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Table of contents entry Based on the precise nano-architecture of bacteriophage M13, a FRET-based fluorescent nanosensor has been formulated for ratiometric sensing of intracellular
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Virus-templated FRET platform for rational design of ratiometric fluorescent nanosensors
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Received (in XXX, XXX) XthXXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x We report here the construction of a bacteriophage M13templated supramolecular nanosystem, i.e. M13-β-CD/AdaFITC/Ada-RhB, which can be used as effective ratiometric fluorescent sensors for intracellular sensing.
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Förster resonance energy transfer (FRET) is a nonradiative process in which an excited state donor transfers energy to a ground state acceptor through long-range dipole-dipole interactions.1 Based on the mechanism of FRET, a series of novel light-harvesting systems, chemosensors and bioprobes have been developed recently.2-4 Conventional FRET-based probes have been designed in the form of small-molecular dyads, where the donor and acceptor fluorophores are connected by an appropriate linker. Recently, FRET mechanism has been adapted for the fabrication of nanoparticle-based probes (i.e. nanoprobes) for ratiometric sensing of ions, biomolecules and other analytes.4 FRET is one of the most widely used sensing mechanisms for ratiometric fluorescent probes. Unlike the one-signal fluorescent probes that can be influenced by probe concentration, probe environment, excitation intensity or any other drifts in the environment and instrument, the ratiometric fluorescent probes can alleviate these shortcomings by self-calibration of two emission bands.5-8 Recently, viral nanoparticles (VNPs) derived from plants and bacteria have attracted great attentions as nanoscale platforms for next-generation materials and biomedical applications,9-17 due to their unique monodisperse, multivalent and modifiable structural features, as well as good biocompatibility. In addition, based on the precise nano-architectures of viruses and virus-like particles (VLPs), FRET-based light-harvesting antennas have been constructed,18-21 where the chromophores are precisely organized within the Förster distance for efficient energy transfer. Bacteriophage M13 is a filamentous virus, 880 nm in length and 6.6 nm in diameter, consisting of a single-stranded DNA enclosed by 2700 copies of the major coat protein P8 and capped with 5 copies of four different minor coat protein (P9, P7, P6 and P3) on the ends. M13 is considered noninfectious and nonhazardous in human and mammalian because of its specific host selectivity, which can guarantee its safety in biological applications. Actually, its good cytocompatibility both in vitro and in vivo has been confirmed by our group and others.22-25 Meanwhile, it has been proven that M13 coating proteins assembled into a fairly rigid structure with all reactive groups being elucidated. In another word, the locations and separation This journal is © The Royal Society of Chemistry [year]
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distances of the reactive amino acid residues are precisely fixed. For example, we have previously shown that the N-terminus alanine residues attribute to the amidation reaction with activated carboxylic acid.26 The distances between two neighboring Nterminus at the exterior surface of M13 are oa ≈ 3.2 nm and ob ≈ 2.4 nm, respectively (Scheme 1A). Thus, after the conjugation of dyes to the N-terminus of M13, the locations of dyes are precisely fixed within the Förster distance, which is in favor of the FRET process occurring.
Scheme 1 (A) The structure of bacteriophage M13. M13 was generated using PyMol (www.pymol.org) with coordinates obtained from the RCSB Protein Data Bank (www.pdb.org). The amino acid residues in blue at the exterior surface of M13 represent N-terminal alanine, to which the β-CD was conjugated. The calculated distances between two neighboring Ntermini are oa ≈ 3.2 nm and ob ≈ 2.4 nm. (B) The structures of M13-β-CD, Ada-FITC and Ada-RhB. (C) Schematic demonstration of the formation of the ratiometric fluorescent pH nanosensor based on M13 and FRET (i.e. M13-β-CD/Ada-FITC/Ada-RhB) via the one-pot self-assembly approach driven by the supramolecular interaction between β-CD and Ada moieties.
[journal], [year], [vol], 00–00 |1
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Limin Chen,a Yehong Wu,a Yuan Lin*a and Qian Wang*ab
ChemComm
The arrows indicate the FRET process between donor and acceptor dyes on the M13 nano-scaffold.
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In this work, M13 was exploited as a scaffold to construct FRET-based ratiometric fluorescent nanoprobes. β-Cyclodextrin (β-CD) were first conjugated to the N-terminus at the exterior surface of M13 using carbodiimide chemistry as our previous report.24 After the conjugation, the locations of β-CD were precisely displayed at the exterior surface of M13 scaffold. Fluorescein isothionate (FITC) (pH sensitive) and rhodamine B (RhB) (pH insensitive) were selected as model donor and acceptor dyes, respectively, which were anchored onto M13 particles upon derivatization with adamantyl (Ada) moieties (Scheme 1B) and sequential supramolecular assembly driven by the molecular recognition between β-CD and Ada moieties (Scheme 1C) according to our previous report.27 As a result, a model M13-templated FRET platform (i.e. M13-β-CD/AdaFITC/Ada-RhB) was achieved via the one-pot self-assembly approach. Bacteriophage M13 was obtained through amplification in E. coil, and M13-β-CD was synthesized using carbodiimide chemistry as our previous report, where β-CD were attached to the N-terminus.24 Ada-RhB was synthesized by esterification reaction, and Ada-FITC was prepared from FITC and 1adamantaneamine. M13-β-CD/Ada-FITC/Ada-RhB was obtained by incubating M13-β-CD with different molar ratios of Ada-FITC and Ada-RhB for 30 min at 4 C, and precipitating with PEG8K and NaCl, which was then confirmed by UV-Vis spectroscopy assay. The UV-Vis spectra show that the absorbance of AdaFITC (490 nm) gradually increases in company with the decrease in the absorbance of Ada-RhB (560 nm) as various molar ratios of Ada-FITC and Ada-RhB from 0 : 6 to 6 : 0 were used (Fig. 1A). Based on the UV-Vis spectrum, the average number of Ada-FITC and Ada-RhB units per M13-β-CD particle can be determined, and the results are given in Table S1, ESI. Transmission electron microscopy (TEM) analysis showed no significant changes in length and diameter of M13 particles after grafted with β-CD and sequential self-assembly (Fig. S1, ESI). A substantial spectral overlap of the donor emission with the acceptor absorption is essential for FRET process occurring. So we first examined the absorption and emission spectra of FITC and RhB upon derivatization with Ada moieties. An efficient spectral overlap between the emission spectrum of Ada-FITC and the absorption spectrum of Ada-RhB was observed (Fig. 1B), suggesting that the FRET process from Ada-FITC to Ada-RhB would take place when they are arranged in an appropriate distance and spatial orientation. To confirm the occurring of FRET process from Ada-FITC to Ada-RhB on the M13-β-CD nano-scaffold, we compared the fluorescence spectra changes of the mixed solution of Ada-FITC and Ada-RhB before and after the addition of M13-β-CD. As shown in Fig. 1C, in the absence of M13-β-CD, the mixed solution of Ada-FITC and Ada-RhB mainly showed the fluorescence emission from Ada-FITC (λ515 nm) when excited at 450 nm; however, in the presence of M13-β-CD, the increase in emission from Ada-RhB (λ580 nm) with concomitant suppression of Ada-FITC emission (λ515 nm) was observed, clearly indicating efficient FRET from Ada-FITC to Ada-RhB in the presence of M13-β-CD. In addition, we also observed that the mixed solution of Ada-FITC and Ada-RhB 2|Journal Name, [year], [vol], 00–00
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without M13-β-CD presented green fluorescence under a 365 nm UV lamp because of no efficient energy transfer from Ada-FITC to Ada-RhB where they are too far apart, whereas it showed yellow fluorescence in the presence of M13-β-CD because part of excitation state energy of Ada-FITC was transferred to the ground state Ada-RhB (Fig. 1C inset). To further confirm the FRET from Ada-FITC to Ada-RhB on the M13-β-CD, we measured the time-resolved fluorescence decay curves of M13-β-CD/Ada-FITC without Ada-RhB and M13-β-CD/Ada-FITC/Ada-RhB. The fluorescence lifetime of Ada-FITC on the M13-β-CD/Ada-FITC was determined to be 3.15 ns while the lifetime of Ada-FITC on the M13-β-CD/AdaFITC/Ada-RhB was shortened to 2.27 ns (Fig. 1D), which also supports the occurrence of FRET from Ada-FITC to Ada-RhB in the presence of M13-β-CD. According to the formula E = 1 – τDA/τD, the FRET efficiency was calculated to be ~ 28 %, where τD represents the lifetime of the donor without the acceptor, and τDA the lifetime of the donor in the presence of the acceptor. Based on the fluorescence spectra and fluorescence delay curves, we hypothesize that Ada-FITC and Ada-RhB should be anchored onto the exterior surface of M13-β-CD in a random manner (Scheme 1C) during the one-pot self-assembly process, which may influence the FRET efficiency on the M13 nano-scaffold.
Fig. 1 (A) UV-Vis spectra of M13-β-CD/Ada-FITC/Ada-RhB with varying molar ratios of Ada-FITC and Ada-RhB (nAda-FITC : nAda-RhB). (B) Normalized absorption spectra of Ada-FITC (green solid) and Ada-RhB (red solid) in pH 7.8 K-phos buffer; normalized fluorescence spectra of Ada-FITC (green dot) and Ada-RhB (red dot) in pH 7.8 K-phos buffer. (C) Fluorescence spectra of the mixture of Ada-FITC and Ada-RhB (nAda-FITC : nAda-RhB= 1 : 1) (green) or M13-β-CD/Ada-FITC/Ada-RhB (nAda-FITC : nAdaRhB = 1 : 1) (red) in pH 7.8 K-phos buffer when excited at 450 nm. The inset shows the photographs of the pH 7.8 K-phos buffer solution containing Ada-FITC and Ada-RhB (nAda-FITC : nAda-RhB = 1 : 1) (right) or M13-β-CD/Ada-FITC/Ada-RhB (nAda-FITC : nAda-RhB = 1 : 1) (left) under 365 nm illumination. (D) Time-resolved fluorescence decay curves of M13-β-CD/Ada-FITC (black) and M13-β-CD/Ada-FITC/Ada-RhB (nAdaFITC : nAda-RhB = 1 : 1) (red) in pH 7.8 K-phos buffer.
To evaluate the potential usage of the M13-templated FRET platform, we examined the ratiometric response of M13-βCD/Ada-FITC/Ada-RhB to hydrogen ion using fluorescence spectroscopy. Hydrogen ion is an important intracellular species, which plays a pivotal role in biological activities, such as cell proliferation, apoptosis, phagocytosis, endocytosis, multidrug This journal is © The Royal Society of Chemistry [year]
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Fig. 2 (A) Fluorescence spectra of M13-β-CD/Ada-FITC/Ada-RhB (nAdaFITC : nAda-RhB = 1 : 2) at varied pH values when excited at 450 nm. (B) Ratiometric pH calibration plot of the fluorescence emission ratio (I515 nm/I580 nm) of M13-β-CD/Ada-FITC/Ada-RhB (nAda-FITC : nAda-RhB = 1 : 2) as a function of pH.
The fluorescence spectra of M13-β-CD/Ada-FITC/Ada-RhB with varied pH values when excited at 450 nm are shown in Fig. 2A. The fluorescence emission intensity of Ada-FITC gradually decreases with decreasing pH due to the protonation of its phenolate moiety which is critical for optimal fluorescence emission. Fig. 2B shows that the emission intensities ratios of Ada-FITC (515 nm) to Ada-RhB (580 nm) changed linearly as a function of pH in the range of 5.0 to 8.4, allowing the detection of pH in a ratiometric manner upon excitation at a single wavelength. This ratiometric nanodevice is more powerful than intensitybased fluorescent probes. In the reversibility experiment, the pH was changed from 5.0 to 8.4 and back to 5.0 twice, and the AdaFITC/Ada-RhB fluorescence emission ratios reached the expected values in all cases (Fig. S2, ESI), indicating excellent reversibility and reproducibility of this M13 nanosensor in reporting pH. Additionally, we noted that the M13 nanosensor showed fast pH response because the Ada-FITC was anchored on the exterior surface of M13 particles with large surface-to-volume ratio. We then evaluated the pH sensitivity of the ratiometric M13 nanosensor in living cells. It has been reported that M13 can enter into many kinds of cells via different endocytosis pathways.39-40 Here, RAW 264.7 macrophages were first incubated with M13-βCD/Ada-FITC/Ada-RhB for 12 h. After cellular uptake, the cells were washed with PBS buffer to remove free M13 particles in solution and on the cell surface, and visualized by confocal laser scanning microscopy (CLSM). As shown in Fig. 3, the green channel was obtained by integrating the spectral region from 510 nm to 520 nm, and the red channel was acquired by integrating the fluorescence signals from 575 nm to 585 nm. The intracellular pH was determined by comparing the ratio between the average fluorescence intensity from 510 nm to 520 nm and the average intensity from 575 nm to 585 nm to the pH calibration plot shown in Fig. 2B. The average pH value based on at least 50 cells using the M13 nanosensor was determined to be ~ 4.7, which is in good agreement with the reported pH range for This journal is © The Royal Society of Chemistry [year]
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the acidic organelles in endocytosis pathways (i.e. pH 4.5~5.0 in lysosomes). This experiment indicates the potential application of the ratiometric M13 nanosensor in fluorescence imaging and sensing of the pH in cellular systems.
Fig. 3 Typical CLSM images of RAW 264.7 macrophages incubated with the M13-β-CD/Ada-FITC/Ada-RhB (nAda-FITC : nAda-RhB = 1 : 2) for 12 h. The excitation wavelength was 488 nm and the images were collected at 510 nm-520 nm (A, green channel) or 575 nm-585 nm (B, red channel). (C) is the overlap of the green and red fluorescence. The scale bars represent 50 μm.
In summary, we report here a bacteriophage M13-templated FRET platform for the development of ratiometric fluorescent nanosensors. Based on the M13-β-CD scaffold, the FRET from Ada-FITC to Ada-RhB could occur. The resulting ratiometric M13 nanosensor with a linear pH sensing range and good reversibility can be also responsive to intracellular pH. More importantly, based on this work, we expect that the precise nanoarchitecture of bacteriophage M13 is applicable to the construction of various other kinds of ratiometric fluorescent nanosensors by changing the FRET components. This work was supported by National Natural Science Foundation of China (21429401 and 21374119).
Notes and references a
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State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P.R. China. [email protected] b Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA [email protected] † Electronic Supplementary Information (ESI) available: Preparation and characterization of M13-β-CD/Ada-FITC/Ada-RhB, experimental details of intracellular imaging experiments. See DOI:10.1039/b000000x/ 1. K. E. Sapsford, L. Berti and I. L. Medintz, Angew. Chem. Int. Edit., 2006, 45, 4562-4588. 2. K. V. Rao, K. K. R. Datta, M. Eswaramoorthy and S. J. George, Chem.-Eur. J., 2012, 18, 2184-2194. 3. L. Yuan, W. Lin, K. Zheng and S. Zhu, Acc. Chem. Res., 2013, 46, 1462-1473. 4. G. Chen, F. Song, X. Xiong and X. Peng, Ind. Eng. Chem. Res., 2013, 52, 11228-11245. 5. A. P. Demchenko, J. Fluoresc., 2010, 20, 1099-1128. 6. K. Kikuchi, H. Takakusa and T. Nagano, Trac.-Trend. Anal. Chem., 2004, 23, 407-415. 7. J. V. Mello and N. S. Finney, Angew. Chem. Int. Edit., 2001, 40, 1536-1538. 8. S. Deo and H. A. Godwin, J. Am. Chem. Soc., 2000, 122, 174-175. 9. K. Li, H. G. Nguyen, X. Lu and Q. Wang, Analyst, 2010, 135, 21-27. 10. Y. J. Lee, H. Yi, W. J. Kim, K. Kang, D. S. Yun, M. S. Strano, G. Ceder and A. M. Belcher, Science, 2009, 324, 1051-1055. 11. R. J. Tseng, C. L. Tsai, L. Ma, J. Y. Ouyang, C. S. Ozkan and Y. Yang, Nat. Nanotechnol., 2006, 1, 72-77. 12. N. F. Steinmetz, Mol. Pharm., 2013, 10, 1-2. 13. N. F. Steinmetz, Nanomed.-Nanotechnol. Biol. Med., 2010, 6, 634641.
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resistance, and signal transduction.28-33 Slightly abnormal pH values can be associated with inappropriate cell activities (e.g. abnormal proliferation and differentiation), which are observed in a variety of diseases, such as cancer, cystic fibrosis and immune dysfunction.34-37 Thus, monitoring of pH distribution and fluctuation with high temporal-spatial resolution in living systems is of great importance for understanding of the relationship between the pH and cell activities, as well as early diagnosis and treatment of diseases.38
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14. M. E. Farkas, I. L. Aanei, C. R. Behrens, G. J. Tong, S. T. Murphy, J. P. O'Neil and M. B. Francis, Mol. Pharm., 2013, 10, 69-76. 15. K. Zhou, F. Li, G. Dai, C. Meng and Q. Wang, Biomacromolecules, 2013, 14, 2593-2600. 16. C. Mao, A. Liu and B. Cao, Angew. Chem. Int. Edit., 2009, 48, 67906810. 17. J. Rong, L. A. Lee, K. Li, B. Harp, C. M. Mello, Z. Niu and Q. Wang, Chem. Commun., 2008, 44, 5185-5187. 18. Y. S. Nam, T. Shin, H. Park, A. P. Magyar, K. Choi, G. Fantner, K. A. Nelson and A. M. Belcher, J. Am. Chem. Soc., 2010, 132, 14621463. 19. R. A. Miller, A. D. Presley and M. B. Francis, J. Am. Chem. Soc., 2007, 129, 3104-3109. 20. M. Endo, M. Fujitsuka and T. Majima, Chem.-Eur. J., 2007, 13, 8660-8666. 21. L. M. Scolaro, M. A. Castriciano, A. Romeo, N. Micali, N. Angelini, C. L. Passo and F. Felici, J. Am. Chem. Soc., 2006, 128, 7446-7447. 22. H. Yi, D. Ghosh, M. H. Ham, J. Qi, P. W. Barone, M. S. Strano and A. M. Belcher, Nano Lett., 2012, 12, 1176-1183. 23. A. Merzlyak, S. Indrakanti and S. W. Lee, Nano Lett., 2009, 9, 846852. 24. L. Chen, X. Zhao, Y. Lin, Z. Su and Q. Wang, Polym. Chem., 2014, 5, 6754-6760. 25. N. Suthiwangcharoen, T. Li, K. Li, P. Thompson, S. You and Q. Wang, Nano Res., 2011, 4, 483-493. 26. K. Li, Y. Chen, S. Li, H. G. Nguyen, Z. Niu, S. You, C. M. Mello, X. Lu and Q. Wang, Bioconjugate Chem., 2010, 21, 1369-1377. 27. L. Chen, X. Zhao, Y. Lin, Y. Huang and Q. Wang, Chem. Commun., 2013, 49, 9678-9680. 28. M. Miksa, H. Kornura, R. Wu, K. G. Shah and P. Wang, J. Immunol. Methods., 2009, 342, 71-77. 29. S. Yao, K. J. Schafer-Hales and K. D. Belfield, Org. Lett., 2007, 9, 5645-5648. 30. S. T. Whitten, B. Garcia-Moreno and V. J. Hilser, Proc. Natl. Acad. Sci. USA., 2005, 102, 4282-4287. 31. D. Lagadic-Gossmann, L. Huc and V. Lecureur, Cell. Death. Differ., 2004, 11, 953-961. 32. M. Lakadamyali, M. J. Rust, H. P. Babcock and X. Zhuang, Proc. Natl. Acad. Sci. USA., 2003, 100, 9280-9285. 33. D. Perezsala, D. Colladoescobar and F. Mollinedo, J. Biol. Chem., 1995, 270, 6235-6242. 34. J. A. Kellum, M. Song and J. Li, Crit. Care., 2004, 8, 331-336. 35. R. J. Gillies, N. Raghunand, M. L. Garcia-Martin and R. A. Gatenby, Ieee. Eng. Med. Biol., 2004, 23, 57-64. 36. H. Izumi, T. Torigoe, H. Ishiguchi, H. Uramoto, Y. Yoshida, M. Tanabe, T. Ise, T. Murakami, T. Yoshida, M. Nomoto and K. Kohno, Cancer. Treat. Rev., 2003, 29, 541-549. 37. R. D. Coakley, B. R. Grubb, A. M. Paradiso, J. T. Gatzy, L. G. Johnson, S. M. Kreda, W. K. O'Neal and R. C. Boucher, Proc. Natl. Acad. Sci. USA., 2003, 100, 16083-16088. 38. A. M. Dennis, W. J. Rhee, D. Sotto, S. N. Dublin and G. Bao, ACS Nano., 2012, 6, 2917-2924. 39. Y. Tian, M. Wu, X. Liu, Z. Liu, Q. Zhou, Z. Niu and Y. Huang, Adv. Healthc. Mater., 2015, 4, 413-419. 40. S. A. Hilderbrand, K. A. Kelly, M. Niedre and R. Weissleder, Bioconjugate Chem., 2008, 19, 1635-1639.
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ChemComm
This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: L. Chen, Y. Wu, Y. Lin and Q. Wang, Chem. Commun., 2015, DOI: 10.1039/C5CC02866C.
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.
www.rsc.org/chemcomm
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DOI: 10.1039/C5CC02866C
Table of contents entry Based on the precise nano-architecture of bacteriophage M13, a FRET-based fluorescent nanosensor has been formulated for ratiometric sensing of intracellular
ChemComm Accepted Manuscript
Published on 14 May 2015. Downloaded by Fudan University on 15/05/2015 01:54:29.
pH.
ChemComm
ChemComm
View Article Online Page 2 of 5 Dynamic Article Links► DOI: 10.1039/C5CC02866C
Cite this: DOI: 10.1039/c0xx00000x
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Virus-templated FRET platform for rational design of ratiometric fluorescent nanosensors
5
Received (in XXX, XXX) XthXXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x We report here the construction of a bacteriophage M13templated supramolecular nanosystem, i.e. M13-β-CD/AdaFITC/Ada-RhB, which can be used as effective ratiometric fluorescent sensors for intracellular sensing.
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Förster resonance energy transfer (FRET) is a nonradiative process in which an excited state donor transfers energy to a ground state acceptor through long-range dipole-dipole interactions.1 Based on the mechanism of FRET, a series of novel light-harvesting systems, chemosensors and bioprobes have been developed recently.2-4 Conventional FRET-based probes have been designed in the form of small-molecular dyads, where the donor and acceptor fluorophores are connected by an appropriate linker. Recently, FRET mechanism has been adapted for the fabrication of nanoparticle-based probes (i.e. nanoprobes) for ratiometric sensing of ions, biomolecules and other analytes.4 FRET is one of the most widely used sensing mechanisms for ratiometric fluorescent probes. Unlike the one-signal fluorescent probes that can be influenced by probe concentration, probe environment, excitation intensity or any other drifts in the environment and instrument, the ratiometric fluorescent probes can alleviate these shortcomings by self-calibration of two emission bands.5-8 Recently, viral nanoparticles (VNPs) derived from plants and bacteria have attracted great attentions as nanoscale platforms for next-generation materials and biomedical applications,9-17 due to their unique monodisperse, multivalent and modifiable structural features, as well as good biocompatibility. In addition, based on the precise nano-architectures of viruses and virus-like particles (VLPs), FRET-based light-harvesting antennas have been constructed,18-21 where the chromophores are precisely organized within the Förster distance for efficient energy transfer. Bacteriophage M13 is a filamentous virus, 880 nm in length and 6.6 nm in diameter, consisting of a single-stranded DNA enclosed by 2700 copies of the major coat protein P8 and capped with 5 copies of four different minor coat protein (P9, P7, P6 and P3) on the ends. M13 is considered noninfectious and nonhazardous in human and mammalian because of its specific host selectivity, which can guarantee its safety in biological applications. Actually, its good cytocompatibility both in vitro and in vivo has been confirmed by our group and others.22-25 Meanwhile, it has been proven that M13 coating proteins assembled into a fairly rigid structure with all reactive groups being elucidated. In another word, the locations and separation This journal is © The Royal Society of Chemistry [year]
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distances of the reactive amino acid residues are precisely fixed. For example, we have previously shown that the N-terminus alanine residues attribute to the amidation reaction with activated carboxylic acid.26 The distances between two neighboring Nterminus at the exterior surface of M13 are oa ≈ 3.2 nm and ob ≈ 2.4 nm, respectively (Scheme 1A). Thus, after the conjugation of dyes to the N-terminus of M13, the locations of dyes are precisely fixed within the Förster distance, which is in favor of the FRET process occurring.
Scheme 1 (A) The structure of bacteriophage M13. M13 was generated using PyMol (www.pymol.org) with coordinates obtained from the RCSB Protein Data Bank (www.pdb.org). The amino acid residues in blue at the exterior surface of M13 represent N-terminal alanine, to which the β-CD was conjugated. The calculated distances between two neighboring Ntermini are oa ≈ 3.2 nm and ob ≈ 2.4 nm. (B) The structures of M13-β-CD, Ada-FITC and Ada-RhB. (C) Schematic demonstration of the formation of the ratiometric fluorescent pH nanosensor based on M13 and FRET (i.e. M13-β-CD/Ada-FITC/Ada-RhB) via the one-pot self-assembly approach driven by the supramolecular interaction between β-CD and Ada moieties.
[journal], [year], [vol], 00–00 |1
ChemComm Accepted Manuscript
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Limin Chen,a Yehong Wu,a Yuan Lin*a and Qian Wang*ab
ChemComm
The arrows indicate the FRET process between donor and acceptor dyes on the M13 nano-scaffold.
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In this work, M13 was exploited as a scaffold to construct FRET-based ratiometric fluorescent nanoprobes. β-Cyclodextrin (β-CD) were first conjugated to the N-terminus at the exterior surface of M13 using carbodiimide chemistry as our previous report.24 After the conjugation, the locations of β-CD were precisely displayed at the exterior surface of M13 scaffold. Fluorescein isothionate (FITC) (pH sensitive) and rhodamine B (RhB) (pH insensitive) were selected as model donor and acceptor dyes, respectively, which were anchored onto M13 particles upon derivatization with adamantyl (Ada) moieties (Scheme 1B) and sequential supramolecular assembly driven by the molecular recognition between β-CD and Ada moieties (Scheme 1C) according to our previous report.27 As a result, a model M13-templated FRET platform (i.e. M13-β-CD/AdaFITC/Ada-RhB) was achieved via the one-pot self-assembly approach. Bacteriophage M13 was obtained through amplification in E. coil, and M13-β-CD was synthesized using carbodiimide chemistry as our previous report, where β-CD were attached to the N-terminus.24 Ada-RhB was synthesized by esterification reaction, and Ada-FITC was prepared from FITC and 1adamantaneamine. M13-β-CD/Ada-FITC/Ada-RhB was obtained by incubating M13-β-CD with different molar ratios of Ada-FITC and Ada-RhB for 30 min at 4 C, and precipitating with PEG8K and NaCl, which was then confirmed by UV-Vis spectroscopy assay. The UV-Vis spectra show that the absorbance of AdaFITC (490 nm) gradually increases in company with the decrease in the absorbance of Ada-RhB (560 nm) as various molar ratios of Ada-FITC and Ada-RhB from 0 : 6 to 6 : 0 were used (Fig. 1A). Based on the UV-Vis spectrum, the average number of Ada-FITC and Ada-RhB units per M13-β-CD particle can be determined, and the results are given in Table S1, ESI. Transmission electron microscopy (TEM) analysis showed no significant changes in length and diameter of M13 particles after grafted with β-CD and sequential self-assembly (Fig. S1, ESI). A substantial spectral overlap of the donor emission with the acceptor absorption is essential for FRET process occurring. So we first examined the absorption and emission spectra of FITC and RhB upon derivatization with Ada moieties. An efficient spectral overlap between the emission spectrum of Ada-FITC and the absorption spectrum of Ada-RhB was observed (Fig. 1B), suggesting that the FRET process from Ada-FITC to Ada-RhB would take place when they are arranged in an appropriate distance and spatial orientation. To confirm the occurring of FRET process from Ada-FITC to Ada-RhB on the M13-β-CD nano-scaffold, we compared the fluorescence spectra changes of the mixed solution of Ada-FITC and Ada-RhB before and after the addition of M13-β-CD. As shown in Fig. 1C, in the absence of M13-β-CD, the mixed solution of Ada-FITC and Ada-RhB mainly showed the fluorescence emission from Ada-FITC (λ515 nm) when excited at 450 nm; however, in the presence of M13-β-CD, the increase in emission from Ada-RhB (λ580 nm) with concomitant suppression of Ada-FITC emission (λ515 nm) was observed, clearly indicating efficient FRET from Ada-FITC to Ada-RhB in the presence of M13-β-CD. In addition, we also observed that the mixed solution of Ada-FITC and Ada-RhB 2|Journal Name, [year], [vol], 00–00
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without M13-β-CD presented green fluorescence under a 365 nm UV lamp because of no efficient energy transfer from Ada-FITC to Ada-RhB where they are too far apart, whereas it showed yellow fluorescence in the presence of M13-β-CD because part of excitation state energy of Ada-FITC was transferred to the ground state Ada-RhB (Fig. 1C inset). To further confirm the FRET from Ada-FITC to Ada-RhB on the M13-β-CD, we measured the time-resolved fluorescence decay curves of M13-β-CD/Ada-FITC without Ada-RhB and M13-β-CD/Ada-FITC/Ada-RhB. The fluorescence lifetime of Ada-FITC on the M13-β-CD/Ada-FITC was determined to be 3.15 ns while the lifetime of Ada-FITC on the M13-β-CD/AdaFITC/Ada-RhB was shortened to 2.27 ns (Fig. 1D), which also supports the occurrence of FRET from Ada-FITC to Ada-RhB in the presence of M13-β-CD. According to the formula E = 1 – τDA/τD, the FRET efficiency was calculated to be ~ 28 %, where τD represents the lifetime of the donor without the acceptor, and τDA the lifetime of the donor in the presence of the acceptor. Based on the fluorescence spectra and fluorescence delay curves, we hypothesize that Ada-FITC and Ada-RhB should be anchored onto the exterior surface of M13-β-CD in a random manner (Scheme 1C) during the one-pot self-assembly process, which may influence the FRET efficiency on the M13 nano-scaffold.
Fig. 1 (A) UV-Vis spectra of M13-β-CD/Ada-FITC/Ada-RhB with varying molar ratios of Ada-FITC and Ada-RhB (nAda-FITC : nAda-RhB). (B) Normalized absorption spectra of Ada-FITC (green solid) and Ada-RhB (red solid) in pH 7.8 K-phos buffer; normalized fluorescence spectra of Ada-FITC (green dot) and Ada-RhB (red dot) in pH 7.8 K-phos buffer. (C) Fluorescence spectra of the mixture of Ada-FITC and Ada-RhB (nAda-FITC : nAda-RhB= 1 : 1) (green) or M13-β-CD/Ada-FITC/Ada-RhB (nAda-FITC : nAdaRhB = 1 : 1) (red) in pH 7.8 K-phos buffer when excited at 450 nm. The inset shows the photographs of the pH 7.8 K-phos buffer solution containing Ada-FITC and Ada-RhB (nAda-FITC : nAda-RhB = 1 : 1) (right) or M13-β-CD/Ada-FITC/Ada-RhB (nAda-FITC : nAda-RhB = 1 : 1) (left) under 365 nm illumination. (D) Time-resolved fluorescence decay curves of M13-β-CD/Ada-FITC (black) and M13-β-CD/Ada-FITC/Ada-RhB (nAdaFITC : nAda-RhB = 1 : 1) (red) in pH 7.8 K-phos buffer.
To evaluate the potential usage of the M13-templated FRET platform, we examined the ratiometric response of M13-βCD/Ada-FITC/Ada-RhB to hydrogen ion using fluorescence spectroscopy. Hydrogen ion is an important intracellular species, which plays a pivotal role in biological activities, such as cell proliferation, apoptosis, phagocytosis, endocytosis, multidrug This journal is © The Royal Society of Chemistry [year]
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Fig. 2 (A) Fluorescence spectra of M13-β-CD/Ada-FITC/Ada-RhB (nAdaFITC : nAda-RhB = 1 : 2) at varied pH values when excited at 450 nm. (B) Ratiometric pH calibration plot of the fluorescence emission ratio (I515 nm/I580 nm) of M13-β-CD/Ada-FITC/Ada-RhB (nAda-FITC : nAda-RhB = 1 : 2) as a function of pH.
The fluorescence spectra of M13-β-CD/Ada-FITC/Ada-RhB with varied pH values when excited at 450 nm are shown in Fig. 2A. The fluorescence emission intensity of Ada-FITC gradually decreases with decreasing pH due to the protonation of its phenolate moiety which is critical for optimal fluorescence emission. Fig. 2B shows that the emission intensities ratios of Ada-FITC (515 nm) to Ada-RhB (580 nm) changed linearly as a function of pH in the range of 5.0 to 8.4, allowing the detection of pH in a ratiometric manner upon excitation at a single wavelength. This ratiometric nanodevice is more powerful than intensitybased fluorescent probes. In the reversibility experiment, the pH was changed from 5.0 to 8.4 and back to 5.0 twice, and the AdaFITC/Ada-RhB fluorescence emission ratios reached the expected values in all cases (Fig. S2, ESI), indicating excellent reversibility and reproducibility of this M13 nanosensor in reporting pH. Additionally, we noted that the M13 nanosensor showed fast pH response because the Ada-FITC was anchored on the exterior surface of M13 particles with large surface-to-volume ratio. We then evaluated the pH sensitivity of the ratiometric M13 nanosensor in living cells. It has been reported that M13 can enter into many kinds of cells via different endocytosis pathways.39-40 Here, RAW 264.7 macrophages were first incubated with M13-βCD/Ada-FITC/Ada-RhB for 12 h. After cellular uptake, the cells were washed with PBS buffer to remove free M13 particles in solution and on the cell surface, and visualized by confocal laser scanning microscopy (CLSM). As shown in Fig. 3, the green channel was obtained by integrating the spectral region from 510 nm to 520 nm, and the red channel was acquired by integrating the fluorescence signals from 575 nm to 585 nm. The intracellular pH was determined by comparing the ratio between the average fluorescence intensity from 510 nm to 520 nm and the average intensity from 575 nm to 585 nm to the pH calibration plot shown in Fig. 2B. The average pH value based on at least 50 cells using the M13 nanosensor was determined to be ~ 4.7, which is in good agreement with the reported pH range for This journal is © The Royal Society of Chemistry [year]
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the acidic organelles in endocytosis pathways (i.e. pH 4.5~5.0 in lysosomes). This experiment indicates the potential application of the ratiometric M13 nanosensor in fluorescence imaging and sensing of the pH in cellular systems.
Fig. 3 Typical CLSM images of RAW 264.7 macrophages incubated with the M13-β-CD/Ada-FITC/Ada-RhB (nAda-FITC : nAda-RhB = 1 : 2) for 12 h. The excitation wavelength was 488 nm and the images were collected at 510 nm-520 nm (A, green channel) or 575 nm-585 nm (B, red channel). (C) is the overlap of the green and red fluorescence. The scale bars represent 50 μm.
In summary, we report here a bacteriophage M13-templated FRET platform for the development of ratiometric fluorescent nanosensors. Based on the M13-β-CD scaffold, the FRET from Ada-FITC to Ada-RhB could occur. The resulting ratiometric M13 nanosensor with a linear pH sensing range and good reversibility can be also responsive to intracellular pH. More importantly, based on this work, we expect that the precise nanoarchitecture of bacteriophage M13 is applicable to the construction of various other kinds of ratiometric fluorescent nanosensors by changing the FRET components. This work was supported by National Natural Science Foundation of China (21429401 and 21374119).
Notes and references a
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State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P.R. China. [email protected] b Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA [email protected] † Electronic Supplementary Information (ESI) available: Preparation and characterization of M13-β-CD/Ada-FITC/Ada-RhB, experimental details of intracellular imaging experiments. See DOI:10.1039/b000000x/ 1. K. E. Sapsford, L. Berti and I. L. Medintz, Angew. Chem. Int. Edit., 2006, 45, 4562-4588. 2. K. V. Rao, K. K. R. Datta, M. Eswaramoorthy and S. J. George, Chem.-Eur. J., 2012, 18, 2184-2194. 3. L. Yuan, W. Lin, K. Zheng and S. Zhu, Acc. Chem. Res., 2013, 46, 1462-1473. 4. G. Chen, F. Song, X. Xiong and X. Peng, Ind. Eng. Chem. Res., 2013, 52, 11228-11245. 5. A. P. Demchenko, J. Fluoresc., 2010, 20, 1099-1128. 6. K. Kikuchi, H. Takakusa and T. Nagano, Trac.-Trend. Anal. Chem., 2004, 23, 407-415. 7. J. V. Mello and N. S. Finney, Angew. Chem. Int. Edit., 2001, 40, 1536-1538. 8. S. Deo and H. A. Godwin, J. Am. Chem. Soc., 2000, 122, 174-175. 9. K. Li, H. G. Nguyen, X. Lu and Q. Wang, Analyst, 2010, 135, 21-27. 10. Y. J. Lee, H. Yi, W. J. Kim, K. Kang, D. S. Yun, M. S. Strano, G. Ceder and A. M. Belcher, Science, 2009, 324, 1051-1055. 11. R. J. Tseng, C. L. Tsai, L. Ma, J. Y. Ouyang, C. S. Ozkan and Y. Yang, Nat. Nanotechnol., 2006, 1, 72-77. 12. N. F. Steinmetz, Mol. Pharm., 2013, 10, 1-2. 13. N. F. Steinmetz, Nanomed.-Nanotechnol. Biol. Med., 2010, 6, 634641.
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resistance, and signal transduction.28-33 Slightly abnormal pH values can be associated with inappropriate cell activities (e.g. abnormal proliferation and differentiation), which are observed in a variety of diseases, such as cancer, cystic fibrosis and immune dysfunction.34-37 Thus, monitoring of pH distribution and fluctuation with high temporal-spatial resolution in living systems is of great importance for understanding of the relationship between the pH and cell activities, as well as early diagnosis and treatment of diseases.38
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14. M. E. Farkas, I. L. Aanei, C. R. Behrens, G. J. Tong, S. T. Murphy, J. P. O'Neil and M. B. Francis, Mol. Pharm., 2013, 10, 69-76. 15. K. Zhou, F. Li, G. Dai, C. Meng and Q. Wang, Biomacromolecules, 2013, 14, 2593-2600. 16. C. Mao, A. Liu and B. Cao, Angew. Chem. Int. Edit., 2009, 48, 67906810. 17. J. Rong, L. A. Lee, K. Li, B. Harp, C. M. Mello, Z. Niu and Q. Wang, Chem. Commun., 2008, 44, 5185-5187. 18. Y. S. Nam, T. Shin, H. Park, A. P. Magyar, K. Choi, G. Fantner, K. A. Nelson and A. M. Belcher, J. Am. Chem. Soc., 2010, 132, 14621463. 19. R. A. Miller, A. D. Presley and M. B. Francis, J. Am. Chem. Soc., 2007, 129, 3104-3109. 20. M. Endo, M. Fujitsuka and T. Majima, Chem.-Eur. J., 2007, 13, 8660-8666. 21. L. M. Scolaro, M. A. Castriciano, A. Romeo, N. Micali, N. Angelini, C. L. Passo and F. Felici, J. Am. Chem. Soc., 2006, 128, 7446-7447. 22. H. Yi, D. Ghosh, M. H. Ham, J. Qi, P. W. Barone, M. S. Strano and A. M. Belcher, Nano Lett., 2012, 12, 1176-1183. 23. A. Merzlyak, S. Indrakanti and S. W. Lee, Nano Lett., 2009, 9, 846852. 24. L. Chen, X. Zhao, Y. Lin, Z. Su and Q. Wang, Polym. Chem., 2014, 5, 6754-6760. 25. N. Suthiwangcharoen, T. Li, K. Li, P. Thompson, S. You and Q. Wang, Nano Res., 2011, 4, 483-493. 26. K. Li, Y. Chen, S. Li, H. G. Nguyen, Z. Niu, S. You, C. M. Mello, X. Lu and Q. Wang, Bioconjugate Chem., 2010, 21, 1369-1377. 27. L. Chen, X. Zhao, Y. Lin, Y. Huang and Q. Wang, Chem. Commun., 2013, 49, 9678-9680. 28. M. Miksa, H. Kornura, R. Wu, K. G. Shah and P. Wang, J. Immunol. Methods., 2009, 342, 71-77. 29. S. Yao, K. J. Schafer-Hales and K. D. Belfield, Org. Lett., 2007, 9, 5645-5648. 30. S. T. Whitten, B. Garcia-Moreno and V. J. Hilser, Proc. Natl. Acad. Sci. USA., 2005, 102, 4282-4287. 31. D. Lagadic-Gossmann, L. Huc and V. Lecureur, Cell. Death. Differ., 2004, 11, 953-961. 32. M. Lakadamyali, M. J. Rust, H. P. Babcock and X. Zhuang, Proc. Natl. Acad. Sci. USA., 2003, 100, 9280-9285. 33. D. Perezsala, D. Colladoescobar and F. Mollinedo, J. Biol. Chem., 1995, 270, 6235-6242. 34. J. A. Kellum, M. Song and J. Li, Crit. Care., 2004, 8, 331-336. 35. R. J. Gillies, N. Raghunand, M. L. Garcia-Martin and R. A. Gatenby, Ieee. Eng. Med. Biol., 2004, 23, 57-64. 36. H. Izumi, T. Torigoe, H. Ishiguchi, H. Uramoto, Y. Yoshida, M. Tanabe, T. Ise, T. Murakami, T. Yoshida, M. Nomoto and K. Kohno, Cancer. Treat. Rev., 2003, 29, 541-549. 37. R. D. Coakley, B. R. Grubb, A. M. Paradiso, J. T. Gatzy, L. G. Johnson, S. M. Kreda, W. K. O'Neal and R. C. Boucher, Proc. Natl. Acad. Sci. USA., 2003, 100, 16083-16088. 38. A. M. Dennis, W. J. Rhee, D. Sotto, S. N. Dublin and G. Bao, ACS Nano., 2012, 6, 2917-2924. 39. Y. Tian, M. Wu, X. Liu, Z. Liu, Q. Zhou, Z. Niu and Y. Huang, Adv. Healthc. Mater., 2015, 4, 413-419. 40. S. A. Hilderbrand, K. A. Kelly, M. Niedre and R. Weissleder, Bioconjugate Chem., 2008, 19, 1635-1639.
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