Lateral Diffusion Dynamics for Single Molecules of Fluorescent

Fumi Hashimoto, Satoshi Tsukahara, and Hitoshi Watarai*. Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka,...
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Langmuir 2003, 19, 4197-4204

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Lateral Diffusion Dynamics for Single Molecules of Fluorescent Cyanine Dye at the Free and Surfactant-Modified Dodecane-Water Interface Fumi Hashimoto, Satoshi Tsukahara, and Hitoshi Watarai* Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan Received October 2, 2002. In Final Form: February 3, 2003 The present study proposed a single molecule probing of transport properties of the nanoregion of liquid-liquid interfaces. Fluorescence from single cyanine dye molecules (DiI) adsorbed at a dodecanewater interface was detected in the absence and presence of surfactants by total internal reflection fluorescence microscopy with a single photon counting devise. Intermittent photon bundles from single DiI molecules were observed in time-resolved photon counting measurements, when the average number of interfacial DiI molecules was less than 1 in the observation area (830 nm in diameter). Photon signals emitted by the same DiI molecule in the observation area were discriminated with the time interval between two photon signals. From the analyses of the photon bundles, the following properties of the interfacial region were obtained: (1) the lateral diffusion coefficient of single DiI molecules from the maximum duration of the photon bundle, (2) the interfacial viscosity from the diffusion coefficient of the single DiI molecules, and (3) the fluorescence quantum yield of single DiI molecules from the density of the photon bundles. The adsorption of anionic or zwitterionic surfactant at the interface reduced the lateral diffusion coefficient of single DiI molecules by an increase in the interfacial viscosity.

Introduction Detection of single molecules is the only ultimate method that enables us to observe the behavior of individual molecules. Some techniques for the detection of single molecules have been developed, most of them utilizing laser-induced fluorescence microscopy. The detection of single molecules in solutions1-4 and at solid-liquid interfaces5,6 has been already reported, but there are no studies on the detection of single molecules at the liquidliquid interface other than ours7 so far. The liquid-liquid interface has been recognized as specific reaction fields in the processes of solvent extraction of metal ions, liquid membrane sensor, and phase transfer catalysis.8-11 The liquid-liquid interface is also considered as a model of biological cell membrane. However, details of the structural properties of the liquid-liquid interface as well as mechanisms of interfacial reactions have not been elucidated at the single molecular level yet. In the laser-induced fluorescence measurement of single molecules, fluorescence signals have to be discriminated from background signals including scattered light. Since fluorescence emitted by single molecules is extremely * To whom correspondence should be addressed. E-mail: [email protected]. (1) Nie, S.; Chiu, D. T.; Zare, R. N. Anal. Chem. 1995, 67, 28492857. (2) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995, 374, 555-559. (3) Tokunaga, M.; Kitamura, K.; Saito, K.; Iwane, A. H.; Yanagida, T. Biochem. Biophys. Res. Commun. 1997, 235, 47-53. (4) Xu, X.-H.; Yeung, E. S. Science 1997, 275, 1106-1109. (5) Wirth, M. J.; Swinton, D. J. Anal. Chem. 1998, 70, 5264-5271. (6) Xu, X.-H. N.; Yeung, E. S. Science 1998, 281, 1650-1653. (7) Hashimoto, F.; Tsukahara, S.; Watarai, H. Anal. Sci. 2001, 17 (Supplement), i81-i83. (8) Watarai, H. Trends Anal. Chem. 1993, 12, 313-318 and references therein. (9) Onoe, Y.; Tsukahara, S.; Watarai, H. Bull. Chem. Soc. Jpn. 1998, 71, 603-608. (10) Volkov, A. G.; Deamer, D. W. Liquid-Liquid Interfaces; CRC Press: Boca Raton, FL, 1996, and references therein. (11) Watarai, H.; Saitoh, Y. Chem. Lett. 1995, 283-284.

weak, the reduction of background is particularly essential. For this purpose, the total internal reflection fluorescence (TIRF) technique2 was adopted in the present study. The dodecane-water interface, where target fluorescent molecules were adsorbed, was prepared in a thin and flat glass cell. When a laser beam was irradiated from the dodecane phase to the interface with an incident angle larger than the critical angle, the beam was totally reflected at the interface and only the evanescent region at the interface was excited. This method enabled us to detect quite weak fluorescence with an extremely low background. As a target fluorescent molecule, 1,1′-dioctadecyl3,3,3′,3′-tetramethylindocarbocyanine (DiI) was employed as shown in Figure 1. It is a monovalent cation possessing two long alkyl chains, and it was expected to be adsorbed substantially at the dodecane-water interface. Indocarbocyanines (cy3) were frequently used as a probe of single molecule studies, dynamics of proteins labeled with cy33,12,13 and adsorption behavior of DiI molecules on a glass surface.5 The purpose of the present study is to develop the method for the detection of single DiI molecules adsorbed at the dodecane-water interface by means of laser-induced fluorescence microscopy for the first time. Analysis of the photon signals of the probe molecules allows us to estimate the lateral diffusion coefficient and the fluorescence quantum yield of the single molecules at the interface. Furthermore, the influence of two kinds of surfactants, that is, sodium dodecyl sulfate (SDS) and dimyristoyl phosphatidylcholine (DMPC), on the lateral diffusion dynamics of single DiI molecules at the interface is investigated. SDS is chosen as a common anionic surfactant and DMPC as a representative phospholipid constituent of bilayers of biological cells. This study (12) Ishijima, A.; Kojima, H.; Funatsu, T.; Tokunaga, M.; Higuchi, H.; Yanagida, T. Cell 1998, 92, 161-171. (13) Ishii, Y.; Yanagida, T. Single Mol. 2000, 1, 5-16.

10.1021/la026644x CCC: $25.00 © 2003 American Chemical Society Published on Web 04/08/2003

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Figure 1. Chemical structure of DiI, which has two long alkyl chains and one positive charge.

demonstrates that the single molecule probing of the interface is a powerful and new means to evaluate the nanoproperties of the interfacial region. Experimental Section Molecular Dynamics Simulation. A molecular dynamics (MD) simulation of DiI in the dodecane-water system was carried out on a workstation (O2, Silicon Graphics) using a DREIDING 2.21 force field by Cerius2 software (MSI, San Diego, CA) as reported previously.14-16 Partial charges of the atoms in all the molecules were distributed by the Gasteiger method. The time step of the calculation was 10 fs, and the total time for the simulation was 100 ps at 300 K in the NPT ensemble. The size of the unit cell of the two-phase system was 24.7 Å × 53.6 Å × 24.5 Å, which contained 1 DiI molecule, 40 dodecane molecules, and 500 water molecules. Prior to the construction of the twophase model, each phase was equilibrated by 100 ps dynamics. Chemicals. The chemicals used in the present study were obtained from commercial sources: DiI perchlorate from Molecular Probes; dodecane (GR), glycerin (GR), and SDS from Nacalai Tesque (Kyoto); DMPC from Funakoshi Co., Ltd. (Tokyo). Water was distilled and purified with a Milli-Q purification system (Milli-Q SP. TOC., Millipore). Solid DiI‚ClO4 was dissolved in dodecane, and the solution was diluted at a concentration of 3 × 10-10 to 1 × 10-6 M. Measurement of TIRF Spectra. Since the reflective indices of dodecane and water are 1.43 and 1.33 at about 500 nm, respectively,17 the critical angle (θc) for the dodecane-water interface is calculated as 69° at this wavelength. When the incident angle of a beam of light from the dodecane phase to the interface is larger than θc, the beam is totally reflected at the interface. TIRF spectra of DiI at the interface were measured using a conventional optical quartz cell (1 cm × 1 cm) with a couple of quartz rectangular prisms (1 cm × 1 cm × 1 cm) attached on the outer walls of the cell as reported previously.18 An aliquot (1.17 mL) of water was introduced in the cell, and then an aliquot (1.00 mL) of a dodecane solution of DiI was quietly added on the water to form an interface (1.0 cm2 in area). An excitation beam propagated horizontally from a spectrofluorometer (650-40, Hitachi) was refracted downward by one prism, passed through the dodecane phase, and irradiated to the interface at an incident (14) Watarai, H.; Gotoh, M.; Gotoh, N. Bull. Chem. Soc. Jpn. 1997, 70, 957-964. (15) Watarai, H.; Onoe, Y. Solvent Extr. Ion Exch. 2001, 19, 155166. (16) Onoe, Y.; Watarai, H. Anal. Sci. 1998, 14, 237-239. (17) Wohlfarth, Ch.; Wohlfarth, B. Refractive Indices of Organic Liquids; Lechner, M. D., Ed.; Springer: Berlin, 1996. (18) Watarai, H.; Funaki, F. Langmuir 1996, 12, 6717-6720.

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Figure 2. Schematic illustration of laser-induced fluorescence microscope under the total internal reflection for the detection of single DiI molecules at the dodecane-water interface: ND, ND filter; λ/2, λ/2 plate for 532 nm; M, mirror; L, lens; C, microcell containing dodecane and aqueous phases; O, objective (60×); F, bandpath filter; P, pinhole; APD, avalanche photodiode detector. angle of about 73°. Light emitted from the interface and the dodecane phase was refracted by the other prism and introduced horizontally to the detector of the spectrofluorometer. After the two phases were allowed to stand for a while, steady-state TIRF excitation and emission spectra were measured. Sample Preparation for Single Molecule Measurement. Since the employed objective had a short working distance (0.21 mm), it was essential to make a specific optical cell possessing a thin container for the aqueous phase. A two-phase microcell was fabricated by stacking a bored (10 mm in diameter) slideglass, a bored (5 mm in diameter) coverslip (0.14 mm in thickness), and another nonbored coverslip in this order.7 Pure water (2.7 µL) was filled in the lower container (0.14 mm in thickness) of the cell in the surfactant-free system. In the SDS system, an aqueous solution of SDS at 5.0 × 10-4 or 5.0 × 10-3 M was filled. In both cases, a dodecane solution of DiI (63 µL) was added on the aqueous layer. A new coverslip was put on the dodecane phase to close the cell, followed by standing for at least 4 h for adsorption equilibrium. DMPC was not dissolved in dodecane or in water, but it was easily dissolved in chloroform. Therefore, DMPC was first dissolved in chloroform at a concentration of 3.5 × 10-5 to 2.1 × 10-4 M, and the solution was mixed with pure diethyl ether at a ratio of 1:19 (chloroform:diethyl ether) by volume to reduce the density. Pure water was filled in the lower container, and then the DMPC solution (5 µL) was quietly spread on the water. After 5 min for evaporation of chloroform and diethyl ether, a dodecane solution of DiI was added on the DMPC layer. Finally, a new coverslip was put on, followed by standing for at least 4 h. Fluorescence Microscope System. The system of the total internal reflection fluorescence microscope for the single molecule detection is shown in Figure 2. The apparatus consisted of an inverted microscope (TE300, Nikon), an oil immersion objective (CFI PlanApo 60×H, NA (numerical aperture) 1.4, working distance 0.21 mm, Nikon), a continuous wave Nd:YAG laser (532 nm, TEM00, 50 mW, 0.3 mm in beam radius, model 4301-050; Uniphase), and an avalanche photodiode detector (APD) (SPCMAQR-16, Perkin-Elmer Optoelectronics), which provided a quantum efficiency of about 65-67% at 570-600 nm with a dead time of 43.2 ns. A pinhole of 50 µm in diameter was attached just in front of the photodiode, which was positioned at the image plane of specimen. The pinhole restricted the observation area to 830 nm in diameter (dobs), which was calculated from the division of the pinhole diameter by magnification of the objective (60). After a laser beam passed through ND (neutral density) filters and a λ/2 plate, it was focused by a lens (focal length, 40 mm) to the interface through a quartz rectangular prism (2 cm × 2 cm × 2 cm) on the cell. The gap between the prism and the upper coverslip was filled with glycerin. The shape of the laser spot at the interface was an ellipse of about 30 µm × 100 µm in size,

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which meant that all of the DiI molecules in the observation area were irradiated by the laser light. The incident angle of the beam was 73° at the interface. Fluorescence emitted by interfacial DiI molecules was collected by the objective, and it was focused on the pinhole after passing through band-pass filters. Time-resolved photon counting was carried out with a multichannel scalar (MCS-plus, EG&G Ortec). The minimum dwell time (td) per one channel was 2 µs, and the maximum number of channels was 8192. In almost all the measurements, td was set to 2 µs or 16 ms, which corresponded to continuous measurements for 16 ms (≈2 µs × 8192) or 130 s (≈16 ms × 8192), respectively. All of the experiments were carried out in a thermostated room at 25 ( 1 °C. The spectrum of the background signal, measured with a streakscope, had peaks at 570 and 630 nm without any optical filters, which were assigned to the Raman scattering of dodecane. For the elimination of the Raman lines, two kinds of optical filters were used: a dichloric mirror (565DM, Nikon) and a band-pass filter (570DF30, path range 555-585 nm; average transmittance, 65%; OD g 4 at 532 nm; Omega Optical), or a band-pass filter (600DF25, path range, 587.5-612.5 nm; average transmittance, 65%; OD g 4 at 532 nm; Omega Optical). The Raman peaks disappeared by using the optical filters. However, the measurements with the APD showed that the background signal with p-polarized excitation light was lower than that with s-polarized one due to the Raman selectivity.19 The following experiments were carried out with p-polarized one. The overall detection efficiency of fluorescence (Qdet) at the interface was calculated20 as 3.3% with the fluorescence collection efficiency of the objective that was obtained from the NA value, the overlap of the optical filters and the emission spectrum of DiI, the transmittances of the objective and microscope, and the quantum efficiency of the APD.

Theoretical Section The average period that it takes one DiI molecule to emit one photon (τd) should be calculated. The average repetition for the excitation of a molecule in a unit of time (kex, in s-1) is expressed as

kex )

103 ln 10  P NA hν S

(1)

where  is the molar absorption coefficient (in M-1 cm-1), P is the power of light (in W), NA is the Avogadro constant, h is the Planck constant, ν is the frequency of incident light, and S is the irradiation area at the interface (in cm2). The 103 is a factor to convert dm3 to cm3. The  value of DiI in dodecane is 7 × 104 M-1 cm-1 at 532 nm. A period for n times excitation is n/kex. A DiI molecule in the excited state was deactivated through a radiative path, a nonradiative path, or an intersystem crossing (triplet state) path. The probability for each path corresponds to its quantum yield (φr, φnr, and φisc), and thus the radiative, nonradiative and intersystem crossing paths occur nφr, nφnr, and nφisc times for n times excitation, respectively (nφr + nφnr + nφisc ) n). The time constants for the radiative and nonradiative paths are 1/kr and 1/knr, respectively, where kr and knr are the intrinsic rate constants (in s-1) for the respective paths. The overall time constant for the path through the triplet state is (1/kisc + 1/kT), where kisc and kT are the rate constants for the intersystem crossing and the deactivation of triplet DiI, respectively. Therefore, a period for the n times excitation and deactivation (τn) is expressed as (19) McCreery, R. L. Raman Spectroscopy for Chemical Analysis; John Wiley & Sons.: New York, 2000. (20) Ishikawa, M.; Hirano, K.; Hayakawa, T.; Hosoi, S.; Brenner, S. Jpn. J. Appl. Phys. 1994, 33, 1571-1576.

τn ) n/kex + nφr/kr + nφnr/knr + nφisc(1/kisc + 1/kT) (2) During this period, the molecule emits nφr photons on average. Thus, an average period for one photon emission (τd) can be written as

τd )

τn 1/kex + φr/kr + φnr/knr + φisc(1/kisc + 1/kT) ) nφr φr (3)

The (kr + knr + kisc) and φr values were reported as 4.0 × 108 s-1 and 0.15, respectively,21 whereas the kisc value was independently obtained as 1.3 × 105 s-1.22 Thus, kr and knr were evaluated as 6.0 × 107 s-1 and 3.4 × 108 s-1, respectively. φisc was equalized to kisc/(kr + knr + kisc). In general, an organic compound in the triplet state (T1) is deactivated by the energy transfer to dissolved oxygen molecule O2(3Σg) to produce itself in the ground state (S0) as23,24

T1 + O2(3Σg) f S0 + O2(1∆g)

(4)

kT ≈ kq[O2],23 where kq was the rate constant for the bimolecular reaction of eq 4. The estimation of the kT value led to (1/kisc + 1/kT) ≈ 1/kisc in the last term in eqs 2 and 3.24,25 Results and Discussion MD Simulation of DiI at the Dodecane-Water Interface. Figure 3 shows a snapshot of the MD simulation after 100 ps, which represents one DiI molecule adsorbed at the dodecane-water interface. It was indicated that the DiI molecule was not released into the dodecane or aqueous phases and that it existed only at the dodecane-water interface. Two alkyl chains of DiI stretch upward to the dodecane phase, and two indoles lie at the interface. Adsorption of DiI on a Glass Surface. For the evaluation of the adsorption of DiI on the glass wall of the microcell, the adsorptivity of DiI on a glass surface from dodecane solution was measured. A coverslip (24 mm × 10 mm in size) was soaked in a dodecane solution of DiI at 3.0 × 10-10 to 7.0 × 10-7 M in a conventional quartz cell, to which the adsorption of DiI was negligible. After being allowed to reach equilibrium overnight, the coverslip was removed and the concentration of DiI remaining in the dodecane solution was measured. The adsorption equilibrium obeyed the Langmuir relationship as

Γ)

ΓsatKgC 1 + KgC

(5)

where Γ was the surface concentration of DiI, Γsat was the saturated surface concentration, Kg was the adsorption constant, and C was the DiI concentration in dodecane solution. The Γsat and Kg values were obtained as 1.9 × 10-10 mol/cm2 and 1.4 × 108 M-1, respectively. Adsorption of DiI at Dodecane-Water Interface. The TIRF spectra in the dodecane-water system were (21) Trautman, J. K.; Macklin, J. J. Chem. Phys. 1996, 205, 221229. (22) Yip, W. T.; Hu, D.; Yu, J.; Bout, D. A. V.; Barbara, P. F. J. Phys. Chem. A 1998, 102, 7564-7575. (23) Kikuchi, K. JOEM Handbook 1 Triplet-triplet Absorption Spectra; Bunshin: Tokyo, 1989. (24) Garner, A.; Wilkinson, F. Chem. Phys. Lett. 1977, 45, 432-435. (25) Silcock, H. L. Solubility of Inorganic and Organic Compounds; Pergamon Press: Oxford, 1979; Vol. 1.

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Figure 3. A partial snapshot of one DiI molecule adsorbed at the dodecane-water interface simulated by molecular dynamics after 100 ps using a DREIDING 2.21 force field with 40 dodecane molecules and 500 water molecules.

measured at the initial DiI concentrations of 2.9 × 10-9, 7.5 × 10-8, 2.6 × 10-7, and 4.9 × 10-7 M. In any case, the fluorescence intensity became constant after 150 min from the formation of the interface. About 20% of DiI remained in the dodecane phase at 4.9 × 10-7 M. However, no DiI existed in the dodecane phase at the other lower concentrations, indicating that all the DiI molecules were adsorbed at the interface. From these observations, the saturated concentration of DiI at the dodecane-water interface was estimated as 3.9 × 10-10 mol/cm2, which was almost equal to that on the glass surface. The peak of emission spectra was in the range of 571-575 nm. Single DiI Molecule Detection. The DiI concentration at the interface was approximately estimated as follows. The two-phase system was prepared in the microcell by changing the initial DiI concentration in a range of 1.0 × 10-8 to 1.1 × 10-6 M. The concentration of DiI remaining in the dodecane phase was calculated with the equilibrium constants for the adsorption on the glass wall. Almost all the DiI molecules in the dodecane phase were adsorbed on the inner glass surface, because the surface area is larger than that of dodecane-water interface. The remaining DiI concentration in the dodecane phase was much lower than that measured in the TIRF experiments, and thus the DiI was almost adsorbed at the interface. For example, when using a dodecane solution of DiI at 1.1 × 10-8 M, the interfacial DiI concentration was estimated to be 2.5 × 10-17 mol/cm2, which corresponded to 0.08 molecules in the observation area. For the confirmation of the estimation, the fluorescence from DiI at the dodecane-water interface was measured at the same time with the laser-induced microscope system. A linear relationship was obtained between the fluorescence intensity and the estimated interfacial concentration. When 11 DiI molecules existed in the observation area on average, photons were continuously observed as Figure 4a in comparison with the background (no DiI in Figure 4c). In the multimolecule system, more than one DiI molecule existed in the observation area in every time as Figure 4d. Figure 4b shows a result in the case that the average number of DiI molecules was less than 1 in the observation area. Different from the result of multimolecule system, intermittent photons were observed. Two

Hashimoto et al.

Figure 4. Examples of photon signals in (a) the multimolecule system, (b) single molecule system, and (c) background. (a) Continuous photons were observed in the 11 DiI molecule system, and (d) a schematic illustration of the situation. (b) Intermittent photon bundles were observed in the 0.02 DiI molecule system, and (e) a schematic illustration of the situation: s-polarized laser beam of 50 mW output power; filter, 565DM + 570DF30; dwell time, 2 µs. The irradiation angle at the interface was 66°, which was less than the critical angle (69°).

photon bundles in the figure strongly indicated that one single DiI molecule existed in the observation area. The situation in this case is illustrated in Figure 4e. In some studies on single molecules, photons emitted by a single molecule suddenly vanished due to its photobleaching, and this was a clear evidence of the single molecule detection. In the present study, DiI at the dodecane-water interface showed a photobleaching, whose rate became faster with increases in the interfacial DiI concentration and laser power. Under the quite low interfacial concentration of DiI with 5 or 10 mW laser power, the contribution of the photobleaching was negligibly small in the time range of about 1 s. The lifetime for photobleaching of DiI, bound to a position-fixed protein, was reported to be about 500 ms in an aqueous solution,2 which was consistent with our results. Photon Signals of the Same Single DiI Molecule. At a dwelling time of 2 µs for the multichannel scalar, only 0 or 1 photon was observed per one channel in almost all the cases. A criterion was necessary whether two photon signals with a time interval (∆t) were caused by one single DiI molecule or not. The τd values were calculated with eq 3 as 7.9 and 4.0 µs for 5 and 10 mW laser power, respectively. The total detection efficiency of the system (Qdet) was 3.3%. After one photon signal was observed, the possibility that no photon signals were observed during mτd was Pnd ) (1 - Qdet)m although the same single DiI molecule continuously emitted photons in the observation area. The Pnd corresponds to the possibility that a phenomenon having a possibility of (1 - Qdet) occurs m times continuously. The mτd agrees with ∆t. The Pnd value was set to 0.05, which corresponded to a 95% confidence level, and ∆t values resulted in 0.70 ms (m ) 88) and 0.35 ms (m ) 88) for 5 and 10 mW laser power, respectively. Νo photon signals during ∆t mean no DiI molecules in the observation area in this time. A continuous measurement for about 16 ms (2 µs × 8192 channels) was carried out 100 times in the surfactant-

Single Molecule Probing

Langmuir, Vol. 19, No. 10, 2003 4201 Table 1. Lateral Diffusion Coefficient (Dl) and Fluorescence Quantum Yield (Of) of Single DiI Molecules, and Apparent Viscosity (η j i) at Surfactant-Free and SDS-Modified Dodecane-Water Interfaces surfactant

Γsa/mol cm-2

Dl/cm2 s-1

φfb

η j i/mPa s

0.12

1.4

2.0 × 10-10 2.5 × 10-10

2.3 × 10-6 2.4 × 10-6c 1.7 × 10-6 1.6 × 10-6

0.12 0.13

1.8 1.9

free SDS

a Interfacial concentration of SDS. b φ ) 0.15 in ethanol.21 f Obtained with a pinhole of 10 µm in diameter, others with 50 µm in diameter.

c

at the single molecule level was calculated as 2.3 × 10-6 cm2 s-1 with

Dl ) dobs2/2tmax

Figure 5. Examples of photon signals of single DiI molecules (a) at the surfactant-free dodecane-water interface and (b, c) at the SDS-modified dodecane-water interface. Axes in (a), (b), and (c) indicate photon bundles. The average numbers of DiI molecules in the observation area were 0.1 for (a-c) and 0 for (d). Interfacial SDS concentration: (a) 0 mol/cm2; (b) 2.0 × 10-10 mol/cm2; (c) 2.49 × 10-10 mol/cm2; (d) 0 mol/cm2 (background). p-Polarized laser beam of 5 mW output power; filter, 565DM + 570DF30; dwell time, 2 µs. The irradiation angle at the interface was 73°, which was larger than the critical angle.

free system, but the photon bundle was scarcely obtained. The total duration of photon bundles was about 10% of total observation period ()2 µs × 8192 channels × 100 times). This implied that the average number of DiI molecules was about 0.1 in the observation area at the interface, supporting the estimation of interfacial DiI concentration. According to the analysis by Poisson distribution, the possibility that more than one molecule existed in it was only 0.46%. Lateral Diffusion Coefficient of Single DiI Molecules. Figure 5a shows a result of single DiI molecules at the dodecane-water interface under the total internal reflection condition with 5 mW laser power. In comparison with the background signal (Figure 5d), a clear photon bundle was observed. DiI is highly adsorbed at the dodecane-water interface, and thus the translational diffusion of DiI is restricted in the lateral direction at the interface. The duration corresponds to the period in which the single DiI molecule stayed in the observation area, because its photobleaching was negligible. One DiI molecule could move through the observation area with various trajectories; for example, it goes straight, passing through the center of the observation area, or it goes around the observation area. It can be assumed that the maximum duration (tmax) of the photon bundles corresponds to the period when the single DiI molecule moves along the diameter of the round observation area. From 400 photon bundles extracted from the data of the 100 times measurements, the tmax value was obtained. The lateral diffusion coefficient (Dl) of DiI

(6)

When a pinhole of 10 µm in diameter was used in measurements instead of 50 µm, a shorter tmax value of 190 µs was obtained. This experiment was carried out with another objective of NA 0.95. In this case, dobs corresponded to the diffraction limit of light, which was calculated as 300 nm with the relationship: dobs ) λ/(2NA), where λ was the observation wavelength (575 nm). The Dl value for the 10 µm pinhole almost agreed with that for the 50 µm pinhole as listed in Table 1, confirming the evaluation method of the diffusion behavior of single DiI molecules. An autocorrelation analysis26 was tried for this case. However, the attempt gave no information of the dynamics of single DiI molecules, because the period when the DiI molecules stayed in the observation area was too short in comparison with proteins of relatively larger molecular weight and the observed photon signals were not high enough. The analysis using tmax shown above could be an alternative method to estimate the diffusion dynamics of molecules of smaller molecular weight. The radius (r) and the diffusion coefficient (D) of a spherical molecule is related to the viscosity (η) of the medium surrounding the molecule with the EinsteinStokes equation as

D ) kT/6πrη

(7)

where k is Boltzmann constant and T absolute temperature. This equation is valid in solution, but the apparent (or average) three-dimensional viscosity of the interface (η j i) can be evaluated with eq 7. The radius of DiI molecule, assumed as a sphere, was estimated as 7.0 × 10-10 m by summing the volume of components of the molecule.27 The η j i value at the surfactant-free dodecane-water interface in Table 1 was as high as that of dodecane (1.4 mPa s), and it was higher than the value for water (0.89 mPa s). This result corresponded to the MD simulation in Figure 3, which suggested that the two long alkyl groups were immersed deeply in the dodecane phase. Fluorescence Quantum Yield of Single DiI Molecules. No energy transfer occurred from an excited DiI to another DiI, because of an extremely lower interfacial DiI concentration. Density of the photon bundles reflected the fluorescence quantum yield of single DiI molecules as eq 3. The φf value at the surfactant-free system in Table 1 was estimated with the average photon density, as(26) Fluorescence Correlation Spectroscopy; Rigler, R., Elson, E. S., Eds.; Springer-Verlag: Berlin, 2001. (27) Barton, A. F. M. CRC Handbook of Solubility Parameters and Other Cohesion Parameters; CRC Press: Boca Raton, FL, 1983; p 64.

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suming that the φisc, kisc, and (kr + knr + kisc) values were constant. The φf value was somewhat lower than that in ethanol.21 Influence of SDS. The adsorption behavior of SDS was quantitatively studied at the dodecane-water interface.28 With the reported adsorption constants, the interfacial concentrations of SDS were calculated as 2.0 × 10-10 and 2.49 × 10-10 mol/cm2 for 5.0 × 10-4 and 5.0 × 10-3 M initial bulk concentrations, respectively, where the saturated interfacial concentration of SDS was 2.5 × 10-10 mol/cm2.28 Since the DiI concentration was much lower than SDS, DiI hardly interfered in the adsorption of SDS. The dodecane solution of DiI was the same as the study in the surfactant-free system, and thus the average number of DiI molecules was 0.1 in the observation area. Parts b and c of Figure 5 represent examples of photon bundles; the duration of the photon bundle was as wide as that in the surfactant-free system. The Dl and φf values of DiI were obtained with about 300-400 photon bundles in the same manner as the free system, as listed in Table 1, They were a little affected by the interfacial SDS regardless of the interface nearly saturated with SDS. The η j i values in the SDS system were somewhat larger than that without SDS. This means that the translational motion of interfacial SDS molecules is similar to that of dodecane molecules. The rotational dynamics of a Rhodamine dye at a toluene-water interface was rather retarded by SDS.29 Viscosity is the principal factor that affects the translational and rotational dynamics of a molecule adsorbed at the interface. Therefore, the interface with toluene of lower viscosity (0.55 mPa s) is more largely affected than that with dodecane of higher viscosity (1.4 mPa s). The φf was slightly affected by the interfacial SDS. Influence of DMPC. Since the phase transition temperature of DMPC is 23 °C,30 DMPC shows liquidlike characteristics under the present experimental temperature (25 °C). The average number of DiI molecules in the observation area was expected to be not higher than 0.1. Parts a and b of Figure 6 show typical examples of observed photon bundles in the DMPC system. The duration of photon bundles was elongated with the increase in the interfacial DMPC concentration. Photon bundles were continued over 16 ms (2 µs × 8192 channels) in some cases at the relatively higher interfacial DMPC concentration, suggesting that the tmax value was larger than 16 ms. Figure 7a displays an example at the highest interfacial concentration of DMPC (2.0 × 10-10 mol/cm2) with a dwell time of 16 ms. The average background signal for 16 ms was 15 ( 5 counts (average ( standard deviation (σ)), and thus counts over 30 (average + 3σ; 99% confidence level) per 16 ms corresponded to photons emitted by single DiI molecules. The background signal of the dodecane-water interface was relatively higher than that in aqueous solutions or at a quartz-water interface, and thus the information on dynamics of the single DiI molecules was limited. A histogram of the photon number per one channel obeyed a Poisson distribution that had a background peak (no DiI) and a signal peak (one DiI) for each DMPC case. The threshold level for the photon signals of single DiI molecules was determined from the Poisson distribution, because the background peak was slightly changed in each (28) Bonfillon, A.; Sicoli, F.; Langevin, D. J. Colloid Interface Sci. 1994, 168, 497-504. (29) Tsukahara, S.; Yamada, Y.; Watarai, H. Langmuir 2000, 16, 6787-6794. (30) Seikagaku jiten (Encyclopedia of Biochemistry), 3rd ed.; Imahori, K., Yamakawa, T., Eds.; Tokyo Kagaku Dojin Co., Ltd.: Tokyo, 1998.

Hashimoto et al.

Figure 6. Examples of photon signals of single DiI molecules at the DMPC-modified dodecane-water interface. Axes in (a) and (b) indicate photon bundles. The average numbers of DiI molecules in the observation area were 0.1 for (a, b) and 0 for (c). Interfacial DMPC concentration: (a) 9.0 × 10-11 mol/cm2; (b) 2.0 × 10-10 mol/cm2; (c) 0 mol/cm2 (background). p-Polarized laser beam of 10 mW output power; filter, 600DF25; dwell time, 2 µs. The irradiation angle at the interface was 73°.

case. For example, the threshold level was set to 50 counts in Figure 7a, which was higher than the 30 counts. Parts b-d of Figure 7 show expanded figures at three points of Figure 7a. In Figure 7b, there were no definite signals beyond the threshold level, indicating that no DiI molecules went across the observation area. In parts c and d of Figure 7, some photon bundles were observed. The duration of the photon bundles increased with the increase in the interfacial DMPC concentration. The dwell times were set to 1.4 and 16 ms for 2.0 × 10-12 mol/cm2 and 2.0 × 10-11 to 2.0 × 10-10 mol/cm2, respectively. The Dl values of DiI were obtained with about 100-900 photon bundles in the same manner as the surfactantfree and SDS systems, as listed in Table 2. The minimum Dl value in the DMPC system is less than that in the surfactant-free system by 2 orders of magnitude. Autocorrelation analysis should be done in this DMPC system, because it was suggested that the period when the single DiI molecules stayed in the observation area was elongated. An autocorrelation function (ACF; G(τ)) at a two-dimensional interface can be expressed as26

G(τ) ) 1 +

1 16Dlτ N 1+ dobs2

(

)

(8)

where τ is ACF time and N is a parameter including the average number of DiI molecules, τd and Qdet. The ACF analysis was done for the data obtained with a dwell time from 0.5 to 16 ms. An example is shown in Figure 8, the fitting curve for eq 8 agreeing well with the observed points. Results for the other DMPC concentrations showed similar tendencies. The obtained Dl values listed in Table 2 were in rough agreement with the Dl values obtained from tmax. At the air-water interface, the Dl value was reported as 4.26 × 10-8 cm2 s-1 at the DMPC concentration of 0.90

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Figure 7. Examples of photon signals of single DiI molecules at the dodecane-water interface modified by 2.0 × 10-10 mol/cm2 DMPC for 16 ms × 8192 channels measurement: (a) overall result; (b-d) expanded samples. Axes in (c) and (d) indicate photon bundles. The average number of DiI molecules in the observation area was 0.1. p-Polarized laser beam of 10 mW output power; filter, 600DF25; dwell time, 16 ms. The irradiation angle at the interface was 73°. Table 2. Effects of DMPC on Lateral Diffusion Coefficient (Dl) and Fluorescence Quantum Yield (Of) of Single DiI Molecules and Apparent Viscosity (η j i) and Intrinsic Viscosity (ηi) at the Dodecane-Water Interface Dl/cm2 s-1 a/

Γs mol cm-2 2.0 × 10-12 2.0 × 10-11 9.0 × 10-11 2.0 × 10-10 a

tmax method

ACF method

2.1 × 10-7 4.3 × 10-8 (8.4 ( 5.4) × 10-8 2.7 × 10-8 (7.6 ( 2.2) × 10-8 1.8 × 10-8 (2.4 ( 1.6) × 10-8

φf 0.05 0.03 0.02 0.05

η j i/ η i/ mPa s mPa s 15 72 120 170

37 270 470 750

Interfacial concentration of DMPC.

Figure 8. An example of autocorrelation function (G(τ)) analysis for the photon signals of single DiI molecules at the interfacial DMPC concentration of 2 × 10-11 mol/cm2 with a dwell time of 16 ms. The solid line represents the fitting curve for eq 8.

nm2 molecule-1.31 This value was in the same order of magnitude as the value of 1.8 × 10-8 cm2 s-1 obtained in the present study at the DMPC concentration of 0.83 nm2 molecule-1 (2.0 × 10-10 mol/cm2). It was also reported that the Dl value at the heptane-water interface was less than that at an air-water interface.32 According to a static (31) Ke, P. C.; Naumann, C. A. Langmuir 2001, 17, 3727-3733.

model, Dl in the presence of phospholipid had empirically a linear relationship with an average area given to one phospholipid molecule (A) at air-water interfaces.32,33 In the present study, the plot of Dl of DiI against A also showed a linear relationship (not shown) with a high correlation coefficient. These results suggested the accuracy of the obtained Dl. The φf values in the DMPC system were less than those in the surfactant-free and SDS systems, as listed in Table 2. One possibility causing the lower φf values is the elongation of the lifetime of the triplet state ()1/kT; kT ≈ kq[O2]). However, a nitrogen bubbling to the dodecane solution of DiI hardly affected the results. A change of the DiI orientation at the interface or an intrinsic decrease in the φf value by the interaction with DMPC is possible at this stage, but further investigations should be done. Interfacial Viscosity in DMPC System. The η j i values in the DMPC system are listed in Table 2. The maximum η j i value was 0.17 Pa s, which was higher than the surfactant-free interface by 2 orders of magnitude. DMPC is a composition of biological cell membrane and it has a self-organizing nature due to a hydrophobic zwitterion. Similar high viscosity was reported at the micelle surfaces of analogous self-associating surfactants, that is, dihexadecyl hydrogen phosphate (DHP) micelle or monoalkyl phosphate micelle,34,35 where a hydrogen-bonding structure was suggested between protonated and deprotonated phosphates.35 The rotational dynamics of a Rhodamine dye was also retarded by DHP adsorbed at a toluenewater interface,29 leading to interfacial viscosity of 12 and 91 mPa s at 6.9 × 10-11 and 1.0 × 10-10 mol/cm2 of interfacial DHP concentration, respectively. These values were in the same order of magnitude as those obtained in the present study. Saffman proposed a theoretical treatment of lateral diffusion of a cylinder having a radius of a and a height (32) Adalsteinsson, T.; Yu. H. Langmuir 2000, 16, 9410-9413. (33) Ke, P. C.; Naumann, C. A. Langmuir 2001, 17, 5076-5081. (34) Rupert, L. A. M.; van Breemen, J. F. L.; Hoekstra, D.; Eugberts, J. B. F. N. J. Phys. Chem. 1988, 92, 4416-4420. (35) Walde, P.; Wessicken, M.; Ra¨dler, U.; Berclaz, N.; CondeFrieboes, K.; Luisi, P. L. J. Phys. Chem. B 1997, 101, 7390-7397.

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h. The intrinsic ηi values listed in Table 2 are about two to four times larger than the respective η j i values, and they increase with the increase in the DMPC concentration. The maximum ηi value is 0.75 Pa s, which is comparable to that of a common viscous liquid, glycerin (0.945 Pa s). Conclusion

Figure 9. A model that the cylindrical DiI molecule undergoes a lateral friction force at the dodecane-water interface. The viscosities of organic phase (upper), interface, and aqueous phase (lower) are ηo, ηi, and ηw, respectively.

of h at an interface as shown in Figure 9.36 The top and bottom of the cylindrical molecule undergo friction forces from the organic and aqueous phases, respectively, and the wall undergoes a friction force from the interfacial layer. From this model, Dl is expressed as36,37

Dl ≈

kT[(ln(2/) - γ] 4π(ηo + ηw)a

)

( )(

(9)

)

a ηo + ηw h ηi

where ηo, ηw, and ηi are the viscosity of organic phase, aqueous phase, and interface, respectively, γ Euler’s constant (≈0.577). The h of DiI was estimated as 1.7 × 10-9 m by the MD simulation, and its a was evaluated as 5.1 × 10-10 m from the calculated molecular volume and (36) Saffman, P. G. J. Fluid Mech. 1976, 73, 593-602. (37) Hughes, B. D.; Pailthorpe, B. A.; White, L. R. J. Fluid Mech. 1981, 110, 349-372.

The present study demonstrated that the single molecule probing was an effective approach for the evaluation of viscosity in the nanospace at the liquid-liquid interface. The photon bundles of single DiI molecules adsorbed at the dodecane-water interface were successfully observed by the total internal reflection fluorescence microscopy. The lateral diffusion coefficient and the fluorescence quantum yield of single DiI molecules at the surfactantfree interface were obtained from photon bundles. Furthermore, the retarding effects of two surfactants on the lateral diffusion of the single DiI molecules were investigated. SDS slightly affected the lateral diffusivity, but DMPC retarded it drastically. The quantum yield was somewhat affected by the surfactants. Further investigations on the dynamics of adsorptiondesorption, chemical reaction, and transfer across the interface of single molecules at the liquid-liquid interface are going on. Acknowledgment. This study was financially supported by a Grant-in-Aid for Scientific Research of Priority Areas (No. 13129204) and Encouragement of Young Scientists (No. 13740422) from the Ministry of Education, Culture and Sports of Japan. LA026644X