS in biological samples. To explore the quenching mechanism, we conducted TEM measurement. As the TEM results shown in Figure 4, the average sizes of HRP-Au NCs were 2.7 ( 0.6, 3.0 ( 0.5, and 3.6 ( 0.7 nm in the absence, presence of 50 μM, and presence of 100 mM H2O2, respectively. In the presence of ROS, Au-S bonding between HRP scaffolds and the encapsulated Au NCs are oxidized to form disulfide product under the catalytic effect of HRP. As a result, fewer HRP molecules are bonded to the Au NCs, and the Au NCs are prone to aggregate to form larger ones without the protection of scaffolds, thus leading to the quenching of fluorescence. As the HRTEM results show, the HRP scaffolds can be clearly seen around the Au NCs in the absence of H2O2, but disappear in the presence of H2O2. Therefore, the decrease of the fluorescence at 650 nm would mainly correlate with the increase of Au NC sizes after the addition of hydrogen peroxide. We also employed X-ray photoelectron spectroscopy (XPS) to monitor the oxidation state of the Au NCs before and after the addition of H2O2. As shown in Figure S7 (Supporting Information), the Au 4f7/2 binding energy (BE) spectrum could be deconvoluted into two components centered at Au(0) BE (83.5 eV, blue dash line) and Au(I) BE (84.7 eV, red dash line). We found that nearly 18.1% Au(I) exists on the

’ ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (No. 21027013), the Innovation Method Fund of China (No. 2008IM040600), and the National High Technology Research and Development Program of China (No. 2009AA03Z321) and the Tsinghua University Initiative Scientific Research Program. ’ REFERENCES (1) Wu, Z.; Jin, R. Nano Lett. 2010, 10, 2568. (2) Negishi, Y.; Takasugi, Y.; Sato, S.; Yao, H.; Kimura, K.; Tsukuda, T. J. Am. Chem. Soc. 2004, 126, 6518. (3) Peyser, L. A.; Vinson, A. E.; Bartko, A. P.; Dickson, R. M. Science 2001, 291, 103. (4) Lin, C.-A. J.; Yang, T.-Y.; Lee, C.-H.; Huang, S. H.; Sperling, R. A.; Zanella, M.; Li, J. K.; Shen, J.-L.; Wang, H.-H.; Yeh, H.-I.; Parak, W. J.; Chang, W. H. ACS Nano 2009, 3, 395. (5) Huang, C.-C.; Chen, C.-T.; Shiang, Y.-C.; Lin, Z.-H.; Chang, H.-T. Anal. Chem. 2009, 81, 875. (6) Guo, W.; Yuan, J.; Dong, Q.; Wang, E. J. Am. Chem. Soc. 2009, 132, 932. (7) Yu, J.; Patel, S. A.; Dickson, R. M. Angew. Chem., Int. Ed. 2007, 46, 2028. (8) Xie, J. P.; Zheng, Y. G.; Ying, J. Y. Chem. Commun. 2010, 46, 961. (9) Wei, H.; Wang, Z. D.; Yang, L. M.; Tian, S. L.; Hou, C. J.; Lu, Y. Analyst 2010, 135, 1406. (10) Liu, Y.; Ai, K.; Cheng, X.; Huo, L.; Lu, L. Adv. Funct. Mater. 2010, 20, 951. (11) Chen, W. B.; Tu, X. J.; Guo, X. Q. Chem. Commun. 2009, 1736. (12) Shiang, Y. C.; Huang, C. C.; Chang, H. T. Chem. Commun. 2009, 3437. (13) Huang, C. C.; Yang, Z.; Lee, K. H.; Chang, H. T. Angew. Chem., Int. Ed. 2007, 46, 6824. 1195

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