Anal. Chem. 1994, 66, 4142-4149
Laser-Induced Fluorescence Detection of a Single Molecule in a Capillary Yuan-Hsiang Lee, Russell G. Maus, Ben W. Smith, and James D. Winefordner* Department of Chemistry, University of Florida, Gainesville, Florida 3261 1-7200
The objective of this work is to develop a novel laserexcited spectrometric method to reliably detect a single molecule in a flowing stream which is confined in a capillary. An argon ion-pumpedTi-sapphire laser tuned to a rubidium atomic transition line at 780.023 nm is used to excite the fluorescence of IR140 molecules in a capillary. A focused laser beam illuminates the whole capillary inner diameter to achieve near unity spatial probing efficiency. A rubidium metal vapor filter, based on the resonance absorption of an atomic metal vapor, absorbs the laser specular scatter from the capillary while passing the molecular fluorescence. The photoelectron signal is measured during the transit time of a single molecule through the 1.05-pL probe volume. A digital weighted quadratic summing filter is used to extract the individual photon bursts from single molecules. The measurement efficiency for the near-infrared dye IR140 is estimated to be near unity. The average number of photoelectronsfrom a single Et140 molecule is estimated to be 5.9, 7.0, and 8.5 for laser powers of 50, 100, and 150 mW, respectively. Detection of a single molecule in a flowing stream has been obtained with a variety of experimental schemes, all involving efficient laser excitation and fluorescent d e t e ~ t i 0 n . l ~As~ a molecule passes through a focused laser beam, it is repeatedly cycled between the ground electronic state and the excited electronic state with the emission of a photon on each cycle, producing a photon burst. This paper addresses an important concept in single-molecule detection, which is how to improve the efficiency of detecting all the molecules in a sample in contrast to just those that pass through the probe volume. In previous experiments for singlemolecule detection, most molecules did not pass through the excitation laser beam and as a result the overall detection efficiency was low. Our unique approach is to confine the molecules in a capillary tube, to irradiate the entire internal diameter of the capillary with the excitation laser beam, and to absorb the laser specular scatter with a metal vapor filter. Sensitive fluorescence detection in solution is limited by the background noise sources from the optics and the solvent. The central problem of isolating the molecular fluorescence from background luminescence and Raman scatter is overcome by reducing the probe volume to the smallest possible size. The (1) Shera, E. B.; Seitzinger, N. K; Davis, L. M.; Keller, R. A.; Soper, S. A Chem. Phjis. Lett. 1990,174, 553-557. (2) Whitten. W. B.; Ramsey, J. M.: Amold, S.; Bronk, B. V.Anal. Chem. 1991, 63, 1027-1031. (3) Soper, S. A,; Mattingly, Q, L.: Vegunta, P.Anal. Chem. 1993,65,740-747.
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reduction in the probe volume can be achieved by optically restricting the illuminated and/or observed volume, by physically decreasing the size of sample confinement, or by a combination of these techniques. Various experimental schemes, such as sheath flow cell and electrodynamic levitated microdroplets, have been applied as the sample confinement for single-molecule detection. By flowing the molecules in a sample stream, Keller et al.' utilized laser-induced fluorescence (LIF) to observe the photon burst from a single molecule. With an electrodynamic trap to confine a single molecule in a microdroplet, Ramsey et aL2were also able to detect a single molecule with a signal-to-noise ratio of 3. Soper et aL3have demonstrated the detection of near-infrared (near-IR) molecules (IR132) traveling through a focused Gaussian laser beam with 97% detection efficiency. Using a very simple capillary-based experimental system, we have used a diode laser to induce fluorescence of near-infrared fluorophors in a flowing stream and have detected 103-104 molecules in a single p r ~ b i n g . ~ , ~ With the development of more versatile laser sources, this capillary-based scheme might be easily applied to practical analytical detection in such methods as capillary zone electrophoresis and capillary liquid chromatography or in any analytical technique involving an extremely small sample volume. However, detection of a single molecule with near 100% measurement efficiency has not been achieved because of the need to decrease the spatial probing efficiency to a value considerably below unity in order to minimize the laser scatter and background noise. In this work, the emphasis is placed on the reliable measurement of a single molecule in a capillary. A major advantage of this approach is that the capillary will facilitate the analysis of extremely small volumes of solution. Furthermore, the extremely small physical dimensions encountered in a capillary column also require that detection be performed on-column, which excludes the utilization of sheathed flow cells and other types of external flow cells. In addition, the use of a near-IR laser as an excitation source for laser-induced fluorescence is attractive since absorption is negligible for almost every type of matter, such as flow cell material, solvent, and most impurities. This is advantageous because background fluorescence from these materials does not impose limitations on achieving a low detection limit. However, studies at the single-molecule level using a quartz ultramicrocapillary tube for sample confinement have not been done, in large part because of the difficulty in discriminating against the high level of laser specular scatter caused by the (4) Johnson, P. A,; Barber, T. E.; Smith, B. W.; Winefordner, J. D. Anal. Chem. 1989,61, 861-863. (5) Lehotay, S. J.; Johnson, P. A; Barber, T. E.; Winefordner, J. D. Appl. Spectrosc. 1990,44, 1577-1579. 0003-2700/94/0366-4142$04.5010 0 1994 American Chemical Society
capillary. Rejection of laser specular scatter has been one of the associated with a /3 - chance of false negative detection (mistaking the presence of analyte .for the blank). Thus, these two a p most important considerations for ultratrace analysis using LIF. An ideal optical filter for laser specular scatter would have to show proaches to detection limit (LOD and LOG) are figure of merits nearly full absorption at the laser wavelength while maintaining describing the detection power of an analytical method. The value transparency over the luminescence band. The metal vapor filter of the detection limit indicates the level of analyte that is discerned from the background noise. If measured carefully, the LOD and (MVF) is a promising optical filter for rejecting narrow-band laser specular scatter based on the resonance absorption of an atomic LOG can give an objective comparison of the detection power of vapor. The MVF is simply a heated quartz cell with parallel different analytical methods. The LOD has a low probability of windows containing an inert gas and a volatile metal with a false positives (a, type I error), 0.14%,but a high probability of resonance atomic absorption line at the laser wavelength. This false negatives @, type I1 error), 50%. The LOG is a more MVF, depending on its composition, is heated to an appropriate conservative analytical figure of merit which has very low temperature in order to produce a sufticient number density of probability of a false positives (a,type I error), below 0.14%,and metal atoms to absorb efficiently all detectable laser specular a low probability of a false negatives @, type I1 error), -0.14%. scatter. Therefore, the metal atoms in the MVF provide a nearly This means that when the analyte is present in the sample at the perfect optical filter for laser specular scatter as long as the laser LOD value, fully 50%of the measurement will fall below x d , even can be tuned to the metal vapor absorption peak and the laser though there is only a 0.14%probability that the measurement of line width is narrower than the absorption bandwidth of the MVF. a blank will give a false positive. On the other hand, if the analyte Compared with conventional laser line rejection filters, such as a is present at the LOG value, measurement of the analyte will be monochromator and notch filter, MVFs offer such advantages as always detected above x d at the 99.86%confidence level. low cost, high absorbance, large signal collection solid angle, The first theoretical treatment for the concept of detection limit narrow absorption bandwidth, and high signal throughput. Rewas developed by Alkemade.gJo In terms of intrinsic and extrinsic detection limits, Winefordner et a1.11,12and Stevenson and cently, several MVFs have been demonstrated to be effective in have shown theoretically that LIF is the only eliminating laser specular scattering for Raman s p e c t r o ~ c o p y . ~ ~ ~Winef~rdner’~-’~ The intent of this study is to develop a novel LIF spectroscopic viable approach for singlemolecule detection. Methods that have technique to reliably detect a single near-IR molecule (IR140) achieved singlemolecule detection using LIF are usually based flowing through a small probe volume within a capillary. The laser on a “cyclic”interaction of molecule and laser, and each molecule is tuned to the rubidium atomic transition line at 780.023 nm and produces a relatively large number of photons during its interfocused onto the uncoated part of a capillary. A heated rubidium action with the laser. An ideal technique for singlemolecule metal vapor filter absorbs the laser specular scatter while passing detection will meet the following demands simultaneously: (i) the molecular fluorescence. The excitation source is a TiIntrinsic noise dominates extrinsic noise. (ii) The efficiency of sapphire ring laser with a line width narrower than the rubidium detection, Ed, and the efficiency of measurement, cm, are both unity. atomic absorption bandwidth. The detector is a singlephoton Noise in a measurement can be classfied as either extrinsic or intrinsic. Extrinsic noise is the excess noise that arises owing avalanche photodiode with high photon detection efficiency and low dark count rate. Because of the importance of the metal vapor to a nonspecific background signal that is present even in the filter, the transmission characteristic of the Rb MVF is evaluated absence of analyte. Extrinsic noise sources are determined by theoretically and experimentally. A weighted quadratic summing such instrumental factors as dark current, source light scatter, background emission from concomitants, and nonselective detecfilter is applied to extract the photon bursts from individual molecules as they pass through the laser beam. Detection of a tion of molecules in blank. Intrinsic noise represents the ultimate single molecule in a capillary with high measurement efficiency limit of an analytical method and is determined by the statistical is demonstrated. nature of the measurement process, such as the discrete nature of matter, fluctuations in the low number of analytes in the probe volume, and shot noise in analyte signal production. As intrinsic CONCEPT OF SINGLE MOLECULE DETECTION The ability to detect single molecules is largely governed by noise arises from the detection of analytes, it cannot be removed. the signal-to-noise ratio (S/N) of the measurement. Kaiser has In a counting measurement, it may be possible to reduce all developed a statistical approach for the detection limit, which is extrinsic noise sources to the extent that the probability of defined as the concentration or amount of an analyte that gives a registering one or more counts from the blank is negligible during S/N of m,where m is the statistical confidence level! The limit the sampling interval. For such an intrinsic noise limit measure of detection (LOD, m = 3) corresponds to a S/N = 3, and the ment, the only noise on the signal is due to the statistical nature limit of guarantee (LOG, m = 6) to a S/N = 6. The reciprocal of of the analyte detection process itself. Signal noise in an extrinsic S/N times 100 is the percentage relative standard deviation (% noise limited experiment is due to contributions from both RSD) of the measurement. It is assumed that the noise is the extrinsic and intrinsic noise sources. same on the blank and on the analyte measurements near the Alkemade defined the detection efficiency as the probability detection limit. The limit of detection X d is associated with an a that any given species appearing in the probe volume produces - chance of false positive detection (mistaking a blank measure(9) Alkemade, C. Th.]. Appl. Spectrosc. 1981,35, 1-14. ment for the analyte). The analyte is present at a concentration (10) Alkamade, C. Th. In Analytical Applications of h e n ; Pipmeier, E. H., Ed.; or amount that gives a signal at the limit of guarantee X, that is Wileyhterscience: New York, 1986; Chapter 4. (6) Indralingam, R; Smeonsson, J. B.; Petrucci, G. A; Smith, B. W.; Winefordner, J. D.Anal. Chem. 1992,64,964-967. (7) Pelletier, M. J. APPI. Spectrosc. 1993,47,69-74. (8) Kaiser, H. Two Papen on the Limit of Detection of a Complete Analytical Procedure; Jafner Publishing: New York, 1969.
(11) Winefordner, J. D.; Stevenson, C. Spectrochim. Acta 1993,44B,757-767. (12)Winefordner, J. D.; Petrucci, G. A; Stevenson, C. L.; Smith, B. W. j.Anal. At. Spectrom. 1994,9, 131-143. (13) Stevenson C. L.; Winefordner, J. D. Appl. Spectrosc. 1992,46,715-724. (14) Stevenson, C. L.; Winefordner, J. D. Appl. Spectrosc. 1992,46,407-419. (15) Stevenson, C. L.; Winefordner, J. D. Appl. Spectrosc. 1991,45,1217-1224.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
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Table 1. Required Sensitivities for Limit of Detection (LOD) and Limit of Guarantee (LOG)
mean blank,
xb (counts) 0.00 0.05 0.25 1.00 5.00 10.00 100.00
mean sensitivity,XI (counts) LOD = 1 molecule LOG = 1 molecule a c 0.0014 /3 = 0.0014 1 2 4 5 9 12 32
6.6 8.9 12.4 15 23 29 69
an event during the transit time?,1° The probe volume was determined by the geometry of the (intersecting) laser beam and by the part of the irradiated region that is “seen”by the detector. In this approach, the transit time was determined by the pulse duration or the width of the time gate in case a time-gating circuit is applied, whichever is the shorter. Multiple probings were obtained by firing many laser shots. In addition, Alkemade was concerned only with those cases when there were no extrinsic noise sources. Winefordner et a1.11J2and Stevenson and Winefordner13-15have developed a theory to take into account the transit time of a single molecule within the probe volume and the presence of extrinsic noise sources. When compared with Alkemade’s concepts, Winefordner’s are more general in two important aspects: (1) Detection efficiency is defined with respect to a transit time of the molecules in the probe volume, not simply over a single “probing” of the laser pulse. (2) Detection efficiency considers the need to detect the signal due to analytes over the extrinsic noise. Therefore, the detection efficiency is defined as the probability that a given analyte appearing in the probe volume produces a signal that is detected above the background noise (if any) during the transit time of the analytes within the observation region. Table 1gives the measurement sensitivity, Xt, required to detect single molecules by using LIF at various mean blank signal levels (a 5 0.0014 for LOD and 6, x 0.0014 for LOG).11J2 For example, at the intrinsic noise limit (Xb = 0), values of Xt = 1count molecule-’ and XI = 6.6 counts molecule-’ are needed to detect a single molecule for the LOD and LOG, respectively. This means that, in order to detect every molecule that crosses the laser with 99.86% confidence (LOG), each molecule must emit enough photons so that, on average, 6.6 counts are registered for the molecule by the detector. As the background level increases, e.g., to 1count, the number of analyte counts from a single molecule above the blank increases to 5 counts molecule-’ (total 5 1 counts molecule-’) at the LOD and 15 counts molecule-’ (total 15 1 counts molecule-’) at the LOG. The overall measurement efficiency, em, is defined as the probability that a given analyte in the sample is detected above the background noise in the observation region. The measurement efficiency, E, is related to the detection efficiency, Ed, by the following equations:
+
Em = E$PEd Ep
+
(1)
=
where ET is the transfer efficiency of the sample from the point of 4144
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
introduction to the probing region and E P is the probing efficiency, which is composed of a spatial probing efficiency, est and a temporal probing efficiency, et. The spatial probing efficiency, es, accounts for the loss of analyte species not passing through the laser excitation and/or observation region. The temporal probing efficiency, Et, is unity for a continuous wave source or a pulsed source that is at a repetition rate sufficiently high such that each analyte interacts with at least one laser pulse while passing through the observation region. Unlike Ed, the value of is related to analyte concentration in the sample and may be less than unity even if Ed = 100%since E,,, accounts for analyte losses between the sample and the detection region and for insufficient spatial and temporal probing of analytes. Even if single-molecule detection with high detection efficiency has been achieved in the sheath flow cell, the measurement efficiency may be far less than unity. For example, a sample stream flowing through a square flow cell has been used to minimize the laser specular scatter caused by the optical materials.16-19 However, the laser beam only probed a small portion of the sample stream to reduce the Raman and background scatter from the solvent and the optical materials. A spatial filter placed before the detector only imaged a small region of probe volume to reduce the amount of laser scatter generated at the air-glass interface. Therefore, the spatial probing efficiency for single-molecule detection in a sheathed flow cell was very low, which means that only a fraction of those molecules passing through the flow cell were detected even though the detection efficiency was near unity. In this work, the laser beam illuminates the whole sample stream, which is confined in a capillary, as compared to many of the previous studies involving sheathed flow cells, where the spatial probing efficiency was much less than unity. Thus, every molecule passing through the capillary flow cell in our case should be detected with near-unity measurement efficiency (~“3. EXPERIMENTAL SECTION
Instrumentation. A schematic diagram for the singlemolecule detection apparatus is shown in Figure 1. All lines output from an argon ion laser (Spectra Physics 2060, Mountain View, CA) are used to pump a single-mode, tunable, continuouswave titanium-sapphire laser in the ring configuration (Schwartz Electro-optics, Inc., Orlando, FL). The laser spectral line width is less than 10 M H Z . ~A~weak, broad-band background fluorescence from the Ti-sapphire crystal is further attenuated with a narrow-band pass interference filter (0.18 nm fwhm at 780.1 nm, Omega Optical, Brattleboro, VT). The sample is contained in a fused silica capillary (inner diameter 11 pm, outer diameter 139 pm, and length 10 cm; Polymicro Technology, Phoenix, AZ), and flow is controlled by a mechanical microliter syringe pump (Harvard Apparatus 975, South Natick, MA). From the known volume of solution introduced with the gas-tight microliter syringe (16) Dovichi, N. J.; Martin, J. C.: Jett, J. H.; Trkula. M.; Keller, R. A Anal. Chem. 1984, 56, 348-354. (17) Nguyen, D. C.: Keller, R. A.; Jett. J. H.; Martin, J. C. Anal. Chem. 1987,59, 2158-2161. (18) Nguyen, D. C.; Keller, R. A.; Trkula, M.J, Opt. SOC. Am. B 1987, 4, 138143. (19) Jett, J. H.; Keller, R. A.; Martin, J. C.; Marrone. B. L.: Moyzis, R IC;Ratliff, R. L.: Seitzinger, V. IC; Shera, E. B.: Stewart, C. C. J , Biomol. Struct., Dyn, 1989, 7, 301-309. (20) Schwartz Electro-optics, Inc.. Titanium-sapphire tunable lasers data sheet, 1990.
Mirror
9
1 1 Counter Timer
830 nm IF
[\71
1
w
u
Capilla~ ~
Beam
Ti:Sapphire
780 nm IF
lF4nterference Filter
FOCUS
Computer
I
I I
Syringe Pump
Beam Dump
I
Lens
MVF-Metal Vapor Filter
Figure 1. Experimental arrangement for detecting a single molecule in a capillary. Heated N2
and time of introduction, the measured volume flow rate is found to be very close to the value specified by the manufacturer. To obtain a stable slow flow rate, a 30-min waiting time is normal before making the measurement. A "fluorescence window" is created by removing a section of the polyimide coating on the capillary with concentrated sulfuric acid at 100 "C. The laser is directed through a 20 x beam expander (Melles Griot, Irvine, CA) and focused by a camera lens (1:1.4,f= 55 mm) onto the uncoated part of the capillary. By translating a razor blade across the laser beam with a microtranslation stage (Newport 461-XY-M, Irvine, CA) and monitoring the laser power on a photodiode, the l/e2 laser beam diameter on the capillary is determined to be 11pm. Since the focused laser beam and the capillary inner diameter are the same, the system is very sensitive to changes in the optical alignment. As an approximation, the probe volume is defined as a cylinder whose radius is equal to the capillary inner radius and whose height is equal to twice the radius of the focused laser beam. From the capillary inner diameter and laser beam diameter, the probe volume is estimated to be 1.05 pL. The fluorescence and the laser scatter are collected at a right angle by using a microscope objective (40 x 0.65, Melles Griot, Irvine, CA) and passed through the Rb MVF. For the absorbance measurements of the Rb MVF at different temperatures, the capillary is replaced with a mirror at 45O in order to direct the laser beam through the Rb MVF. The commercially available metal vapor cell (Opthos, Rockville, MD) consisted of a quartz cylinder (length 10 cm, diameter 2.5 cm) with parallel quartz windows on both ends. An excess of rubidium metal (500 mg) was distilled into the evacuated cell such that sufficient metal would be present to give saturated metal vapor at all temperatures used in this work. Nitrogen gas at 200 Torr (at room temperature) was used as a fill gas in order to quench the atomic resonance fluorescence and broaden the absorption bandwidth of the Rb MVF. To avoid window discoloring due to the attack of hot Rb metal vapor, the Rb MVF is held inside a cylindrical forced hot nitrogen heating device, as shown in Figure 2. The nitrogen, which is heated by a furnace (Lindberg 55035, Watertown, WI) whose temperature is controlled to within
F!"3l .
Win
Figure 2. Cutaway view for the design of rubidium metal vapor filter.
f l "C, flows past both windows and recycles back to the body of the MVF. In this way, the flat window faces of the MVF are kept warmer than the center of the MVF, preventing metal vapor from condensing onto the windows, which would reduce the signal throughput and the transmittance of the MVF. The Rb MVF used in this work has functioned flawlessly for over 1year. A bandpass interference filter (10 nm fwhm at 830 nm, Corion, Holliston, MA) is placed after the Rb MVF to block the Raman scattering at 860 nm from the solvent.21 With a second microscope objective (40 x 0.65, Melles Griot, Irvine, CA), the output from the Rb MVF is then refocused onto a fiber pigtail (100-pm core diameter, Canstar, ON, Canada), which is prealigned to the avalanche photodiode. The avalanche photodiode (EG&G OptoelectronicsCanada SPCM200, Vaudreuil, Canada) is a single-photon counting module with 25%photon detection efficiency at 830 nm and a dark count rate of 25 counts ~ - 1 .The ~ ~ digital output pulses from the avalanche photodiode are interfaced to a computer-controlled counter/timer board (Keithley Metrabyte CTM-05,Tauntor, MA) for further data analysis. To tune the Ti-sapphire laser to the Rb transition line at 780.023 nm, the Rb MVF is heated to 100 "C and laser scatter (21) Lehotay, S. J. Ph.D. Dissertation, University of Florida, Gainsville, FL, 1992. (22) EG&G SPCM-200-PQ-F830 data sheet, 1993.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4145
7
I
il I"
,
I
I
I
I
779.95
780.00
780.05
780.10
WAVELENGTH (nm)
Figure 3. Calculated rubidium Voigt absorption profile of the 780.023-nm rubidium line at different temperatures. Path length is 10 cm, and nitrogen pressure is 200 Torr at 25 "C.
from the capillary is passed through the Rb MVF onto the avalanche photodiode. The Ti-sapphire laser is then tuned to maximum absorbance of the Rb MVF by slowly adjusting the intracavity birefringent filter. Fine tuning is done by tilting the intercavity Fabry Perot etalon to further maximize absorbance. The maximum absorbance is more like a plateau than a peak because the Fabry Perot etalon can be adjusted sigdicantly before the laser is tuned off-wavelength and the absorbance decreased sharply. The calculated laser line width (fwhm) at 780.023nm is less than 0.081 pm when the Ti-sapphire laser is operated in the ring configuration with a 10-MHz spectral line width.I0 Reagents and Chemicals. The IR140 [Exciton Chemical Co., Inc., Dayton, OH; chemical name, 5,5'-dichlor~-ll-(diphenylamino)3,3'-diethyl-l0,12-ethylenethiatricarbocyanineperchlorate; CAS no. 5365517-7; fw = 7791 is dissolved in methanol (Optima grade, Fisher Scientific, Orlando, FL). Sample solutions at different concentrations are prepared from serial dilution of a 1.5 x M stock solution and analyzed on the same day. The fluorescence maximum for IR140 in methanol is 833 nm with excitation at 780.023nm. RESULTS AND DISCUSSION
Characterization of Rb Metal Vapor Filter. Because of its role in absorbing the laser specular scatter, the Rb MVF is one of the most critical components for detecting single molecules in a capillary. The rubidium atom has two main isotopes, *5Rband *7Rb, which exist naturally in the ratio of 0.727:0.273.23 Each isotope has its own hyperfine structure. The melting point for rubidium is 39 "C, so it is easy to produce a suf6cient number density of atoms at a moderate temperature. From the oscillator strength and number density of each electronic t r a n ~ i t i o nthe ,~~ absorption profile of Rb can be simulated at various conditions. Based on Puerta and Martin's approximati~n,~~ Figure 3 shows the calculated Voigt absorption profile, a convolution of Gaussian and Lorentzian profiles, of Rb at 27,50,and 100 "C. As Figure 3 shows, increasing the cell temperature has a large effect on the (23) Beacham, J. R.; Andrew, K. L. /. Opt. SOC.Am. 1971,61 (2), 231-235 (24) Gallagher, A; Lewis, E. L. /. Opt. SOC.Am. 1973,63 (3), 864-869. (25) Puerta, J.; Martin, P. Appl. Opt. 1981,20 (221, 3923-3928.
4146 Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
01 0
>
IC, 20
40
I
1
1
,
80
60
.
I
100
I
,
120
*
1
140
.
8
160
1
180
.
'0
TEMPERATURE ( C ' )
Figure 4. Experimental results for absorption of rubidium metal vapor filter at 780.023 nm. Path length is 10 cm, and nitrogen pressure is 200 Torr at 25 "C.
absorbance, which essentially mirrors the increasing Rb metal vapor number density. The presence of more than one peak for each absorption profile is due to the hyperfine structure of the overall transition. The Voigt absorption bandwidth is estimated to be 21 pm at 780.023nm, which is close to the measured value.z6 Ideally, once the narrow laser line is tuned to the absorption peak of the Rb MVF at 100 "C, an absorbance of several thousand could be reached. By calibrating with neutral density filters, the transmitted signal through the Rb MVF is measured at different temperatures. The measured transmittance is then converted to absorbance. The result is shown in Figure 4, where the absorbance of the laser scatter is plotted as a function of temperature. The maximum absorbance of -8 at 170"C is believed to be limited by a weak, broad-band laser background from the Ti-sapphire crystal. Single-Molecule Detection. To achieve single-molecule detection with near unity measurement efficiency, the capillary must be fully illuminated with the focused laser beam. Dovichi et a1.16 and Soper et aL3 have described the algorithm for the parabolic flow within the sample stream. The average linear flow velocity of molecules flowing through the capillary can be estimated from the volume flow rate and the cross-sectional area of the capillary (9.5x cmz). The average molecular transit time through the probe volume can be simply determined using the equationI6
average transit time =
n x laser beam diameter 4 x average linear flow velocity (3)
The calculated transit time is -0.8 ms when the volume flow rate of the syringe pump is set at 0.061 pL/min. Possible deviations from the molecular transit time in the probe volume are the uncertainties of the small capillary inside diameter, distorted laser beam shape within the cylindrical capillary, and Brownian diffusion. Desorption of molecules from the capillary walls, left over from a previous experiment at high concentrations, could lead to more detected molecules than expected. Altema(26) Barber, T.E.Ph.D. Dissertation, University of Florida, Gainsville, FL, 1992.
,
Average no. of molecules in probe volume per transit time ,08 I:* , , , , lY', , , ,
,,y
.
, , , l?' ,lY' , , , lf3 , , ,l l '
, ,,
Table 2. Probability for Different Numbers of Molecules Occupying the Probe Volume at Any Given Time.
no. of molecules in probe vol
probability
0
0.887 0.106 0.006 -0.000
1087
1 2
3
10' 'I
106
a
.
[IR140] = 1.9 x
M; probe volume, 1.05 pL.
io5:
(4)
IO':
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Id !
I 1 1 1 1 . q
7
.1.111.I
10'12
I
.'."',I
*
1-*,1*-,
. - m s - T
10"~ [IR140], M
10-11
10'
-
.-.~--.l
lod
.---"I
10"
Figure 5. log-log plot of the IR140 fluorescence emission versus the sample concentration with a transit time of 0.8 ms at different laser powers: 150 (O), 100 (0),and 50 mW(0).
tively, the number of molecules detected would be less than the number of molecules expected due to sample adsorption on the walls of the capillary. The average photodestruction probability of the flowing molecules can be evaluated by measuring the dependence of fluorescence intensity on the flow velocity of fluorescent molecules through a laser beam.27 When the laser power is 150 mW, the fluorescence signal from the IR140 decreases to 85% of the maximum value when the volume flow rate is decreased from 1.26 to 0.061 pL/min. The corresponding transit time of IR140 molecules in the probe volume increases from 0.039 ms to 0.8 ms when the volume flow rate is decreased over this range, thus indicating that the majority of the molecules are not photodestructed during their travel through the laser beam. Figure 5 shows the log-log calibration curves of IR140 over various concentration ranges. These calibration curves are obtained at three different laser powers while the molecular transit time is 0.8 ms. The signal from IR140 molecules is linear for at least 4 orders of magnitude from 1.9 x 10-l2 to 1.9 x lo-* M. Within this linear concentration range, the average number of molecules present in the probe volume per transit time are from M and 1.2 to 12 000. For an IR140 concentration of 1.9 x a transit time of 0.8 ms, there are -1500 molecules that actually travel through the probe volume within a 1-s integration time. To assure that only one molecule resides within the probe volume at any given time, the probability of two or more than two molecules occupying the probe volume must be minimized. Assuming the number of molecules in the probe volume during the transit time follows the Poisson distribution, the probability that n molecules occupy the probe volume simultaneously is given by ~~~
~
~
~
(27) Mathies, R A., Stryer. L. In Applications of Fluorescence in the Biomedical Sciences; Tylor, D. L., Waggoner, A. S., Lanni, F., Murphy, R F., Birge, R, Eds.; Alan R Liss, Inc.: New York, 1986; pp 129-140.
where Nt is the number of molecules arrived at time t, k is the number of time segment per transit time, and ;Ip is the average number of molecules in probe volume per time segment. M, the average At an IR140 concentration of 1.9 x number of molecules in the probe volume (1.05 pL) is 0.12. For the present experimental parameters, since the transit time of single IR140 molecules through the probe volume is 0.8 ms, therefore k = 4 and ;Ip = 0.03 for a counter with a 200-ps time segment (integration time). In other words, when a single molecule flows through the probe volume with a 0.8-ms transit time, the emitted photons should be distributed over four consecutive time segments. Table 2 gives the probability for different numbers of molecules occupying the probe volume at any given time. Most of time, there is no molecule in the probe volume (P(n=O) = 0.887). The probability of one molecule occupying the probe volume is P(n=l) = 0.106 while the probability of multiple occupancy (P(n z 2) = 0.006) is relatively low and can be neglected. It is possible to estimate the average number of events expected as a single IR140 molecule passes through the focused laser beam. Here, the number of events is the number of time segments with any signal counts from single IR140 molecules. The number of events, N,, for all molecules traveling through the probe volume during the measurement time T can be estimated from
where Ne, is the number of events from molecules during the measurement time, Np is the average number of molecules in probe volume, and tr is the transit time of a single molecule in probe volume. The calculated total number of events, N,, is 120 during a typical experimental run ([IR140] = 1.9 x M, Np = 0.12, T = 200 ms, k = 4, and t, = 0.8 ms). Within the 200-ms measurement time, -30 molecules will pass through the probe volume. To obtain photon bursts from individual single molecules, various signal processing methods have been proposed for the continuous detection and monitoring of molecules entering the probe vol~me.~,~.13-15,28,2~ The individual photon bursts are analyzed and searched within the data stream in order to obtain direct (28) Soper, S. A; Davis, L. M.; Shera, E.B. /. Opt. Soc. Am. B 1992,9, 17611769.
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9ool
1000 Blank
A
800 h
10
5 v)
0
l
IR140
201
20
40
80
60
100
120
140
B
160
180
200
Time (ms)
Figure 6. Photon burst data for the methanol blank (A) and for a subjected to the weighted quadratic 1.9 x l O - I 3 M IR140 solution (8) summing (WQS) filter algorithm with k = 4. The IR140 molecules is excited with 100-mW laser power at a flow rate of 0.061 pUmin (transit time, 0.8 ms). The raw data are taken in a 200-ps integration time. \iilithin the 200-ms sampling time, -30 molecules pass through the probe volume. The discriminator threshold (dashed lines) was set at S(t)= 5. The number of events that exceeded this threshold for the dye solution is found to be 110.
evidence for single-molecule sensitivity. To enhance the visibility of photon bursts from single molecules, the weighted quadratic summing (WQS) filter, a modified sliding sum method, has been evaluated and demonstrated to be an efficient photon burst indicator of a passing single molecule in previous single-molecule detection experiments. The raw data are squared and processed with the WQS filter algorithm, which is given by k-I
w ( z ) d(t
S(t) =
+ t)'
r=O
where k covers time intervals that are approximately equal to the transit time of a single molecule through the probe volume, w ( t ) is a weighing factor, and d(t t) is the raw data point at time t 5 . Due to the lack of knowledge in the actual laser excitation profile within the capillary and the photon emission profile from single molecules, different weighing factors might give an optimized S/N ratio depending on the experimental conditions. As the molecules cross the laser beam, the fluorescence signal increases slowly, followed by an abrupt cessation, when the molecule is photodecomposed. To best discriminate the timecorrelated fluorescence signal from the random background noise, the weighing factor, tu(.), is chosen as an asymmetric triangular
+
+
(7)
Figure 6 shows the WQS filtered data for a 1.9 x M IR140 solution and the methanol blank. The largeamplitude bursts from (29) Soper, S. A; Davis, L. M.; Fairfield, F. R; Hammond, M. L.; Harger, V. A;
Jett. J. H.; Keller, R. A; Martin, J. C.; Nutter, H. L.; Shera, E. B.; Simpson, D. J. In Optical Methodsfor Ultrasensitive Detection and Analysis: Techniques and Applications; Feary,B. L., Ed. Proc. SPIE-Int. SOC.Opt. Instncm. Eng. 1991.1435. 168-178.
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l 0
W
0
5
10
15
20
25
k 30
Discriminator level Figure 7. Cumulative number of events exceeding the discriminator M IR140 solution threshold versus discriminator for a 1.9 x ( 0 )and for the methanol blank (0).The experimental conditions are the same as those in Figure 6. When the discriminator level is set to 5, there are 110 events detected from the IR140 molecules while only 1 event from the methanol blank.
IR140 solution are clearly evident for individual single molecules passing through the center of laser beam. The variation in the burst amplitude arises from the molecules flowing through different parts of the Gaussian intensity laser profile. When the molecules travel through the edges of the laser beam, they experience fewer excitation cycles and thus emit fewer photons. Variation in the solvent blank photoelectron bursts is due to fluctuations of background and dark counts. In order to evaluate the detection efficiency for the near-IR dye molecules, a plot of the cumulative number of WQS filtered events exceeding a discriminator threshold is constructed for the solvent blank and the dye solution. Figure 7 shows the cumulative number of filtered events exceeding a discriminator threshold for a 1.9 x M IR140 solution and the methanol blank when excited with 100-mW laser power. Since the spatial probing efficiency is near unity, the measurement efficiency is defined as the ratio of measured to expected total number of events for molecules flowing through the capillary. To minimize the false positive probability, the discriminator threshold is set at S(t) = 5, indicated as the dashed iine in Figure 6. The number of events that exceed the threshold for the IR140 molecules is found to be 110, while 1 event is due to blank. Since the expected number of events during this 200-ms measurement time is -120, the measurement efficiency is calculated to be slightly less than loo%, which is believed due to errors in the estimation of analyte concentration and the probe volume and/or due to sample adsorption on the walls of the capillary. To designate photon bursts from individual single molecules, the raw data are compared with WQS filtered data. The corresponding four consecutive raw data points with WQS amplitude higher than the specific discriminator threshold are binned to four (200 ps x 4) within the 200-ms measurement time. The total photoelectron counts of these four consecutive raw data points give the size of the photon burst from this speci6c single molecule. Table 3 summarizes the total number of events and average detected photo-
Table 3. Number of Fluorescent Events at Different Laser Powers
Table 4. Comparison of Near-Infrared Single-Molecule Detection for IR132 and IR140
parameter
IR132"
IR140
excitation wavelength (nm) observed wavelengthb (nm) photon detection efficiency probe volume @L) transit time (ms) av photoelectron per molecule detection efficiency (%) spatial probing efficiency (%) measurement efficiency (%)
800 840 (30)
780 830 (10) 0.007c 1.05 0.8 8.5d 97 100 97
laser power (mw)
50 discriminator measured events expected events av photoelectrons per molecule"
3.1 112 120
5.9
100
5.0
150
7.1
110
116
120 7.0
120 8.5
" Average number of photoelectrons per molecule is calculated as
E C J / N , where N is the number of molecules with WQS amplitude higher than the discriminator level and Ci is the photon counts from an individual molecule using the raw data.
0.007 0.8 10
18 (h4) 97 (*7) 0.01
0.0097
Data taken from ref 3. *The number in parenthesis is fwhm spectral bandpass. The photon detection efficiency is calculated from the product of the geometric collection efficiency of M 0 1 (lo%),the transmission efficiency of M 0 1 (go%), the transmission efficiency of rubidium metal vapor filter (go%), the transmission efficiency of the band-pass a t e r for the fluorescence emission of the dye (50%),the geometric collection efficiency of M 0 2 (go%), the transmission efficiency of M02 (90%),the transmission efficiencyof optical fiber (90%), and the quantum efficiency of the avalanche photodiode at 830 nm. Laser power, 150 mW. (I
electrons from a single molecule with excitation at different laser powers. A higher discriminator threshold is required when the laser power is increased while maintaining similar detection efficiency. It is found that the average number of photoelectrons detected from a single IR140 molecule while traveling through the probe volume per transit time are 8.5, 7.0, and 5.9 for 150-, lo@, and 50-mW laser powers. Ideally, when a single molecule passes through the laser beam, the distribution of emitted photons should mirror the Gaussian profile of the laser beam. Unfortunately, if the size of photon burst from a single molecule is small, as in this work, the Poisson noise will severely distort the Gaussian distribution. The other complication with the present experimental configuration is that some molecules pass through the edge of the laser beam. This makes the exact counting of single molecules more difficult. Since the average number of background counts per transit time is less than 1 count within these three laser powers, 5 or more signal counts justify the use of the LOD concept, but do not justify the case of the LOG. Table 4 compares the near-IR single-molecule detection of IR132 and IR140. Although the average photon yield per molecule is lower for IR140 with similar photon detection efficiency, the measurement efficiency is 4 orders of magnitude higher because the whole capillary inner diameter is fully illuminated with the laser beam to assure near unity spatial probing efficiency. CONCLUSIONS In summary, this work has demonstrated the photon burst detection of single near-IR molecules (IR140) with high efficiency. There are several components that are interesting and relevant in the area of single-molecule detection. For example, the use of the metal vapor filter and the high spatial probing efficiency produced by irradiating the entire inner diameter of the capillary are unique. This approach depends heavily on the use of a novel
metal vapor filter to minimize laser scatter from the capillary reaching the detector. The measured absorbance of the rubidium metal vapor filter at 170 "Cis determined to be -8. The maximum absorbance is believed to be limited by the background fluorescence from the Ti-sapphire laser. The use of near-IR excitation and detection will benefit ultrasensitive fluorescence detection due to the reduced background noise sources from impurities. The fluorescence signal versus concentration plot has good linearity for at least 4 orders of magnitude from 1.9 x 10-l2 to 1.9 x lo-* M. To enhance the detection of single molecules, a digital weighted quadratic summing filter is applied to extract the individual photon bursts from the raw data. The development of oncolumn capillary detection with single-molecule-levelsensitivity would have a major impact on numerous analytical applications, including cell sorting and sizing, immunoassay, DNA sequencing, and miniaturized chemical separation techniques involving capillary electrophoresis, packed capillary liquid chromatography, and open tubular capillary liquid chromatography. ACKNOWLEDGMENT The authors thank Dr. D. Hueber and Dr. S. J. Lehotay for helpful discussions in this work. We also acknowledge the University of Florida, Division of Sponsored Research, for partial financial support of this work. Received May 26, 1994. Accepted September 7,1994.@ Abstract published in Advance ACS Abstracts, October 15, 1994.
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