Plasmonic Imaging of Brownian Motion of Single DNA Molecules

Apr 2, 2013 - Single AgNPs can be observed under dark-field illumination with incoherent .... images using Meta Imaging Software (Molecular Devices,. ...
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Plasmonic Imaging of Brownian Motion of Single DNA Molecules Spontaneously Binding to Ag Nanoparticles Ken Hirano,*,†,‡ Tomomi Ishido,†,# Yuko S. Yamamoto,†,# Norio Murase,† Masatoshi Ichikawa,§ Kenichi Yoshikawa,§,∥ Yoshinobu Baba,†,‡,⊥ and Tamitake Itoh*,†,‡ †

Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa 761-0395, Japan ‡ FIRST Research Center for Innovative Nanobiodevices, Nagoya University, Chikusa, Nagoya 464-8603, Japan § Department of Physics, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan ∥ Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Kyoto 610-0394, Japan ⊥ Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan S Supporting Information *

ABSTRACT: We find the spontaneous binding of single DNA molecules to uncoated silver nanoparticles (AgNPs) in aqueous solution with Mn2+ (3 mM). From dark-field optical microscopic imaging of AgNPs bound to DNA molecules, we demonstrate analysis of the Brownian motion of single DNA molecules via plasmon resonance elastic light scattering. Our results provide that the plasmonic imaging technique is free from photobleaching and blinking and thus is useful in long-time observations of single-molecule DNA dynamics. KEYWORDS: Silver nanoparticle, plasmon resonance, single DNA molecule, DNA labeling, video-rate imaging, nanoparticle tracking

T

demonstrated the manner of Brownian motion on DNA intrachain fluctuations and translational motions by using DNA/AgNPs complex in aqueous solution through the combination of plasmonic imaging of AgNPs and fluorescent imaging of YOYO-1 stained single DNA molecules. In order to investigate the labeling AgNPs onto DNAs via metal ions we used UV−vis spectroscopy (V-630, JASCO) to monitor DNA/AgNPs complex formation. We used T4 dc DNA (166 kbp; Nippon Gene) and AgNPs with an average diameter of ∼40 nm. A suspension of AgNPs was prepared by the Lee and Meisel method.15 The sample solution was prepared as 13 × 1013 molecules/L T4 DNA, 5.8 × 1013 particles/L AgNPs ([T4 DNA]/[AgNP] = 1:4.4) in a 5 mM Tris (pH 7.4) buffer. Figure 1a,b shows the absorption spectra of DNA/AgNPs solutions at various Mn2+ concentrations [Mn2+] at times of 1 and 60 min. This result clearly shows a drastic decrease in plasmon absorption maxima with increasing [Mn2+]. At 20 and 50 mM concentrations, the intensity of the plasmon resonance peak at 410 nm almost disappeared after 60 min. On the other hand, at low concentrations (≤8 mM) it essentially remained constant over 60 min. This drastic decrease in plasmon absorption maxima indicates that collapsing of electric double-layer of AgNP surfaces by Mn2+ with higher concentration and subsequent aggregation and precipitation of AgNPs.16 Figure 1c,d shows that the color of

he imaging of single DNA dynamics in aqueous solution is important for clarification of biomolecular mechanisms and thus vital in molecular biology, biophysics, and polymer physics.1−6 For analyzing the dynamics of single DNA molecules, fluorescent labels, such as dye molecules and quantum dots (QDs), have been developed.7,8 However, bleaching of fluorescent labels imposes limits on DNA imaging, as it restricts the observation time and pH range. Although QDs diminish the effect of photobleaching, they may cause breakages/damages on DNA under photoillumination as reported by Anas et al.9 As alternatives to fluorescent labels, Au or Ag nanoparticles (NPs), which have strong plasmon resonances in the visible to near-infrared region, are promising candidates. Single AgNPs can be observed under dark-field illumination with incoherent white light.10−12 Thus, AgNPs bound to single DNA molecules (DNA/AgNPs complexes) are useful for stable, quench-free, and pH-independent imaging. Previously, DNA/AgNPs complexes have been studied by darkfield and electron microscopies.13,14 However, in these systems the AgNPs are chemically linked to DNA and are also immobilized on substrates. Thus, a more convenient method is required for in situ imaging of the dynamics of single intact DNA molecules in solution. In the present study, we propose a convenient method for video-rate imaging of single native DNA molecules labeled with AgNPs. To label AgNPs onto single DNA molecules, we also proposed, examined, and discussed a simple method of metal ion interaction between uncoated AgNPs and native DNAs. Using this stable labeling method via Mn 2+ ion, we © 2013 American Chemical Society

Received: July 6, 2012 Revised: March 29, 2013 Published: April 2, 2013 1877

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Figure 1. UV−vis spectra (a,b) and corresponding photographs (c,d) of DNA/AgNPs/Mn2+ solutions at Mn2+ concentrations of 0, 3, 8, 20, and 50 mM for incubation times of (a,c) 1 min and (b,d) 60 min. (e) Schematic representation of AgNPs binding and DNA conformation change depending on [Mn2+].

the solution depends on [Mn2+]. The color change from yellow to dark gray also indicated aggregation and subsequent precipitation of AgNPs, which is consistent with the decrease in the plasmon resonance peaks. Interestingly, the DNA/ AgNPs solution with 3 mM [Mn2+] does not show a remarkable color change, indicating that AgNPs and DNA molecules form a quasi-stable state assisted by Mn2+. Figure 1e shows the quasi-stable state of the DNA/AgNPs complex. The unchanged spectra in Figure 1a1,b1 indicate that DNA molecules and AgNPs coexist in solution without forming complexes, suggesting that Mn2+ plays an essential role in the formation of the complexes. We here discuss the role of Mn2+ in the formation of DNA/ AgNPs/Mn complexes. The specific interaction between Mn2+ and AgNPs is unclear for us. Thus, to examine the specific interaction besides electrostatic interactions, we investigated the formation of DNA/AgNPs complexes with other metal cations such as Mg2+, Co2+, Ni2+, Li+, and Na+ (data not shown). Mg2+, Co2+ and Ni2+ binds AgNPs to DNA at concentrations >4 mM, whereas Li+ and Na+ did not exhibit remarkable changes in solutions. By observing DNA/AgNPs/ [metal ion] solutions, we found that AgNPs on DNA molecules independently aggregate in the presence of Mg2+ and less numbers of AgNPs were bound to Co2+ and Ni2+ compared with Mn2+, although AgNPs were bound to noncondensed

DNA molecules when the metal ion concentrations were 4 mM. The DNA/AgNPs formed aggregates in the presence of >10 mM concentrations of Mg2+, Co2+, and Ni2+. These metal ions also enhanced the nonspecific binding of DNA/AgNPs to the surface of the cover glass compared with Mn2+. The Co2+ and Ni2+ solutions absorb visible light; such absorbance makes the observation unclear because excitation and emission/ scattering by white and optional visible light are largely affected by the absorbance of solution. Note that we cannot find the explicit relationship between formation of DNA/AgNPs complex and hydrated radius of metal ion.17 The lack of the relationship suggest that quasi-stable DNA/AgNPs/[metal ion] complexes are dependent on both the electrostatic and “chemical” interactions. This point should be further explored using density functional theory (DFT) calculation and detailed ζ-potential measurement.17 In short, we examined DNA/ AgNPs complexes using various kinds of metal ion, and only Mn2+ with 3 mM realized the quasi-stable DNA/AgNPs complexes without aggregation of both DNA molecules and AgNPs. To microscopically investigate the quasi-stable state of single DNA/AgNPs complexes we observed the dark-field and fluorescence microscopic images of DNA/AgNPs solutions. Figure 2a,b illustrates the experimental setup for dark-field and fluorescence imaging, respectively. For dark-field imaging 1878

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Figure 2. Setup for (a) dark-field optical images and (b) fluorescence images of quasi-stable DNA/AgNPs complexes.

collimated unpolarized white light from a 50 W halogen lamp was introduced into an inverted microscope (IX71, Olympus, Japan) through a dark-field condenser (U-DCW with IXADUC, Olympus, Japan) to the sample solution. The scattered elastic light from the DNA/AgNPs complex was collected with an objective lens (100×, N.A. 0.5−1.2), and sequential images, captured with a digital camera (XZ-1, Olympus, Japan), were recorded on digital videotape in video rate. Fluorescence images of single DNA molecules were obtained using an inverted microscope equipped with an object lens (100×, N.A. 1.3) and a highly sensitivity EM-CCD camera (ImagEM, Hamamatsu Photonics, Japan). Fluorescence images were directly recorded onto a PC in video rate, which was connected to the camera. The samples for fluorescent observation were simply mixed with a 0.1 μM YOYO-1 dye solution with DNA solution, [base-pair]/[YOYO] = 360:1. YOYO-1 intercalates via the insertion of a planar polycyclic aromatic ring between adjacent bases of double-stranded DNA. The prepared sample solutions were incubated at room temperature for ∼10 min and sandwiched between coverslip and glass slides for the optical microscopy measurements. Before staring optical microscopic measurement of DNA/ AgNPs complexes, we directly observed the morphology of the DNA/AgNPs complexes using dark-field optical and scanning electron microscopies (SEM) (JSM-6700F, JEOL). To improve the visibility of the complex for SEM, the concentration of AgNPs in solution was 4-fold higher than that for the subsequent optical microscopy experiments. Note that DNA molecules cannot be directly observed by SEM; thus, the alignment of a large number of AgNPs on the DNA molecules is important for observing DNA/AgNPs complexes. Figure 3a,b shows the dark-field and SEM images of the same DNA/AgNPs complex, respectively. Figure 3c,d shows the magnified SEM images of the same DNA/AgNPs complex. We easily identify that a large number of AgNPs are aligned in tandem with DNA molecules. These images show direct evidence for the formation of a DNA/AgNPs complex assisted by Mn2+. We observed single DNA/AgNPs complexes by the plasmonic imaging in solution. Figure 4a−d shows dark-field optical microscopic images of DNA/AgNPs in the presence of 3 mM [Mn2+] (Supporting Information Movies S1 and S2). Several AgNPs can bind and align to single DNA molecules (Figure 4a−c) and fluctuating single DNA chains are observed in real-time through the motion of bound AgNPs (Figure 4d). The AgNPs are different in colors because of their different shapes and sizes.18 This color variation enables clear observation of each bound AgNPs position on single DNA

Figure 3. (a) Dark-field optical microcopy images of T4 DNA labeled with AgNPs in the presence of 8 mM Mn2+. (b−d) Corresponding magnified SEM images.

molecules during Brownian motion. Additionally, we found that DNA/AgNPs complexes stained with YOYO-1 dye molecules sometimes exhibit anomalous bright spots (data not shown). This phenomenon may be due to surface-enhanced fluorescence from the dye molecules located in the plasmonic fields around the AgNPs.19 By observing a large number of individual DNA molecules (approximately 170), we build a histogram of the number of AgNPs bound on single DNA molecules in Figure 4e. The histogram suggests that the formation of DNA/ AgNPs complexes follows a Poisson process, showing the majority of the complexes are formed by the addition of first one then two and three particles, resulting in many AgNPs aligned along a single DNA molecule. It is important to examine the stability of DNA/AgNPs complex after removing Mn2+ for biological applications. Thus, we investigated the stability of the DNA/AgNPs complex in the presence of a metal ion chelating agent. We measured the number of bound AgNPs on single DNA molecules in the presence of 3 mM [Mn2+] and EDTA. Figure 4e shows the distribution change of the number of bound AgNP(s) on single DNA molecules with/without EDTA (10 mM or 20 mM). The number of AgNPs adhered to DNA, slightly decreases upon EDTA addition to DNA/AgNPs solution after 60 min. The DNA/AgNPs complexes which had more than four AgNPs on DNA, were especially affected by EDTA. The DNA/AgNPs/ Mn complex was stable irrespective of Mn2+ depletion. The stability of the DNA/AgNPs complex in the absence of Mn2+ in aqueous solution enables greater flexibility in designing future biological experiments. To obtain physical quantities from direct observation using dark-field and fluorescence microscopy, we investigated the hydrodynamic radii RH for the DNA/AgNPs complex. The hydrodynamic radius RH is predicted from Einstein-Stokes equation as follows D=

kBT 6πηsRH

(1)

where D is translational diffusion constant, kB is the Boltzmann constant, T is temperature, and ηs is the viscosity of the solvent (1.002 mPa s for pure water at T = 293 K). D is obtained from 1879

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Table 1. RH of the bound AgNPs compared with free AgNPs and DNA molecules no. of AgNP on DNA free AgNP AgNP on DNA

DNA

1 2 3

RH (nm) 42.7 438.6 449.4 488.1 916.7

± ± ± ± ±

15.4 165.3 174.1 162.1 297.8

are almost the same regardless of the number of bound AgNPs; this infers that the bound AgNPs on single DNA chains may not interact with each other. Figure 5 shows the calculated RH of single DNA molecules dependence on [Mn2+]. RH decreases with increasing [Mn2+],

Figure 4. Dark-field optical microscopy images of AgNP-labeled T4 DNA in the presence of 3 mM Mn2+, (a) two AgNPs, (b) four AgNPs, and (c) many AgNPs aligned along single DNA molecules. (d) Sequential dark-field optical images at 0.1 s intervals of three AgNPs on a single DNA molecule (white lines indicate that the three AgNPs were tethered to the same single DNA molecule). Scale bars 2 μm. (e) Distribution of the number of bound AgNP(s) to single DNA molecules with and without 10 mM and 25 mM EDTA.

Figure 5. Hydrodynamic radius RH of single DNA molecules with AgNPs at various [Mg2+]. Each RH is measured and calculated from the trajectory of the center of mass of a whole single DNA molecule using fluorescence microscopy. In each photograph, the DNA/AgNPs complex is observed by fluorescence microscopy for DNA visualization and dark-field microscopy for AgNPs visualization. Each data point is obtained by averaging over 20 molecules. Scale bars 5 μm.

the mean square displacement of the center of mass of an object, here, a single DNA molecule for fluorescence imaging or single AgNP for dark-field imaging at time t as follows 2

⟨(R⃗(t ) − R⃗(0)) ⟩ = 4Dt

indicating that more tightly confined DNA/AgNPs complexes form at higher Mn2+ concentrations. To identify the formation of a single DNA/AgNPs complex we observed the dark-field and fluorescence images of DNA/AgNPs solutions at each Mn2+ concentration (see photographs above graph in Figure 5). In the absence of Mn2+, the Brownian motion of AgNPs in the presence of DNA (Movie S3 in Supporting Information) is similar to that of free AgNPs. In contrast, in the presence of Mn2+, the AgNPs are bound to single DNA molecules. At 2−4 mM Mn2+, the AgNPs are aligned on single DNA molecules (Supporting Information Movies S1 and S2) and this alignment indicates the formation of DNA/AgNPs complexes. At >10 mM [Mn2+], AgNPs aggregation is dependent on Mn2+ concentration. The aligned AgNPs were observed by both fluorescence and dark-field microscopies. The location of the aligned AgNPs coincides with that of the DNA, directly

(2)

where, R = (Rx, Ry) is the position of the center of mass at time t. Rx and Ry were obtained from video frames of recorded images using Meta Imaging Software (Molecular Devices, U.S.A.). In the analyzed images single DNA molecules or AgNPs was in focus during the observation period of 1 s. Table 1 shows RH of the bound AgNPs compared with free AgNPs and intact single DNA molecules. The RH values of bound AgNPs are between the values of the free AgNPs and single DNA molecules. The value is reasonable, as RH of the bound AgNPs is larger than that of free AgNPs since the AgNPs bind to DNA molecules and is smaller than that of single DNA molecules as the AgNPs induce intramolecular fluctuations in single DNA chains. Interestingly, the RH values 1880

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confirming the existence of DNA/AgNPs complexes. At 2−4 mM [Mn2+], the fluorescence from the DNA molecules was not condensed (Supporting Information Movie S4). Upon aggregation of AgNPs (>10 mM Mn2+), the fluorescence from the DNA molecules also compacted. The [Mn2+] for the noncondensation of DNA/AgNPs complexes exhibits similar value to that for stable complexes from spectroscopic bulk experiments (cf. Figure 1). On the basis of these similarities, we focus on 3 mM [Mn2+] to investigate the properties and dynamics of the DNA/AgNPs complex. We here discuss the mechanism of the formation of DNA/ AgNPs complexes. The trend of decreasing in RH for DNA/ AgNPs/Mn in Figure 5 is similar to that of the shrinkage change for DNA/Mn in the absence of AgNPs (Figure S1 in Supporting Information). Thus, it is expected that Mn2+ binds to DNA with specific interactions and plays a role in neutralizing the DNA charges and compacting the DNA chain over a range of several tens of mM of Mn 2+ concentrations. Indeed, such metal-ion binding to DNA is known using Ag+, whose phenomena is used for Ag coating of DNA through Ag+ bound to DNA.20 The ζ-potential of colloidal AgNPs used in the present study was ∼−60 mV. It is known that both cationic and anionic NPs can form aggregates with DNA and condensing agents, even without specific interactions.21,22 Thus, we consider that Mn2+ reduces the thickness of the electric double layer of both DNA and AgNPs, resulting in an increase in DNA and AgNPs collision frequency and subsequently increase in probability of formation of DNA/ AgNPs complexes. We also quantitatively analyzed the Brownian motion of the AgNPs in the DNA/AgNPs complexes from the dark-field time-resolved images. Figure 6a,b shows the fluctuation in the distance between two AgNPs in the mixture solution of AgNPs and DNA molecules in the absence and presence of Mn2+, respectively. Figure 6c shows the distance d between two AgNPs in the mixture solution of AgNPs and DNA molecules. The d was calculated from each AgNPs’ position of the center of mass at time t. The position of the x−y axis was obtained from recorded video-frame images using imaging software. In the presence of Mn2+, d varies within a confined range, whereas in the absence of Mn2+, d increases with time. As d changes over time, this indicates that the Brownian motion of the AgNPs is restricted with DNA/AgNPs complex formation. Figure 6d shows the histogram of the maximum d values during a 4 s period between two AgNPs with and without Mn2+ (N ∼ 100 for each condition). In the presence of Mn2+, d is less than ∼4.5 μm and fluctuates within a narrow range, indicating that the two AgNPs are tethered to the same DNA molecule. In the absence of Mn2+, the maximum d between two AgNPs exceeds the long-axis length of the T4 DNA molecule, owing to the independent diffusion of each AgNPs through Brownian motion. The spatial expansion W of a semiflexible polymer in good solvent (dashed line in Figure 6e) is theoretically predicted as follows: W ∼ λN3/5, where λ is Kuhn length (100 nm for B-form DNA chain) and N is a segment number of DNA chain.23 The spatial expansion WT4 for T4 DNA molecule in aqueous buffered solution is 4.46 μm. This theoretical value of WT4 is reasonable compared with the experimental values in our study because the fluctuating d of two AgNPs on a single DNA molecule is confined to this WT4 range (Figure 6c,d). These results clearly indicate that two AgNPs can be bound to the same DNA molecule.

Figure 6. Dark-field optical images showing the tracking of two AgNPs in the mixture solution of AgNPs and T4 DNA molecules with/ without Mn2+ (white lines added to indicate d). Sequential images (0.1 s intervals) of the Brownian motion of two AgNPs in the mixture solution of AgNPs and DNA molecules with (a) and without (b) 3 mM Mn2+. Scale bars 2 μm. (c) Temporal fluctuations in the distance between two AgNPs in the mixture solution of AgNPs and DNA molecules with/without Mn2+. (d) Histogram of the distance between two AgNPs in the mixture solution of AgNPs and T4 DNA molecules with and without 3 mM Mn2+. Maximum distance d measured during 4 s. The AgNPs selected for measurement have an initial separation distance of less than 2 μm at 0 s. (e) Schematic representation of a single DNA molecule fluctuating within a confined volume during Brownian motion.

The labeling and imaging of AgNPs on DNA molecules benefits a long time observation that is free from AgNPs photobleaching and blinking. The trajectory motion of a single AgNP on a DNA molecule can be traced for >7 min (Supporting Information Figure S2). AgNPs can be temporarily defocused during the long observation times, however as the AgNPs are extremely bright they can be easily distinguished and their center of mass can be traced. During the long measurement times information of various physical quantities (Brownian motion, thermal fluctuations, and so forth) can be obtained simultaneously from the data. Two AgNPs on the same DNA molecule are detected as a plateau of relative mean square displacement (Supporting Information Figure S3); from the fluctuation of the AgNPs on the same DNA molecule, we estimate the contour distance between two AgNPs along the DNA chain (average d = 1.19 μm corresponds to 26.9 μm in contour distance for Supporting Information Figure S4). In conclusion, we have reported a method for binding uncoated AgNPs on intact DNA molecules in the presence of Mn2+ and utilizing the AgNPs as a probe to obtain time1881

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(2) Yokota, H.; Saito, K.; Yanagida, T. Phys. Rev. Lett. 1998, 80 (20), 4606−4609. (3) Kinosita, K., Jr.; Yasuda, R.; Noji, H.; Ishiwata, S.; Yoshida, M. Cell 1998, 93, 21−24. (4) Eid, J.; Fehr, A.; Gray, J.; Luong, K.; Lyle, J.; Otto, G.; Peluso, P.; Rank, D.; Baybayan, P.; Bettman, B.; et al. Science 2009, 323, 133−138. (5) Yeh, J.-W.; Taloni, A.; Chen, Y.-L.; Chou, C.-F. Nano Lett. 2012, 12, 1597−1602. (6) Hirano, K.; Ichikawa, M.; Ishido, T.; Ishikawa, M.; Baba, Y.; Yoshikawa, K. Nucleic Acids Res. 2012, 40, 284−289. (7) Biju, V.; Itoh, T.; Anas, A.; Sujith, S.; Ishikawa, M. Anal. Bioanal. Chem. 2008, 391, 2469−2495. (8) Bruchez, M. P. Curr. Opin. Chem. Biol. 2011, 15, 775−780. (9) Anas, A.; Akita, H.; Harashima, H.; Itoh, T.; Ishikawa, M.; Biju, V. J. Phys. Chem. B 2008, 112, 10005−10011. (10) Optical Properties of Metal Clusters; Kreibig, U., Vollmer, M., Eds.; Springer Series in Materials Science, Vol. 25; Springer: New York, 1995. (11) Michaels, A. M.; Nirmal, M.; Brus, L. E. J. Am. Chem. Soc. 1999, 121, 9932−9939. (12) Itoh, T.; Asahi, T.; Masuhara, H. Appl. Phys. Lett. 2001, 79, 1667−1669. (13) Sun, L.; Sun, Y.; Xu, F.; Zhang, Y.; Yang, T.; Guo, C.; Liu, Z.; Li, Z. Nanotechnology 2009, 20, 125502. (14) Busson, M. P.; Rolly, B.; Stout, B.; Bonod, N.; Larquet, E.; Polman, A.; Bidault, S. Nano Lett. 2011, 11, 5060−5065. Kim, J. Y.; Lee, J. S. Nano Lett. 2009, 9, 4564−4569. and Lee, J. S.; Lytton-Jean, A. K. R.; Hurst, S. J.; Mirkin, C. A. Nano Lett. 2007, 7, 2112−2115. (15) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86 (17), 3391−3395. (16) Burda, C.; Chen, X.; Narayanan, R.; El-Sayed, M. A. Chem. Rev. 2005, 105, 1025−1102. (17) Mukherjee, B.; Weaver, J. W. Environ. Sci. Technol. 2010, 44 (9), 3332−3338. (18) Yoshida, K.; Itoh, T.; Tamaru, H.; Biju, V.; Ishikawa, M.; Ozaki, Y. Phys. Rev. B 2010, 81, 115406. (19) Anger, P.; Bharadwaj, P.; Novotny, L. Phys. Rev. Lett. 2006, 96, 113002. (20) Patolsky, F.; Weizmann, Y.; Lioubashevski, O.; Willner, I. Angew. Chem., Int. Ed. 2002, 41 (13), 2323−2327. (21) Rudiuk, S.; Yoshikawa, K.; Baigl, D. J. Colloid Interface Sci. 2012, 368 (1), 372−377. (22) Zinchenko, A. A.; Luckel, F.; Yoshikawa, K. Biophys. J. 2007, 92 (4), 1318−1325. (23) Yoshikawa, K.; Yoshikawa, Y. Physical Chemistry of DNACarrier Complexes. In Non-Viral Gene Therapy: Gene Design and Delivery; Taira, K., Kataoka, K., Niidome, T., Eds.; Springer-Verlag: Tokyo, 2005; pp 11−18.

resolved dark-field optical microscopy images. The effectiveness of this method was examined by fluorescence microscopy, SEM, and UV−vis spectroscopy. Because plasmon resonance elastic light scattering is not susceptible to bleaching and blinking, and no radical scavengers or blinking suppression chemicals are needed, the proposed method overcomes the drawbacks of fluorophores and QDs, thereby enabling longer observation times and pH-independent observations. Furthermore, the DNA molecules and AgNPs do not have to undergo biochemical treatment. The plasmonic imaging based on darkfield optical microscopy is also performed using inexpensive instruments relative to fluorescence microscopic imaging because it is does not require high sensitivity CCD camera and fluorescence excitation lamp unit. Using the method, the plasmonic imaging of the AgNPs acquires lack of information on various DNA chain properties such as the local fluctuations inside the DNA chain although ordinary fluorescence imaging would be only analyzed against whole DNA chain. We expect that this study will contribute to investigations of singlemolecule DNA dynamics and DNA/protein interactions. We are currently investigating the possibility of measuring the motion of single DNA chains in detail using smaller plasmonic metal NPs.

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ASSOCIATED CONTENT

S Supporting Information *

The following four figures are provided: (1) conformational changes in single DNA molecules in the presence of [Mn2+], (2) long-time trace of single AgNP trajectory bound to a single DNA molecule, (3) mean square displacement of the relative position of two AgNPs bound to the same DNA molecule, and (4) estimation of distance between two bound AgNPs along DNA molecule. Four movies are also provided: (1) dark-field microscopy observations of unbound AgNPs and (2,3) two types of AgNPs bound to DNA by Mn2+, and (4) fluorescence microscopy observations of a single DNA molecule with bound AgNPs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: (T.I.) [email protected]; (K.H.) hirano-ken@ aist.go.jp. Author Contributions #

T.I. and Y.S.Y. contributed equally to this article.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the Industrial Technology Research Program from the New Energy and Industrial Technology Development Organization (NEDO), Japan. This work was partly supported by “Kiban C (20510111)” from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST) Program”.

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REFERENCES

(1) Yanagida, T.; Nakase, M.; Nishiyama, K.; Oosawa, F. Nature 1984, 307, 58−60. 1882

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