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Technical Notes
Confinement and Detection of Single Molecules in Submicrometer Channels William A. Lyon and Shuming Nie*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Detection channels with submicrometer dimensions (500600 nm i.d.) have been produced directly on electrophoresis capillaries for confinement and detection of single molecules at room temperature. These restrictive channels markedly reduce the Brownian motion of low molecular weight analytes and allow detailed studies of single molecules in solution. With a confocal fluorescence microscope, single molecules have been observed for an extended period of ∼60 ms, which is 50-100 times longer than the bulk diffusion time. These molecules have also been manipulated by an electrokinetic force and detected on-line with high signal-to-noise ratios. Ultrasensitive measurements at the single-molecule level have potential applications in many research areas, such as chemical analysis, DNA sequencing, molecular dynamics, and nanostructured materials.1-5 Recent advances have allowed the detection, identification, and dynamic study of single molecules in lowtemperature solids,6,7 on dielectric surfaces,8-12 and in roomtemperature liquids.13-16 Current methods for single-molecule detection are primarily based on laser-induced fluorescence (LIF) because of its broad applicability and high sensitivity.17 However, (1) Moerner, W. E. Science 1994, 265, 46-53. Skinner, J. L.; Moerner, W. E. J. Phys. Chem. 1996, 100, 13251-13262. (2) Keller, R. A.; Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Martin, J. C.; Wu, M. Appl. Spectrosc. 1996, 50, 12A-32A. Goodwin, P. M.; Ambrose, W. P.; Keller, R. A. Acc. Chem. Res. 1996, 29, 607-613. (3) Nie, S.; Zare, R. N. Annu. Rev. Biophys. Biomol. Struct. 1997, 26, 565594. (4) Xie, X. S. Acc. Chem. Res. 1996, 29, 598-606. (5) Funatsu, T.; Harada, Y.; Tokunaga, M.; Saito, K.; Yanagida, T. Nature 1995, 374, 555-559. (6) Orbit, M.; Bernard, J.; Personov, R. I. J. Phys. Chem. 1993, 97, 1025610268. (7) Guttler, F.; Irngartinger, T.; Plakhotnik, T.; Renn, A.; Wild, U. P. Chem. Phys. Lett. 1994, 217, 393-397. (8) Betzig, E.; Chichester, R. J. Science 1993, 262, 1422-1425. (9) Xie, X. S.; Dunn, R. C. Science 1994, 265, 361-364. (10) Ambrose, W. P.; Goodwin, P. M.; Martin, J. C.; Keller, R. A. Science 1994, 265, 364-367. Ambrose, W. P.; Goodwin, P. M.; Martin, J. C.; Keller, R. A. Phys. Rev. Lett. 1994, 72, 160-163. (11) Trautman, J. K.; Macklin, J. J.; Brus, L. E.; Betzig, E. Nature 1994, 369, 40-42. Macklin, J. J.; Trautman, J. K.; Harris, T. D.; Brus, L. E. Science 1996, 272, 255-258. (12) Ha, T.; Enderle, Th.; Ogletree, D. F.; Chemla, D. S.; Selvin, P. R.; Weiss, S. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6264-6268. (13) Shera, E. B.; Seitzinger, N. K.; Davis, L. M.; Keller, R. A.; Soper, S. A. Chem. Phys. Lett. 1990, 174, 553-557. (14) Barnes, M. D.; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 1995, 67, 418A423A. (15) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5740-5747. (16) (a) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266, 1018-1021. (b) Nie, S.; Chiu, D. T.; Zare, R. N. Anal. Chem. 1995, 67, 2849-2857.
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the fluorescence emission rate of a single molecule is intrinsically limited by its excited-state lifetime, and optical saturation occurs at high laser intensities. To extract detailed information, it is necessary to interrogate a single molecule for an extended period of time so that enough photons are accumulated. This requirement poses a significant problem for single-molecule studies in solution because the analyte molecules undergo constant Brownian motion. With confocal or evanescent wave excitation, single molecules are detected only for a short transit period (about 0.52.0 ms), during which an analyte molecule diffuses in and out of a small probe volume. We report the use of submicrometer detection channels and confocal fluorescence microscopy for prolonged observation of single molecules in solution at room temperature. Confocal fluorescence detection allows real-time observation of single molecules with ultrahigh sensitivity, while ultrasmall channels provide a spatial confinement effect that can be used to extend the observation time. This work is different from previous approaches for confinement and detection of single molecules in solution. Ramsey and co-workers18 pioneered the use of levitated microdroplets, which allows individual trapped molecules to be interrogated until photobleaching. Moerner and co-workers19 employed the nanometer-scale pores of polyacrylamide gels to reduce Brownian motion; single dye molecules trapped in these pores were imaged in three dimensions with evanescent wave excitation. Xu and Yeung20 combined an intensified CCD camera and evanescent wave excitation to increase the data acquisition rate and observed single-molecule diffusion and photodecomposition in free solution. A novel feature of this work is that detection channels are prepared directly on electrophoresis capillaries, which makes it possible to manipulate the motion of single molecules by electrokinetic or hydrodynamic forces. These channels generally have inner diameters of 500-600 nm and can greatly reduce the Brownian motion of analyte molecules in solution. We demonstrate that single rhodamine 6G (R6G) molecules can be confined in femtoliter-sized volumes for an average time of ∼60 ms. (17) Other methods have also been used for single-molecule detection, including frequency-modulated optical absorption at low temperatures (Moerner, W. E.; Kador, L. Phys. Rev. Lett. 1989, 62, 2535), electrochemistry (Fan, F.-R. F.; Bard, A. J. Science 1995, 267, 871-874), and surface-enhanced Raman scattering (Nie, S.; Emory, S. R. Science 1997, 275, 1102-1106). (18) Barnes, M. D.; Ng, K. C.; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 1993, 65, 2360. (19) Dickson, R. M.; Norris, D. J.; Tzeng, Y.-L.; Moerner, W. E. Science 1996, 274, 966-969. (20) Xu, X. H.; Yeung, E. S. Science 1997, 275, 1106-1109. S0003-2700(97)00074-7 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Schematic diagram of the confocal fluorescence microscope coupled with capillary electrophoresis for single-molecule detection. See text for detailed discussion.
Physical confinement in such small channels opens new opportunities for biochemical and biophysical studies of single molecules. EXPERIMENTAL SECTION Instrumentation. A confocal fluorescence microscope was constructed by using a Nikon Diaphot inverted microscope and a photon-counting avalanche photodiode (APD) for ultralow light detection (Figure 1). This microscope was equipped with a thermoelectrically cooled CCD camera (Princeton Instruments, Trenton, NJ) or a video-rate intensified CCD (ICCD) camera (Photon Tech. Intl., South Brunswick, NJ) for wide-field imaging. The overall design was similar to that of Nie et al.,16 but modifications were made to improve the photon detection efficiency. In particular, an aspheric lens (f ) 11 mm, NA ) 0.25; Thorlabs, Newton, NJ) was used to focus the fluorescence light on the photodiode, and the optical components outside the microscope were made into a single assembly. This compact, detachable assembly is about 20 cm long and contains (i) a 100µm pinhole (Melles Griot, Irvine, CA), (ii) a high-performance optical filter (Omega Optical Inc., Brattleboro, VT), (iii) an aspheric focusing lens, (iv) a photon-counting detector (SPCM200, EG&G, Vaudreuil, Canada), and (v) two XYZ translation
stages (Newport Corp., Irvine, CA). The modified microscope was able to detect 4-5 photons for every 100 photons emitted, as estimated under the experimental conditions of laser irradiance, absorption cross section, and fluorescence quantum yield. The achieved photon detection efficiency was about 4-5%, considerably higher than the 1-2% efficiency reported earlier.16 In dilute R6G ethanol solution, the improved microscope yielded ∼400 photons molecule-1 ms-1, while the previous apparatus detected only ∼100 photons under similar conditions. The use of confocal fluorescence microscopy for singlemolecule detection in electrophoresis capillaries has two additional problems. First, refractive index differences and the capillary shape cause significant light scattering at the capillary inner and outer walls. This problem was solved by immersing the capillary in an index-matching oil (nD ) 1.517; Fluka, Ronkonkoma, NY). However, the laser beam profile inside the capillary could still deviate from the ideal, diffraction-limited case because the indexmatching might not be perfect. We examined this issue by comparing the photon burst widths of single molecules in bulk solution and in a capillary. The width of a photon burst indicates the diffusion time that a single molecule moves across the probe volume and thus provides an estimate of the laser beam size. Our Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
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Figure 2. Microphotographs of submicrometer detection channels produced on electrophoresis capillaries. (A) Schematic illustration of the capillary pulling process. (B) Transmission microphotograph of the tapered region of a pulled capillary. (C) Fluorescence microphotograph of a detection channel. Capillaries were filled with a dilute dye solution for inner diameter measurement. (D) Line plot of the detection channel.
results suggest that the probe volume inside a capillary is on the order of 1-2 fL, only slightly larger than that in bulk solution.16 The second problem arises from the need to isolate the microscope system so that electrical discharges do not occur when high voltages are applied to the capillary. We solved this problem by using an electrically insulated microscope stage. The hydrocarbon immersion oil also helped to prevent electrical discharge at the metal-framed objective. Preparation and Characterization of Tapered Capillaries. Submicrometer detection channels were prepared on electrophoresis capillaries by using a CO2 laser-based micropipet puller (Sutter Instruments, Novato, CA). Unlike the pulling of near-field fiber-optic probes or patch-clamp micropipets,21,22 electrophoresis capillaries (150-µm o.d., 2-, 5-, or 10-µm i.d.) were gently pulled without breaking (Figure 2). Two retaining blocks were placed in the path of the pulling arms to prevent capillary breakage. This manual procedure was not completely reproducible; the usable channels were individually selected by microscopic inspection and capillary electrophoresis current flow. Transmission optical microscopy showed that a pulled capillary had two tapered regions, connected by a relatively long (∼1-2 mm) and uniform channel (Figure 2). To determine the channel inner diameter, we filled the capillary with a dilute R6G solution and obtained fluorescence images with an oil-immersion objective and a CCD camera. A cross-sectional line plot shows that the fluorescence signal spans 6-7 CCD pixels, corresponding to an inner diameter of 500-600 nm. Independent calibration results reveal that approximately 12 detector pixels correspond to 1.0µm physical distance on the sample, with an error of ∼10%. We note that the channel dimensions approach the diffraction limit of far-field optical microscopy, and some narrower or irregular regions could not be resolved. (21) Valaskovic, G. A.; Morrison, G. Appl. Opt. 1995, 34, 1512-1528. (22) Brown, K. T.; Flaming, D. G. Advanced Micropipette Techniques for Cell Physiology; Wiley and Sons: New York, 1986.
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Procedures and Reagents. Capillary electrophoresis (CE) was used primarily as an electrokinetic “pump” to regulate liquid flow in the detection channel.23 Fresh sample solutions of rhodamine 6G (10-8-10-10 M) were prepared in ethanol or 10 mM borate buffer immediately before use via serial dilution from an ethanol stock solution. Similarly, fresh sample solutions of TMR-5-dUTP were prepared in 10 mM borate buffer from the stock solution. Fluorescent labeling of coliphage T4 DNA was achieved by using the intercalating dye POPO-3.24 The DNA was labeled at a ratio of 4 base pairs per dye molecule by mixing an aliquot of DNA sample with a specific volume of freshly prepared 1 × 10-7 M POPO-3 (in 10 mM Tris buffer; diluted from stock solution in dimethyl sulfoxide). A small amount of the DNA mixture was diluted into a solution of 10 mM Tris buffer, to obtain a final concentration of ∼10-10 M DNA. All chemicals and biochemicals used in this work were obtained from commercial sources: rhodamine 6G perchlorate, sodium borate, TRIZMA base (tris[hydroxymethyl]aminomethane), TRIZMA hydrochloride (tris[hydroxymethyl]aminomethane hydrochloride), 2-mercaptoethanol, coliphage T4 DNA, and HPLC grade ethanol from Sigma Chemical Co. (St. Louis, MO); dimethyl sulfoxide (DMSO) from Fisher Scientific (Fair Lawn, NJ); tetramethylrhodamine-5-deoxyuridine-5′-triphosphate (TMR-5-dUTP) and POPO-3 from Molecular Probes (Eugene, OR). Ultrapure water was prepared by a Milli-Q purification system (Millipore, Bedford, MA). RESULTS AND DISCUSSION Fluorescence photon burst data were obtained at millisecond resolution, both in a 2-µm-i.d. electrophoresis capillary and in a pulled detection channel (Figure 3). Similar to confocal detection in bulk solution,15,16 single R6G molecules in the standard capillary yield short photon bursts (0.5-2.0-ms width) as they diffuse across (23) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (24) Glazer, A. N.; Rye, H. S. Nature 1992, 359, 859-861.
Figure 4. Fluorescence signals observed from single rhodaminenucleotide conjugate molecules in a 2-µm-i.d. capillary (A) and in a pulled detection channel (B). The capillary was filled with 10-11 M tetramethylrhodamine-5-deoxyuridine-5′-triphosphate (TMR-5-dUTP) in 10 mM borate buffer. Other conditions as in Figure 3.
Figure 3. Fluorescence photon bursts detected from single R6G molecules in a 2-µm-i.d. capillary (A) and in a pulled detection channel (B). (C) Expanded views of selected detection events. Capillaries were filled with 10-10 M R6G in borate buffer (pH 9.2) by pressure injection and were held in air to stop liquid flow. Blank data were obtained from pure borate buffer solution under otherwise identical conditions. Excitation wavelength, 514.5 nm; laser power, 1 mW; integration time, 1 ms.
the focused laser beam. A drastically different behavior is observed for single R6G molecules in the submicrometer channel. As shown in Figure 3B, each detection event consists of a cluster of fluorescence signals extended over an average time of ∼60 ms. This observation time is about 50-100 times longer than the diffusion time in a standard capillary. Fluctuations in signal intensity indicate that the molecules are not fixed or adsorbed on the capillary wall but are dynamically confined in the channel. The total number of fluorescence photons in each detection event is ∼3000-5000 in water solution and reaches ∼10 000 in ethanol. The end of a detection event signifies that a single molecule either has diffused out of the laser beam (along the channel axis) or has been damaged photochemically. In the latter case, the fluorescence signal drops abruptly to the baseline (see peaks 1 and 5 in Figure 3C). The observed confinement indicates that the effective diffusion coefficient (D) of R6G in the pulled detection channels is about 50-60 times lower than the bulk value (2.8 × 10-6 cm2 s-1).25 This reduction could arise from several factors, such as size restriction, zigzag motion of single analyte molecules, and (25) Rigler, R.; Mets, U.; Widengren, J.; Kask, P. Eur. Biophys. J. 1993, 22, 169-175.
electrostatic interactions of the analyte with the capillary wall. Considering that the detection channels are considerably larger than the size of R6G molecules, steric hindrance or size restriction is unlikely to be important. The hard-sphere theory of restricted diffusion26 also indicates the absence of such an effect. We believe that single R6G molecules make repetitive collisions with the channel wall and are unable to move out of the detection zone. This motion is significantly non-Brownian, because the positively charged R6G molecules are expected to experience an attractive electrostatic force when they diffuse into the electrical double layer of the negatively charged capillary wall. The ζ potential of the silica glass surface is ∼-150 mV, and the thickness of the double layer is about 5-10 nm at the electrolyte concentration used.27 To examine further the effect of electrostatic forces on singlemolecule motion, we studied the behavior of a rhodaminenucleotide molecular conjugate (TMR-5-dUTP). This nucleotide conjugate is negatively charged and should experience repulsive electrostatic interactions and, thus, shorter confinement times. As shown in Figure 4, the average confinement time is only ∼20 ms for the conjugate molecules. Electrostatic forces are also present when a charged molecule approaches a macroscopic glass surface, but the effect is enhanced in small domains by frequent molecule-wall encounters. Indeed, the confinement effect observed in this work is considerably larger than that reported by Xu and Yeung.20 Their real-time video imaging data show that the diffusion coefficients of R6G and a R6G-oligonucleotide conjugate are reduced by 7 and 2.6 times, respectively, when interacting with a flat, negatively charged quartz surface. Moerner and co-workers19 have confined single molecules for much longer periods of time by using polyacrylamide gels. This confinement, however, is likely to arise from size (26) Bean, C. P. In Membrane; Eisenman, G., Ed.; Dekker: New York, 1972; pp 1-54. (27) Rieger, P. H. Electrochemistry, 2nd ed.; Chapman and Hall: New York, 1994; Chapter 2.
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Figure 5. On-line detection of single TMR-5-dUTP molecules flowing across a detection channel by electrokinetic forces. (A) 0 V, no flow; (B) 100 V; (C) 500 V; (D) 1000 V; and (E) 5000 V. Other conditions as in Figure 3.
restriction because polyacrylamide is not charged and the pores have molecular dimensions. These gel pores are not uniform in size or shape, and the physical nature of the pores’ walls is unclear. This work uses much larger channels prepared from silica glass capillaries. These channels have solid walls and can be individually characterized for single-channel measurements. A major advantage of these detection channels is, perhaps, the ability to manipulate the motion of single molecules such as fluorescent dyes and DNA by electrokinetic or hydrodynamic forces. Figure 5 shows on-line detection of single R6G molecules when electrical voltages are applied across the capillary to induce liquid flow. Because of the strong electroosmotic force, single molecules all move across the detection channel in one direction. As the voltage increases, more molecules are detected per second (faster flow speed), but the signal intensities become lower (less interrogation time). Figure 6 shows fluorescence signals detected from single T4 DNA fragments that are fluorescently labeled with an intercalation dye (POPO-3). The observed peaks show an elongated shape and contain some interesting features, such as spikes at one or both ends. This indicates that the T4 DNA molecules are not completely stretched at the flow speed used and may form coils at one or both ends. These preliminary results 3404 Analytical Chemistry, Vol. 69, No. 16, August 15, 1997
Figure 6. (A) Schematic illustration of single DNA fragments moving through a detection channel in an extended conformation under hydrodynamic flow conditions. (B) Fluorescence signals observed from T4 DNA fragments fluorescently labeled with the intercalation dye POPO-3. (C) Expanded views of elongated peaks and fine features. The data integration time was reduced to 0.1 ms. Other conditions as in Figure 3.
demonstrate the potential use of pulled capillaries for on-line, single-molecule studies. CONCLUDING REMARKS We have observed a spatial confinement effect for single molecules in submicrometer channels at room temperature. These detection channels are produced directly on electrophoresis capillaries and could be used for high-efficiency counting of single molecules in solution. When combined with spectroscopic identification, this approach could be useful to the DNA sequencing scheme of Keller et al., in which ordered nucleotide molecules are detected and identified sequentially in a flow stream.28 This work also opens new possibilities for studying the dynamics and reactions of individual biomolecules in solution. For example, a single enzyme molecule may be confined in a femtoliter-sized volume, and its conformational states and chemical reactions are probed with a confocal laser beam with millisecond temporal resolution. The behavior of different enzyme molecules can be studied sequentially by flushing out one molecule and introducing another. Previous studies have only examined the time-averaged (28) Ambrose, W. P.; Goodwin, P. M.; Jett, J. H.; Johnson, M. E.; Martin, L. C.; Marrone, B. L.; Schecker, J. A.; Wilkerson, C. W.; Keller, R. A. Ber. Bunsenges. Phys. Chem. 1993, 97, 1535-1542.
activities of single enzyme molecules.29,30 With further reduction in size and chemical modification to achieve size or charge selectivity, pulled capillaries could be used as artificial ion channels. Electrical measurement of biological ion channels has recently been applied to count and characterize single biopolymers such as poly(ethylene glycol) and nucleic acids.31,32 Singlemolecule detection using artificial nanochannels will be important (29) Xue, Q.; Yeung, E. S. Nature 1995, 373, 681-682. (30) Craig, D. B.; Arriaga, E. A.; Wong, J. C. Y.; Lu, H.; Dovichi, N. J. J. Am. Chem. Soc. 1996, 118, 5245-5253. (31) Berzrukov, S. M.; Vodyanoy, I.; Parsegian, V. A. Nature 1994, 370, 279281. (32) Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 13770-13773.
not only for chemical analysis but also for fundamental studies of molecular transport across biological ion channels or cell membrane pores. ACKNOWLEDGMENT S.N. acknowledges the Whitaker Foundation for a Biomedical Engineering Award and the Beckman Foundation for a Beckman Young Investigator Award. This work was supported by Indiana University Startup Funds. Received for review January 22, 1997. Accepted May 29, 1997.X AC9700742 X
Abstract published in Advance ACS Abstracts, July 1, 1997.
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