Anal. Chem. 1999, 71, 4460-4464
Ultrasensitive Cross-Correlation Electrophoresis on Microchip Devices Julius C. Fister, III, Stephen C. Jacobson, and J. Michael Ramsey*
Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142
Cross-correlation electrophoresis with fluorescence detection on a microchip device was used to resolve a mixture of 63 pM fluorescein and 70 pM dichlorofluorescein. The signal-to-noise ratios of analyte peaks in a correlogram derived from a 9-bit pseudorandom injection sequence were ∼17 for dichlorofluorescein and ∼6 for fluorescein. In contrast, neither peak was detected in a conventional electropherogram obtained on the microchip. Detection limits estimated from a correlogram derived from the average of 12 7-bit correlograms were 6.5 pM dichlorofluorescein and 21 pM fluorescein. These are the lowest detection limits reported for a microchip separation device using analog detection and the first example of crosscorrelation electrophoresis on a monolithic device. The small injection volumes, injection reproducibility, and rapid separations enabled by microchip devices make cross-correlation multiplex injection schemes a viable approach for achieving enhanced sensitivity. A variety of chemical separations procedures have been achieved using electrokinetically driven microfabricated fluidic networks.1-10 Microfabricated instruments are more versatile than capillaries because reagent mixing2,11 and separation can be performed rapidly under computer control. However, detection limits in both microfabricated electrophoresis channels and capillaries are often higher than desired because the minute dimensions limit both the injection volume and the path length (1) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (2) Jacobson, S. C.; Koutny, L. B.; Hergenro ¨der, R.; Moore, A. W.; Ramsey, J. M. Anal. Chem. 1994, 66, 3472-3476. (3) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (4) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 2369-2373. (5) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642. (6) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118. (7) Ekstro ¨m, B.; Jacobson, G.; O ¨ hman, O.; Sjo¨din, H. International Patent WO 91/16966 in Adv. Chromatogr. 1993, 33, 1-66. (8) Moore, A. W., Jr.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1995, 67, 4184-4189. (9) Ocvirk, G.; Verpoorte, E.; Manz, A.; Grasserbauer, M.; Widmer, H. M. Anal. Methods Instrum. 1995, 2, 74. (10) Jacobson, S. C.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1995, 67, 2059-2063. (11) Jacobson, S. C.; Hergenro ¨der, R.; Moore, A. W., Jr.; Ramsey, J. M. Anal. Chem. 1994, 66, 4127-4132.
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of the detection zone. Cross-correlation chromatography12 in which multiple injections are performed in a continuous but random sequence addresses this limitation by providing a multiplex advantage.12-14 For example, using a microfabricated injector coupled via epoxy to a conventional electrophoresis capillary, S/N enhancements of ∼8 were obtained in ∼2.8× the time required for a single electropherogram.15 However, because the migration time was ∼11 min,15,16 achieving significantly greater S/N enhancements would require >1 h. Long injection sequences are problematic because both detector drift and migration time drift create artifacts in the correlated data.15-18 Microfabricated electrophoresis devices appear to be ideal platforms to exploit the benefits of the cross-correlation technique because rapid, reproducible injections can be performed with no moving parts.2,5,6,15 Furthermore, because analysis times in microfabricated electrophoresis channels tend to be shorter than in standard capillaries, injection sequences that are long relative to a single electropherogram would require only several minutes. Consequently, significant S/N enhancements could be obtained within a relatively modest time. In this work, we report the use of cross-correlation electrophoresis with fluorescence detection for resolving dilute dye solutions on monolithic microfabricated electrophoresis devices. EXPERIMENTAL SECTION Microchip Fabrication. A cross-shaped channel network was machined in a glass substrate using photolithographic and wet chemical etching techniques described previously.6 The channel dimensions, measured with a surface profiler, were 9.5 µm deep, 47.0 µm wide at the top, and 31.0 µm wide at the bottom. The length of the separation channel was 3.5 cm. A 150-µm-thick glass cover slip was bonded to the machined substrate to seal the channels.6 Glass reservoirs were attached to the channel openings using epoxy. Injection Voltages. To minimize artifacts due to injections, the potential at the injection cross should remain constant throughout the pseudorandom injection sequences, i.e., as the (12) Smit, H. C. Chromatographia 1970, 3, 515-518. (13) Annino, R. J. Chromatogr. Sci. 1976, 14, 265-270. (14) Phillips, J. B. Anal. Chem. 1980, 52, 468A-478A. (15) van der Moolen, J. N.; Poppe, H.; Smit, H. C. Anal. Chem. 1997, 69, 42204225. (16) van der Moolen, J. N.; Louwerse, D. J.; Poppe, H.; Smit, H. C. Chromatographia 1995, 40, 368-374. (17) Mulder, R.; Hoogerbrugge, P. J.; Smit, H. C. Chem. Intell. Lab. Syst. 1987, 1, 243-264. (18) Mulder, R.; Mars, C.; Smit, H. C. Chemom. Intell. Lab. Syst. 1991, 12, 155168. 10.1021/ac990853d CCC: $18.00
© 1999 American Chemical Society Published on Web 09/09/1999
microchip toggles between injection and separation (not-inject) modes. Channel resistances were determined by measuring the current flow between reservoirs with a microammeter and applying Ohm’s law. During the pseudorandom injection sequences, independent computer-controlled high-voltage power supplies controlled the potential at each reservoir; the field strength in the separation channel was maintained at ∼400 V cm-1 during the separations. Injection Sequences. Spreadsheet logic functions were used to generate 7- and 9-bit pseudorandom binary sequences (PRBS) of 1’s and 0’s.12,19 Because of the logic statements used to derive the PRBS, each N-bit sequence contains exactly one more 1 than 0’s and a total of 2N - 1 points; therefore, the duty cycle of any practical sequence is indistinguishable from 50%. Injection profiles were derived from the PRBS by assigning reservoir potentials corresponding to the inject state to 1’s and potentials corresponding to the “separation” or not-inject state to 0’s. During each N-bit sequence, exactly 2N-1 injections are performed, for the remaining 2N-1 - 1 clock periods the reservoir potentials are in the notinject state. A 0.25-s clock period was assigned to each point in the injection sequences so that the total duration of a single sequence was 31.75 (7-bit) or 127.75 s (9-bit). Fluorescence Detection. Fluorescence from pseudorandom injection sequences was monitored using a custom-built instrument similar to those described previously.20 Briefly, a ∼3-mW laser beam from an argon ion laser (502 nm, Coherent Innova I-90) was focused by a plano-convex lens to a ∼20-µm diameter in the separation channel. Fluorescence was collected by a 20× 0.42 NA microscope objective situated below the microchip. The fluorescence was focused onto a photomultiplier tube (PMT; Hamamatsu R928) and digitized by a 16-bit data acquisition board (National Instruments) which was connected to the output of the PMT. Rayleigh and Raman scattering were rejected using an interference filter (Omega Optical). Chemicals. The buffer for all experiments was a solution of 4 mM sodium borate (Sigma Aldrich) in water (Barnstead Nanopure). Dichlorofluorescein and fluorescence (Sigma Aldrich) were used as received without further purification. Prior to performing separations, the channels were conditioned with a solution of 0.1 N NaOH (Sigma Aldrich) followed by a rinse with pure water. RESULTS AND DISCUSSION Cross-correlation electrophoresis was compared to traditional electrokinetic separations using a solution containing 70 pM dichlorofluorescein and 63 pM fluorescein. (For thorough presentations of cross-correlation theory as applied to chromatographic systems see publications by Annino13 and Smit et al.16,18) Using a microchip device with computer-controlled reservoir potentials, 13 successive 7-bit pseudorandom injection sequences were executed. Immediately following the final pseudorandom sequence, the reservoir potentials were switched to the not-inject state for 30 s; then, an individual 0.25-s gated injection was performed to obtain the impulse response, i.e., electropherogram. The gated injection width matched the 0.25-s clock period of the pseudorandom sequences to allow a valid comparison between (19) Horowitz, P.; Hill, W. The Art of Electronics; Cambridge University Press: New York, 1980; Chapter 9. (20) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723.
the correlograms and electropherogram.16 The fluorescence signal was digitized at 100 Hz using the custom-built instrument described above. During the series of 13 pseudorandom injection profiles, the detected fluorescence intensity assumes a steady-state pseudorandom pattern, which repeats every 31.75 s, i.e., the length of a single injection sequence.12,16,18 Correlograms were derived from the detector output by circularly cross-correlating the 7-bit pseudorandom injection profile with successive 31.75-s segments of data. (The sampling density of the injection profiles matched the 100-Hz data acquisition rate such that there were 25 points per 0.25-s clock period.) Because correlograms derived from data acquired before slower compounds have reached the detector or after faster compounds have eluted from the channel may contain biased peak intensities and baseline artifacts,12,16,18 the starting point of the first 31.75-s segment was chosen to be ∼15 s after the arrival of the faster compound. Since the migration time difference between the two compounds is only ∼2.0 s, this ensures that each 31.75-s segment of detector output contains a complete pseudorandom sequence. However, because of the discarded data, only 12 complete correlograms could be resolved from the 13 injection sequences. In an additional step performed prior to crosscorrelation, the injection profile was modified by setting the first point of each clock period to either 1 for the inject state or -1 for the not-inject state and setting the remaining 24 points in each clock period to zero. These modifications18 prevent temporal broadening of correlogram features because the autocorrelation function of the modified 1:25 duty cycle injection profile is a ∆ function. In Figure 1a, which shows one of the 12 7-bit correlograms, two distinct peaks appear at migration times of 20.6 (dichlorofluorescein) and 22.0 s (fluorescein). In contrast, although the conventional electropherogram shown in Figure 1b exhibits evidence of an analyte peak at ∼22 s, the intensity is well below the detection limit. Figure 1c shows the average of all 12 7-bit correlograms and is discussed below. The intensities of peaks in the correlogram, estimated by subtracting the average baseline intensity from the peak maximums, were IDCF ) 0.17 ( 0.02 and IF ) 0.07 ( 0.02 for dichlorofluorescein and fluorescein, respectively. All of the correlograms presented here are expressed in arbitrary units. The uncertainties are given by 21/2σ, where σ is the standard deviation of the baseline. Migration time drift or irreproducible injections could lead to peak broadening and introduce baseline artifacts,17 decreasing the performance advantage of the multiplex approach. To avoid these effects, the migration time jitter should be small relative to the width of the analyte peaks. For the dichlorofluorescein peaks in the 12 7-bit correlograms, the ratio of the standard deviation of the migration time and the average full width (4σ) of the peaks was ∼0.1. The reproducibility of the 7-bit correlograms was evaluated by finding the average intensity and sample standard deviation of the peaks in the 12 correlograms: IDCF ) 0.15 ( 0.02 and IF ) 0.06 ( 0.01. The agreement between the sample standard deviations of the averaged peak intensities and the standard deviation predicted from the baseline noise in a single correlogram indicates that migration time drift or irreproducible injections are not significant sources of variance in the correlograms. Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
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Figure 1. Electropherogram and correlograms of 70 pM dichlorofluorescein and 63 pM fluorescein: (a) an individual 7-bit correlogram; (b) electropherogram from a single 0.25-s gated injection; (c) the average of 12 7-bit correlograms.
When random noise on the detector output is the dominate source of variance, the S/N enhancement of cross-correlation can be predicted from the length of the injection sequence.16 As described in the Experimental Section, 2N-1 injections are performed during an N-bit injection sequence. However, the crosscorrelation step integrates noise from all of the clock periods. If we ignore the missing “not-inject” clock period and assume that each sequence contains a total of 2N clock periods, the S/N enhancement is given by
S/N =
2N-1
x2N
)
x2N 2
(1)
For example, an ∼5.6× enhancement is predicted for a 7-bit injection sequence. In the correlograms described above, the average S/Ns of the peaks are calculated to be 9.0 ( 1.0 for dichlorofluorescein and 4.0 ( 1.0 for fluorescein. The S/N of the peaks within each correlogram is given by the ratio of the peak intensity to the standard deviation of the baseline. Unfortunately, the S/N enhancement cannot be evaluated because, as shown in Figure 1b, no peaks appear in the electropherogram. Nonetheless, the S/Ns of the underlying peaks can be estimated by taking the ratio of the average S/Ns of the 12 correlograms and the predicted S/N enhancement: ∼1.6 for dichlorofluorescein and ∼0.7 for fluorescein. These estimates are consistent with the absence of distinct peaks in the electropherogram. Given the indefinite impulse response, one could better evaluate the performance of cross-correlation electrophoresis on 4462 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 2. Comparison of averaged 7- and 9-bit correlograms: (a) an individual 9-bit correlogram; (b) the average of four 7-bit correlograms; (c) a 9-bit correlogram of a solution containing ∼600 pM rhodamine B, 70 pM dichlorofluorescein, and 63 pM fluorescein.
microchips by comparing correlograms resolved from a series of 9-bit injection sequences to the 7-bit correlograms. Since the duration of a 9-bit pseudorandom injection sequence is 4 times longer than a 7-bit sequence, the theoretical S/N enhancement is 2 times greater or ∼11.3. Three consecutive 9-bit injection sequences were performed using the same analyte solutions described above for comparison of experiment with theoretical expectation. Figure 2a shows one of two correlograms resolved from the detector output. (Parts b and c of Figure 2 are discussed below.) The average intensities of the resolved peaks were IDCF ) 0.54 ( 0.04 and IF ) 0.18 ( 0.04. The uncertainties are given by 21/2σ, where σ is the average standard deviation of the baselines. As predicted, the measured S/Ns, ∼17 for dichlorofluorescein and ∼6 for fluorescein, are ∼2 times higher than for the 7-bit sequences, showing that the device is delivering performance near the theoretical limit. Despite the improved S/N achieved through use of a 9-bit injection sequence, an important drawback of longer injections is that drift is more difficult to detect because fewer correlograms are obtained per unit time. Detector drift15 or migration time drift can seriously distort correlograms resolved from electrophoresis data. Ideally, the duration of each pseudorandom injection sequence would be just slightly longer than the migration time separating the fastest and slowest components. In this situation, the greatest number of correlograms would be available to detect drift. Successive correlograms would be averaged to increase the S/N. The S/N benefit of averaging correlograms was evaluated using the 12 7-bit correlograms described above: 3 new correlo-
grams were created by averaging groups of 4 consecutive correlograms. In the absence of drift or significant correlation noise, the average of four 7-bit correlograms should exhibit the same S/N as a single 9-bit correlogram. Compare Figure 2b, which shows one of the averaged 7-bit correlograms, to Figure 2a, which shows a single 9-bit correlogram. The average S/N of the resolved peaks in the three averaged 7-bit correlograms was 18.0 ( 1.6 for dichlorofluorescein and 6.3 ( 1.6 for fluorescein. Within the precision of the data, the S/Ns of the 9-bit correlograms and of the average of four 7-bit correlograms are indistinguishable. Averaging all 12 7-bit correlograms should further increase the S/N. If random noise in the raw data is the dominant contribution to the variance of each correlogram, the S/N of the average of 12 correlograms is predicted to be 121/2 or ∼3.5 times greater than the individual 7-bit correlograms described above or ∼31 ( 3.5 (dichlorofluorescein) and ∼13.5 ( 3.5 (fluorescein). Figure 1c shows the average of all 12 7-bit correlograms; the estimated S/Ns of the resolved peaks are 29.3 (dichlorofluorescein) and 9.8 (fluorescein). Again, within the precision of the data, there is reasonable agreement between the observed and predicted S/N enhancements. Based on these results, the estimated detection limits for a S/N of 3 are calculated to be 6.5 pM dichlorofluorescein and 21 pM fluorescein. Another useful figure of merit is the relative time required to achieve a given S/N enhancement by averaging successive electropherograms compared to a multiplexed approach. For example, a S/N enhancement of n1/2 can be achieved by averaging n electropherograms; the time required is given by ntm, where tm is the migration time of the slowest component. Using eq 1, it can be shown that a pseudorandom sequence containing 2n injections results in a theoretical S/N enhancement of n1/2; given the ∼50% duty cycle of pseudorandom injection sequences, 2n injections require a time of 4n∆t where ∆t is the clock period. Assuming that the total duration of the injection sequence(s) is long compared to the migration time, the relative time advantage of the correlation approach is given by tm/(4∆t). On the basis of the ∼25-s migration time observed in these experiments and the 0.25-s clock period used in the pseudorandom sequences, the predicted time advantage is ∼25×. For example, the complete series of 7-bit injection sequences required ∼7 min; a signal averaging approach would require ∼2.9 h. One source of baseline noise in correlograms arises from proportional noise in the detected fluorescence. Because fluorescence from various analytes overlaps one another, proportional noise from more intense fluorophores may obscure weaker peaks in a correlogram, i.e., a multiplex disadvantage similar to that observed in Fourier transform spectroscopies. Irreproducible injections will further degrade precision because the consequent baseline artifacts that appear in the correlograms are proportional to the intensities of the resolved peaks.17,18 Potential applications of correlation electrophoresis that might be impacted by these effects include ultrasensitive immunoassays and detection of labeled proteins and amino acids. In these applications, weak signals from small concentrations of analyte must be resolved from stronger signals due to the labeling agent. To evaluate the effect of a strong concomitant fluorophore on correlogram S/N, ∼600 pM rhodamine B was added to the analyte solution used above and a series of three 9-bit pseudorandom
Figure 3. Fifteen-second segments of the detector output from solutions containing (a) 70 pM dichlorofluorescein and 63 pM fluorescein, (b) ∼600 pM rhodamine B, 70 pM dichlorofluorescein, and 63 pM fluorescein, and (c) a segment of the corresponding 9-bit pseudorandom injection profile.
injection sequences performed. Compare the detector output from the analyte solution containing only dichlorofluorescein and fluorescein, which is shown in Figure 3a, to the detector output from the analyte solution containing added rhodamine B, which is shown in Figure 3b. For the solutions containing only dichlorofluorescein and fluorescein, the detector output exhibits little structure. In contrast, Figure 3b shows that strong fluorescence from rhodamine B overwhelms the more dilute analytes and the highly structured detector output directly corresponds to the 9-bit pseudorandom injection profile shown in Figure 3c. Despite the strong concomitant fluorescence, Figure 2c shows that both dilute analytes produce clearly resolved peaks in the correlogram derived from the solution containing rhodamine B. However, because of increased proportional noise, the baseline variance increases and the S/N ratio is reduced by ∼2 times compared to the 9-bit correlogram derived from a solution containing only dichlorofluorescein and fluorescein, which is shown in Figure 2a. The S/N enhancements gained through cross-correlation electrophoresis can be compared to other approaches for improving sensitivity on microfabricated devices. A stacking injection technique21 improved detection limits of dansylated amino acids by between ∼18 and 30 times when compared to pinched sample injections performed on the same microchip device. These improvements are comparable with the theoretical S/N enhancements observed in the current work. Despite the advantage of achieving enhanced S/N without increasing the analysis time, the stacking injection technique reduced plate numbers by as much (21) Jacobson, S. C.; Ramsey, J. M. Electrophoresis 1995, 16, 481-486.
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as ∼40% compared to a pinched injection. In contrast, the similarity of the peak widths observed in correlograms derived from a single 7-bit sequence (Figure 1a) and from the average of 12 7-bit sequences (Figure 1c) indicates that performing long or repeated correlation sequences does not degrade significantly peak resolution when compared to shorter sequences. This suggests that the correlation approach described here could be extended to even longer sequences to reduce detection limits further. As an alternative to correlation techniques, Fourier approaches for improving detection limits on microfabricated devices have also been investigated.22 However, significantly enhanced detection limits have not been reported. CONCLUSION The results presented here demonstrate that cross-correlation electrophoresis with analog fluorescence detection delivers significantly higher sensitivity than a traditional electrophoretic separation of ultradilute dye solutions using a microchip format. Because separations on microchip devices can be faster than separations performed using conventional instruments, dramatic S/N enhancements can be achieved in modest times. For example, Figure 1b shows that taking the average of 12 consecutive 7-bit correlograms, which require only ∼7 min to acquire, elicits two distinct peaks from the noise that dominates the electropherogram shown in Figure 1a. An additional benefit of the rapid migration times observed on microchip devices is that cross-correlation electrophoresis could be used to follow chemical reactions generating minute quantities of fluorescent products. Rapid migration times are (22) Crabtree, H. J.; Kopp, M. U.; Manz, A. Anal. Chem. 1999, 71, 2130-2138. (23) Valentin, J. R.; Hall, K. W.; Becker, J. F. J. Chromatogr. 1990, 518, 199206. (24) Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, J. M. Anal. Chem. 1998, 70, 3476-3480.
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required because the duration of a single pseudorandom injection sequence must be long with respect to the time scale of composition changes in the sample; otherwise, artifacts and baseline drift appear in the resulting correlograms.23 Given the performance reported here, one could track reaction products on a tens of second time scale; higher field strengths and shorter electrophoresis channels could reduce this figure by ∼3 orders of magnitude.24 Finally, cross-correlation electrophoresis on microchip devices could be readily coupled to more universal detection techniques such as UV-visible absorption,15 refractive index, or indirect detection. These techniques exhibit higher detection limits than fluorescence spectroscopy, a condition exacerbated by the short path lengths and small sample volumes found on microchip devices. Cross-correlation electrophoresis could reduce the microchip detection limits of these techniques by more than 1 order of magnitude. ACKNOWLEDGMENT This research was sponsored by the Office of Research and Development in the U.S. Department of Energy. Oak Ridge National Laboratory (ORNL) is managed by Lockheed Martin Energy Research Corp. for the U.S. Department of Energy, under Contract DE-AC05-96OR22464. This research was sponsored in part by an appointment for J.C.F. to the ORNL Postdoctoral Research Associates Program, which is administered jointly by the Oak Ridge Institute for Science and Education and ORNL. We thank Justin E. Daler for fabrication of the microchips and Christopher T. Culbertson for programming assistance.
Received for review August 2, 1999. Accepted August 3, 1999. AC990853D
