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Highly Sensitive Fluorescence Molecular Switch for the Ratio Monitoring of Trace Change of Mitochondrial Membrane Potential Caixia Wang, Ge Wang, Xiang Li, Kui Wang, Jing Fan, Kai Jiang, Yuming Guo, and Hua Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02781 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 2017

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Analytical Chemistry

Highly Sensitive Fluorescence Molecular Switch for the Ratio Monitoring of Trace Change of Mitochondrial Membrane Potential Caixia Wang,† Ge Wang,‡ Xiang Li,†,§ Kui Wang,† Jing Fan,†,§ Kai Jiang,†,§ Yuming Guo,† Hua Zhang*,† † Key Laboratory of Green Chemical Media and Reactions, Ministry of Education; Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals; School of Chemistry and Chemical Engineering, Henan Normal University; Xinxiang, 453000, P.R. China. ‡ Xinxiang Medical University; Xinxiang, 453000, P.R. China. § Key Laboratory for Yellow River and Huai River Water Environment and Pollution Control, Ministry of Education; School of Environment, Henan Normal University; Xinxiang, 453000, P.R. China. Corresponding Author Email: [email protected], Tel/Fax: +86-373-3329030. ABSTRACT: As one of the earliest events in apoptosis, trace change of mitochondrial membrane potential (MMP) greatly affects cell health. The local MMP, as an intracellular variable factor, differs considerably from one area to another in an extremely fine cell structure—mitochondrial membrane, which increases the difficulty for the real-time monitoring of MMP trace change in living cells. More regrettably, so far no ratio fluorescence probe for MMP is available. Such probe is a kind of precision analysis tools that detect trace change of MMP in the complex biological systems at subcellular level. In this study, a molecular switch (hemicyanine derivative, TPP-CY) was reported as ratio fluorescence probe for real-time detection of trace change of MMP in living cells. Given the formation of “C-O” bond in the probe molecule, the probe exhibits a remarkable ratio fluorescence intensity change (I563/I663) within seconds during the response process for MMP, that is, TPP-CY transforming to TPP-SP. Furthermore, TPP-CY at a low concentration (0.23 µM) can present extremely high sensitivity for the trace change of MMP in living cell. The detection limit can be as low as -0.16 mV. More importantly, trace change of MMP and mitochondrial morphology at subcellular level during cell apoptosis can be accurately monitored by TPP-CY with the excellent selectivity and high resolution. TPP-CY could be used as a potential tool for evaluating cell health.

INTRODUCTION Mitochondrion usually appears negative potential difference (approximately -180 to -200 mV), namely mitochondrial membrane potential (MMP, ∆ψm), which is a key indicator of cell health. Stable MMP maintains the normal physiological activities, such as mitochondria for ATP synthesis and proton gradient across the lipid bilayer.1 These characteristics are vital to normal physiological functions, such as signal transduction,2 cell division,2 and cell apoptosis.3 Changes of MMP, even trace change, greatly affect mitochondrial functions.4 More seriously, the unusual change of MMP would further cause mitochondrial disorders, for keratitis,5,6 parkinson’s disease (PD),7,8 and cancer.9,10 Substantial data indicated that MMP can be frequently used as an early signal event for irreversible cell apoptosis.6,11 Thus, MMP has become a new focus in cytobiology and chemical biology. Herein, an interesting thing to note is that MMP in living organisms mainly located in mitochondrial membrane that is a kind of extremely thin intracellular membranes, only 60-75 Å. Furthermore, given its constant fluctuation during intracellular physiological processes, local MMP, as an intracellular

variable factor, differs considerably from one area to another in mitochondrial membrane, which largely increases the difficulty for the real-time detecting of MMP in living cells. Therefore, it is challenging and highly desired to prepare the precise imaging approaches with high resolution for the realtime, ratio detection and in situ detecting of MMP at subcellular level. Fluorescence micro-imaging approach combined with fluorescent molecular probes presents remarkable opportunity for detecting and imaging target objects that possess charge, such as MMP, in living cells because they present good biocompatibility and high selectivity.12-17 Recently, there have been reported some fluorescent sensors or probes with positive charge for the imaging of MMP in living cells,11, 18-21 such as tetramethylrhodamine methyl ester (TMRM), tetramethylrhodamine ethyl ester (TMRE), Rhodamine 123 (Rhod123), and JC-1. They can accumulate into the mitochondrial membrane matrix space and thus facilitate the imaging of MMP via different recognition mechanisms. However, existing probes appear some insufficients. For example, TMRE, TMRM and Rhod123 only appeared fluorescence change at single wavelength for MMP. Although JC-1 emits the ratio fluorescence for different MMPs, it is often affected

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by the environment of the mitochondrial substrate. Thus, these probes are unsuitable for precise in situ analysis in complex biological systems. More regrettably, the existing probes are incapable of in situ and real-time detection of trace change of MMP at ultrahigh resolution during disease-related processes. Therefore, there urgently need to develop a mitochondriatargeted fluorescent probe with high resolution for the precise imaging and measuring of MMP in living cells on real time and in situ mode. With these in mind, a molecular switch (hemicyanine derivative, TPP-CY) was reported as a fluorescent probe for the imaging and quantification of MMP in living cells in this work. Triphenylphosphine (TPP) as the recognition unit of the probe results in TPP-CY appearing excellent selectivity and sensibility for mitochondrial MMP. More importantly, TPP can be used as an adjustment unit for molecular structure to alter the “C-O” bond during the recognition process. On the basis of this design strategy, 0.23 µM of TPP-CY presented two different fluorescence signals for the trace change of MMP in living cells at high-resolution mode. The signals had high signal-to-noise ratios at two sites (563 and 663 nm) within seconds. According to structural analyses, spectral analysis and whole-cell assay result, this fluorescent molecular switch with high resolution can be an excellent potential tool for realtime and in situ imaging of trace change of MMP during apoptosis of living cells. EXPERIMENTAL SECTION Chemicals and materials. All solvents and reagents used were reagent grade. Silica gel (200-300 mesh) and aluminum oxide (activated, neutral, approximately 150 mesh) were used for flash column chromatography. Commercial fluorescent dye-MitoTracker® Red CMXRos was used in the colocalization experiment. Detailed synthesis methods are given below. NMR spectra were obtained using a Varian Inova 400 MHz (or a Bruker Avance II 400 MHz) spectrometer. Fluorescence spectra were obtained with a spectrophotometer, absorption spectra with a UV-vis spectrophotometer.

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minimum essential medium (WelGene) supplemented with penicillin/streptomycin and 10% fetal bovine serum (FBS; Gibco) were used to culture cells in a 5.0 wt%/vol CO2 incubator at 37 °C. Cell lines were seeded into a 35 mm dish with 20 mm well glass bottomed dish (Mat Tek) one day before imaging. Afterward, the cells were stained with TPP-CY. Confocal microscopic imaging. Live cells were first stained with MitoTracker® Red CMXRos (1.0 µM) 15 min and then stained with 0.23 µM of TPP-CY for 5 min. Olympus spectral confocal multi-photon microscope (FV1200) with the modelocked titanium-sapphire laser source (MaiTai, SpectraPhysics, USA) was used to image cell on one/two-photon mode. These imaging results were obtained at the following parameters: internal PMTs are at 16 bit and 1024×1024 pixels, excitation wavelength: 515 nm, scan range: 520-620 nm (green channel), 620-720 nm (red channel). RESULTS AND DISCUSSION Design strategy of fluorescence probe TPP-SP. This study aimed to design a fluorescence molecular switch (TPP-CY) as a probe with ultra-sensitivity and high selectivity for the ratio imaging of MMP trace change in living cells. In molecular designing, spiropyrans (SP), an important photochromic compound, was selected as the fluorophore, which shows reversible structural isomerization between colorless SP and colored hemicyanine under excitation by different lasers. The most remarkable innovation in the present design strategy is that TPP was introduced into the probe to adjust “C-O” bond energy. This design strategy could further adjust molecular structural change only under different potentials, that is, colored hemicyanine (TPP-CY) to colorless TPP-SP. Based on this design strategy, TPP-CY would exhibit excellent optical switch performance for different potentials (Scheme 1). More importantly, TPP was introduced into “N” atom of indolium group, which further improved the stability and specificity of S P-b a s e d p ro b e . Th e n a t u re a n d p o s i t i o n o f t h e

Fluorescence quantum yield measurements. Fluorescence quantum yield (Φ) for MMC in double distilled water (3.3 µmol/L) was calculated using the following equation (1): Φx = Φs (Fx/Fs) (As/Ax) (λexs/λexx) (nx/ns)2

(1)

where Φ is the quantum yield, F is the integrated area under the corrected emission spectrum (in Ep units), A is the absorbance at the excitation wavelength, λex is the excitation wavelength, n is the refractive index of the solution, and the subscripts x and s refer to the unknown and the standard, respectively. Rhodamine B (Φs = 0.97) in methanol was used as the reference standard. In all spectral experiments, the final solutions contained DMSO (< 5‰). Each experiment was carried out in five replicates (n=5). Cell culture and cytotoxicity. NIH3T3, HepG2, MCF-7, 293T, and HeLa cell lines were obtained from the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences. Dulbecco's modified Eagle's (WelGene) and Eagle's

Scheme 1. The response mechanism of TPP-CY for MMP. The molecular structure of fluorescent probe (TPP-CY and TPP-SP) and the intermediate products (TPP and SP).


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substituents of indole moiety spiropyrane influence the photochromic performance.22,23 Alkyl chain length between indolium and TPP is critical on molecular switching. To obtain the appropriate photochromic performance, two-carbon chain was introduced to link TPP and indolium group.24 Furthermore, TPP and indolium group19 as dual recognition units result in TPP-CY showing excellent selectivity and sensibility for MMP, because TPP has a more positive electrostatic isosurface than the indolinium group.19 Probe was synthesized on the basis of the route (Figure S1). Fluorescent probe and the intermediate products were clearly characterized by NMR and ESI-MS (attached Figure 1 in Supporting Information).

is at 0 mM and 3.3 mM, respectively. However, the fluorescence intensity of TPP-CY was continuously enhanced at 563 nm, and at this point fluorescence intensity at 663 nm was essentially constant (Figure 1b) when the zeta potential decreased from -8.57 to -15.5 mV, that is, the concentration of SDS was between 3.3 mM and 7.8 mM (Figure 1b). Accordingly, the fluorescence quantum yield (Φ) increased to 0.11. An excellent linear relationship existed between the ratio fluorescent emission intensity (I563/I663) at these two wavelengths (563 and 663 nm) with different negative potentials (Figure 1d). The detection limit of TPP-CY for potential was approximately -0.16 mV, which indicated that TPP-CY was beneficial to detect the trace change of potential (< 0.01%).

Spectral properties of probe to the negative potential change. To obtain the different negative potentials in solution, sodiumdodecyl sulfate (SDS) was added into the phosphate buffer (PBS, pH 7.4) . 25 The value of the different negative potentials under different concentrations of SDS was detected by the zeta potential. The spectral properties and specific response of TPP-CY to the negative potential were determined in this kind of testing environment. Data indicated that TPP-CY exhibited the dramatic fluorescence changes under the different negative potential changes within short response time (within seconds, Figure S2a). Under free state, TPP-CY appeared a mainly fluorescence peak at 663 nm (3.3 µM, ε = 1.4 × 104) with an extremely low fluorescence (Φfree state = 0.030) when it was excited at 540 nm. But, the fluorescent spectral data (Figure 1a) showed that there gradually appeared two fluorescence signals at 563 and 663 nm within seconds in the presence of the negative potential in testing system. This result indicated that TPP-CY would possess potential positioning capability in the mitochondrial matrix. More interestingly, the fluorescence intensity of TPP-CY was also enhanced at 663 nm and 563 nm when the zeta potential ranged from 0 to -8.57 mV. That is, the concentration of SDS

Ratio fluorescence response mechanism for the trace change of the negative potential. The structures of TPP-CY and SP were optimized to elucidate the mechanism of ratio fluorescence response process. The optimized result showed that the bond length of “C-O”was 1.431 Å. Given the introduction of TPP into the probe, the bond length of “C-O” in spiro-ring became longer, and the bond energy became weaker in the TPP-CY molecule, than those in SP (1.429 Å, Figure S3). Based on the above results, TPP-CY mainly existed in an open-form (hemicyanine dye) under free state. Fluorescence also appeared at 663 nm. However, the recognition group (TPP) of probe TPP-CY could combine with the negative potential, which changed the C-O bond of spiro-ring. The bond length of “C-O” in spiro-ring changed from 1.431 Å to 1.429 Å. Therefore, probe mainly existed in close-form (TPPSP) with short wavelength, fluorescent also occurred at 563 nm. On account of chemical bond switching with different charges, TPP-CY possessed potential function applied to image trace change of negative potential on ratio fluorescence mode through molecular switch.

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Figure 1. (a) TPP-CY (3.3 µM) recognize the different concentrations of SDS (0-4.2 mM). (b) Emission spectra of 3.3 µM TPPCY mixtures with different concentrations of SDS (4.2-9.6 mM). (c) Emission spectra of 3.3 µM TPP-CY mixtures with different zeta potential of SDS (0 to -15.5 mV). (d) The linear fluorescent response of TPP-CY to zeta potential (0 to -15.5 mV) in secondary water. I563/I663 = -6.28 - 1.52 zeta potential (R2 = 0.9960).

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Figure 2. (a) Specific selectivity of TPP-CY for MMP. 1, Al3+ (0.10 mM) and NO3- (0.30 mM); 2, Ni2+ (0.10 mM) and Cl- (0.20 mM); 3, Mg2+ (0.10 mM) and SO42- (0.10 mM); 4, Cu2+ (0.10 mM) and SO42- (0.10 mM); 5, Li+ (0.10 mM) and CO32- (0.050 mM); 6, Na+ (0.10 mM) and CO32- (0.050 mM); 7, K+ (0.10 mM) and H2PO4- (0.10 mM); 8, Hg2+ (0.10 mM) and Cl- (0.20 mM); 9, Ca2+ (0.10 mM) and Cl- (0.20 mM); 10, K+ (0.10 mM) and Cl- (0.10 mM); 11, SDS (0.10 mM) (n = 5). (b) Photo-stability TPP-CY in solution under 540 nm with visible light in different time intervals. Solution: PBS buffer at 25°C.


Analytical Chemistry and cationic surfactant (Benzalkonium Chloride, zeph) were selected as interfering substances (Figure S4). Figure 2a shows no obvious fluorescent changes (I563/I663) when the interfering ions were added into the test system.

Fluorescence recognition of MMP trace change of living cells. The trace change of MMP is an important indicator for the irreversible apoptosis in cells.5,6,11 Thus, the fluorescent recognition ability of TPP-CY for the trace change of MMP was first verified in mitochondria of living cells. Colocalization experiment was firstly performed to evaluate the TPP-CY specific locating in mitochondria due to the trace change of MMP occurring in the mitochondria of living cells. Confocal microscopic imaging (Figure 3a) showed that a specific subcellular distribution of TPP-CY in the living cells. The commercial MitoTracker® Red CMXRos, a red mitochondria fluorescent dye, was used in colocalization experiment. The stained area in cells by TPP-CY (Figure 3a)

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Figure 3. Localization images in living HepG2 cells. (a) Stained with TPP-CY (0.23 µM). (b) MitoTracker® Red CMXRos (1.0 µM). TPP-CY: excitation wavelength: 509 nm; scan range: 520600 nm (green channel); MitoTracker® Red CMXRos: excitation wavelength: 559 nm; scan range: 592-720 nm (red channel); (c) the merged image of a and b; (d) Intensity profile of regions of interest in HepG2 cells, green channel: TPP-SP and red channel: MitoTracker® Red CMXRos. Images and data are representative of replicate experiments (n = 5).

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Prior to ratio imaging of MMP in living cells, the photostability of TPP-CY, as one of the evaluation parameters, was evaluated by residual fluorescence intensity. To detect the photo-stability of TPP-CY, it was monitored in solution under iodine-tungsten lamp in different time intervals. TPP-CY retained more than 80% of fluorescence intensity in water solution after they were continuously irradiated by an iodinetungsten lamp for 7.0 h. These data showed (Figure 2b) that TPP-CY possessed stronger photostability than those of some existing probes (Figure S5). These optical properties and specific selectivity showed that TPP-CY can be used to image MMP specifically on ratio mode in living cells.

Intensity

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Figure. 4. (a) Excitation wavelength: 515 nm; scan range: 520620 nm (green channel), 620-720 nm (red channel); (b) The fluorescence intensity of living cells in Figure a I (red line) and II (black line).

was in accordance with that area stained by MitoTracker® Red CMXRos (Figure 3b). The Pearson correlation coefficient between TPP-CY and MitoTracker® Red CMXRos was 0.91 (Figure 3c), which indicated the high selectivity of TPP-CY for MMP. And it located in the mitochondrial matrix. Furthermore, the existing probes for MMP detection need a comparatively high concentration,11,18-21 thereby resulting in severe cytotoxicity for mitochondrial morphology in living organisms. To our surprise, TPP-CY at considerably low concentration (0.23 µM) detected MMP at extremely low cytotoxicity (Figure S6). And then, we further examined TPP-CY staining in HepG2 cells treated with the membrane-potential uncoupler 3chlorophenylhydrazone (CCCP, 0.050 mM), which can decrease the MMP.26,27 Confocal microscopic imaging (Figure 4a I) showed that there only emitted fluorescence in green channel (520-620 nm) and remarkably low fluorescence in red channel (620-720 nm) when HepG2 cells were untreated with CCCP. However, the intensity of green channel (520-620 nm) decreased, whereas that of red channel (620-720 nm) increased (Figure 4a II) when HepG 2 cells were treated with CCCP (0.050 mM) for 5.0 min. This result was attributed to that CCCP can decrease MMP. The actual spectral variations were extracted from the living cell imaging (Figure 4b), which agreed with the fluorescence changes in the solution (Figure 1). Notable trace change of MMP is showed in the Supporting Information (Video 1). Dynamic monitoring of the trace change of MMP and mitochondrial morphology during living cell apoptosis. The trace change of MMP is an important indicator for the irreversible apoptosis in cells. In this work, different catechol concentration (0.10 mM, 0.50 mM and 2.0 mM; incubate time: 36 h)


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were selected as an apoptosis inducer to establish apoptotic model in HepG2 cells.28 TPP-CY (0.23 µM) was used for real-time detection of the trace change of MMP at subcellular level in the early apoptosis of cells.29-31 Confocal microscopic imaging (Figure 5I) showed that there only emitted fluorescence in green channel (520-620 nm) and considerably low fluorescence in red channel (620-720 nm) when HepG2 cells were treated with catechol (0.1 mM). Nevertheless, the intensity of green channel (520-620 nm) decreased (Figure 5 II and III) and that of red channel (620-720 nm) increased when HepG2 cells were treated with catechol (0.50 mM and 2.0 mM). This result (Figure 5) was similar to the fluorescence intensity with CCCP treatment (Figure 4), which indicated that MMP decreased during cell apoptosis in early stage. Figure 5 II shows the mitochondrial morphology change during induced apoptosis in living cells. The shape changed from filaments into ball, which was similar to that in literature.29 Therefore, Figure 5 indicates the feasibility of real-time detection of the trace change of MMP and evaluation of the health degree of living cells at subcellular level by cell morphology31 in early stage of apoptosis.

toxicity and extremely high sensitivity (the lowest detection limit for MMP: -0.16 mV) for the negative potential. Consequently, TPP-CY responds to MMP in living cell experiments at substantially low concentration (0.23 µM). The trace change of MMP and mitochondrial morphology at subcellular level during cell apoptosis process can be accurately monitored by TPP-CY. Thus, the probe can be used to evaluate cell health through mitochondrial morphology at subcellular level and accurately monitor trace change of MMP in living cells, thus showing great potential in chemical biology research and medical diagnosis of mitochondrial diseases.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed list of all utilized standards including: experimental details, synthesis, NMR and mass spectra, and additional spectroscopic data. (i) Synthetic procedures of TPP-SP and intermediates. (ii) Spectral properties of probe to sodium-dodecyl sulphate. (iii) Quantum calculations. (iv) Specific selectivity of TPP-SP for sodium-dodecyl sulphate. (v) The photo-stability of TPP-SP and MitoTracker® Red CMXRos. (vi) Cell viability of TPP-SP. (vii) The spectrum extraction of different concentration with catechol in living cancer cells. (viii) pH effect. (ix) Incubation and staining of living cells with TPP-SP. Attached: 1H and 13C NMR spectra of TPP-SP and intermediates. (PDF) The Supporting InformationVideo 1 is the dynamic imaging of HepG2 cells that were treated with the membrane-potential uncoupler 3-chlorophenylhydrazone (CCCP, 0.05mM) with AVT (*. avi).

AUTHOR INFORMATION Corresponding Author [email protected] [email protected]

Author Contributions

Figure 5. HepG2 cells were stained with 0.23 µM of TPP-CY for 5.0 min. And after, they were stained with the same time of catechol by 36 h. (I) HepG2 cells were stained with 0.10 mM of catechol; (II) HepG2 cells were stained with 0.50 mM of catechol; (III) HepG2 cells were stained with 2.0 mM of catechol. Excitation wavelength: 515 nm; scan range: 520-620 nm (green channel), 620-720 nm (red channel).

CONCLUSIONS In conclusion, we reported a molecular switch (TPP-CY) as a fluorescent probe to detect trace changes of MMP in living cells. TPP-CY exhibits a remarkable ratio fluorescence for trace change of negative potential within a short response time (seconds). The ratio fluorescence change (I563/I663) results from the formation of C-O bond under its different length when the probe encounters the different negative potentials. Furthermore, TPP-CY possesses excellent selectivity and low cyto-

All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by National Natural Science Foundation of China (21402043 and 21722501), Program for Science Technology Innovation Talents and Team in Universities of Henan Province (18HASTIT001 and 18IRTSTHN002), Key Project of Science and Technology of Henan Province (162102210267), Key Scientific Research Project of Higher Education of Henan Province (16B150004 and 18A150046). The High Performance Computing Center of Henan Normal University.

REFERENCES



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(1) LaNoue, K. F.; Duszynski, J. J. Bioenerg. Biomembr. 1992, 24, 499-506. (2) Kbeirts, P. Sci. 1999, 283, 1475-1481. (3) Kroemer, G.; Zamzami, N.; Susin, S. A. Immunol. Today 1997, 18, 44-51. (4) Chen, L. B. Annu. Rev. Cell Biol. 1988, 4, 155-181. (5) Green, D. R.; Reed, J.C. Sci. 1998, 281, 1309-1312. (6) Gandre-Babbe, S.; Bliek, A. M. Mol. Biol. Cell 2008, 19, 2402-2412. (7) Moore, D. J.; West, A. B.; Dawson, V. L.; Dawson, T. M. Annu. Rev. Neurosci. 2005, 28, 57-87. (8) Keeney, P. M.; Xie, J.; Capaldi, R. A.; Bennett, J. P. J. Neurosci. 2006, 26, 5256-5264. (9) Nunnari, J.; Suomalainen, A. Cell 2012, 148, 1145-1159. (10) Yousif, L. F.; Stewart, K. M.; Kelley, S. O. Chem. Bio. Chem. 2009, 10, 1939-1950. (11) Roberg, K.; Johansson, U.; Ollinger, K. Free Radic. Biol. Med. 1999, 27, 1228-1237. (12) Niu, W. F.; Guo, L.; Li, Y. H.; Shuang, S. M.; Dong, C.; Wong, M. S. Anal. Chem. 2016,88, 1908-1914. (13) Masanta, G. M.; Lim, C. S.; Kim, H. J.; Han, J. H.; Kim, H. M.; Cho, B. R. J. Am. Chem. Soc. 2011, 133, 5698-5700. (14) Lee, M. H.; Park, N.; Yi, C.; Han, J. H.; Hong, J. H.; Kim, K. P.; Kang, D. H.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2014, 136, 14136-14142. (15) Hou, J. T.; Wu, M. Y.; Li, K.; Yang, J.; Yu, K. K.; Xie, Y. M.; Yu, X.Q. Chem. Commun. 2014, 50, 8640-8643. (16) Chalmers, S.; Caldwell, S. T.; Quin, C.; Prime, T. A.; James, A. M.; Cairns, A. G.; Murphy, M. P.; McCarron, J. G.; Hartley, R. C. J. Am. Chem. Soc. 2012, 134, 758-761.

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(17) Denisov, S. S.; Kotova, E. A.; Plotnikov, E. Y.; Tikhonov, A. A.; Zorov, D. B.; Korshunova, G. A.; Antonenko, Y. N. Chem. Commun. 2014, 50, 15366-15369. (18) Zhao, N.; Chen, S. J.; Hong, Y.; Tang, B. Z. Chem. Commun. 2015, 51, 13599-13602. (19) Zhang, L.; Liu, W. W.; Huang, X. H.; Zhang, G. X.; Wang, X. F.; Wang, Z.; Zhang, D. Q.; Jiang, X. Y. Analyst 2015, 140, 5849-5854. (20) Yuan, H. S.; Cho, H.; Chen, H. H; Panagia, M.; Sosnovik, D. E.; Josephson, L. Chem. Commun. 2013, 49, 10361-10363. (21) Scaduto Jr. R. C.; Grotyohann, L. W. Biophys. J. 1999, 76, 469-477. (22) Li, X. L.; Li, J. L.; Wang, Y. M.; Matsuura, T.; MengLi, J. J. Photochem. Photobiol. A: Chem. 2004, 161, 201-213. (23) Görner, H. Phys. Chem. 2001, 3, 416-423. (24) Prager, S.; Burghardt, I.; Dreuw A. J. Phys. Chem. A, 2014, 118, 1339-1349. (25) Islam, M. F. Nano. Let. 2003, 3, 269-273. (26) Harriman, A.; Mallon, L. J.; Ulrich, G.; Ziessel, R. ChemPhys. 2007, 8, 1207-1214. (27) Russell, Jr. C. S.; Lee, W. G. Biophys. J. 1999, 76, 469477. (28) Ibuki, Y.; Goto, R. Biochim. Biophs. Acta. 2004, 1690, 11-12. (29) Bereiter-Hahn, J.; Voth, M. Microsc. Res. Tech. 1994, 27, 198-219. (30) Tanaka A.; Youle, R. J. Mol. Cell 2008, 29, 409-410. (31) Chen, H.; Chan, D. C. Curr. Top. Dev. Biol. 2004, 59, 119-144.


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