NANO LETTERS
Clay Nanoparticle-Supported Single-Molecule Fluorescence Spectroelectrochemistry
2009 Vol. 9, No. 2 655-658
Chenghong Lei,† Dehong Hu,† and Eric Ackerman*,‡ Pacific Northwest National Laboratory, Richland, Washington 99352 Received October 2, 2008; Revised Manuscript Received December 12, 2008
ABSTRACT Here we report that clay nanoparticles allow formation of a modified transparent electrode, spontaneous adsorption of fluorescent redox molecules on the clay layer, and thus the subsequent observation of single-molecule fluorescence spectroelectrochemistry. We can trace single-molecule fluorescence spectroelectrochemistry by probing the fluorescence intensity change of individually immobilized single redox molecules modulated via cyclic voltammetric potential scanning. This work opens a new approach to explore interfacial electron transfer mechanisms of redox reactions.
Interfacial electron transfer processes play an important role in many chemical and biological processes.1-8 However, interfacial electron transfer processes are usually very complex due to high dependence on its local environments. Reaction rates vary from site to site and from time to time. These spatial and temporal inhomogenities are difficult to dissect with ensemble-averaged measurements and consequently sometimes yield different experimental results for interfacial electron transfer rate constants. Single-molecule spectroscopy revealed complex reaction dynamics involving photoinduced, excited-state intramolecular, and interfacial electron transfers.9-13 Single-molecule studies of photoinduced electron transfers in the enzyme flavin reductase revealed multiple interconverting conformers.12 Singlemolecule studies of photosensitized electron transfers on dyecontaining nanoparticles also showed blinking and fluctuation dynamics varying from molecule to molecule and time to time.14 Recently developed single-molecule spectroelectrochemistry extends single-molecule approaches to groundstate interfacial electron transfer by simultaneously modulating the electrochemical potential while detecting single molecule fluorescence.15,16 Here we develop clay nanoparticles-supported single-molecule fluorescence spectroelectrochemistry to investigate interfacial electron transfer reaction dynamics. At the clay-modified transparent electrode, we can trace single-molecule fluorescence spectroelectrochemistry, one immobilized redox molecule at a time, by probing the fluorescence intensity change of individually * To whom correspondence should be addressed. E-mail: eackerm@ sandia.gov. Fax: 1-505-284-1323. Current address: Sandia National Laboratory. † These authors contributed equally to this work. ‡ Currentaddress:SandiaNationalLaboratory.E-mail:
[email protected]. 10.1021/nl802998e CCC: $40.75 Published on Web 01/13/2009
2009 American Chemical Society
immobilized single redox molecules modulated via cyclic voltammetric potential scanning. Sodium Montmorillonite (SM) with its inherent layered inorganic nanostructure is a member in the smectite group of clays. SM has the advantages of high chemical stability with high surface-area nanostructural features yielding special binding and sorptive properties for many substances, including electron transfer mediators such as cation dyes.17-20 Sodium montmorillonite colloid (SMC) is composed of nanoscaled SM particles dispersed in water as individually charged sheets sized in tens to a few hundreds of nanometers. Although clay-modified electrodes have been extensively studied,2,4,18,21-27 one of the unique characteristics of the nanoscaled SM particles is that they can readily form a transparent thin film on a transparent ITO/glass coverslip. The clay-modified coverslip as a working electrode allows not only adsorption of the probe dye molecules, but also provides transparency necessary for the electrochemical cell to couple with a fluorescence confocal microscopy to study possible single-molecule fluorescence spectroelectrochemistry. Cation dyes of phenazines, phenoxazines, and phenothiazines have been extensively used as electron transfer mediators for enzymatic redox reactions and biosensor development.17-20 As a phenoxazine dye, cresyl violet is special because it is also strongly fluorescent.28 Intermittent single-molecule electron transfer of cresyl violet on a TiO2 nanoparticle interface without electrochemical potential scanning was observed using single-molecule fluorescence spectroscopy.14 Cresyl violet molecules adsorbed on semiconductor surfaces are also efficient electron donors under photoexicitation.29-31 Single-molecule fluorescence spectro-
Figure 1. (A) AFM height images of SM on the glass coverslip. (B) SEM image of sodium montmorillonite nanoparticles. (C) Cyclic voltammogram of cresyl violet/SM/ITO prepared by incubating SM/ITO in 36 µM cresyl violet for 30 min and then rinsing 5 times with buffer. (D) Cyclic voltammogram of cresyl violet/SM/ITO prepared by incubating SM/ITO in 3.6 nM cresyl violet for 10 min and then rinsed 5 times with buffer (one cycle shown). (E) Plots of voltage E (V) vs time and fluorescence intensity vs. time in synchronization with three continuous CV scans (one cycle displayed in panel D). Buffer: pH 6.2, 5 mM sodium phosphate. Scan rate: 100 mV/s. Potential scan range: 0.1 to -0.7 V.
electrochemistry of cresyl violet in solution has been studied using cyclic voltammetry (CV)-coupled single-molecule fluorescence spectroscopy (CV-SMFS).16 That work showed cresyl violet has an oxidized fluorescent state and a reduced nonfluorescent state correlating with CV potential scanning. In this work, to account for the inhomogeneous rates of the interfacial electron transfer originating from complex dynamical reaction processes we employed CV-SMFS to investigate interfacial electron transfer dynamics of single dye molecules using the clay-modified transparent ITO/glass coverslip as the working electrode (Supporting Information). 656
This new approach allows us to detect the fluorescence spectroelectrochemistry of single immobilized dye molecules adsorbed on the clay-modified surface rather than single free dye molecules diffusing into the laser focal volume in solution.16 We observe single-molecule fluorescence intensity fluctuations and blinking of the individual immobilized molecules. We attribute these phenomena to intermittency of the interfacial electron transfer processes modulated by CV potential scanning. Figure 1A shows the height image of the clay nanoparticles by atomic force microscope on the glass coverslip (AFM). Nano Lett., Vol. 9, No. 2, 2009
Figure 2. (A) Image of laser focused area on the SM/ITO electrode. (B) Cyclic voltammogram of SM/ITO electrode in 120 pM cresyl violet solutions. (C) Voltage E (V) vs time plot of three CV scans and plot of fluorescence intensity of two single cresyl violet molecules adsorbed on SM/ITO vs time during three CV scans. The molecule in the lower panel appears to exhibit photobleaching after ∼30 s. (D) Average of 15 single molecules adsorbed on SM/ITO from the same acquired image. Buffer: pH 6.2, 5 mM sodium phosphate. Scan rate: 100 mV/s. Potential scan range: 0.1 to -0.7 V.
Scanning electron microscopy (SEM) displayed the characteristic three-dimensional raglike textural surface formed by the deposition and the imperfect stacking of colloidal clay nanoparticles (Figure 1B). Figure 1C shows the typical cyclic voltammogram of cresyl violet/SM/ITO in pH 6.2, 5 mM sodium phosphate buffer. Under the experimental conditions, the cyclic voltammetry displayed ∆Ep values (the differences between the reduction and the oxidation peak potentials) of ∼200 mV at a scan rate of 100 mV/s, where the electron transfer rate was 0.29 s-1 estimated by Laviron method.32 As estimated from the integration of the charge under the oxidation peak in Figure 1C, there were ∼4.6 × 10-10 mole·cm-2 of electroactive dye molecules on the claymodified electrode surface. The fluorescence of reduced and oxidized cresyl violet adsorbed on clay was studied on the combined cyclic voltammetry and fluorescence microscope. The excitation laser from the fluorescence microscope was focused on the SM/ITO surface. Following an adsorption process by incubation of SM/ITO surface in only 3.6 nM dye, the adsorbed cresyl violet was electrochemically undetectable (Figure 1D). Nonetheless, the electrode reaction occurred as the redox dye’s fluorescence intensity changes Nano Lett., Vol. 9, No. 2, 2009
synchronously with CV potential scanning (Figure 1E). The electrode reaction is proposed as16 -e-
Cresyl violet (non-fluorescent) {\} Cresyl violet+ (fluorescent) +e-
(1)
The ratio of the fluorescence intensity levels at high/low voltages is ∼2:1 (Figure 1E), indicating that at low potentials not all adsorbed cresyl violet molecules on clay displayed redox electroactivity. To examine the electrochemical dynamics of single molecules, we conducted cyclic voltammetry-single molecule fluorescence experiments. CV-SMFS experiments with 120 pM cresyl violet were done by focusing on the SM/ITO surface with 0.4 µW excitation. Figure 2A shows the image of the laser-focused SM/ITO area. The image demonstrates that there were many cresyl violet molecules immobilized on the clay-modified surface for a long period (longer than seconds). Although cresyl violet molecules were adsorbed on the SM/ITO surface, they were not electrochemically detectable at such a low concentration (Figure 2B). However, the fluorescence intensity trajectory of single molecules show 657
the change of fluorescence intensity of single cresyl violet molecules adsorbed on the SM/ITO in synchronization with the electrochemical potentials at the surface (Figure 2C). The long trajectories permit detailed statistical analyses of the on-off emission intensity of single molecules. Each emission on-off event, corresponding to fluorescence turning on and off between the oxidized and reduced states of cresyl violet, might result from redox chemical reactions or intrinsic single molecule blinking. The main difference between these two possibilities is that redox reactions should correlate with the changing electrochemical potential, while intrinsic blinking is unrelated. To investigate how single molecule intensity trajectories were modulated by CV potential scanning, the sum of 15 single molecule trajectories with respect to the potential oscillation period of 16 s is shown in Figure 2D. Higher potentials were correlated to higher fluorescence intensity and vice versa, demonstrating that electron transfers provided a major contribution to the fluorescence intensity changes of cresyl violet. The intensity at the end of 16 s period did not recover to its initial level due to the photobleaching of some molecules. Figure 2D shows that the ratio of high to low intensity is ∼2:1, which is in agreement with that exhibited in the ensemble-averaged experiment (Figure 1E). This result is also consistent with the ensemble experimental finding that not all adsorbed single cresyl violet molecules on clay underwent redox electrochemical reaction. The inhomogeneity of the adsorbed single molecules might be due to the adsorption sites on the three-dimensional raglike textural clay surface (Figure 1A,B), yielding differences in molecular orientations and distances of individual adsorbed molecules from the ITO electrode surface. Using a simple first order reaction model for the electron transfer process, the electron transfer rate constants can be calculated assuming every on/off switching event is caused by electron transfer. The statistical analysis shows that a single cresyl violet molecule’s average “off” state lifetime is 3.5 s. Thus oxidation electron transfer occurs after 3.5 s on average, converting a cresyl violet molecule from its “off” state to the fluorescent “on” state. The oxidation electron transfer rate constant is 1/0.35 ) 0.28 s-1. Cresyl violet molecules on average remain fluorescent for 2.9 s before turning “off”, so the reduction electron transfer rate is 1/2.9 ) 0.34 s-1. These results are close to the ensemble averaged electron transfer rate of 0.29 s-1 (Figure 1C). In conclusion, we observed single-molecule fluorescence spectroelectrochemistry of cresyl violet adsorbed on the claymodified surface. The fluorescence intensity of single molecules exhibits on/off switching due to redox reaction controlled by cyclic voltammetry. By analyzing the on/off state lifetime, the measured oxidation/reduction electron transfer rates of single molecules of cresyl violet adsorbed on SM/ITO are in agreement with the ensemble-averaged electron transfer rate. The method developed in this work could open up a new approach to explore interfacial electron transfer mechanisms of chemical or biological oxidation/reduction reactions using electron transfer mediators such as redox dyes.
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Acknowledgment. We gratefully acknowledge funding of this work by the U.S. Department of Energy Office of Basic Energy Sciences under Contract DE-AC06-RLO1830. A portion of the research described in this paper was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy’s Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Supporting Information Available: Experimental details. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Simonneaux, G.; Bondon, A. Chem. ReV. 2005, 105 (6), 2627–2646. (2) Ghosh, P. K.; Bard, A. J. J. Am. Chem. Soc. 1983, 105 (17), 5691– 5693. (3) Fox, M. A.; Dulay, M. T. Chem. ReV. 1993, 93 (1), 341–357. (4) Fitch, A. Clays Clay Miner. 1990, 38 (4), 391–400. (5) Adams, D. M.; Brus, L.; Chidsey, C. E. D.; Creager, S.; Creutz, C.; Kagan, C. R.; Kamat, P. V.; Lieberman, M.; Lindsay, S.; Marcus, R. A.; Metzger, R. M.; Michel-Beyerle, M. E.; Miller, J. R.; Newton, M. D.; Rolison, D. R.; Sankey, O.; Schanze, K. S.; Yardley, J.; Zhu, X. Y. J. Phys. Chem. B 2003, 107 (28), 6668–6697. (6) Brown, G. T.; Darwent, J. R.; Fletcher, P. D. I. J. Am. Chem. Soc. 1985, 107 (23), 6446–6451. (7) Duonghong, D.; Ramsden, J.; Gratzel, M. J. Am. Chem. Soc. 1982, 104 (11), 2977–2985. (8) Gratzel, M. Nature 2001, 414 (6861), 338–344. (9) Holman, M. W.; Liu, R. C.; Adams, D. M. J. Am. Chem. Soc. 2003, 125 (41), 12649–12654. (10) Liu, R. C.; Holman, M. W.; Zang, L.; Adams, D. M. J. Phys. Chem. A 2003, 107 (34), 6522–6526. (11) Weiss, S. Science 1999, 283 (5408), 1676–1683. (12) Yang, H.; Luo, G. B.; Karnchanaphanurach, P.; Louie, T. M.; Rech, I.; Cova, S.; Xun, L. Y.; Xie, X. S. Science 2003, 302 (5643), 262– 266. (13) Lu, H. P.; Xie, X. S. J. Phys. Chem. B 1997, 101 (15), 2753–2757. (14) Biju, V.; Micic, M.; Hu, D. H.; Lu, H. P. J. Am. Chem. Soc. 2004, 126 (30), 9374–9381. (15) Palacios, R. E.; Fan, F. R. F.; Bard, A. J.; Barbara, P. F. J. Am. Chem. Soc. 2006, 128 (28), 9028–9029. (16) Lei, C.; Hu, D.; Ackerman, E. J. Chem. Commun. 2008, 5490–5492. (17) Kubota, L. T.; Gorton, L. Electroanalysis 1999, 11 (10-11), 719– 728. (18) Lei, C. H.; Deng, J. Q. Anal. Chem. 1996, 68 (19), 3344–3349. (19) Ruan, C. M.; Yang, F.; Lei, C. H.; Deng, J. Q. Anal. Chem. 1998, 70 (9), 1721–1725. (20) Yang, F.; Ruan, C. M.; Xu, J. S.; Lei, C. H.; Deng, J. Q. Fresenius’ J. Anal. Chem. 1998, 361 (2), 115–118. (21) Fitch, A.; Lee, S. A. J. Electroanal. Chem. 1993, 344 (1-2), 45–59. (22) Fitch, A.; Du, J.; Gan, H. M.; Stucki, J. W. Clays Clay Miner. 1995, 43 (5), 607–614. (23) Fitch, A.; Krzysik, R. J. J. Electroanal. Chem. 1994, 379 (1-2), 129– 134. (24) Edens, G. J.; Fitch, A.; Lavyfeder, A. J. Electroanal. Chem. 1991, 307 (1-2), 139–154. (25) Ege, D.; Ghosh, P. K.; White, J. R.; Equey, J. F.; Bard, A. J. J. Am. Chem. Soc. 1985, 107 (20), 5644–5652. (26) Kamat, P. V. J. Electroanal. Chem. 1984, 163 (1-2), 389–394. (27) King, R. D.; Nocera, D. G.; Pinnavaia, T. J. J. Electroanal. Chem. 1987, 236 (1-2), 43–53. (28) Magde, D.; Brannon, J. H.; Cremers, T. L.; Olmsted, J. J. Phys. Chem. 1979, 83 (6), 696–699. (29) Parkinson, B. A.; Spitler, M. T. Electrochim. Acta 1992, 37 (5), 943– 948. (30) Liu, D.; Kamat, P. V. J. Chem. Phys. 1996, 105 (3), 965–970. (31) Liu, D.; Fessenden, R. W.; Hug, G. L.; Kamat, P. V. J. Phys. Chem. B 1997, 101 (14), 2583–2590. (32) Laviron, E. J. Electroanal. Chem. 1979, 101 (1), 19–28.
NL802998E
Nano Lett., Vol. 9, No. 2, 2009
