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Received 00th January 2012, Accepted 00th January 2012
Smart nanoprobes for the detection of alkaline phosphatase activity during osteoblast differentiation Eun-Kyung Lim,ab Joo Oak Keem,b Hui-suk Yun,d Jinyoung Jung,*abc Bong Hyun Chung,*abc
DOI: 10.1039/x0xx00000x
Gold nanoparticle-conjugated fluorescent hydroxyapatite (AuFHAp) was developed as a smart nanoprobe for alkaline phosphatase (ALP) activity. AuFHAp showed NIR fluorescence due to hydrolysis of their phosphate groups by ALP. In addition, gold nanoparticles on them help reduce the nonspecific signal by absorbing nonspecific fluorescence. Through in vitro tests, we confirmed that AuFHAp probe was capable of detecting ALP levels related to osteoblast activity in living cells with high fluorescence intensity. Alkaline phosphatase (ALP) is an important marker for osteoblast activity during early osteoblast differentiation. ALP activity in serum is routinely used as a diagnostic indicator of several bone diseases, e.g., osteoblastic cancer. ALP levels rise with the increased bone activity. 1,2 Although ALP has been extensively studied, its diverse range of physiological and pathological functions have not been fully elucidated. Hence, ALP activity is required to directly measure in bone tissue with anatomical accuracy. There are currently no noninvasive methods for detecting ALP activity in vitro/in vivo.3 Thus, an appropriate probe is needed for detecting ALP activity in living systems to diagnose physiologically relevant conditions. In general, biological tissues, e.g., bone tissue, have high absorbance and autofluorescence in the ultraviolet and visible ranges. However, in the near-infrared (NIR) region (700 ~ 900 nm), the absorbance spectra for all biomolecules is minimized.4 Herein, we describe the development of a smart nanoprobe enabling ALP activity detection during osteoblast differentiation in live cells. This smart nanoprobe was formulated using indocyanine green (ICG)-loaded hydroxyapatite with gold nanoparticles attached to its surface, designated as gold nanoparticle-conjugated fluorescent hydroxyapatite (AuFHAp) (Fig. 1). ICG is an FDA-approved imaging dye used in medical diagnostics, possessing absorption and fluorescence peaks in the NIR region. Hydroxyapatite (HAp) has been widely used in clinics as a biocompatible material because its structure and composition are similar to those of bonded minerals.5,6 Additionally, HAp plays an important role in maintaining the mechanical properties of natural bone while offering a favorable environment for osteoblast proliferation and protein adhesion. Here, we attempt to reduce the nonspecific fluorescence signal of FHAp by attaching gold nanoparticles as acceptors of ICG fluorescence on its surface, taking advantage of the fluorescence resonance energy
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transfer (FRET) effect. 7,8 The properties of AuFHAp were investigated to assess its capability as a smart nanoprobe.
Fig. 1. Preparation of gold nanoparticle-conjugated FHAp (AuFHAp) for the detection of alkaline phosphatase (ALP) activity. To prepare the AuFHAp, FHAp was first synthesized by chemical precipitation (ESI†). Its ellipsoidal morphology was confirmed using TEM and SEM (Fig. 2a).5 We modified the hydroxyl groups on the surface of the FHAp with amine groups using 3aminopropyltrimethoxysilane (APTMS) to attach the gold nanoparticles. The amine groups were verified by FT-IR and XPS (Fig. S1).9-12 As shown in Fig. S1, the amine-functionalized FHAp has characteristic bands for amine groups at 3,550 cm-1 (N-H stretching), 3,000 cm-1 (C-N stretching) and 1,600 cm-1 (N-H banding).12,13 ). In addition, only the N spectra is present for XPS in contrast to the XPS spectra of FHAp, confirming the presence of amine groups on the surface of the FHAp as binding sites for the gold nanoparticles. The gold nanoparticles were synthesized as seeds using the gold salt reduction method (ESI†). They were attached to the amine-functionalized FHAp by covalent bonding between the gold and the amine groups to produce the AuFHAp. Their morphology were confirmed using TEM and SEM (Fig. 2b).9-12,14 The AuFHAp had a diffraction peak corresponding to gold nanoparticles (111) at 2θ = 38.2, (200) at 2θ =44.4, (220) at 2θ = 64.6 and (311) at 2θ = 77.5. The gold nanoparticles were present at a
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weight ratio of 2.6 % compared with the FHAp (Fig. S2).15-18 Furthermore, the optical properties of the AuFHAp and FHAp were analyzed using UV-vis and fluorescence spectrometry (Fig. 2c). The FHAp possessed the optical properties (absorption and fluorescence spectra) of the corresponding ICG dye.19 As shown in Fig. 2c, the AuFHAp showed strong absorbance in the NIR region due to SPR effect of the gold nanoparticles (Fig. S3). This absorption range partially overlapped with the fluorescence spectrum of the FHAp. The AuFHAp did not produce fluorescence because the gold nanoparticles absorbed the fluorescence (Fig. 2c).9,10
Journal Name DOI: 10.1039/C4CC09620G Based on these characteristics, we measured ALP activity using AuFHAP from the cell lysates of MC3T3-E1 undergoing osteogenic differentiation (ESI†).28-36 The ALP activity generally increased in accordance with the extent of differentiation (Fig. 4a and S4), whereas it was hardly detected when MC3T3-E1 cells (preosteoblast) were cultured for 21 days without differentiation inducing agents (Fig. S4). AuFHAp and FHAp exhibited biocompatibility in the target cells (pre-osteoblasts and osteoblasts, respectively) below concentrations of 10 µg/mL (Fig. S5).28,37,38 Then, to directly detect ALP activity in live cells during osteoblast differentiation without cell lysis, we exposed the nanoprobes (AuFHAp and FHAp, respectively) to the cells and detected cellular NIR fluorescence using fluorescence microscopy.2 As shown in Fig. 4b and S6, in vitro ALP activity was observed using AuFHAp with high fluorescence at 17 and 21 days.24,29,39 In the FHAp-treated cells, there was not sufficient fluorescence intensity for detection, even after 21 days. The fluorescence of the images followed a similar pattern to the cell lysate data (Fig. 4).
Fig. 2. The morphology of (a) FHAp and (b) AuFHAp (i: TEM and ii: SEM images). (c) The optical properties of FHAp (green) and AuFHAp (dark red) (dash line: absorbance and solid line: fluorescence intensity) Next, the ALP activity was measured using AuFHAp at various concentrations. As a control, FHAp was used in same manner. 20-26 Fig. 3a shows the increase in fluorescence intensity with increasing AuFHAp concentration at the same ALP enzyme concentrations (5 µg/mL) (IntensityALP+) by releasing ICG dye from the AuFHAp as ALP degrades the material by hydrolysis of the phosphate groups (ESI†). There was a slight increase in the fluorescence intensity of the FHAp that subsequently decreased to produce a weak signal. This was caused by non-specific fluorescence intensity in the absence of ALP (IntensityALP-). We further investigated the ability of AuFHAp to detect ALP activity in the presence of ALP inhibitors (Levamisol).2,27 Inhibitors were pre-incubated with the ALP enzyme, which was then added to the AuFHAp. The enzymatic reaction was detected by measuring the fluorescence intensity. As shown in Fig. 3b, the fluorescence intensity of the AuFHAp corresponding to the ALP activity was clearly inhibited as inhibitor concentrations were increased, demonstrating the applicability of AuFHAp as a nanoprobe for the detection of ALP activity.
Fig. 3. (a) Measurement of ALP activity using AuFHAp (black) and FHAp (gray) at various concentrations (△ △Intensity = IntenstiyALP+-IntensityALP-). (b) Inhibition assay of ALP activity using AuFHAp.
2 | J. Name., 2012, 00, 1-3
Fig. 4. Detection of in vitro alkaline phosphatase (ALP) activity during osteoblast differentiation (a) after cell lysis using AuFHAp (black) and FHAp (gray), respectively (△ △Intensity = Intensity – Intensity0day) and (b) in living cells. Merged images are presented using a red filter for fluorescence of AuFHAp and a blue filter for labelled cell nuclei. In conclusion, we have developed a gold nanoparticle-conjugated fluorescent hydroxyapaptite (AuFHAp) as smart nanoprobes and demonstrated its potential for detecting ALP activity in live cells. AuFHAp showed weak nonspecific fluorescence in the absence of ALP because the gold nanoparticles absorbed the fluorescence of the ICG from the FHAp by FRET. However, in the presence of ALP, the fluorescence markedly increased in the NIR region. Thus, this probe provides a feasible and powerful tool for ALP detection as a marker for osteoblast activity. Based on these features, we expect that AuFHAp can be used for non-invasive monitoring of ALP activity related with bone disease in vivo model.
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Journal Name The authors acknowledge the financial support by BioNano HealthGuard Research Center funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as Global Frontier Project (HGUARD_2013 M3A6B2078950), National Research Foundation of Korea (NRF) grants (NRF-2012R1A1A2043991), and KRIBB Research Initiative Program, Republic of Korea.
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a
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 305-806, Daejeon, Republic of Korea. b BioNano Health Guard Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 305-806, Daejeon, Republic of Korea. c Nanobiotechnology Major, School of Engineering, University of Science and Technology (UST), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea d Powder & Ceramics Division, Korea Institute of Materials Science (KIMS) 797 Changwondaero, Seongsangu, Changwon 642-831, Republic of Korea E-mail: [email protected]; [email protected]; Fax: +82-42-879-8594; Tel: +82-42-579-8456 † Electronic Supplementary Information (ESI) available: [Experimental details, Characterization (FT-IR, XPS, XRD and TGA analysis), alkaline phosphatase (ALP) activity assay, In vitro tests (Osteoblast differentiation, Biocompatibility tests, ALP activity assay)]. See DOI: 10.1039/c000000x/ 1
A. Sabokbar, P. J. Millett, B. Myer and N. Rushton, Bone and Mineral, 1994, 27, 57-67. 2 T. I. Kim, H. Kim, Y. Choi and Y. Kim, Chem. Commun., 2011, 47, 9825-9827. 3 T. P. Gade, M. W. Motley, B. J. Beattie, R. Bhakta, A. L. Boskey, J. A. Koutcher and P. Mayer-Kuckuk, PloS one, 2011, 6, e22608. 4 A. Zaheer, R. E. Lenkinski, A. Mahmood, A. G. Jones, L. C. Cantley and J. V. Frangioni, Nat. Biotechnol., 2001, 19, 1148-1154. 5 H. J. Lee, H. W. Choi, K. J. Kim and S. C. Lee, Chem. Mater., 2006, 18, 5111-5118. 6 Z. Hong, X. Qiu, J. Sun, M. Deng, X. Chen and X. Jing, Polymer, 2004, 45, 6699-6706. 7 N. T. Chen, S. H. Cheng, C. P. Liu, J. S. Souris, C. T. Chen, C. Y. Mou and L. W. Lo, Int. J. Mol. Sci., 2012, 13, 16598-16623. 8 D. Ghosh, Optics and Photonics Journal, 2013, 03, 18-26. 9 J. Kang, J. Yang, J. Lee, S. J. Oh, S. Moon, H. J. Lee, S. C. Lee, J.-H. Son, D. Kim, K. Lee, J.-S. Suh, Y.-M. Huh and S. Haam, J. Mater. Chem., 2009, 19, 2902. 10 J. Lee, J. Yang, H. Ko, S. Oh, J. Kang, J. Son, K. Lee, S. W. Lee, H. G. Yoon, J. S. Suh, Y. M. Huh and S. Haam, Adv. Funct. Mater., 2008, 18, 258-264. 11 R. D. Ross and R. K. Roeder, J. Biomed. Mater. Resear. Part A, 2011, 99, 58-66. 12 S. Wang, S. Wen, M. Shen, R. Guo, X. Cao, J. Wang and X. Shi, Int. J. Nanomed., 2011, 6, 3449-3459.
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COMMUNICATION DOI: 10.1039/C4CC09620G 13 M. Mir, F. L. Leite, P. S. d. P. H. Junior, F. L. Pissetti, A. M. Rossi, E. L. Moreira and Y. P. Mascarenhas, Materials Research, 2012, 15, 622-627. 14 N. R. Jana, L. Gearheart and C. J. Murphy, Langmuir, 2001, 17, 6782-6786. 15 L. Wang, S. He, X. Wu, S. Liang, Z. Mu, J. Wei, F. Deng, Y. Deng and S. Wei, Biomaterials, 2014, 35, 6758-6775. 16 F. Chen, P. Huang, Y. J. Zhu, J. Wu and D. X. Cui, Biomaterials, 2012, 33, 6447-6455. 17 M. Ansari, S. M. Naghib, F. Moztarzadeh and A. Salati, CeramicsSilikaty, 2011, 55, 123-126. 18 J. C. Fricain, S. Schlaubitz, C. Le Visage, I. Arnault, S. M. Derkaoui, R. Siadous, S. Catros, C. Lalande, R. Bareille, M. Renard, T. Fabre, S. Cornet, M. Durand, A. Leonard, N. Sahraoui, D. Letourneur and J. Amedee, Biomaterials, 2013, 34, 2947-2959. 19 E. I. A. lu, T. J. Russin, J. M. Kaiser, B. M. Barth, P. C. Eklund, M. Kester and J. H. Adair, ACS Nano, 2008, 2, 2075-2084. 20 G. R. Beck, E. C. Sullivan, E. Moran and B. Zerler, J. Cell. Biochem., 1998, 68, 269-280. 21 K. Isama and T. Tsuchiya, Biomaterials, 2003, 24, 3303-3309. 22 L. Kyllonen, S. Haimi, B. Mannerstrom, H. Huhtala, K. M. Rajala, H. Skottman, G. K. Sandor and S. Miettinen, Stem. Cell. Res. Ther., 2013, 4, 17. 23 H. J. Lee, A. N. Koo, S. W. Lee, M. H. Lee and S. C. Lee, J. Control. Release., 2013, 170, 198-208. 24 K. Oya, Y. Tanaka, H. Saito, K. Kurashima, K. Nogi, H. Tsutsumi, Y. Tsutsumi, H. Doi, N. Nomura and T. Hanawa, Biomaterials, 2009, 30, 1281-1286. 25 J. L. Santos, E. Oramas, A. P. Pego, P. L. Granja and H. Tomas, J. Control. Release., 2009, 134, 141-148. 26 Y. Tsukamoto, S. Fukutani and M. Mori, J. Mater. Sci., 1992, 3, 180183. 27 H. V. Belle, Clin. Chem., 1976, 22, 972-976. 28 Y. F. Chou, W. Huang, J. C. Dunn, T. A. Miller and B. M. Wu, Biomaterials, 2005, 26, 285-295. 29 A. Gao, R. Hang, X. Huang, L. Zhao, X. Zhang, L. Wang, B. Tang, S. Ma and P. K. Chu, Biomaterials, 2014, 35, 4223-4235. 30 H. Kim, A. Tabata, T. Tomoyasu, T. Ueno, S. Uchiyama, K. Yuasa, A. Tsuji and H. Nagamune, J. Bone Miner. Metab., 2014. 31 N. M. Moore, N. J. Lin, N. D. Gallant and M. L. Becker, Biomaterials, 2010, 31, 1604-1611. 32 R. Shu, R. McMullen, M. J. Baumann and L. R. MaCabe, J. Biomed. Mater. Resear. Part A, 2003, 67, 1196-1204. 33 K. Wang, L. Cai, L. Zhang, J. Dong and S. Wang, Adv. Healthc Mater., 2012, 1, 292-301. 34 L. Yang, S. Perez-Amodio, F. Y. Barrere-de Groot, V. Everts, C. A. van Blitterswijk and P. Habibovic, Biomaterials, 2010, 31, 2976-2989. 35 Y. Yao, X. Shi and F. Chen, J. Nanosci. Nanotechno., 2014, 14, 4851-4857. 36 H. J. Yoon, C. R. Seo, M. Kim, Y. J. Kim, N. J. Song, W. S. Jang, B. J. Kim, J. Lee, J. W. Hong, C. W. Nho and K. W. Park, Nutr. Res., 2013, 33, 1053-1062. 37 E. K. Lim, H. O. Kim, E. Jang, J. Park, K. Lee, J. S. Suh, Y. M. Huh and S. Haam, Biomaterials, 2011, 32, 7941-7950.
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38 T. Yuasa, Y. Miyamoto, K. Ishikawa, M. Takechi, Y. Momota, S. Tatehara and M. Nagayama, Biomaterials, 2004, 25, 11591166. 39 K. J. Cha, J. M. Hong, D. W. Cho and D. S. Kim, Biofabrication, 2013, 5, 025007.
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This article can be cited before page numbers have been issued, to do this please use: E. Lim, J. O. Keem, H. Yun, J. Jeong and B. H. Chung, Chem. Commun., 2015, DOI: 10.1039/C4CC09620G.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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Received 00th January 2012, Accepted 00th January 2012
Smart nanoprobes for the detection of alkaline phosphatase activity during osteoblast differentiation Eun-Kyung Lim,ab Joo Oak Keem,b Hui-suk Yun,d Jinyoung Jung,*abc Bong Hyun Chung,*abc
DOI: 10.1039/x0xx00000x
Gold nanoparticle-conjugated fluorescent hydroxyapatite (AuFHAp) was developed as a smart nanoprobe for alkaline phosphatase (ALP) activity. AuFHAp showed NIR fluorescence due to hydrolysis of their phosphate groups by ALP. In addition, gold nanoparticles on them help reduce the nonspecific signal by absorbing nonspecific fluorescence. Through in vitro tests, we confirmed that AuFHAp probe was capable of detecting ALP levels related to osteoblast activity in living cells with high fluorescence intensity. Alkaline phosphatase (ALP) is an important marker for osteoblast activity during early osteoblast differentiation. ALP activity in serum is routinely used as a diagnostic indicator of several bone diseases, e.g., osteoblastic cancer. ALP levels rise with the increased bone activity. 1,2 Although ALP has been extensively studied, its diverse range of physiological and pathological functions have not been fully elucidated. Hence, ALP activity is required to directly measure in bone tissue with anatomical accuracy. There are currently no noninvasive methods for detecting ALP activity in vitro/in vivo.3 Thus, an appropriate probe is needed for detecting ALP activity in living systems to diagnose physiologically relevant conditions. In general, biological tissues, e.g., bone tissue, have high absorbance and autofluorescence in the ultraviolet and visible ranges. However, in the near-infrared (NIR) region (700 ~ 900 nm), the absorbance spectra for all biomolecules is minimized.4 Herein, we describe the development of a smart nanoprobe enabling ALP activity detection during osteoblast differentiation in live cells. This smart nanoprobe was formulated using indocyanine green (ICG)-loaded hydroxyapatite with gold nanoparticles attached to its surface, designated as gold nanoparticle-conjugated fluorescent hydroxyapatite (AuFHAp) (Fig. 1). ICG is an FDA-approved imaging dye used in medical diagnostics, possessing absorption and fluorescence peaks in the NIR region. Hydroxyapatite (HAp) has been widely used in clinics as a biocompatible material because its structure and composition are similar to those of bonded minerals.5,6 Additionally, HAp plays an important role in maintaining the mechanical properties of natural bone while offering a favorable environment for osteoblast proliferation and protein adhesion. Here, we attempt to reduce the nonspecific fluorescence signal of FHAp by attaching gold nanoparticles as acceptors of ICG fluorescence on its surface, taking advantage of the fluorescence resonance energy
This journal is © The Royal Society of Chemistry 2012
transfer (FRET) effect. 7,8 The properties of AuFHAp were investigated to assess its capability as a smart nanoprobe.
Fig. 1. Preparation of gold nanoparticle-conjugated FHAp (AuFHAp) for the detection of alkaline phosphatase (ALP) activity. To prepare the AuFHAp, FHAp was first synthesized by chemical precipitation (ESI†). Its ellipsoidal morphology was confirmed using TEM and SEM (Fig. 2a).5 We modified the hydroxyl groups on the surface of the FHAp with amine groups using 3aminopropyltrimethoxysilane (APTMS) to attach the gold nanoparticles. The amine groups were verified by FT-IR and XPS (Fig. S1).9-12 As shown in Fig. S1, the amine-functionalized FHAp has characteristic bands for amine groups at 3,550 cm-1 (N-H stretching), 3,000 cm-1 (C-N stretching) and 1,600 cm-1 (N-H banding).12,13 ). In addition, only the N spectra is present for XPS in contrast to the XPS spectra of FHAp, confirming the presence of amine groups on the surface of the FHAp as binding sites for the gold nanoparticles. The gold nanoparticles were synthesized as seeds using the gold salt reduction method (ESI†). They were attached to the amine-functionalized FHAp by covalent bonding between the gold and the amine groups to produce the AuFHAp. Their morphology were confirmed using TEM and SEM (Fig. 2b).9-12,14 The AuFHAp had a diffraction peak corresponding to gold nanoparticles (111) at 2θ = 38.2, (200) at 2θ =44.4, (220) at 2θ = 64.6 and (311) at 2θ = 77.5. The gold nanoparticles were present at a
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weight ratio of 2.6 % compared with the FHAp (Fig. S2).15-18 Furthermore, the optical properties of the AuFHAp and FHAp were analyzed using UV-vis and fluorescence spectrometry (Fig. 2c). The FHAp possessed the optical properties (absorption and fluorescence spectra) of the corresponding ICG dye.19 As shown in Fig. 2c, the AuFHAp showed strong absorbance in the NIR region due to SPR effect of the gold nanoparticles (Fig. S3). This absorption range partially overlapped with the fluorescence spectrum of the FHAp. The AuFHAp did not produce fluorescence because the gold nanoparticles absorbed the fluorescence (Fig. 2c).9,10
Journal Name DOI: 10.1039/C4CC09620G Based on these characteristics, we measured ALP activity using AuFHAP from the cell lysates of MC3T3-E1 undergoing osteogenic differentiation (ESI†).28-36 The ALP activity generally increased in accordance with the extent of differentiation (Fig. 4a and S4), whereas it was hardly detected when MC3T3-E1 cells (preosteoblast) were cultured for 21 days without differentiation inducing agents (Fig. S4). AuFHAp and FHAp exhibited biocompatibility in the target cells (pre-osteoblasts and osteoblasts, respectively) below concentrations of 10 µg/mL (Fig. S5).28,37,38 Then, to directly detect ALP activity in live cells during osteoblast differentiation without cell lysis, we exposed the nanoprobes (AuFHAp and FHAp, respectively) to the cells and detected cellular NIR fluorescence using fluorescence microscopy.2 As shown in Fig. 4b and S6, in vitro ALP activity was observed using AuFHAp with high fluorescence at 17 and 21 days.24,29,39 In the FHAp-treated cells, there was not sufficient fluorescence intensity for detection, even after 21 days. The fluorescence of the images followed a similar pattern to the cell lysate data (Fig. 4).
Fig. 2. The morphology of (a) FHAp and (b) AuFHAp (i: TEM and ii: SEM images). (c) The optical properties of FHAp (green) and AuFHAp (dark red) (dash line: absorbance and solid line: fluorescence intensity) Next, the ALP activity was measured using AuFHAp at various concentrations. As a control, FHAp was used in same manner. 20-26 Fig. 3a shows the increase in fluorescence intensity with increasing AuFHAp concentration at the same ALP enzyme concentrations (5 µg/mL) (IntensityALP+) by releasing ICG dye from the AuFHAp as ALP degrades the material by hydrolysis of the phosphate groups (ESI†). There was a slight increase in the fluorescence intensity of the FHAp that subsequently decreased to produce a weak signal. This was caused by non-specific fluorescence intensity in the absence of ALP (IntensityALP-). We further investigated the ability of AuFHAp to detect ALP activity in the presence of ALP inhibitors (Levamisol).2,27 Inhibitors were pre-incubated with the ALP enzyme, which was then added to the AuFHAp. The enzymatic reaction was detected by measuring the fluorescence intensity. As shown in Fig. 3b, the fluorescence intensity of the AuFHAp corresponding to the ALP activity was clearly inhibited as inhibitor concentrations were increased, demonstrating the applicability of AuFHAp as a nanoprobe for the detection of ALP activity.
Fig. 3. (a) Measurement of ALP activity using AuFHAp (black) and FHAp (gray) at various concentrations (△ △Intensity = IntenstiyALP+-IntensityALP-). (b) Inhibition assay of ALP activity using AuFHAp.
2 | J. Name., 2012, 00, 1-3
Fig. 4. Detection of in vitro alkaline phosphatase (ALP) activity during osteoblast differentiation (a) after cell lysis using AuFHAp (black) and FHAp (gray), respectively (△ △Intensity = Intensity – Intensity0day) and (b) in living cells. Merged images are presented using a red filter for fluorescence of AuFHAp and a blue filter for labelled cell nuclei. In conclusion, we have developed a gold nanoparticle-conjugated fluorescent hydroxyapaptite (AuFHAp) as smart nanoprobes and demonstrated its potential for detecting ALP activity in live cells. AuFHAp showed weak nonspecific fluorescence in the absence of ALP because the gold nanoparticles absorbed the fluorescence of the ICG from the FHAp by FRET. However, in the presence of ALP, the fluorescence markedly increased in the NIR region. Thus, this probe provides a feasible and powerful tool for ALP detection as a marker for osteoblast activity. Based on these features, we expect that AuFHAp can be used for non-invasive monitoring of ALP activity related with bone disease in vivo model.
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Journal Name The authors acknowledge the financial support by BioNano HealthGuard Research Center funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as Global Frontier Project (HGUARD_2013 M3A6B2078950), National Research Foundation of Korea (NRF) grants (NRF-2012R1A1A2043991), and KRIBB Research Initiative Program, Republic of Korea.
Notes and references
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a
BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 305-806, Daejeon, Republic of Korea. b BioNano Health Guard Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 305-806, Daejeon, Republic of Korea. c Nanobiotechnology Major, School of Engineering, University of Science and Technology (UST), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Republic of Korea d Powder & Ceramics Division, Korea Institute of Materials Science (KIMS) 797 Changwondaero, Seongsangu, Changwon 642-831, Republic of Korea E-mail: [email protected]; [email protected]; Fax: +82-42-879-8594; Tel: +82-42-579-8456 † Electronic Supplementary Information (ESI) available: [Experimental details, Characterization (FT-IR, XPS, XRD and TGA analysis), alkaline phosphatase (ALP) activity assay, In vitro tests (Osteoblast differentiation, Biocompatibility tests, ALP activity assay)]. See DOI: 10.1039/c000000x/ 1
A. Sabokbar, P. J. Millett, B. Myer and N. Rushton, Bone and Mineral, 1994, 27, 57-67. 2 T. I. Kim, H. Kim, Y. Choi and Y. Kim, Chem. Commun., 2011, 47, 9825-9827. 3 T. P. Gade, M. W. Motley, B. J. Beattie, R. Bhakta, A. L. Boskey, J. A. Koutcher and P. Mayer-Kuckuk, PloS one, 2011, 6, e22608. 4 A. Zaheer, R. E. Lenkinski, A. Mahmood, A. G. Jones, L. C. Cantley and J. V. Frangioni, Nat. Biotechnol., 2001, 19, 1148-1154. 5 H. J. Lee, H. W. Choi, K. J. Kim and S. C. Lee, Chem. Mater., 2006, 18, 5111-5118. 6 Z. Hong, X. Qiu, J. Sun, M. Deng, X. Chen and X. Jing, Polymer, 2004, 45, 6699-6706. 7 N. T. Chen, S. H. Cheng, C. P. Liu, J. S. Souris, C. T. Chen, C. Y. Mou and L. W. Lo, Int. J. Mol. Sci., 2012, 13, 16598-16623. 8 D. Ghosh, Optics and Photonics Journal, 2013, 03, 18-26. 9 J. Kang, J. Yang, J. Lee, S. J. Oh, S. Moon, H. J. Lee, S. C. Lee, J.-H. Son, D. Kim, K. Lee, J.-S. Suh, Y.-M. Huh and S. Haam, J. Mater. Chem., 2009, 19, 2902. 10 J. Lee, J. Yang, H. Ko, S. Oh, J. Kang, J. Son, K. Lee, S. W. Lee, H. G. Yoon, J. S. Suh, Y. M. Huh and S. Haam, Adv. Funct. Mater., 2008, 18, 258-264. 11 R. D. Ross and R. K. Roeder, J. Biomed. Mater. Resear. Part A, 2011, 99, 58-66. 12 S. Wang, S. Wen, M. Shen, R. Guo, X. Cao, J. Wang and X. Shi, Int. J. Nanomed., 2011, 6, 3449-3459.
This journal is © The Royal Society of Chemistry 2012
View Article Online
COMMUNICATION DOI: 10.1039/C4CC09620G 13 M. Mir, F. L. Leite, P. S. d. P. H. Junior, F. L. Pissetti, A. M. Rossi, E. L. Moreira and Y. P. Mascarenhas, Materials Research, 2012, 15, 622-627. 14 N. R. Jana, L. Gearheart and C. J. Murphy, Langmuir, 2001, 17, 6782-6786. 15 L. Wang, S. He, X. Wu, S. Liang, Z. Mu, J. Wei, F. Deng, Y. Deng and S. Wei, Biomaterials, 2014, 35, 6758-6775. 16 F. Chen, P. Huang, Y. J. Zhu, J. Wu and D. X. Cui, Biomaterials, 2012, 33, 6447-6455. 17 M. Ansari, S. M. Naghib, F. Moztarzadeh and A. Salati, CeramicsSilikaty, 2011, 55, 123-126. 18 J. C. Fricain, S. Schlaubitz, C. Le Visage, I. Arnault, S. M. Derkaoui, R. Siadous, S. Catros, C. Lalande, R. Bareille, M. Renard, T. Fabre, S. Cornet, M. Durand, A. Leonard, N. Sahraoui, D. Letourneur and J. Amedee, Biomaterials, 2013, 34, 2947-2959. 19 E. I. A. lu, T. J. Russin, J. M. Kaiser, B. M. Barth, P. C. Eklund, M. Kester and J. H. Adair, ACS Nano, 2008, 2, 2075-2084. 20 G. R. Beck, E. C. Sullivan, E. Moran and B. Zerler, J. Cell. Biochem., 1998, 68, 269-280. 21 K. Isama and T. Tsuchiya, Biomaterials, 2003, 24, 3303-3309. 22 L. Kyllonen, S. Haimi, B. Mannerstrom, H. Huhtala, K. M. Rajala, H. Skottman, G. K. Sandor and S. Miettinen, Stem. Cell. Res. Ther., 2013, 4, 17. 23 H. J. Lee, A. N. Koo, S. W. Lee, M. H. Lee and S. C. Lee, J. Control. Release., 2013, 170, 198-208. 24 K. Oya, Y. Tanaka, H. Saito, K. Kurashima, K. Nogi, H. Tsutsumi, Y. Tsutsumi, H. Doi, N. Nomura and T. Hanawa, Biomaterials, 2009, 30, 1281-1286. 25 J. L. Santos, E. Oramas, A. P. Pego, P. L. Granja and H. Tomas, J. Control. Release., 2009, 134, 141-148. 26 Y. Tsukamoto, S. Fukutani and M. Mori, J. Mater. Sci., 1992, 3, 180183. 27 H. V. Belle, Clin. Chem., 1976, 22, 972-976. 28 Y. F. Chou, W. Huang, J. C. Dunn, T. A. Miller and B. M. Wu, Biomaterials, 2005, 26, 285-295. 29 A. Gao, R. Hang, X. Huang, L. Zhao, X. Zhang, L. Wang, B. Tang, S. Ma and P. K. Chu, Biomaterials, 2014, 35, 4223-4235. 30 H. Kim, A. Tabata, T. Tomoyasu, T. Ueno, S. Uchiyama, K. Yuasa, A. Tsuji and H. Nagamune, J. Bone Miner. Metab., 2014. 31 N. M. Moore, N. J. Lin, N. D. Gallant and M. L. Becker, Biomaterials, 2010, 31, 1604-1611. 32 R. Shu, R. McMullen, M. J. Baumann and L. R. MaCabe, J. Biomed. Mater. Resear. Part A, 2003, 67, 1196-1204. 33 K. Wang, L. Cai, L. Zhang, J. Dong and S. Wang, Adv. Healthc Mater., 2012, 1, 292-301. 34 L. Yang, S. Perez-Amodio, F. Y. Barrere-de Groot, V. Everts, C. A. van Blitterswijk and P. Habibovic, Biomaterials, 2010, 31, 2976-2989. 35 Y. Yao, X. Shi and F. Chen, J. Nanosci. Nanotechno., 2014, 14, 4851-4857. 36 H. J. Yoon, C. R. Seo, M. Kim, Y. J. Kim, N. J. Song, W. S. Jang, B. J. Kim, J. Lee, J. W. Hong, C. W. Nho and K. W. Park, Nutr. Res., 2013, 33, 1053-1062. 37 E. K. Lim, H. O. Kim, E. Jang, J. Park, K. Lee, J. S. Suh, Y. M. Huh and S. Haam, Biomaterials, 2011, 32, 7941-7950.
J. Name., 2012, 00, 1-3 | 3
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38 T. Yuasa, Y. Miyamoto, K. Ishikawa, M. Takechi, Y. Momota, S. Tatehara and M. Nagayama, Biomaterials, 2004, 25, 11591166. 39 K. J. Cha, J. M. Hong, D. W. Cho and D. S. Kim, Biofabrication, 2013, 5, 025007.
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