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Effects of Supramolecular Encapsulation on Photophysics and Photostability of a 9,10-Bis(arylethynyl)anthracene-Based Chromophore Revealed by Single-Molecule Fluorescence Spectroscopy Masaaki Mitsui,*,† Koji Higashi,‡ Yohei Hirumi,‡ and Kenji Kobayashi‡ †

Department of Chemistry, College of Science, Rikkyo University, 3-34-1, Nishiikebukuro, Toshima-ku, Tokyo, 171-8501, Japan Department of Chemistry, Faculty of Science, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan

‡

S Supporting Information *

ABSTRACT: The effects of supramolecular encapsulation on the photophysics and photostability of a highly fluorescent dimeric derivative of 2,6-diacetoxy-9,10-bis(arylethynyl)anthracene (G2) were investigated by single-molecule fluorescence spectroscopy (SMFS). The fluorescence properties of free-G2 and its self-assembled boronic ester encapsulation complex, G2@(Cap)2, were compared in solution and a glassy polymer film. The fluorescence spectral characteristics and theoretical calculations suggest that the environment affects the excited-state conformation and subsequent fluorescence emission of G2@(Cap)2. In particular, in the liquid and polymer environments, G2@(Cap)2 emits a fluorescence photon in the planar and twist conformation, respectively, whereas the fluorescence-emitting conformation of free-G2 is planar in both environments. The luminous conformation differences between free-G2 and G2@(Cap)2 in polymer are reflected in the intersystem crossing (ISC) parameters (the ISC quantum yield and triplet lifetime), as determined by fluorescence autocorrelation analysis. The photobleaching yield revealed a 3-fold enhancement in the photostability of encapsulated G2 (relative to free-G2). Under the SMFS measurement conditions, the photostability of the encapsulation complex was independent of the guest’s photostability and appeared to be dominated by the thermal stability of the Cap host molecule.

1. INTRODUCTION Supramolecular encapsulation is a promising strategy for photostabilizing fluorescent dyes.1 Among the extensive range of molecular containers are cyclodextrins,1−4 cucurbit[n]urils,5,6 rotaxanes,2,3,7 and self-assembled molecular capsules.8−12 As is well-known, the inclusion of the dye alters the microenvironmental parameters (e.g., polarity and polarizability)1 and restricts the conformation of the guest within the host cavity.13,14 Consequently, the spectral properties, photophysics, and photostability of the guest molecules are radically altered by encapsulation within the host. The effects of encapsulation on the guest’s photophysical properties have been usually investigated by ensemble spectroscopic methods. Singlemolecule investigations remain rare,6,14 despite the rapidly growing demand for fluorescence probes that are sufficiently stable and bright for inclusion in fluorescence-based singlemolecule techniques such as single-molecule fluorescence spectroscopy (SMFS),15,16 single-molecule orientation imaging,15−17 and single-molecule super-resolution imaging.18 Recently, Kobayashi et al.13 reported the supramolecular encapsulation of highly fluorescent 2,6-diacetoxy-9,10-bis(arylethynyl)anthracene derivatives in a self-assembled boronic © XXXX American Chemical Society

ester cavitand capsule (Cap). Two of these derivatives (hereafter designated as G1 and G2) are shown in Figure 1a. The Cap is formed through dynamic boronic esterification between the cavitand tetraboronic acid I and the bis(catechol)linker II (Figure 1b). Kobayashi et al. showed that the Cap enhances the photostability of the encapsulated guests by enclosing them in a protective nanocavity. In our recent study, the photophysics and photostability of G1 and its encapsulation complex (G1@Cap) in a glassy polymer matrix were quantitatively examined by SMFS.14 We found that the inhomogeneity of the fluorescence emission and intersystem crossing (ISC) processes of G1 were constrained by conformational restriction and reduced heterogeneity in the surrounding environment. In a comparison of the photobleaching yields (Φb) of G1@Cap and free-G1, the encapsulated G1 was found to be nearly 10 times more photostable than its free counterpart. Furthermore, the G1@Cap was >30-fold more Received: August 29, 2016 Revised: October 7, 2016

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DOI: 10.1021/acs.jpca.6b08734 J. Phys. Chem. A XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry A

Figure 1. (a) Chemical structures of two 2,6-diacetoxy-9,10-bis(arylethynyl)anthracene derivatives, G1 and G2. (b) Encapsulation of G2 guest by two boronic ester cavitand capsules (Cap) self-assembled by I and II. The alkyl chains of Cap are replaced by methyl groups.

2. EXPRIMENTAL SECTION

photostable than rhodamine 6G (R6G), a widely used fluorescent dye in single-molecule studies. The present study reports an SMFS study of the π-extended guest G2 bearing two 2,6-diacetoxy-9,10-bis(arylethynyl)anthracene units (Figure 1a). G2 has a larger molar absorption coefficient (εmax = 104 100 M−1 cm−1 at 514 nm in toluene) than G1 (εmax = 45 800 M−1cm−1 at 494 nm in toluene), and a high fluorescence quantum yield (Φf = 0.92 in toluene).13 Intriguingly, encapsulation further enhances the molar absorption coefficient and photostability of G2 (εmax = 116 600 M−1cm−1 at 539 nm for G2@(Cap)2 in toluene), without altering the fluorescence quantum yield (Φf = 0.92 in toluene).13 Thus, the G2 guest should be approximately twice as bright as the G1 guest. Moreover, the encapsulation complex of G2 has an excellent two-photon absorption property.13 Stimulated by these findings, we here investigate the effect of supramolecular encapsulation on the photophysics and photostability of G2. To this end, we conduct an SMFS study of G2@(Cap)2, and compare the fluorescence spectral properties of G2@(Cap)2 and free-G2 in a rigid polymer environment. The different properties are then interpreted by theoretical calculations. The results confirm the effectiveness of G2@(Cap)2 as a long-lasting fluorescent probe that is sufficiently bright for detection at the single-molecule level.

2.1. Sample Preparation. The nonpolar glassy polymer Zeonex (Tg = 123 °C, Zeon Chemicals), methylcyclohexane (MCH), and toluene (spectroscopic grade, Wako) were used as received. The dielectric constant of MCH (2.02) is similar to that of Zeonex (2.3). The ensemble absorption spectra of G2 and G2@(Cap)2 in MCH were recorded on a spectrometer (Lambda 650, Perkin−Elmer), and the fluorescence spectra were obtained by a RF-5300PC fluorometer (Shimadzu) equipped with a liquid-nitrogen-cooled charge-coupled device (CCD) camera coupled to a polychromator. G2 and Cap were synthesized as described elsewhere.13 Although the association constant (Ka) of G2@(Cap)2 is not reported in the literature, the Ka of G1@Cap was extremely large (2.12 × 107 M−1 in C6D6 at 313 K),13 so the Ka of G2@(Cap)2 should be comparably large. In fact, G2 (approximately 10−10 M) was almost completely encapsulated at low concentrations of Cap (10−100 μM) in toluene. Samples for the SMFS experiments were typically prepared by spin-coating one drop of G2 or G2@(Cap)2 (approximately 10−10 M) and Zeonex (10 mg/ mL) in toluene onto thoroughly cleaned cover glasses. The thickness of the doped polymer films, measured by atomic force microscopy (SPM-9700, Shimadzu), was 200 nm. B


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parameter in similar π-conjugated systems.20,21 If not otherwise mentioned, we thus optimized ground-state geometries at the HF/6-31G(d) level with no symmetry constraints. We also calculated the harmonic vibrational frequencies of the optimized geometries to ensure that they represented the true potential minima. To predict the vertical transition energies from the optimized ground-state geometry to the excited singlet and triplet states, we calculated the excited-state electronic structures, hybrid B3LYP functional and 6-31G(d) basis set, i.e., the TD-B3LYP/6-31G(d)//HF/6-31G(d) levels of theory. This theoretical approach reproduced well the experimental electronic excitation energies of similar πconjugated molecules in previous studies.21,22 All calculations were performed using the GAUSSIAN 09 package.23

2.2. Single-Molecule Fluorescence Spectroscopy. Our homemade SMFS apparatus is thoroughly described elsewhere.19 Briefly, the dye molecules were excited by a continuous 488 nm argon ion laser (177G, Spectra Physics). After elimination of the residual plasma lines by laser-line filters, the excitation light was spatially filtered, collimated, and passed through a Glan−Thompson polarizer. The expanded and linearly polarized laser beam was then reflected by a dichroic mirror (LPD01-488RU-25, Semrock) and guided into an oil immersion objective (100× , NA 1.4, Olympus), where it was focused to a diffraction-limited spot (approximately 210 nm fwhm) in the sample plane. The average excitation intensities were 100−300 W/cm2, corresponding to an excitation rate (kex) of approximately 105 s−1 for G2 and G2@(Cap)2. The sample substrates were mounted onto an O-ring, forming the top face of a small vacuum chamber. During the roomtemperature SMFS measurements, the sample side was placed in a high vacuum ( 10−3 is much higher in G2 than in G2@(Cap)2. Although the distribution widths of τT are almost identical in G2 and G2@(Cap)2 (Figure 4b), the average τT, denoted by ⟨τT⟩, increases from 1.12 ms in G2 to 1.44 ms in G2@(Cap)2. Contrarily, supramolecular encapsulation effected no change in the ⟨τT⟩ of G1 (see Table 1), suggesting very weak electronic coupling between the BPEA moiety and Cap.14 Hence, we suspect that, in G2, the encapsulation effects on ΦISC and τT originate from a geometric factor of the G2 guest, as discussed later. The enhanced photostability of encapsulated G2 (and also G1) has been qualitatively confirmed in ensemble measurements.13 Here, we quantify this enhancement at the singlemolecule level. In single-molecule measurements under a lowpowered excitation laser (approximately 200 W/cm2), the average survival lifetime ⟨tsur⟩ of G2@(Cap)2 was 78.3 s,

Figure 3. Time traces of the fluorescence intensity (a, b) and fluorescence intensity autocorrelation curves (c, d) for free-G2 and (b) single G2@(Cap)2 molecules embedded in a Zeonex film.

Zeonex, respectively. Note that the insets already shown in Figure 2c,d are the corresponding fluorescence spectra, recorded simultaneously with the time traces. Both traces exhibit fluorescence blinking, which is unresolved at the bintime of 10 ms, and one-step irreversible photobleaching. Previously, fluorescence blinking of 9,10-bis(phenylethynyl)anthracene and G1 in Zeonex was attributed to triplet blinking originating from ISC toward and away from the triplet state (T1).14,19 Thus, we also assign the observed blinking of G2 and G2@(Cap)2 to the ISC process. To verify this assignment, we analyzed the fluorescence intensity autocorrelation function (ACF) curve (i.e., the second-order correlation function g(2)(t)) obtained, assuming the three-state model of S0, S1, and T1. The D


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Figure 4. Histograms of (a) ISC quantum yields and (b) triplet lifetimes obtained from 70 G2 and (b) 112 G2@(Cap)2 molecules in a Zeonex film.

Figure 5. Histograms of photobleaching yields of (a) G2 (225 molecules) and (b) G2@(Cap)2 (200 molecules) in a Zeonex film.

approximately 4.6 times longer than that of bare G2 (⟨tsur⟩ = 17.1 s). However, the survival time strongly depends on the excitation laser power, and does not account for the number of excitation−de-excitation cycles. To quantify the effect of encapsulation on the photostability of a single G2 molecule, we evaluated the photobleaching quantum yield Φb, defined as Φb = ξΦf/N.30−32 In this expression, ξ represents the overall detection efficiency of the current microscope setup (approximately 4%), Φf is the fluorescence quantum yield, and N is the total number of detected photons emitted by a single molecule before photobleaching. The G2 and G2@(Cap)2 molecules in toluene yielded the same Φf (0.92);13 this value was assumed in the estimation of Φb. The Φb histograms of G2 and G2@(Cap)2 under 200 W/cm2 excitation power are shown in panels a and b of Figure 5, respectively. Assuming a Poisson distribution of the photobleaching events over time, the probability density function of a molecule with quantum photobleaching yield Φb is given by P(Φb) = C exp(−Φ0/Φb)/ Φb2, where Φ0 is the photostability of a single molecule, and C is a normalization constant.32 This function was fitted to the histograms in Figure 5. From the fitting, we obtained Φ0 = 8.7 × 10−8 and 2.8 × 10−8 for G2 and G2@(Cap)2, respectively, revealing a 3-fold increase in the photostability of the encapsulated G2. These results are summarized in Table 1. Under the same experimental conditions, the Φ0 of R6G, a commonly used fluorescent dye in ensemble and singlemolecule spectroscopy studies,6,33,34 was determined as ≥7.9 × 10−7.14 Therefore, G2@(Cap)2 is at least 28-fold more stable than R6G.

form in Zeonex. However, this conclusion is negated by the pronounced encapsulation effects on the photophysics and photostability of G2, which strongly indicate that the G2@(Cap)2 complexes maintain their encapsulated form in the polymer matrix. This raises the following question: Why is the fluorescence emission wavelength of G2@(Cap)2 so widely different in liquid and solid environments? The answer was sought for in theoretical calculations. Figure 6 shows the two optimized geometries of G2 obtained at the HF/6-31G(d) level of theory; one is a planar structure, and the other is a twist structure. The energies of these conformers differ by less than 0.4 kcal/mol, with or without zero-point vibrational energy (ZPVE) corrections. Therefore, the planar and twist conformation are nearly isoenergetic. Note that comparable energy differences (approximately 0.2 kcal/mol) were also obtained at the HF/6-31G(d,p) level. The computational results are summarized in Table 2 (see also Supporting Information). Unfortunately, the CPU cost was too expensive to perform the HF/6-31G(d) level of calculation on G2@(Cap)2. Thus, we conducted the HF/6-31G(d) calculation on the G1@Cap complex instead of G2@(Cap)2. Remarkably, the conformations of the twist conformer of G2 resemble those of the acetoxy groups of G1@Cap, in which the acetoxy groups are largely twisted relative to the π-framework. Since the two Cap moieties in G2@(Cap)2 are well-separated, the steric hindrance between the two Cap moieties may not add too much to the energy difference between planar and twist conformations (∼0.4 kcal/mol). As can be identified at the twist conformation in Figure 6, however, the twist of acetoxy groups occurs concertedly with the twist between two 9,10-bis(arylethynyl)anthracene units. This suggests that a planar conformation having the twisted acetoxy groups is more unstable than the twist conformation of G2@(Cap)2. Accordingly, we postulate that the twist conformation is the most stable structure of G2@(Cap)2.

4. DISCUSSION 4.1. Encapsulation Effects on Photophysics. As seen in Figure 2d, the liquid and rigid polymer environments altered the ⟨λem max⟩ values of the encapsulation complex, but not those of free-G2 (Figure 2c). Moreover, the λem max distributions of free-G2 and G2@(Cap)2 were quite similar. At a glance, these results suggest that the G2@(Cap)2 complexes lose their encapsulated E


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Figure 6. Optimized structures (i.e., “planar” and “twist” conformations) of G2 obtained at the HF/6-31G(d) level. For comparison, an optimized structure of G1@Cap obtained at the same level of theory is also shown. The conformations of the acetoxy groups in the twist conformation of G2 and G1@Cap are remarkably similar.

arylethynyl groups) also occurs in the rigid polymer matrix. Such conformational relaxation has been frequently observed in dye molecules embedded in glassy polymers.14,19,35−38 In particular, it has been found that the amorphous Zeonex polymer contains sufficiently microscopic free spaces that facilitate the structural relaxation of 9,10-bis(phenylethynyl)anthracene.19 In contrast, most of the G2@(Cap)2 complexes in S0 would exist in the conformationally stable twisted form and undergo vertical excitations (central panel of Figure 7). In the liquid phase, this twist conformation can relax to the planar one in S1. Such conformational change in S1 is supported by the comparable Stokes shift between free-G2 and G2@(Cap)2 in MCH solution (i.e., 26 nm for both G2 and G2@(Cap)2). However, in the rigid polymer environment, this conformational relaxation should be highly restricted by the bulky Cap structures, which present large activation barriers to rotations of the arylethynyl groups (Figure 7, right panel). The suppressed conformational rearrangement of G2@(Cap)2 in Zeonex is responsible for the average emission wavelength of ⟨λem max⟩ = 542 nm, which is only slightly red-shifted (+8 nm) from the absorption maximum of G2@(Cap)2 in MCH (i.e., λabs max = 534 nm). The λabs max value of G2@(Cap)2 in Zeonex is expected to be comparable or slightly larger than that in MCH, because of their comparable polarity and the rigidity of the Zeonex matrix. Indeed, such a red-shift phenomenon has been observed for 9,10-bis(phenylethynyl)anthracene,19 and thus, the actual Stokes shift is likely less than 8 nm. In either case, a very

Table 2. Calculated Total Energies, Vertical Transition Energies from S0 to S1 and from S0 to T1, and Energy Gaps between S1 and T1 States, Obtained at the HF/6-31G(d) and TD-B3LYP/6-31G(d)//HF/6-31G(d) Levels G2

ΔEea kcal/mol

ΔE0a kcal/mol

planar twist

0 +0.17

+0.37 0

E(S1) eV E(T1) eV ΔE(S1 − T1)b eV 2.33 2.41

1.54 1.57

0.79 0.84

a

Total energy. The subscripts 0 and e denote ZPVE corrections and no corrections, respectively. bΔE(S1 − T1) = E(S1) − E (T1).

On the basis of the computational results, we proposed photoabsorption and fluorescence emission schema for G2 and G2@(Cap)2 in liquid and rigid polymer environments. The schema are presented in Figure 7. The arylethynyl groups of free-G2 can rotate almost freely in liquid. Consequently, the photoabsorption process of G2 in liquid is contributed by various twisted confirmations. However, the vertically excited multiple conformations rapidly relax to the most stable planar conformation in S1, which is chiefly responsible for the subsequent fluorescence emission process.14,19 In the polymer, G2 molecules can exist in both planar and twist conformations, because the calculated energy stability was comparable in both conformations. Given the comparable ⟨λem max⟩ of free-G2 in liquid and polymer environments, we infer that excited-state structural planarization (i.e., rotations or twists of the

Figure 7. Proposed photoabsorption and fluorescence emission schema in free and encapsulated G2 in liquid and rigid polymer environments. F


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may be affected by the fluorescence-emitting conformation, which differs between G2 and G2@(Cap)2 in the polymer. However, we lack quantitative information on this effect. 4.2. Encapsulation Effects on Photostability. The increased photostability of the encapsulated G2 is attributed to the cavitand capsule, which shields the anthracene framework from reactive species, most likely from singlet oxygen O2(1Δg). As the lowest triplet energy of G2 (1.5−1.6 eV) is much higher than the energy of O2(1Δg) (0.97 eV), single oxygen molecules can be produced by quenching of the long-lived triplet state of G2 (approximately 1 ms). Ultimately, all of the single G2@(Cap)2 molecules examined in this work became photobleached. Remarkably, although free-G2 is twice as photostable as free-G1, the photostabilities of G2@(Cap)2 and G1@Cap are almost the same (Φ0 = 2.8 × 10−8 versus Φ0 = 2.3 × 10−8) (see Table 1). Therefore, we suspect that the high photostability of the encapsulation complexes is conferred by the highly stable Cap host molecule. As the Cap molecule does not absorb the excitation laser light (488 nm), it does not decompose at this wavelength. During the SMFS measurements, every excitation−de-excitation cycle of the guest molecule (approximately 105 s−1) is accompanied by thermal dissipation of the excess energies, which probably dissociates the boronic ester bonds in Cap (either partially or completely). These bonds are dynamic covalent bonds, meaning that they reversibly form and dissociate under thermodynamic control.9−13 When the host cavity collapses, the G2 guest can react with singlet oxygen, forming a 9,10-endoperoxide in the anthracene moiety. To further improve the photostability of the encapsulation complex, we must therefore suppress the dissociation of the boronic ester bonds in Cap. However, given the inherently dynamic nature of these bonds, strengthening them is a difficult task. As another strategy, we could suppress the triplet blinking of the encapsulated dye, because formation of the excited triplet state is a major source of photobleaching of the guest dyes. This might be achieved by attaching an effective triplet quencher, such as cyclooctatetraene, to the guest or Cap moiety.40,41

small Stokes shift supports the restriction of conformational relaxation of G2@(Cap)2 in Zeonex. Consequently, the individual G2@(Cap)2 complexes fluoresce in the higherenergy (i.e., metastable) twist conformation in S1. The fluorescence spectra of single G2@(Cap)2 complexes in Zeonex exhibit a sharp, vibrationally resolved feature and smaller λem max values (reflecting the larger S1 → S0 transition energies) than the fluorescence spectra of G2@(Cap)2 in MCH solution (see Figure 2d, inset). As the range of λem max values in free-G2 is maintained in the encapsulated G2 (Table 1), we infer that the individual G2@(Cap)2 complexes in Zeonex adopt various degrees of twist conformations. Such a phenomenon was not observed in G1. As mentioned before, the fluorescence spectrum of G2@(Cap)2 in MCH is broader than that of G2 in MCH (see Figure 2a,b). Then, we tentatively performed the geometry optimization of G2@(Cap)2 at the HF/6-31G level with no harmonic vibrational analysis, suggesting that the π-conjugated framework of G2 is slightly bent upon encapsulation. Thus, the planarization of G2@(Cap)2 in S1 may be also accompanied by not only “twist-to-planar” structural rearrangement but also “bent-to-planar” rearrangement. These structural relaxation effects should be reflected in both the absorption and fluorescence spectra. However, it is clearly discernible in only the fluorescence spectrum (Figure 2b). A possible explanation for this result is that the broadening effect on an absorption spectrum by the bent-to-planar conformational rearrangement in G2@(Cap)2 may be partly counteracted by the spectral sharpening effect from the highly restricted rotation of the arylethynyl groups in the encapsulation complex.13,14 In fact, the sharpening of the absorption spectrum upon encapsulation is much less prominent in G2, compared to G1.14 As depicted in Figure 4, encapsulation reduced the ΦISC and increased the τT of G2. We presume that the S1 → T1 and T1 → S0 ISC processes occur in the planar confirmation of free-G2 in Zeonex, and in the twist conformations in the G2@(Cap)2/ Zeonex system (see Figure 7). We suspect that this conformational difference largely accounts for the observed encapsulation effect. We then calculated the vertical transition energies from the planar or twist ground-state geometry to the excited singlet and triplet states. The results are listed in Table 2. Both S0 → T1 vertical transition energies of free-G2, i.e., E(T1) and the S1−T1 energy gap ΔE(S1−T1), were slightly larger in the twist conformation than the planar conformation. As the π-conjugated framework of G2 has a D2h-like symmetry, no direct spin−orbit coupling (SOC) is expected between S1 and T1 (also S0 and T1).19 Instead, SOC in G2 should occur by Herzberg−Teller vibronic coupling. In this scenario, when the density-of-states of the vibronic levels in the T1 (or S0) state that are isoenergetic with S1 (or T1) lies within the statistical limit, the ISC rate constants reduce, as the energy gap between the singlet and triplet states increases.39 Hence, the computational results qualitatively suggest smaller T1 → S0 and S1 → T1 ISC rate constants (i.e., longer triplet lifetimes and smaller ISC yields) in the twist than in the planar conformation. This suggestion is compatible with the observed encapsulation effects on the ISC parameters. Given the reported fluorescence quantum yields (0.92 for both G2 and G2@(Cap)2 in toluene13), the quantum yield of nonradiative pathways in both G2 and G2@(Cap)2 should be 0.08, 1 or 2 orders of magnitude larger than the ΦISC values obtained herein. Thus, the main nonradiative pathway in G2 and G2@(Cap)2 should be the internal conversion from S1 to S0. Hence, the IC process

5. CONCLUSIONS We revealed the effects of supramolecular encapsulation on the photophysics and photostability of highly fluorescent 2,6diacetoxy-9,10-bis(arylethynyl)anthracene dimeric guest, G2. In both liquid and polymer matrix, free-G2 fluoresces in the planar conformation. In contrast, within the rigid confines of the polymer environment, the excited-state planarization of G2@(Cap)2 is prevented by the bulky Cap host. Consequently, G2@(Cap)2 in polymer is forced to emit in the twist conformation. Such a phenomenon was absent in the monomeric guest G1, because the torsional degree of freedom between two 2,6-diacetoxy-9,10-bis(arylethynyl)anthracene monomeric units is absent in G1. Despite the different photostabilities of the free guests, the photostabilities of their encapsulation complexes were remarkably similar, suggesting that the thermal stability of the boronic ester bond dissociations in the host (rather than the photostability of the guest) dominates the photostability mechanism. The excellent photostability and brightness of the encapsulated 9,10-bis(arylethynyl)anthracene derivatives within the self-assembled boronic ester cavitand capsule could be exploited in singlemolecule fluorescence-based spectroscopy and assay, advancing the applicability of these complexes to fluorescence-based single-molecule detection. G


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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.6b08734. Additional data, including results of theoretical calculations for G2 obtained at HF/6-31G(d), HF/6-31G(d,p), and TD-B3LYP/6-31G(d)//HF/6-31G(d) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +81-3-3985-2364. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.M. is grateful to Mr. Takeo Saito from Shimadzu Corporation who is acknowledged for experimental work with AFM. The computations were performed using the Research Center for Computational Science, Okazaki, Japan. This work is supported by Grants-in-Aid for Scientific Research (C), nos. 24550018 and 15K05398.

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