Anal. Chem. 2001, 73, 943-950
Phase-Sensitive Fluorescence Lifetime Detection in Capillary Electrophoresis Yan He and Lei Geng*
Department of Chemistry, University of Iowa, Iowa City, Iowa 52242
A simple and highly sensitive fluorescence lifetime detection method for capillary electrophoresis has been introduced. The detection scheme is based on the integrated phase-sensitive fluorescence intensity. The integrative nature of the method results in high sensitivity of lifetime detection. The limit of detection is 7.8 amol of fluorescein injected, representing a 2 orders of magnitude improvement over the detection limits previously reported in the UV-visible region. Rayleigh scattering, Raman scattering, and background fluorescence can be effectively suppressed by setting the detector out of the phase from the background signal. Fluorescence background can be eliminated whether the fluorescence lifetime of the background is longer or shorter than the solute molecules of interest. The signal-to-noise ratio of measurements is optimized by varying the modulation frequency and the detector phase angle. Fluorescence lifetime detection has been actively pursued in chemical separations as a method for species identification,1-15 peak resolution,16-20 and background reduction,21-25 especially for (1) Desilets, D. J.; Kissinger, P. T.; Lytle, F. E. Anal. Chem. 1987, 59, 18301834. (2) Lieberwirth, U.; Arden-Jacob, J.; Drexhage, K. H.; Herten, D. P.; Muller, R.; Neumann, M.; Schulz, A.; Siebert, S.; Sagner, G.; Klingel, S.; Sauer, M.; Wolfrum, J. Anal. Chem. 1998, 70, 4771-4779. (3) Chang, K.; Force, R. K. Appl. Spectrosc. 1993, 47, 24-29. (4) Soper, S. A.; Legendre, B. L., Jr.; Willams, D. C. Anal. Chem. 1995, 67, 4358-4365. (5) Nunnally, B. K.; He, H.; Li, L.-C.; Tucker, S. A.; McGown, L. B. Anal. Chem. 1997, 69, 2392-2397. (6) Li, L.-C.; He, H.; Nunnally, B. K.; McGown, L. B. J. Chromatogr., B 1997, 695, 85-92. (7) Flanagan, J. H., Jr.; Owens, C. V.; Romero, S. E.; Waddell, E.; Kahn, S. H.; Hammer, R. P.; Soper, S. A. Anal. Chem. 1998, 70, 2676-2684. (8) He, H.; Nunnally, B. K.; Li, L.-C.; McGown, L. B. Anal. Chem. 1998, 70, 3413-3418. (9) Li, L.; McGown, L. B. J. Chromatogr., A 1999, 841, 95-103. (10) Burt, J. A.; Dvorak, M. A.; Gillispie, G. D.; Oswald, G. A. Appl. Spectrosc. 1999, 53, 1496-1501. (11) Dvorak, M. A.; Oswald, G. A.; Van Benthem, M. H.; Gillispie, G. D. Anal. Chem. 1997, 69, 3458-3464. (12) Imasaka, T.; Ishibashi, K.; Ishibashi, N. Anal. Chim. Acta 1982, 142, 1-12. (13) Ishibashi, K.; Imasaka, T.; Ishibashi, N. Anal. Chim. Acta 1985, 173, 165175. (14) Kawabata, Y.; Sauda, K.; Imasaka, T.; Ishibashi, N. Anal. Chim. Acta 1988, 208, 255-262. (15) Li, L.-C.; McGown, L. B. Anal. Chem. 1996, 68, 2737-2743. (16) Cobb, W. T.; McGown, L. B. Appl. Spectrosc. 1987, 41, 1275-1279. (17) Smalley, M. B.; Shaver, J. M.; McGown, L. B. Anal. Chem. 1993, 65, 34663472. (18) Smalley, M. B.; McGown, L. B. Anal. Chem. 1995, 67, 1371-1376. (19) Cobb, W. T.; McGown, L. B. Appl. Spectrosc. 1989, 43, 1363-1367. 10.1021/ac001197g CCC: $20.00 Published on Web 01/26/2001
© 2001 American Chemical Society
base calling in DNA sequencing.2-9 Fluorescence lifetime is an intrinsic property of the molecule that is determined by the radiative and nonradiative decay rates and does not vary with the concentration and thus the fluorescence intensity. The measured fluorescence lifetimes across an elution peak are generally sharply distributed with a small standard deviation. Consequently, there is almost no overlap between species in fluorescence lifetime detection for applications such as sequencing. Species identification can be potentially achieved in lifetime detection with high accuracy. Fluorescence lifetime detection can be performed in both the time domain and the frequency domain. In the time domain, a pulsed laser excites the fluorescent molecules. The fluorescence decay is collected with a time-correlated single photon counting method (TCSPC),2,4,7 with a gated integrator,22-25 or with a gigahertz oscilloscope.1,10-14,21 The signal observed in the time domain is the fluorescence intensities at various times after the excitation pulse, for each migration time of the electropherogram or chromatogram. In the frequency domain, a sinusoidally modulated laser provides the excitation. Collection of complete information about the fluorescence decay at each migration time is facilitated by exciting the molecules traversing the detection zone with a series of harmonics of a fundamental frequency.5,6,8,9,15,17,18 The phase delays and demodulation factors are collected at all harmonic frequencies (multiharmonic Fourier transform method, MHF). Alternatively, a pulsed laser can be used for excitation. The Fourier components of the pulse train provide the harmonic frequencies for the frequency domain experiments. The data collected in either the time or the frequency domain are subjected to statistical analysis, with the nonlinear leastsquares (NLLS), the maximum likelihood estimator (MLE), or the maximum entropy (MEM) methods, to yield the fluorescence lifetimes throughout an electrophoresis run. Both the time domain and the frequency domain methodologies have been successfully applied to CE and HPLC. The existing methods of lifetime measurements are dispersive by nature. The fluorescence photons are dispersed into many channels, time bins in the time domain and frequencies in the (20) Cobb, W. T.; McGown, L. B. Anal. Chem. 1990, 62, 186-189. (21) Miller, K. J.; Lytle, F. E. J. Chromatogr. 1993, 648, 245-250. (22) Latva, M.; Ala-Kleme, T.; Bjennes, H.; Kankare, J.; Haapakka, K. Analyst 1995, 120, 367-372. (23) Richardson, J. H.; Larson, K. M.; Haugen, G. R.; Johnson, D. C.; Clarkson, J. E. Anal. Chim. Acta 1980, 116, 407-411. (24) Furura, N.; Otsuki, A. Anal. Chem. 1983, 55, 2407-2413. (25) Iwata, T.; Senda, M.; Kurosu, Y.; Tsuji, A.; Maeda, M. Anal. Chem. 1997, 69, 1861-1865.
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frequency domain, to form the decay curve. The requirement to construct accurate photon statistics in TCSPC mandates a low counting rate to collect one photon out of 50-100 excitation pulses,26-29 introducing a further loss of the fluorescence intensity. The low counting rate is used to avoid pulse pileup that can result in apparently shortened decay times and nonexponential decays.26-29 The consequence of photon dispersion is a reduction in the detection sensitivity. The lowest mass of solute detected in the visible spectral region, 1 fmol of fluorescein injected, has been reported in capillary electrophoresis.5 Integration of photons can potentially improve the detection sensitivity and the signal-to-noise ratio (SNR) of the fluorescence lifetime detection. The reduction of the background signal, including the Rayleigh and Raman scattering and the background fluorescence, is an advantage of time-resolved fluorescence detection in HPLC and CE.21-25 In the time domain, the scattering and the fluorescence background can be effectively suppressed by gated detection. The gated integration has been shown to significantly improve the SNR of the fluorescence detection. The reduction of fluorescence background with gated detection is not complete due to the exponential nature of the background. In the frequency domain lifetime detection using multiharmonic excitation, the background signals are collected along with the solute fluorescence during data acquisition. Background signals are separated from the solute fluorescence through NLLS fitting of the globally linked data to multiexponential decays. Background signals that contain more than one decay component may pose difficulties since the data quality in the fluorescence lifetime measurements generally limits the number of exponential components that can be included in the fit. A new method of fluorescence lifetime detection based on phase-sensitive fluorescence is introduced in this paper, for capillary electrophoresis. The fluorescence intensity is integrated within a square wave that is synchronously modulated at the same frequency as the excitation, leading to much improved detection sensitivity. Complete suppression of complex background signals can be achieved by setting the square wave out of phase from the background. Instrumentation for lifetime detection is substantially simplified with the phase-sensitive fluorescence detection. EXPERIMENTAL SECTION Chemicals. Chemicals were obtained at the highest purity available and were used as received. Ultrapure Milli-Q (Millipore, Bedford, MA) water was used to prepare all solutions. Fluorescein and rhodamine B solutions at various concentrations were prepared by serial dilution from stock solutions prepared at millimolar concentration range. Sodium tetraborate buffer (4 mM) at pH 8.5 was used throughout the experiments. Capillary Electrophoresis. The CE system was built in-house and interfaced with the sample chamber of the spectrofluorometer. A Teflon block with two vial holders was machined to enclose the injection end of the capillary and the output of the high-voltage (26) Harris, C. M.; Sellinger, B. K. Aust. J. Chem. 1979, 32, 2111-2129. (27) O′Connor, D. V.; Phillips, D. Time-correlated Single Photon Counting; Academic Press: Orlando, FL, 1984. (28) Demas, J. N. Excited-State Lifetime Measurements; Academic Press: New York, 1983. (29) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer/Plenum: New York, 1999.
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cable. Both the buffer and the sample vials were standard 1.8-mL vials with prescored snap caps to prevent solvent evaporation. The outlet end of the capillary was placed in the sample compartment of the spectrofluorometer, which was connected to the ground of the high-voltage power supply (CZE1000, Spellman, Hauppauge, NY). Polyimide-coated capillaries ( 50 µm i.d., 375 µm o.d.; Polymicro Technologies, Phoenix, AZ) were used in the CE experiments. The total column length was 42 cm and the effective length between the inlet and the detection window was 25 cm, unless stated otherwise. The samples were introduced into the capillary by electrokinetic injection at 15 kV for 5 s. The separations were run at 25 kV of potential. The capillary was held in an aluminum bracket holder that was attached to a miniaturized three-dimensional translation stage fixed inside the sample chamber. The capillary was mounted at an angle of ∼20° with respect to the incident laser beam to minimize scattering off the capillary walls.30,31 Phase-Sensitive Fluorescence Detection. An SLM48000MHF spectrofluorometer (Spectronics Instruments Inc., Rochester, NY) was used for the phase-sensitive fluorescence measurements. An air-cooled argon laser provided 42 mW of excitation at 488 nm. The laser beam was sinusoidally modulated in intensity with a Pockels cell and focused with an f/2 lens into the capillary. In the static experiments with a cuvette, the fluorescence emission was collected with another f/2 lens and selected through a 515nm long-pass filter. In CE detection, the original f/2 lens in the emission channel was replaced by an f/0.8 biconvex fused-silica lens (Oriel, Stratford, CT), which provided high collection efficiency and a long working distance. In addition to the 515-nm long-pass filter, a 488-nm holographic notch filter (Kaiser Optical Systems, Ann Arbor, MI) was added in the fluorescence emission path to reject scattered laser light. In the phase-sensitive measurements, the instrument was operated at the single-frequency mode where the Pockels cell was driven directly with the frequency generator. The modulation frequency was optimized during the experiments for the best SNR. The detector was synchronously modulated at the same frequency as the laser. The phase angle of the detector, or the phase delay of the detector from the excitation, was calibrated by setting the phase-sensitive fluorescence intensity to zero for scattered light. A laser line filter was used to isolate the scattered light from the buffer solution. The detector phase angle was 90° when the phase-sensitive intensity for the scattered light was minimized. The phase-sensitive fluorescence, the ac and the dc intensities, were recorded simultaneously during a CE run. Methodology of the Phase-Sensitive Fluorescence Lifetime Detection. The principles of phase-sensitive fluorescence have been extensively described in the context of spectroscopy and imaging (chapter 22 of ref 29, and references therein). This paper describes the phase-resolved fluorescence as a detection principle in CE. When a multicomponent sample is illuminated with a sinusoidally modulated light at an angular modulation frequency of ω, the phase-sensitive fluorescence intensity observed at a detector phase angle of φD is29 (30) Yeung, E. S.; Wang, P.; Li, W.; Giese, R. W. J. Chromatogr. 1992, 608, 73-77. (31) Lee, T. T.; Yeung, E. S. J. Chromatogr. 1992, 595, 319-325.
FPR(φD) )
∑I M cos(φ - φ i
i
i
D)
(1)
i
where Ii, Mi, and φi are the intensity contribution, modulation, and phase delay of the ith fluorescent component, respectively. When φi - φD ) 90°, the intensity contribution of the ith component to the signal FPR is zero and the observed intensity will be the sum of the contributions of the remaining species. Generally, the apparent phase lifetime can be calculated from the ratio of two phase-sensitive measurements at different detector phase angles by assuming that the system does not change between the two measurements. For example, with two measurements at φD ) 0° and φD ) 90°,
τ ) ω-1(FPR(90°)/FPR(0°))
(2)
This assumption can be realized experimentally in most static systems but is hardly possible in CE, where two separate electrophoresis runs of the sample are conducted to acquire the 0° and 90° data. Although it is not uncommon to achieve a runto-run standard deviation of less than 1%, it is difficult, if not impossible, to perform two CE runs that exactly overlay each other due to the variation in sample injection and the interaction between the analyte and the inner wall of the capillary. This difficulty is alleviated by calculating τp from a single CE run, by measuring both FPR(φD) (the PR signal) and the root-mean-squares of FAC(t) (the AC signal) simultaneously using a phase-sensitive detector. The fluorescence lifetime at each elution time is evaluated from the PR and the AC signals
τ ) ω-1 tan[cos-1(PR/AC) + φD]
(3)
In the phase-sensitive fluorescence lifetime detection (PSLD), if a fluorescence background is present, such as the fluorescence of impurities in the buffer or the gel in capillary gel electrophoresis, the directly measured fluorescence lifetime is an apparent lifetime with contributions from both the background and the solute of interest. The average lifetime of the background can be evaluated from the background measurement first to facilitate the calculation of the real lifetime of the eluting species. The real lifetime of the sample can be recovered by (Supporting Information)
[
τ ) ω-1 tan cos-1
PRS - PRB
xACS2 + ACB2 - 2ACSACB cos∆φ
+ φD
]
(4)
where the subscripts S and B denote signals of the sample and the background, respectively. The phase difference between the sample and the background is found by
PRS PRB ∆φ ) cos-1 - cos-1 ACS ACB
(5)
RESULTS AND DISCUSSION Fluorescence Lifetime Electropherograms. To generate a fluorescence lifetime electropherogram, the ac intensity (AC) and
Figure 1. The DC, AC, and PR electropherograms of an injection of 10 nM fluorescein collected during a CE run (A) and the calculated fluorescence lifetime electropherogram (B). DC, dc intensity; AC, ac amplitude; PR(90°), phase-sensitive fluorescence intensity measured at a detector phase angle of 90°. Open circles: fluorescence lifetimes calculated without the instrument factor correction. Filled squares: fluorescence lifetimes calculated with the instrument factor correction. The total length of the capillary was 45 cm, with an effective length of 27 cm from the inlet to the detection window. Electropherograms are displaced on the intensity axis for clarity of display.
the phase-sensitive fluorescence intensity (PR) were monitored during an electrophoresis run. In addition, the integrated dc intensity (DC) was collected. Three electropherograms were obtained by plotting these signals versus the migration time (Figure 1A). The DC curve is identical to the electropherogram collected with conventional fluorescence intensity-based detection and has the highest SNR among the three signals. In practice, the AC and the PR signals are collected with two separate electronic circuits. As the two circuits have difference sensitivities of response, an instrument factor k is introduced to account for the sensitivity difference in the recovery of the fluorescence lifetimes.
τp ) ω-1 tan[cos-1(k(PR/AC)) + φD]
(6)
The instrument factor k needs to be evaluated using a fluorescent molecule with a known lifetime. Fluorescein was used as the Analytical Chemistry, Vol. 73, No. 5, March 1, 2001
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Figure 2. Electropherogram of a mixture of rhodamine B and fluorescein. The AC and PR intensities were collected at a modulation frequency of 30 MHz and a phase detector angle of 90° from scattering. The concentrations for rhodamine B (first peak) and fluorescein (second peak) were 2 and 0.5 µM, respectively.
standard fluorophore with a lifetime of 4.1 ns. The k factor is fairly constant at high intensities and decreases sharply as the AC intensity decreases. The intensity dependence of the instrument factor was fit by an empirical function for the convenience of calculation:
k ) 2.164 + 0.099 log AC + 0.012 log2 AC + 0.120 log3 AC - 0.022 log4 AC - 0.014 log5 AC (7) With this function, the fluorescence lifetime electropherogram was calculated according to eq 6. Without the correction, the fluorescence lifetime was scattered but fairly constant throughout the electropherogram (Figure 1). The elution peak was not observed in the uncorrected lifetime electropherogram. After the instrument factor correction, the elution peak of fluorescein is clearly observed in the lifetime trace. The lifetime in the peak region (six points across the elution peak), however, is 3.88(0.15) ns, shorter than the fluorescein lifetime determined in a static measurement in a cuvette (4.1 ns). This is caused by the contribution of the background in the AC signal. After this background contribution is considered (vida infra), the lifetime across the peak is evaluated to be 4.02(0.14) ns, in close agreement with the fluorescein lifetime measured in bulk. All fluorescence lifetimes shown in the rest of this paper are corrected with the intensity-dependent instrument factor. Figure 2 shows the electropherogram of rhodamine B and fluorescein. The fluorescence lifetimes of the two molecules were evaluated to be 1.60(0.06) and 4.14(0.09) ns across the peaks, respectively, in good agreement with their lifetimes. The fluorescence lifetimes obtained for elution peaks can be used to identify the solutes. The modulation frequency is one of the experimental factors that determine the SNR of the lifetime measurement. The phasesensitive electropherograms of fluorescein at various modulation frequencies have shown a linear correlation between the intensity 946 Analytical Chemistry, Vol. 73, No. 5, March 1, 2001
Figure 3. Minimization of the scattered light at a detector phase angle of 90°. The PR(0°) electropherogram is displaced upward by one intensity unit for the clarity of presentation. The injected sample was 0.5 µM fluorescein. Both the notch filter and the long-pass filter were removed from the emission channel during the electrophoresis run.
and sin2φ, as expected. The highest intensity is obtained when the phase delay is 45°, when the angular modulation frequency is the reciprocal of the fluorescence lifetime of the solute. Reduction of the Scattered Light and Noise Analysis. When the detector phase angle is 90° out of phase from the scattered light, scattering is minimized, as illustrated in Figure 3. A high intensity of the scattered laser light is observed in both the DC and the AC electropherograms. This electrophoresis run is chosen especially to show the sharp spike that appeared in the DC electropherogram. It can be caused either by the scattered laser light by large particles traversing the detection zone or by the fluorescence of the impurities in the sample. When the detector was set to be collinear with the scattered light (0° detector phase angle), the elution peak was completely overwhelmed by the noise in the background and was not observed. The phasesensitive electropherogram collected at the 90° detector phase angle showed a zero background. The elution peak was clearly observed and the sharp spike was completely suppressed, indicating that the scattered laser irradiation rather than impurity fluorescence is responsible for the spike. Phase-sensitive detection is an effective method for the elimination of superfluous peaks caused by the scattering. The reduction of scattering background by tuning the detector to 90° out of phase should also reduce the associated shot noise, thus improving the SNR of the lifetime measurement. The effects of the detector phase angle on the scattered light intensity and noise are demonstrated in Figure 4. A high background is observed when the detector phase is set at 0.5°. The projection factor of the scattered light, or the cosine between the scattering and the detector, is close to unity under this condition. When the detector phase angle is increased incrementally, the intensity of the scattered light is gradually suppressed and completely eliminated at 90°. A general reduction of the background noise is observed with increasing detector phase angle in Figure 4A before it reaches a fairly constant value at large detector phase angles. The noise characteristics of the phase-sensitive detection can be analyzed with data presented in Figure 4A. The signal observed
S/N ) S/xS + N2E
(10)
At high signal levels, the shot noise is predominant in the variance and the SNR is directly proportional to the square root of the signal intensity
S/N ) xS
(11)
A log-log plot of the SNR versus the signal should display a straight line with a slope of 0.5. At the limit of low signal intensities, the electronic noise exceeds the contribution from the shot noise. A plot of log(SNR) as a function of log S should be a linear line with a slope of 1
S/N ) S/NE
Figure 4. Effects of the detector phase angle on the scattered light intensity and the associated noise. No notch filter or long-pass filter was used in the emission channel. (A) Electropherogram displaying the PR intensity of the buffer while the detector phase angle was varied. (B) Relationship between the signal-to-noise ratio and the PR intensity. Inset: variation of the PR intensity as a function of cosφ.
is the projection of the scattered light onto the detector phase vector
S ) Iscatter cos φD
(8)
The background shot noise NS and the electronic noise NE are the main contributors to the variance in this signal
N2 ) N2S + N2E ) S + N2E
(9)
The shot noise is directly proportional to the square root of the phase-sensitive intensity S. The SNR of the phase-sensitive scattering intensity is determined by the signal level and the electronic noise
(12)
Figure 4B depicts the variation in SNR when the signal level changes. The phase-sensitive scattering intensity increases linearly with the cosine of the detector phase angle. The log-log plot displays a linear relationship with a slope of 0.51 in the highintensity range, corresponding to a shot noise controlled SNR. At lower intensities, a slope of 1.03 was found. The noises in the electronic modules are the main contributors to the variance in the signal in this range. Clearly, the electronic noise contributes significantly in the phase-sensitive fluorescence detection and determines the limit of detection of the method. Reduction of the Fluorescence Background. (1) Background Decaying Faster Than the Solutes. The fluorescence background can be minimized in a way similar to that described for the scattered light, as demonstrated by injecting a fluorescein peak over a constant flow of rhodamine B (Supporting Information). In the phase-sensitive electropherogram collected at a detector phase angle of 90°, a high fluorescence background is generated by the rhodamine B emission. When the detector was set out of phase from the rhodamine B fluorescence, the electropherogram showed a zero background. The fluorescence lifetime electropherogram, however, displays an incorrect lifetime close to 3.2 ns at the fluorescein peak. The discrepancy between this observed lifetime and the fluorescein lifetime of 4.1 ns is a result of the rhodamine B contribution to the total AC intensity. Although the contribution from rhodamine B is completely reduced in the phase-sensitive intensity, the AC intensity is a sum of contributions from both fluorescein and rhodamine B. The ratio of PR to AC leads to a lowered phase angle and thus a fluorescence lifetime that is shorter than that of fluorescein. The correct fluorescence lifetime of 4.22(0.05) ns was calculated with eq 4. (2) Background Decaying Slower Than the Solutes. In the electropherogram described above, the background fluorescence was emitted by rhodamine B, which has a shorter fluorescence lifetime of 1.6 ns than the solute fluorescein. The principles of background reduction in PSLD can also be used to suppress background fluorescence that decays at a slower rate than the molecules of interest. The electrophoresis of a mixture of rhodamine B and fluorescein shown in Figure 5 was generated by injecting the mixture into a buffer containing a high concentration of fluorescein. Fluorescein in the buffer created a constant fluorescence background. Solutes rhodamine B and fluorescein Analytical Chemistry, Vol. 73, No. 5, March 1, 2001
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When the detector angle was tuned to suppress fluorescein, a large scattering background was left. The compounded background from fluorescein and the scattered light can be completely suppressed by setting the detector at 90° from the vectorial sum of the scattering and the fluorescein emission (Supporting Information). For a background that contains n emitting species, the background fluorescence is a weighted sum of all component contributions29 n
IB )
∑[A + B sin(ωt - φ )] i
i
(14)
i
i)1
where Ai, Bi, and φi are the dc intensity, ac amplitude, and phase delay of the ith background component. The overall effect of the sum is a sine wave with an apparent phase angle Φ determined by the fluorescence emission of all background components:29 Figure 5. Reduction of slow-decaying fluorescence background. The background was 5 µM fluorescein. The sample was a mixture of 1 µM rhodamine B and 5.5 µM fluorescein.
in the mixture were separated and eluted at 102 and 191 s, respectively. Both solute peaks appear in the DC, the AC, and the PR electropherogram collected when the detector phase angle was set at 90°, as well as the high-fluorescence background. The phase-sensitive signal with detector angle set out of phase from fluorescein, however, showed a negligible fluorescence background, and only one elution peak, the rhodamine B peak, is observed. This sample demonstrates the versatility of background reduction by the phase-sensitive fluorescence detection. Fluorescence background can be completely removed regardless of its decay rate compared to the solutesswhether the background fluorescence decays faster or slower than the solute fluorescence. In PSLD, the background fluorescence is eliminated by detecting the emission with a detector out of phase from the background. The percentage of the solute fluorescence that is usable for the evaluation of the lifetime is
IPR(φD) ) IM sin(φB - φ)
(13)
The same usable percentage of the solute fluorescence is collected for all elution times in the electropherogram because the angle between the detector and the solute will not change when the intensity changes across an elution peak. At the modulation frequency of 30 MHz, 36% of the fluorescein emission is used for the fluorescence lifetime calculation throughout the elution peak. (3) Simultaneous Suppression of Both Longer and Shorter Lived Background Signals. An even more complex background contains both a fast-decaying background from the scattered light, which displays the instrument response function, and a slowdecaying fluorescence background from fluorescein. The solute rhodamine B has a lifetime longer than the scattered light and shorter than fluorescein. When the detector phase angle was set 90° from the scattering, the background fluorescence persisted. 948
Analytical Chemistry, Vol. 73, No. 5, March 1, 2001
(
Φ ) tan-1
n
)
∑(ωR τ /x1 + ω τ ) 2 i i
2 2 i
i)1
n
∑(ωτ /x1 + i
i)1
ω2τ2i )
(15)
Ri ) Ai/A is the contribution of the ith component to the background intensity. The sinusoidal form of the overall intensity presents the opportunity for background suppression using the phase-sensitive fluorescence detection. When the detector phase angle is set to 90° out of phase from the overall background signal (φD ) Φ + 90°), the phase-sensitive intensity of the background becomes zero. The observed intensity of the solute peak is the projection of its total AC intensity onto this detection angle. The reduction of both the scattering and the fluorescein emission backgrounds demonstrates two important properties of the phase-sensitive fluorescence detection. First, a background with complex multiexponential decays can be suppressed. Second, both the longer and the shorter lived background signals can be suppressed simultaneously. The background signal in complex modes of capillary electrophoresis, such as capillary electrochromatography and capillary gel electrophoresis, can be multiexponential in nature. Phase-sensitive fluorescence detection provides an effective solution in such situations. It is noted that the suppression of a multiexponential fluorescence background using the detector phase angle can also substantially reduce the fluorescence intensity of the solute, by a factor of sin(φB - φ) (eq 13). In other words, background cannot be minimized by lifetime-based detection methods when the fluorescence lifetime of the background is similar to that of the solute. Background reduction generally has two desirable effects in fluorescence detection: to remove the background peaks that can overwhelm the solute peaks and to improve the SNR by reducing the noise in the background. Time gating has been used to effectively suppress the scattering and short-lived fluorescence background in chemical separations. Seitzinger et al. substantially improved the SNR in the measurement of rhodamine B (τ ) 2.1 ns in deionized water) by reducing the long-lived fluorescence of Ru(bpy)33+ (τ ) 286 ns) with a gating window located at early
times of the fluorescence decay.32 This strategy can be potentially applied to CE detection. Partial reduction of the background is achieved in time gating methods due to the exponential nature of the decays. By completely suppressing the fluorescence background, phase-sensitive fluorescence detection should provide much improved SNR if the measurements are shot noise limited. The current instrumentation, however, is limited by the electronic noises. The SNR advantage could be fully achieved in the future when digital detection is employed. The background reduction in the current form can be quite useful in biological and environmental samples21,23 where the intense background peak can overlap or even overwhelm the solute peak. Removal of these interfering background peaks, rather than reduction of the shot noise, is of interest here. In phase-sensitive detection, an optimum frequency can be chosen to maximize the phase deference between the solute and the background. This theoretically calculated optimum frequency, however, could be quite high. A high frequency can reduce the modulation of the laser and thus lower the detected fluorescence intensity. The choice of modulation frequency should thus be done experimentally to optimize the SNR. Sensitivity and the Detection Limit. In both time domain and frequency domain lifetime detection, the fluorescence intensity is dispersed into many channels to facilitate calculation of the fluorescence lifetime. In the time domain, the channels are the time bins in TCSPC or the time points along the decay profile displayed on an oscilloscope. In the MHF mode of the frequency domain, the channels are the different harmonic frequencies of modulation. Phase-sensitive fluorescence detection, in contrast, is integrative and measures an integrated intensity at each migration time. This integrative nature should lead to higher sensitivity in lifetime detection. The limit of detection (LOD) is not easily defined in the fluorescence lifetime since the lifetime is an intrinsic parameter of the solute molecule that does not vary with the concentration. Three versions of LOD have been reported in the literature for fluorescence lifetime detection in HPLC and CE: (1) an LOD calculated from the total intensity, (2) an LOD estimated to be the lowest concentration that generates a fluorescence lifetime that is within three standard deviations from the mean across an elution peak, and (3) an LOD defined as the lowest experimentally injected concentration that allows the reliable recovery of the fluorescence lifetime. The lowest detection limit reported to date in the visible region is the lifetime detection of 1 fmol of fluorescein injected in CE.5 The concentration profile of the phase-sensitive fluorescence lifetime detection is depicted in Figure 6, for fluorescein concentrations of 10 µM, 1 µM, 100 nM, 10 nM, and 1 nM. The DC electropherograms are displayed with the corresponding lifetimes across the peak. The statistics of replicate injections are listed in Table 1. Twelve CE runs were performed at 1 nM concentration, for two sample solutions. A fluorescence lifetime of 3.99(0.41) ns was obtained with seven injections of sample 1 and a lifetime of 4.25(0.23) ns was recovered from five injections of sample 2. The average fluorescein lifetimes obtained at all concentrations are in close agreement with the 4.1 ns measured in bulk solutions. The (32) Seitzinger, N. K.; Hughes, K. D.; Lytle, F. E. Anal. Chem. 1989, 61, 26112615.
Figure 6. Fluorescence lifetimes evaluated for electropherograms of fluorescein at various concentrations. Table 1. Fluorescence Lifetime of Fluorescein Determined by Phase-Sensitive Fluorescence Detection injected concn
average lifetime (ns)
standard deviation (ns)
10 µM 1 µM 100 nM 10 nM 1 nM (A) 1 nM (B)
4.20 4.18 4.12 4.07 3.99 4.25
0.04 0.11 0.10 0.05 0.41 0.23
LOD, or the lowest concentration experimentally injected for reliable lifetime measurement (definition 3), is 1 nM for our phasesensitive fluorescence lifetime detection. With an injection volumn of 7.8 nL, the mass detection limit is 7.8 amol of fluorescein injected, representing a 2 orders of magnitude improvement over LODs reported in the literature in the visible region. Using the criterion of three standard deviations, the LOD of PSLD is 3.5 amol. The dynamic range of the method, defined as the range of concentration when the fluorescence lifetime can be reliably determined, is 4 orders of magnitude from 1 nM to 10 µM. The usage of near-IR dyes and digital detection systems may further improve the LOD of the fluorescence lifetime detection. In the near-IR, the background fluorescence is greatly minimized since few molecules emit in this spectral region. The scattered light is also reduced due to the lower Raman cross sections. Soper has demonstrated LODs significantly better than in the ultraviolet and the visible regions.4 The existence of peak overlap can be indicated by a changing average lifetime across an elution peak.20 Since only the average lifetime is measured in phase-sensitive fluorescence detection during electrophoresis, overlapping elution peaks cannot be resolved using NLLS fits as possible in the frequency domain MHF detection. A simple and effective solution is the twodimensional fluorescence correlation analysis,33 which is based on phase-sensitive fluorescence detection. We have demonstrated that strongly overlapping peaks with a resolution of 0.28 in a conventional one-dimensional electropherogram can be resolved by fluorescence correlation. (33) Wang, G.; Geng, L. Anal. Chem. 2000, 72, 4531-4542.
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The approach of phase-sensitive fluorescence lifetime detection can be accomplished with a single detection channel and a lockin amplifier, simplifying the instrumentation for the lifetime detection. In the time domain, two optical paths and two electronic channels are necessary to provide the start and stop pulses in the measurement. In the MHF mode of the frequency domain lifetime measurement, additional harmonic comb generators are needed for the modulation of the excitation source and two fluorescence detection channels for the sample and the reference. In the phase-sensitive fluorescence detection presented in this paper, both required signals, the ac and the phase-sensitive intensity, are collected with the same detection and electronic channel. The detection can be accomplished with a single lock-in amplifier, significantly simplifying the instrumentation. The sensitivity of detection is much improved compared to the existing methods that disperse fluorescence photons into separate time or frequency channels. As demonstrated in Figure 4, the measurement is limited by the electronic noise with the current instrumentation. A quiet digital lock-in amplifier can further improve the detection limit of the technique. CONCLUSIONS We have introduced a new approach to fluorescence lifetime detection for capillary electrophoresis based on the phase-sensitive (34) Schlag, E. W.; Selzle, H. L.; Schneider, S.; Larsen, J. G. Rev. Sci. Instrum. 1974, 45, 364-367.
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fluorescence. The integrative nature of the method leads to high detection sensitivity. The improvement of LOD from the femtomole to the attomole range is desirable and perhaps necessary since many biochemical applications such as DNA sequencing require subfemtomole sensitivity. The methodology of PSLD can be potentially extended to multiplexing applications, such as capillary array electrophoresis, when an intensified CCD is modulated. Complete background reduction is achieved by setting the detector phase angle 90° out of phase from the background, whether it decays faster or slower than the solutes, and even when the background decay is multiexponential. The LOD of the technique is limited by the electronic noises currently. The benefit of background reduction will be fully appreciated when quieter digital detection systems, such as digital lock-in amplifiers, or photon counting34 are employed. ACKNOWLEDGMENT The work was partially supported by the National Institutes of Health and the University of Iowa through a Central Investment Fund for Research Enhancement (CIFRE) grant. SUPPORTING INFORMATION AVAILABLE Selected data. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 4, 2000. AC001197G
October
11,
2000.
Accepted
