Report
Detecting Single Molecules in Liquids
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n important challenge facing analytical chemists today is the efficient detection and identification of single molecules. The ability to make such highly sensitive and selective measurements could lead to enormous breakthroughs in DNA analysis, groundwater monitoring, fluorescence immunoassay, chemical separations, and fundamental studies of physical and chemical phenomena at the single-molecule level. For example, given single-molecule sensitivity, analyte quantitation can be performed in a discrete or "digital" manner, thus greatly reducing initial mass requirements for some of these applications. Over the past few years, several fluorescence-based techniques have evolved with sufficient sensitivity to detect single molecules. The best known of these are the methods developed by Moerner (1,2) and Orrit (3,4), in which single molecules of impurities in cryogenic solids are probed using narrowband dye lasers in
M i c h a e l D. Barnes William B. W h i t t e n J . Michael Ramsey Oak Ridge National Laboratory 418 A
Analytical Chemistry, July 1, 1995
photodiode detection (14). Using fluoresThe ability to identifycence correlation techniques, researchers have demonstrated single-molecule desingle molecules can tection by illuminating a very small vol(~ 1 f L) in a drop of solution susreduce sample size ume pended below the objective of a confocal microscope (15). In a similar setup, conforequirements for cal fluorescence microscopy was recently used to detect single molecules dia variety rectly (16). A technique developed in our laboratory uses either levitated or fallingof applications droplet streams of micrometer-sized
the far wings of an inhomogeneously broadened transition. Recently, several groups have reported detection of single molecules on room-temperature surfaces using near-field microscopy techniques {5-7) that, in addition to being highly sensitive, reveal interesting information on dipole orientation and surface dynamics. Of greatest interest to analytical chemists are techniques for single-molecule detection in liquids. These techniques, based on time-gated detection in a flowing stream, were pioneered by Keller (8-10) and Mathies (11, 12). Recent improvements in Keller's technique include near-IR excitation (13) and avalanche-
droplets (17-19). There are advantages and disadvantages associated with each technique. The principal trade-off is sample throughput versus S/N. The technique of choice for a particular application would of course depend on the priorities of the experiment. In this Report, we discuss the nature of this trade-off by considering the intrinsic sensitivity limitations involved in singlemoleculefluorescencedetection in liquids and give some examples of different detection schemes. Sensitivity limitations
Several factors limit sensitivity in fluorescence detection of single molecules and limit the fraction of molecules in the sam0003-2700/95/0367-418A/$09.00/0 © 1995 American Chemical Society
pie that can actually be detected (molecu lar detection efficiency). The fluores cence process begins with absorption of a photon, which drives the molecule from the ground electronic state S0 to the first excited electronic singlet state Sj (Figure 1). The rate constant for absorption &abs is the product of the absorption cross sec tion σ (cm2) and the laser photon flux F (cm-2 s_1). Absorption rates are usually limited only by the laser intensity. Fol lowing dissipation of a small fraction of the absorbed laser energy in a fast (< 1ΓΓ14 s) nonradiative relaxation step, emission of a fluorescence photon occurs in a few nanoseconds for most ionic dyes. The mol ecule has now returned to the ground electronic state and is prepared for an other absorption-emission cycle. At satu ration excitation intensities, the absorp tion-emission cycle time is limited by the fluorescence lifetime of the molecule so that, for high quantum efficiency dyes, mil lions of fluorescence photons per second may be emitted. An advantage of ionic dyes for singlemolecule detection is that fluorescence quantum efficiencies are near unity. How ever, for some combinations of dye and solvent, singlet-triplet intersystem cross ing rates knr (to T:) can be significant, re sulting in a substantial reduction in fluo rescence count rates. Absorption cross sections for most dyes (~ 10~16 cm2) are typically many orders of magnitude larger than other inelastic light-scattering pro cesses such as Raman scattering. The temporal and spectral properties of fluorescence are also advantageous to single-molecule detection. Fluorescence emission is typically red shifted signifi cantly with respect to the excitation fre quency and is generally a much slower process, occurring in nano- to microsec onds, than Rayleigh or Raman scattering, which occurs on a femtosecond scale. These properties facilitate separation of the signal from background in both the time and frequency domains.
There are several important limitations on the magnitude of the signal that can be obtained from a single molecule. The flu orescence count rate, defined approxi mately by the absorption-emission cycle time, is limited at saturation by the finite fluorescence lifetime lA4spont. In addi tion to limitations imposed by the finite flu orescence lifetime, dyes also have a finite photochemical lifetime that limits the aver age number of absorption-emission cy cles the molecule may undergo before photochemical destruction or "bleaching" takes place. Photobleaching competes with sponta neous emission such that the total num ber of fluorescence photons emitted per molecule is proportional to the spontane ous emission rate divided by the photobleaching rate (20). Thus, even in an ex periment in which the laser-analyte inter action time is essentially infinite, there will be a finite number of fluorescence events. The photochemical lifetime can be ex pressed as the product of the excitation rate k.ahs and the photobleaching quantum yield Φ, which is the probability of photo bleaching per excitation cycle. The photo bleaching quantum yield is a function of
both dye and solvent (21) with typical val ues ranging from ~ 1ΓΓ5 to 10"7. The av erage number of absorption-emission cy cles that the molecule may undergo be fore photobleaching occurs is 1/Φ, or about 106. In the saturation limit (feabs ~ 108/s), the photochemical lifetime is on the order of 10 ms. What matters most, however, is not the signal but the S/N and the magnitude of the background signal. Source noise (from Rayleigh and Raman scattering from the solvent andfluorescencefrom impurities in the solvent) in these types of experi ments usually dominates the background. Fluorescence from impurities can often be minimized by using ultrapure solvents, but preparation and handling of extremely dilute solutions is still not a trivial prob lem. For example, dilute solutions of dye can undergo photodegradation in room light, or dye molecules can stick to con tainer walls. The contribution of Rayleigh and Ra man scattering can also be significant be cause of the finite out-of-band rejection of bandpass filters typically used in singlemolecule detection experiments. Be cause the magnitude of the background signal scales with the probe volume, mini mizing the illuminated volume is critical. As a rule of thumb, solvent scattering places an upper limit on the probe vol ume (~ 10 pL). Because photon counting is universally used for single-molecule detection experi ments, it is important to maximize the photon detection efficiency. Fluorescence emission is isotropic in space; typically only 10-20% is collected by the detection optics. Combined with finite filter trans mission of < 50% and detector quantum efficiency of 10-50%, typical photon detec tion efficiencies range from ~ 0.5 to 5%. Combining the photon detection effi ciency with the finite single-molecule signal (resulting from photobleaching) yields a maximum of a few thousand de tectable fluorescence photons. Analytical Chemistry, July 1, 1995 4 1 9 A
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count distributions are characterized by the average number of signal counts and the noise ab, respectively, molecular detection efficiencies can be quantified for specified values of the average S/N or /ob. Figure 3 shows the dependence of molecular detection probability on average S/N for single-chromophore molecules such as R6G. The threshold for each value of /ob is chosen to minimize the total error probability (false positive + false s, negative). Because the photocount probability distribution for single-chromophore molecules is a decaying exponential, overlap with the background distribution decreases slowly with increasing S/N such that the unit molecular detection efficiency is only an asymptotic limit. Even with an average S/N of 50, the molecular detection efficiency is only about 95%. Figure 1 . Energy-level schematic typical of most solvated ionic dyes. The average S/N therefore provides an upThe dashed line AabB represents excited-state absorption and may be important at high per bound with respect to error probabilintensities. After fast intramolecular relaxation, the molecule may spontaneously radiate ity. Other experimental factors such as laA. , or undergo intersystem crossing /cnr. ser intensity inhomogeneity or molecular diffusion could cause molecular detection efficiencies to be much lower than those Detection criteria tions for single-chromophore molecules given by the average S/N. Detection of a single molecule is usually such as rhodamine 6G (R6G) with a defined by a threshold criterion similar to threshold set at an arbitrary position. The Molecular detection probabilities are the operation of a discriminator circuit probability of spurious detection resultinfluenced by the shape of the signal photothat outputs a logic pulse when the ampli- ing from statistical fluctuations in the back- count distribution as well as the average tude of an (analog) input signal exceeds ground (a false positive) is represented S/N. The photocount statistics associated some predefined threshold level. In a typi- by the dark green area. Likewise, the prob- with fluorescence from a single molecule cal experiment, the threshold level is ability that a true single-molecule event is are highly dependent on the number of inusually ~ 3 times the standard deviation in ignored (a false negative) is represented dependent chromophores (23). Figure 4 the nonfluorescent background signal. by the light green area. shows background and signal photoFor Poisson-distributed (shot-noise limChoosing a high threshold value of 5 or ited) background signals, detection is 6 o b ensures a vanishingly small probabilachieved when the fluorescence signal ity of spurious detection; however, it also generated from the molecule of interest implies that a large fraction of molecules exceeds ~ 3 times the square root of the will not be detected because the signal Background mean background signal. Unfortunately, failed to exceed the threshold. Conmost of the "proof" that a single moleversely, lowering the threshold value incule has been detected in liquids is based creases the fraction of detectable moleon S/N, supported by small probabilicules with a concomitant increase in the ties of more than one molecule occupying probability of error. Thus, the molecular the probe volume in a given measuredetection efficiency, or the probability that ment interval. The relatively small numthe signal amplitude generated from a ber of fluorescence photons emitted from single molecule will exceed the threshold, 0 5 10 molecules in liquids has precluded is related to amplitude probability distriPhotocounts toh definitive tests, such as photon antibunch- butions for the background and signal and Figure 2. Fluorescence and ing, observed in cryogenic solids (22). the extent of overlap between them. background photocount probability Although the selection of a threshold distributions for R6G. value is arbitrary, this setting strongly inMolecular detection efficiency Detection threshold is indicated by the vertical fluences molecular detection efficiencies The overlap between signal and backline. The false-positive error probability is represented by the dark green area, and the and concentration detection limits. Figground photocount distributions affects false-negative error probability is represented ure 2 shows typical signal and backthe probability of molecular detection. Be- by the light green area (which corresponds to ground photocount probability distributhe fraction of molecules undetected). cause the signal and background photo420 A
Analytical Chemistry, July 1, 1995
order of a few nanoseconds, and Rayleigh and Raman scattered light from the sol vent occurs essentially instantaneously. Thus, background radiation and fluores cence are separated in time as well as in frequency. The arrival time distribution for Rayleigh and Raman scattering is mapped by the detector response func tion (typically Gaussian-like with fullwidth half-maximum of about 500-750 ps), and the temporal profile for fluorescence is a decaying exponential (with a typical time constant of τ ~ 3.5 ns) convoluted with the instrument response function. Thus, by choosing a suitable counting win dow in time, the number of background counts can be reduced by rejecting photocathode events that occur outside the de fined window. Time-correlated photon counting in strumentation is used in this technique. An analog voltage pulse with an amplitude proportional to the arrival time is gener ated for each photocathode event. Pulseheight discrimination can then be used to accept or reject the pulse, thereby reduc ing the background signal. The major ad vantage of this technique is speed and high sample throughput. Used with a simple flow cell or flow cytometry appara tus, single molecules can be detected with laser-analyte interaction times on the
—
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molecules of β-phycoerythrin shows welldefined peaks corresponding to one, c φ two, and three molecules. In the case of Ε 0.90single-chromophore molecules, the simi / * Φ c larity between the photocount distribu '•g 0.80tions precludes a clear determination of Φ the number of molecules from the fluores Φ •σ cence burst amplitude. However, if the laS 0.70- ( Ο ser-analyte interactiontimeis long Φ enough to measure the photobleaching ki S 0 60 0 20 40 60 80 netics, it is possible to distinguish be Average S/N tween one or more molecules based on the time dependence of the fluorescence Figure 3. Molecular detection efficiencies for single-chromophore count rate versus time (19). molecules as a function of average Often the laser-analyte interaction time S/N. is much less than the average photo The threshold for each point is chosen to bleaching time and the fluorescence sig minimize the total error (false positive + false nal is limited by the transit time of the negative) probability. molecule through the detection volume. In these cases, usually only the burst ampli tude is measured, which makes the dis count distributions (complete photobleaching) for molecules with 1,2, and 34 tinction between one or two singlechromophore molecules more obscure. independent chromophores, all with the same average S/N. However, the differ The fraction of total signal bursts ex pected from two molecules is the Poisson ence in overlap among the three differ probability of having two molecules in ent signals and background distributions implies very different molecular detection the probe volume divided by the probabil ity of there being one molecule in the probabilities. probe volume—there can be a significant For example, β-phycoerythrin, a large fraction of two-molecule bursts even if the molecule with 34 independent chro mophores, has a detection probability ap probability of there being two molecules proaching unity for a very modest average present is very small. Complications such S/N of 10 whereas the detection probabil as excitation intensity inhomogeneity, ity for R6G is only 0.77 for the same aver molecular diffusion, and other processes can also obscure the distinction between age S/N. The signal photocount distribu one or two molecules. tion also depends on the fraction of molecules that undergo photobleaching (24). In other words, when the laserSome examples analyte interaction time is much less than The experimental techniques that have the average photochemical lifetime, only evolved for single-molecule detection in a fraction of the molecules will undergo liquids share several common characteris photobleaching. However, incomplete tics—they all illuminate and/or image a photobleaching predominantly affects the small volume to minimize background part of the distribution that does not scattering from the solvent, use highoverlap with the background and there numerical aperture objectives to maxi fore does not significantly influence molec mize light collection efficiency, and use ular detection probabilities. some means, such astightspatial and opti cal filtering, to further reduce background signals. Aspects of four of these tech How many? How accurately can a distinction be made niques are examined here. Time-gated detection. This tech among zero, one, two, or more mole cules? Just as molecular detection efficien nique, developed by Keller (9), is based on the fact that the time scales for fluores cies are highly dependent on photocount cence and Raman and Rayleigh scattering statistics, so is the ability to clearly deter mine the number of molecules in a given differ by several orders of magnitude. Fluorescence lifetimes in the visible re probe volume. As demonstrated by Ng (18), the distribution of signal bursts from gion of the spectrum are usually on the & 1-00
0
5 10 15 Photocounts / ch
20
Figure 4. Fluorescence and back ground photocount probability distributions for molecules containing 1 , 2, and 34 independent chromophores. The overlap with the background distribution is much different for the three types of molecules even though the average S/N is the same. Molecular detection efficiencies are much higher for multiple-chromophore molecules than for single-chromophore molecules that have the same average S/N. Analytical Chemistry, July 1, 1995 421 A
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order of a few milliseconds with photon burst amplitudes of ~ 10-20 counts. However, there are some disadvan tages. In a simple flow cell without hydrodynamic focusing, only ~ 0.01% of the analyte molecules are actually interro gated in the probe volume (13). There are also significant detector dead-time limita tions imposed on the single-molecule fluorescence count rate because of the fi nite processing time of time-to-amplitude converters (TAC) used in these experi ments. Typical TAC dead times are about 5 ps, which effectively limits input count rates to < 100 kHz. Some of these problems have been ad dressed (14) by inserting a small-bore cap illary (~ 0.3 μπι diameter) into a sheath flow cuvette just above a focused laser beam. This configuration significantly re duced diffusive losses for sulforhodamine 101 as evidenced by the molecular detection efficiency of 80%, which is close to the theoretical limit based on an average S/N of ~ 10. S/N ratios were also improved by using a subnanosecond anti coincidence gate as a temporal prefilter in front of the TAC to reduce the amount of time spent processing background events. Fluorescence correlation spec troscopy and confocal microscopy. Confocal fluorescence microscopy tech niques (14) are well suited for singlemolecule detection. The high-numerical aperture objective (typically > 1) used for both fluorescence collection and laser beam focusing allows for very efficient light collection and an extremely small illu mination volume of ~ 1 fL, which re sults in small background signals and satu ration intensities (for R6G) that can be reached with average powers < 1 mW. In fluorescence correlation spectros copy (25,26), a confocal microscope mea sures average fluctuations in the fluores cence signal using digital autocorrelation analysis of time-resolved fluorescence sig nals (27). The benefit of this type of ap proach is that the autocorrelation function carries information on diffusion con stants that can be related to the size of the analyte (i.e., distinguish a small polypep tide from a large protein or DNA frag ment). Zare (16) recently showed that single-molecule detection can also be ac complished directly using confocal fluores cence microscopy without autocorrela 422 A
Analytical Chemistry, July 1, 1995
tion analysis. S/N ratios of ~ 10 against a shot-noise limited background of ~ 6 counts were obtained for R6G and fluores cein in a 1-ms integration period. From the standpoint of ultradilute solu tion analysis, perhaps the biggest limita tion of confocal fluorescence microscopy is the extremely small imaging volume. In a typical experimental configuration, a drop of solution is placed on a microscope slide and the number of photon bursts per unit time is controlled by diffusion of the analyte into and out of the illumination region. If a molecule does not undergo photobleaching, it can return several times to the laser beam, producing more than one photon burst. Thus, the number of photon bursts may exceed the actual num ber of individual molecules interrogated. Furthermore, because of the small probe
bleaching time, providing the maximum possible signal. Third, it has been shown that the radiative properties, both in the frequency and time domains, of atoms or molecules may be significantly modified through cavity quantum electrodynamic effects in microdroplets (29,31). In partic ular, both the spontaneous emission rate and integrated fluorescence yield for R6G are significantly enhanced in 4-pm glyc erol droplets (31). Thus, the microdroplet approach offers several advantages in terms of the total single-molecule signal level. In earlier work (19), single R6G mole cules were detected in levitated glycerol microdroplets with S/N ratios of 10-40 and the integrated fluorescence signals ranged from ~ 150 to 2200 photons us ing continuous wave (cw) laser excitation and ordinary photomultiplier tube detec tion. The distribution of signals agreed well with theoretical predictions. Much of the sensitivity associated with the levi tated droplet technique, however, is ob tained at the expense of speed. The time required for lévitation, fluorescence measurement, and size determination is typically several minutes, making the approach impractical for applications that demand high throughput. In particular, analysis of ultradilute solutions using digital molecular detection techniques (18) requires ~ 104-106 measurements. A high-speed version of microdroplet fluorometry is therefore highly desirable. dimensions, confocal microscopy is not well suited for applications involving flow To improve the throughput capacity of ing sample streams, such as CE, where microdroplet fluorescence, we have develthe capillary dimension perpendicular to oped instrumentation for single-molethe flow direction is typically > 10 pm, al cule detection in a stream of free-falling lowing a large fraction of analyte mole droplets. Droplets fall through the excules to pass undetected. tended cavity of a high-power Ar+ laser, Microdroplets. The microdroplet ap leaving the tip of the droplet generator at proach to single-molecule detection in liq ~ 1 m/s. Because of this high speed, a large beam width to maximize laseruids is unique in several respects. First, the 1-pL probe volume is defined by the analyte interaction time and extremely high average power to maximize the fluodroplet instead of by the laser beam, which means that, assuming negligible va rescence count rate are required. Average cw powers of ~ 20 kW and a beam width por pressure, analyte molecules are con fined to the probe region without diffusive of ~ 1.5 mm are required to saturate the S0->Si transition for R6G. losses, every molecule present can be in terrogated, and the molecular detection ef Typically, the average intracavity ficiency is limited only by the average power for an Ar+ laser generating 8 W at S/N. Second, by levitating droplets in an 514.5 nm is only about 150 W. We added a electrodynamic trap (28), the laser- custom high reflector with a transmisanalyte interaction time can be made sion of 5 χ 10~5, resulting in a maximum cw much larger than the average photopower of ~ 10 kW. Without forming a
The microdroplet approach offers several advantages in terms of the total single-molecule signal level.
waist inside the cavity, we can generate in tensities at the droplet of ~ 300-500 kW/ cm 2 . An advantage of high-intensity cw ex citation is that fluorescence count rates are limited only by the fluorescence life time of the analyte. In addition, the inten sity distribution within the droplet gener ated by standing-wave excitation becomes much more uniform than the internal in tensity distribution associated with travel ing-wave excitation. In the current con figuration, we have determined a detec tion limit for R6G of ~ 10 molecules. Improvements currently being made will allow us to reach the single-molecule de tection level with rates of 5-10 kHz. Down the road The past several years have seen great ad vances in both instrumentation and meth odology for single-molecule detection in liquids. Application of these techniques is now moving into areas such as DNA frag ment sizing (34) and CE (35). Future re search will most likely concentrate on increasing speed and sensitivity to pro vide efficient discrimination among differ ent types of molecules and to lower con centration detection limits below the femtomolar level.
(13) Soper, S. A; Mattingly, Q. L.; Vegunta, P. Anal. Chem. 1993, 65, 740. (14) Li, L. Q.; Davis, L. M. Rev. Sci. Instrum. 1993, 64,1524. (15) Eigen, M.; Rigler, R. Proc. Natl. Acad. Sci. USA 1994, 91,5740. (16) Nie, S.; Chiu, D. T.; Zare, R. N. Science 1994, 266,1018. (17) Whitten, W. B.; Ramsey, J. M.; Arnold, S.; Bronk, B. V. Anal. Chem. 1991, 63,1027. (18) Ng, Κ C; Whitten, W. B.; Arnold, S.; Ram sey, J. M. Anal. Chem. 1992, 64,2914. (19) Barnes, M. D.; Ng, Κ C; Whitten, W. B.; Ramsey, J. M. Anal. Chem. 1993, 65, 2360. (20) Hirschfeld, T. Appl. Opt. 1976,15,3135. (21) Soper, S. Α.; Nutter, H. L.; Keller, R. A; Davis, L. M.; Shera, Ε. Β. Photochem. Photobiol. 1993,57,972. (22) Basché, T.; Moerner, W. E.; Orrit, M.; Talon, H. Phys. Rev. Lett. 1992, 69,1516. (23) Whitten, W. B.; Ramsey, J. M. Appl. Spectrosc. 1992, 46,1587. (24) Kollner, U.Appl. Opt. 1993,32,806. (25) Magde, D.; Elson, E.; Webb, W. W. Phys. Rev. Lett. 1972,29,705. (26) Schneider, M. B.; Webb, W. W. Appl. Opt. 1981,20,1382. (27) Koppel, D. E. Phys. Rev. A 1974,10,1938. (28) Arnold, S.; Folan, L. M. Rev. Sci. Instrum. 1986,57, 2250. (29) Barnes, M. D.; Whitten, W. B.; Arnold, S.; Ramsey, J. M.J. Chem. Phys. 1992, 97, 7842.
(30) Benner, R. E.; Barber, P. W.; Owen, J. F.; Chang, R. K. Phys. Rev. Lett. 1980, 44, 475. (31) Barnes, M. D.; Whitten, W. B.; Ramsey, J. M. / Opt. Soc. Am. Β 1994,11,1297. (32) Barnes, M. D.; Whitten, W. B.; Ramsey, J. M. Chem. Phys. Lett. 1994,227,628. (33) Lin, H-B.; Eversole, J. D.; Merrit, C. D.; Campillo, A. J. Phys. Rev. A 1992, 45, 6756. (34) Castro, Α.; Fairfield, F. R.; Shera, Ε. Β. Anal. Chem. 1993, 65,849. (35) Lee, Y-H.; Maus, R. G.; Smith, B. W.; Winefordner.J. O.Anal. Chem. 1994,66, 4142. Michael D. Barnes, staff scientist, conducts research on ultrasensitive fluorescence de tection and photophysics of microparticles. William B. Whitten, senior staff scientist, fo cuses on applying microparticle techniques to chemical analysis. J. Michael Ramsey, group leader, conducts research on minia ture chemical instrumentation, nonlinear spectroscopies, real-time microparticle characterization, and ultrasensitive laserbased detection techniques. Address corre spondence to Ramsey at the Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, 77V 37831-6142.
This research was sponsored by the U.S. De partment of Energy, Office of Basic Energy Sci ences and Office of Research and Develop ment with Martin Marietta Energy Systems. References (1) Moerner, W. E. Science 1994,265,46. (2) Moerner, W. E.; Kadar, L. Anal. Chem. 1989, 61,1217 A. (3) Orrit, M.; Bernard, J. Phys. Rev. Lett. 1990, 65,2716. (4) Orrit, M.; Bernard, J.; Personov, R.J. Phys. Chem. 1993,97,10256. (5) Betzig, E.; Chichester, R. J. Science 1993, 262,1422. (6) Xie, X. S.; Dunn, R. C. Science 1994,265, 361. (7) Ambrose, W. P.; Goodwin, P. M.; Martin, J. C; Keller, R. A. Phys. Rev. Lett. 1994, 72, 160. (8) Nguyen, D. C; Keller, R. A. Anal. Chem. 1987,59,2158. (9) Shera, Ε. Β.; Seitzinger, Ν. Κ; Davis, L. M.; Keller, R. Α.; Soper, S. A. Chem. Phys. Lett. 1990,774,553. (10) Wilkerson, C. W.; Goodwin, P. M.; Am brose, W. P.; Martin, J. C; Keller, R. A Appl. Phys. Lett. 1993, 62,2030. (11) Peck, K; Stryer, L; Glazer, A N.; Mathies, R. A Proc. Natl. Acad. Set. USA 1989,86, 4087. (12) Mathies, R. A; Peck, K; Stryer, L. Anal. Chem. 1990, 62,1786.
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