CHEMPLUSCHEM COMMUNICATIONS DOI: 10.1002/cplu.201300241
Recyclable, Biocompatible, Magnetic Titanium Dioxide Nanoparticles with Immobilized Enzymes for Biocatalysis Linjun Cai,*[a] Lei Chen,[b] Lufeng Zhang,[a] Guolong Lu,[a] Xinyue He,[a] Si Zhang,[a] Hang Sun,[a] Zhenning Liu,[a] and Bing Zhao*[b] In recent years, magnetic nanoparticles (MNPs) have attracted increasing interest in wide range of disciplines, including biomedicine, catalysis, and environmental control.[1, 2] Owing to their inherent magnetic properties, MNPs can be easily collected and separated with an external magnet, which allows efficient, for example, drug delivery,[3] catalytic industrial processes,[4] and removal of pollutants.[5] An attractive way to fabricate biocatalysts is to covalently immobilize enzymes (e.g., glucose oxidase[6] and chloroperoxidase[7]) on MNPs; this enables the catalyst to be conveniently and quickly separated from the reaction products. With a silica or polymer coating, MNPs are capable of increasing enzyme stability and activity.[7, 8] According to studies into such enzyme-immobilized MNPs, the improvement of techniques for biocompatible immobilization of enzymes is still an important research goal and another challenge is how to deal with the loss of enzyme activity after several cycles. Titanium dioxide (TiO2) is a nontoxic, environmentally friendly, and biocompatible material that has various applications in biology and biomedicine.[9] A recent study demonstrated that magnetic TiO2 (Fe3O4@TiO2) microspheres were capable of noncovalently capturing phosphopeptides through bridging bidentate bonds between the phosphoric acid group of phosphopeptides and the TiO2 surfaces, and thus, the phosphopeptides could be enriched for further identification and characterization based on mass spectrometry.[10] Owing to outstanding properties in photocatalysis, TiO2 is also widely used in the degradation of organic pollutants[11] and bacteria photodegradation.[12] Thus, the photocatalysis-induced self-cleaning property provides a convenient and efficient way for TiO2 recycling. Herein, we developed a biocatalyst based on TiO2 MNPs, which allow biocompatible enzyme immobilization, and are recyclable with exposure to UV light owing to the self-cleaning surface. As shown in Scheme 1, Fe3O4@TiO2 nanoparticles (NPs) were fabricated and mesoporous anatase shells formed by a hy[a] Dr. L. Cai, L. Zhang, G. Lu, X. He, S. Zhang, Dr. H. Sun, Prof. Z. Liu Key Laboratory of Bionic Engineering Ministry of Education, Jilin University 5988 Renmin Street, Chang Chun 130022 (P.R. China) Fax: (+ 86) 431-85095575-888 E-mail:
[email protected] [b] Dr. L. Chen, Prof. B. Zhao State Key Laboratory of Supramolecular Structure and Materials Jilin University, 2699 Qianjin Street Chang Chun 130012 (P.R. China) Fax: (+ 86) 431-85193421 E-mail:
[email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cplu.201300241.
2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Scheme 1. Fe3O4@TiO2 NP based horseradish peroxidase (HRP) immobilization, catalysis, and recycling. GA = glutaraldehyde.
drothermal method. HRP was covalently attached to TiO2 with GA as a linker and used as a model enzyme to examine the loading capability of Fe3O4@TiO2 NPs. The native structure of the catalytic center, the heme group of HRP, was preserved on the anatase TiO2, which enabled the enzyme to provide a similar enzyme activity to that of free HRP. Moreover, with an external magnet, the HPR-immobilized Fe3O4@TiO2 NPs can be easily collected and reused in a new solution of substrate, and thus, the enzyme-catalyzed reaction can be easily controlled as well. Furthermore, recycling of Fe3O4@TiO2 NPs was achieved by TiO2-induced photodegradation of the adsorbents upon exposure to UV radiation. Fe3O4 NPs were synthesized by a well-known solvothermal approach[4, 13] and, as shown in Figure 1 a, their morphology was spherical with an average diameter of 200 nm. To increase the enzyme-loading and photodegradation capabilities, the TiO2 shells were changed into mesoporous anatase during a hydrothermal procedure.[10] Accordingly, the amorphous, black TiO2-coated Fe3O4 suspension became brown after the hydrothermal procedure and, as shown in Figure 1 b, the Fe3O4 NPs were surrounded by mesoporous shells with an estimated thickness of 50 nm. The anatase TiO2 shells were confirmed by X-ray diffraction (XRD) and Raman spectroscopy. Figure 2 A shows XRD spectra of Fe3O4 and Fe3O4@TiO2 NPs, in which a strong band corresponding to the anatase (101) surface and two other bands corresponding to the anatase (004) and (200) surfaces appeared after the TiO2 coating (spectrum b in Figure 2 A). Furthermore, three typical Raman bands corresponding to anatase B1g, A1g, and Eg modes[14, 15] appeared in spectrum b in Figure 2 B; this is consistent with the XRD results. Thus, all of these results indicated that anatase shells were successfully coated onto the Fe3O4 NPs. The porous structure of anatase was able to highly enhance the surface areas of the Fe3O4@TiO2 nanocomposites,[10] and thus, allowed their high loading capability of enzymes. The Fe3O4@TiO2 NPs can be functionalized with 3-aminopropyltrimethoxysilane (APTMS), resulting in a NH2 layer, and ChemPlusChem 2013, 78, 1437 – 1439
1437
CHEMPLUSCHEM COMMUNICATIONS
www.chempluschem.org
silver surfaces (Figure 3 c); this is attributed to a nonnative five-coordinated high-spin state. In contrast, the Raman spectrum of HRP on Fe3O4@TiO2 NPs (Figure 3 a) is almost the same as that of the solution of HRP; this indicates that the native configuration of heme, known as a catalytic center of HRP, is preserved. The photocatalytic property of anatase TiO2 enables it to be capable of degrading adsorbents on the surface, which gives TiO2 self-cleaning characteristics.[19–21] Accordingly, TiO2 can be reused or recycled Figure 1. Transmission electron microscope (TEM) images of Fe3O4 (a) and Fe3O4@TiO2 (b) with UV radiation. To investigate the photodegradaNPs. tion capability of anatase, the HRP-immobilized Fe3O4@TiO2 NPs (Figure 3 a) were exposed to UV irradiation (UV lamp, l = 365 nm) for 5 h and the NPs were subsequently separated and washed with a magnet. As shown in Figure 3 d, HRP was not detectable, which indicated the possibility of HRP detachment from the Fe3O4@TiO2 NPs. Re-immobilization of new HRP samples on the recycled Fe3O4@TiO2 NPs is feasible, according to the Raman spectrum of HRP shown in Figure 3 e. To further confirm recyclability for other enzymes, cytochrome c (another heme protein with some peroxidase activity) was immobilized on the recycled Fe3O4@TiO2 NPs (Figure 3 f). Typical Raman bands of cytochrome c[22] clearly appeared, which demonstrated the feasibility Figure 2. XRD (A; label A = anatase) and Raman spectra (B) of Fe3O4 (a) and Fe3O4@TiO2 (b) NPs. of the proposed Fe3O4@TiO2 NPs for versatile enzyme immobilization. The catalytic activity of the HRP-immobilized Fe3O4@TiO2 therefore, proteins can be immobilized by using GA as a crosslinker between amine groups of the proteins (i.e., lysine resiNPs was examined by using 3,3’,5,5’-tetramethylbenzidine dues) and APTMS surface groups.[16, 17] The immobilization of (TMB) as a model substrate. TMB is a chromogen that yields a blue color upon oxidization, as a result of oxygen radicals HRP on Fe3O4@TiO2 NPs was confirmed by the Raman spectra produced by the hydrolysis of hydrogen peroxide with HRP. As of HRP. As seen in Figure 3 a, a typical Raman fingerprint of shown in the inset of Figure 4 A, the color change of the prodHRP was observed after HRP immobilization. ucts was visible after the catalysis of HRP-immobilized Direct binding of proteins to metal nanomaterials usually Fe3O4@TiO2 NPs in the presence of H2O2. In contrast, no color causes conformational changes in proteins, which results in partial denaturalization and loss of protein functions.[18] For change was observed in the substrate solution without HRPa six-coordinated high-spin configuration of heme, the n3 band immobilized Fe3O4@TiO2 NPs or with the as-prepared appears at 1506 cm 1 in the Raman spectrum of a solution of Fe3O4@TiO2 NPs (blue and black lines in Figure 4 A); this conHRP (Figure 3 b), but the band shifted to 1491 cm 1 on the firmed the catalytic capability of the HRP-immobilized Fe3O4@TiO2 NPs. Moreover, the Fe3O4@TiO2 NPs can be easily collected, separated by an external magnet (Figure 4 A, inset), and can be re-dispersed in a new solution of substrate, and thus, the enzyme-catalyzed reactions are easy to control. After the reaction, two typical bands at l = 369 and 651 nm, which correspond to the products of TMB,[23, 24] appeared (red line in Figure 4 A), and the product concentration increased with time, as shown in Figure 4 B. Figure 4 C shows the time-dependent reaction catalyzed by the same amount of immobilized and free HRP. In a control reaction, the same amount of free HRP as that immobilized on the Fe3O4@TiO2 NPs was used for a comparison of the enzyme activity. Thus, Figure 3. Raman spectra of HRP-immobilized Fe3O4@TiO2 NPs (a), HRP solution (b), HPRthe ratio of the specific activity of free HRP versus adsorbed Ag NPs (c), HRP-immobilized Fe3O4@TiO2 NPs after 5 h exposure to UV irradiathat of immobilized HRP can be simplified to the tion (d), recycled Fe3O4@TiO2 NPs with re-immobilized HRP (e), and recycled Fe3O4@TiO2 ratio of the reaction rate of free HRP versus that of NPs with immobilized cytochrome c (f). 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2013, 78, 1437 – 1439
1438
CHEMPLUSCHEM COMMUNICATIONS
www.chempluschem.org activity. Moreover, the Fe3O4@TiO2 NPs could be recycled, as a result of the photodegradation and self-cleaning properties of TiO2 shells. All results presented herein indicated that the magnetic TiO2 NPs, with a combination of magnetic and photocatalytic properties, could be widely applicable in not only biocatalysis, but also biosenors and magnetic, targeted, drugdelivery systems.
Acknowledgements This study was supported by the Development Program of the Science and Technology of Jilin Province (20130206011YY), the Scientific Foundation for Young Scientists of Jilin University, and a Research Program of the Key Laboratory of Bionic Engineering, Ministry of Education, Jilin University.
Figure 4. A) A photograph (inset) and UV/Vis spectra of products catalyzed by HRP-immobilized Fe3O4@TiO2 NPs (red), a control spectrum without HRPimmobilized Fe3O4@TiO2 NPs (blue), and the as-prepared Fe3O4@TiO2 NPs without HRP immobilization (black). B) Time-dependent UV/Vis spectra of the products catalyzed by the HRP-immobilized Fe3O4@TiO2 NPs (time interval is 2 min). C) Time-dependent absorbance bands at l = 651 nm of the products catalyzed by free HRP (red) and the HRP-immobilized Fe3O4@TiO2 NPs (black). D) Reuse of the HRP-immobilized Fe3O4@TiO2 NPs for five cycles (35 min of reaction time for each cycle).
immobilized HRP. From Figure 4 C, the DA/min values (the average change in absorbance at l = 651 nm per minute) for free and immobilized HRP within 20 min is 0.008 and 0.007, respectively, and thus, the activity yield is 1.14. These results suggested that most molecules of HRP retained their enzyme activity after immobilization. Therefore, the Fe3O4@TiO2 NP-based biocatalysis combines the advantages of high enzyme activity, easy magnetic operation, biocompatibility, and enzyme recyclability. Furthermore, the recyclability of the nanobiocatalyst was determined for peroxidation of TMB. After each cycle, the Fe3O4@TiO2 NPs were magnetically collected and added to a new solution of substrate containing H2O2. As shown in Figure 4 D, the catalytic activity of the Fe3O4@TiO2 NPs remains near 80 % after 5 cycles. Therefore, the HRP-immobilized Fe3O4@TiO2 NPs can be reused for multiple reactions. Moreover, as mentioned above, the Fe3O4@TiO2 NPs themselves are recyclable and the immobilized enzymes that may lose most activity after several cycles can be decomposed by UV-induced degradation and new enzymes can then be re-immobilized on the recycled Fe3O4@TiO2 NPs treated with APTMS and GA to give a fresh and active nanobiocatalyst. Thus, the proposed Fe3O4@TiO2 NPs provide an efficient way for enzyme–substrate separation, enzyme reuse, and nanomaterial recycling. In summary, magnetic TiO2 NPs were synthesized, characterized, and used as a novel nanobiocatalyst with peroxidase immobilization. Magnetic cores enabled the nanobiocatalyst to be conveniently controlled for enzyme–substrate separation by using an external magnet. Anatase shells were able to preserve the protein native structure and minimize the loss of catalytic 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Keywords: biocatalysis · enzymes · magnetic properties · nanoparticles · titanium [1] J. Liu, S. Z. Qiao, Q. H. Hu, G. Q. Lu, Small 2011, 7, 425 – 443. [2] J. Gao, H. Gu, B. Xu, Acc. Chem. Res. 2009, 42, 1097 – 1107. [3] A. Singh, F. Dilnawaz, S. Mewar, U. Sharma, N. R. Jagannathan, S. K. Sahoo, ACS Appl. Mater. Interfaces 2011, 3, 842 – 856. [4] Y. Deng, D. Qi, C. Deng, X. Zhang, D. Zhao, J. Am. Chem. Soc. 2008, 130, 28 – 29. [5] P. Wang, Q. Shi, Y. Shi, K. K. Clark, G. D. Stucky, A. A. Keller, J. Am. Chem. Soc. 2009, 131, 182 – 188. [6] L. M. Rossi, A. D. Quach, Z. Rosenzweig, Anal. Bioanal. Chem. 2004, 380, 606 – 613. [7] W. Wang, Y. Xu, D. I. Wang, Z. Li, J. Am. Chem. Soc. 2009, 131, 12892 – 12893. [8] X. Gao, K. M. Kerry Yu, K. Y. Tam, S. Chi Tsang, Chem. Commun. 2003, 2998 – 2999. [9] J. Park, S. Bauer, K. von der Mark, P. Schmuki, Nano Lett. 2007, 7, 1686 – 1691. [10] W. F. Ma, Y. Zhang, L. L. Li, L. J. You, P. Zhang, Y. T. Zhang, J. M. Li, M. Yu, J. Guo, H. J. Lu, C. C. Wang, ACS Nano 2012, 6, 3179 – 3188. [11] A. Fujishima, T. N. Rao, D. A. Tryk, J. Photochem. Photobiol. C 2000, 1, 1 – 21. [12] T.-S. Wu, K.-X. Wang, G.-D. Li, S.-Y. Sun, J. Sun, J.-S. Chen, ACS Appl. Mater. Interfaces 2010, 2, 544 – 550. [13] L. Chen, W. Hong, Z. Guo, Y. Sa, X. Wang, Y. M. Jung, B. Zhao, J. Colloid Interface Sci. 2012, 368, 282 – 286. [14] D. Regonini, A. Jaroenworaluck, R. Stevens, C. R. Bowen, Surf. Interface Anal. 2010, 42, 139 – 144. [15] X. Xue, W. Ji, Z. Mao, H. Mao, Y. Wang, X. Wang, W. Ruan, B. Zhao, J. R. Lombardi, J. Phys. Chem. C 2012, 116, 8792 – 8797. [16] L. Chen, X. X. Han, Z. Guo, X. Wang, W. Ruan, W. Song, B. Zhao, Y. Ozaki, Anal. Methods 2012, 4, 1643 – 1647. [17] X. X. Han, Y. Kitahama, Y. Tanaka, J. Guo, W. Q. Xu, B. Zhao, Y. Ozaki, Anal. Chem. 2008, 80, 6567 – 6572. [18] M. Feng, H. Tachikawa, J. Am. Chem. Soc. 2008, 130, 7443 – 7448. [19] U. Vukicevic, S. (C.) Ziemian„ A. Bismarck, M. S. P. Shaffer, J. Mater. Chem. 2008, 18, 3448 – 3453. [20] I. Alessandri, J. Am. Chem. Soc. 2013, 135, 5541 – 5544. [21] I. K. Konstantinou, T. A. Albanis, Appl. Catal. B 2004, 49, 1 – 14. [22] D. H. Murgida, P. Hildebrandt, Acc. Chem. Res. 2004, 37, 854 – 861. [23] D. Bhattacharya, A. Baksi, I. Banerjee, R. Ananthakrishnan, T. K. Maiti, P. Pramanik, Talanta 2011, 86, 337 – 348. [24] A. K. Dutta, S. K. Maji, D. N. Srivastava, A. Mondal, P. Biswas, P. Paul, B. Adhikary, J. Mol. Catal. A: Chem. 2012, 360, 71 – 77. Received: June 25, 2013 Published online on October 11, 2013
ChemPlusChem 2013, 78, 1437 – 1439
1439
