Anal. Chem. 2000, 72, 3678-3681
Single-Molecule Analysis of DNA Immobilized on Microspheres Mark A. Osborne, W. Scott Furey, David Klenerman,* and Shankar Balasubramanian*
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW U.K.
The formation and analysis of single molecules of fluorescently labeled DNA immobilized on polystyrene microspheres is described. Analysis by confocal fluorescence microscopy revealed single-step photobleaching, characteristic of a single fluorophore. Microspheres provide a means of locating single molecules by bright-field microscopy, prior to single-molecule detection. This allows the interrogation of single molecules without suffering the limitations of premature photobleaching. Statistical analysis of fluorescence intensities for >100 microspheres suggests attachment of DNA to micropsheres to be consistent with Poisson statistics. Recent advances in optical detection have lead to the efficient detection of single molecules at ambient temperatures by fluorescence microscopy.1-6 Single-molecule detection (SMD) permits the study of properties of single molecules that include proteins, nucleic acids, and small organic molecules. The approach can also probe the interactions between such molecules in a regime that can resolve molecular detail that is hidden by the analysis of ensembles, which generates only an averaged signal. This paper describes the formation, localization, and interrogation by SMD of one bead-one molecule substrates. Previous studies carried out by SMD have included enzyme-catalyzed reactions,7-9 the dynamics of molecules small and large,10-14 and the detection of binding interactions between molecules.15,16 A significant consideration in single-molecule experiments is the means of displaying * To whom correspondence should be sent: (tel) +44-1223-336347; (fax) +44-1223-336913; (e-mail)
[email protected]. (1) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553-557. (2) Ambrose, W. P.; Goodwin, P. W.; Martin, J. C.; Keller, R. A. Phys. Rev. Lett. 1994, 72, 160-163. (3) Xie, X. S.; Dunn, R. C. Science 1994, 265, 361-364. (4) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018-1021. (5) Schmidt, Th.; Schutz, G. J.; Baumgartner, W.; Gruber, H. J.; Schindler, H. J. Phys. Chem. 1995, 99, 17662-17668. (6) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995, 374, 555-559. (7) Xue, Q.; Yeung, E. S. Nature 1995, 373, 681-683. (8) Craig, D. B.; Arriaga, E. A.; Jerome, J. C. Y.; Wong, C. Y.; Lu, H.; Dovichi, N. J. J. Am. Chem. Soc. 1996, 118, 5245-5253. (9) Lu, H. P.; Xun, L.; Xie, X. S. Science 1998, 282, 1877-1882. (10) Widengren, J.; Mets, U.; Rigler, R. J. Phys. Chem. 1995, 99, 13368-13379. (11) Macklin, J. J.; Trautman, J. K.; Harris, T. D.; Brus, L. E. Science 1996, 272, 255-258. (12) Ha, T.; Enderle, Th.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Phys. Rev. Lett. 1996, 77, 3979-3982. (13) Ying, L.; Xie, S. J. Phys. Chem. 1998, 102, 10399-10409. (14) Ha, T.; Ting, A. Y.; Liang, J.; Caldwell, W. B.; Deniz, A. A.; Chemla, D. S.; Shultz, P. G.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 893-898.
3678 Analytical Chemistry, Vol. 72, No. 15, August 1, 2000
Figure 1. Schematic of the DNA-microsphere construct. A 13-mer with a 5′-biotin link and a single TMR fluorescent label are coupled to a streptavidin-functionalized 0.5-µm polystyrene microsphere. The microsphere is attached to a biotinylated glass cover slip prior to the DNA attachment.
the subject molecule for analysis. Display formats have included simply studying a dilute solution,1,4,10 the localization of molecules in a matrix (or gel),17 constraining solutions to a one molecule/ well format using nanoscopic vials,18 and by adsorption of a dilute solution of molecules onto a surface.2,3,6 Interest in the use of microspheres as a handle for single molecules has grown with the development of flow cytometric DNA analysis techniques. In general, these systems use fragmented DNA of considerable size (72 kbp) attached to beads that are stained with intercalating dyes.19 We present conclusive data showing the attachment of DNA molecules of a single length with a single fluorescent label to follow Poisson statistics. Knowing where single molecules are in a sample (whether on a surface or in solution) before making an observation has advantages. By locating our molecule prior to exposure to laser light, we maximize the observation time and (15) Harada, Y.; Funatsu, T.; Murakami, K.; Nonoyama, Y.; Ishihama, A.; Yanagida, T. Biophys. J. 1999, 76, 709-715. (16) Trabesinger, W.; Shutz, G. J.; Gruber, H. J.; Schindler, H.; Schmidt, T. Anal. Chem. 1999, 71, 279-283. (17) Dickson, R. M.; Norris, D. J.; Tzeng, Y. L.; Moerner, W. E. Science 1996, 274, 966-969. (18) Tan, W. H.; Yeung, E. S. Anal. Chem. 1997, 69, 4242-4248. (19) Shecker, J. A.; Goodwin, P.; Affleck, R. L.; Wu, M.; Martin, J. C.; Jett, J. H.; Keller, R. A.; Harding, J. D. Proc. SPIE-Int. Soc. Opt. Eng. 1995, 2386, 4-12. 10.1021/ac000129r CCC: $19.00
© 2000 American Chemical Society Published on Web 07/01/2000
Figure 2. Time profiles of the fluorescence from two different microspheres each containing a single 13-mer DNA molecule labeled with a single TMR. (a) Continuous fluorescence before a single-step photobleaching characteristic of a single molecule. (b) Fluorescence showing a transient dark state typical of a single fluorophore undergoing rotational or spectral diffusion. The laser was switched on 500 ms after the start of the scan.
fluorescence collection before photobleaching the label. This is particularly important if one wants to synchronize changes in external conditions with the observation of single-molecule fluorescence. While this point has been addressed by Ha and coworkers, it was recognized that the spectroscopic technique they employed in locating molecules relied on selection rules that neglected weakly emitting molecules.20 We have explored the immobilization of single molecules onto microscopic beads that are clearly resolvable by bright-field microscopy allowing singlemolecule location without imposing selection criteria. EXPERIMENTAL SECTION A 13-mer of sequence 5′-TCGCAGCCGUCCA-3′ with a 5′-biotin link and an internal propargylamino uridine base was postsynthetically modified by reaction with TMR succinimide ester.21 A glass cover slip (No. 0, 22 mm × 64 mm, Chance Propper Ltd.) was biotinylated by incubation for 8 h with biotin-BSA conjugate (1 mg/mL, in 0.01 M phosphate-buffered saline (PBS) solution, pH 7.4). A 1-µL drop of streptavidin-functionalized microspheres (Polysciences, Inc.) of 500-nm diameter suspended in 100 mM NaCl (pH 7.4) at 0.1% volume was deposited onto the biotinylated cover slip, incubated for 1 h, and washed in deionized water to remove unbound microspheres. This procedure generated a surface coverage of ∼1 sphere per 10 µm × 10 µm square. At SMD sensitivities, the nonfluorescent microspheres exhibited residual fluorescence at 580 nm which was removed by photobleaching a small area (3 mm2) of beads on the cover slip with a frequency-doubled Nd:YAG laser (1 W, 532 nm) for 2 h prior to coupling the DNA. Control samples of photobleached beads showed no measurable fluorescence. A 50-µL sample of DNA (0.1 pM in 100 mM NaCl, 100 mM Tris, pH 7.4) was deposited over the photobleached microspheres and incubated for 30 min before washing with deionized water to remove unbound DNA. A (20) Ha, T.; Chemla, D. S.; Enderle, Th.; Weiss, S. App. Phys. Lett. 1997, 70, 782-784. (21) Furey, W. S.; Joyce, C. M.; Osborne, M. A.; Klenerman, D.; Peliska, J. A.; Balasubramanian, S. Biochemistry 1998, 37, 2979-2990.
schematic of the construct is shown in Figure 1. The concentrations employed were optimized such that on average there was one DNA molecule per microsphere.22 The beaded array of DNA was analyzed using a modified confocal fluorescence microscope.23 In brief, a high numerical aperture objective lens (NA ) 1.3, 100× oil immersion) is used to obtain a diffraction-limited focus (φ ∼520 nm) of the excitation laser (CW Argon Ion Laser Technology RPC50) and collect fluorescence. Laser scatter is rejected by a dichroic beam splitter (540DRLP, Omega Optics Inc.) before the fluorescence is spatially filtered through a 50-µm pinhole placed in the image plane. The emitted light was finally focused through a band-pass filter (580 ( 15 nm) onto a single-photon avalanche diode detector (SPAD QE ) 60%, EG&G). Data were recorded on a PC-implemented multichannel scalar (MCS-PLUS, EG&G Ortec). The diameter of the microspheres, 500 nm, was chosen to fit within diffractionlimited focus of the microscope. The microspheres provided an optically resolvable marker by which the associated single molecules of DNA could be located prior to exposure to laser light. The microscope was operated in the bright-field mode using lowlevel illumination (
