Anal. Chem. 1995, 67, 2284-2287
Manipulation of Sample Fractions on a Capillary Electrophoresis Chip Carlo 8. Eftenhauser,* Andreas Manz, and H. Michael Wldmer CorporateAnalytical Research, Ciba Ltd., CH-4002 Basel, Switzerland
A microfabricated capillary electrophoresis chip fias been used for selective isolation of fraction zones after fast size separation of a synthetic mixture of fluorescentphosphorothioate oligonucleotides. Withdrawal of preselected sample zones is achieved by simple automated switching of the applied potentials to a channel system which has been etched into a glass plate by conventional microfabrication techniques. Both single sample zones and groups of zones can be selectivelyisolatedfrom unwanted sample components on a time scale of a few minutes. A single sample component, originally injected into a volume of -90 pL prior to the separation, was still confined within a volume of -300 pL after the separation and removal procedure; Le., extensive dilution is avoided. Complex timing protocols allow for the withdrawal of any subset in the sequence of separatedfractions. The results demonstrate the high degree of sample control that can be obtained on gel-filledmicrostructureswith samples in the pico- to nanoliter range. They are of particular relevance for future developments of micromanipulation and microanalysis systems for biochemical analysis. Over the past few years, a number of experimental procedures has been described in the literature for the collection of sample fractions separated by capillary electrophoresis (CE), either for micropreparative purposes or to subject the selected fraction to a subsequent The major difficulty is that the outlet end of the capillary has to be part of a closed electrical circuit to be able to perform the separation,while at the same time it should provide some means for the withdrawal of the separated bands. In the stopped-flow techniques?+ the separation potential has to be interrupted and the end of the separation capillary has to be transferred from the buffer vial into a fraction collection vial. Switching the voltage on again for a brief period of time then drives the sample zone of interest into the collection vial. (1) Hjerten, S.; Zhu, M:D. J. Chromatogr. 1985,327,157. (2) Rose, D.J.; Jorgenson, J. W. J. Chromatogr, 1988,438,23. (3) Cohen, A S.;Najarian, D. R.; Paulus, A; Guttman, A; Smith, J. A; Karger, B. L. Proc. Natl. Acad. Sci. U.S.A. 1988,85, 9660. (4) Guttman, A;Cohen, A; Heiger, D. N.; Karger, B. L. Anal. Chem. 1990, 62,137. (5) Albin, M.; Chen, S.-M.; Louie, A; Pairaud, C.; Colbum, J.; Wiktorowicz, J. Anal. Biochem. 1992,206,382. (6) Huang, X.;Zare, R N.J. Chmmatogr. 1990,516, 185. (7) Guzman, N. A; Trebilcock, M. A; Advis, J. P. Anal. Chim. Acta 1991, 249,247. (8) Fujimoto, C.; Muramatsu, Y.; Suzuki, M.; Jinno, IC J. High Resolut. Chromatogr. 1991, 14,178. (9) Camillen, P.; Okafo, G. N.; Southan, C. Anal. Biochem. 1991, 196, 178. (10) Cheng, Y.-F.; Fuchs, M.; Andrews, D.; Carson, W. J. Chromatogr. 1992, 608,109. (11) Eriksson, IC-0.; Palm, A; Hjerten, S. Anal. Biochem. 1992,201, 211. 2284 Analytical Chemistry, Vol. 67, No. 73,July 7, 7995
Although this technique has been successfully applied, it usually leads to considerable dilution of the collected sample zones. In another experimentalapproach, the capillary is elechidy grounded before the outlet orifice,6-* e.g., by means of an on-column frit or a specially designed grounding tee assembly in a way similar to the techniqes used for electrochemical detection in CE and for CE-MS coupling. This allows for reduction in the extent of sample dilution, but manufacturing of these devices is not an easy task since dead volumes have to be kept at a sufficiently low level. Recently, elegant fraction collection methods, by means of moving surfaces such as filter papeF or membranes?JO have been reported. In the present article, we describe a novel approach to sample fraction manipulation in CE which is based on the use of microfabricated planar devices. It has been demonstrated in a number of recent publications that column switching operations on planar glass structures can be simply controlled by switching the electric p ~ t e n t i a l s . ~As ~ - a~ consequence ~ of the photolithographic fabrication process, dead volumes at capillary intersections can be kept at a negligibly low level. Micromachmed CE devices should therefore be ideally suited for the sample manipulation operations required for fraction collection with samples in the picoto nanoliter range. We report here on the first sample manipulation experiments carried out on glass chips with gel-filled channel systems, which complement our previously published results on high-speed separations of anti-sense oligonucleotides.18 In this context, we would like to point out that very recently high-speed separations of DNA fragments on similar devices have been rep01-ted.l~ EXPERIMENTAL SECTION Most details of the experimental procedure and the detection and data acquisition system were described in two earlier publi~ations.'~J~ Therefore, only some relevant new aspects are described here. The layout of the channel system on the glass chip is identical to the one described previously13and is reproduced in Figure 1. The channel system was filled with a noncross-linked polymerized solution containing 10%polyacrylamide in a 100 mM Tris, 100 mM boric acid, 2 mM EDTA, and 7 M (12) Harrison, D.J.; Flun, IC; Seiler, IC;Fan, Z.; Effenhauser, C. S.; Manz, A Science 1993,261,895. (13) Effenhauser, C. S.; Manz, A; Widmer, H. M. Anal. Chem. 1993,65,2637. (14) Seiler, IC; Harrison, D. J.; Manz, A Anal. Chem. 1993,65,1481. (15) Seiler, IC;Fan, Z. H.; Fluri, K; Harrison, D. J. Anal. Chem. 1994,66,3485. (16) Jacobson, S. C.; Hergenrbder, R; Koutny, L B.; Ramsey, J. M. Anal. Chem. 1994,66,1114. (17) Burggraf, N.;Manz, A; Verpoorte, E.; Effenhauser, C. S.; Widmer, H. M.; de Rooij, N. F. Sew. Actuators E 1994,20, 103. (18) Effenhauser, C. S.; Paulus, A; Manz, A; Widmer, H. M. Anal. Chem. 1994, 66,2949. (19) Woolley. A T.; Mathies, R A Proc. Nafl.Acad. Sci. U.S.A.1994,91,11348. 0 1995 American Chemical Society 0003-2700/95/0367-2284$9.00/0
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Figure 2. Electropherogram of a fluorescein-labeled phosphorothioate oligonucleotide mixture PS pd(T)10-2~mixture recorded at 1130 V/cm in a 10% Tnon-cross-linked polyacrylamide matrix (buffer: 100 mM Tris, 100 mM boric acid, 2 mM EDTA, 7 M urea, pH 8.5). Separation length, 7.0 cm. The chain length of the oligonucleotides is tentatively assigned. Figure 1. Layout of the glass chip with integrated sample injector. Channel cross sections: 50 x 12 (thin channels) and 250 x 12 p m (broad channels), respectively. After application of a high voltage between reservoirs 1 (sample) and 4 (injection waste), the geometrically defined injection volume (double-'T injection, shaded area) is filled by electrophoretic migration of the sample ions. After loading is completed, application of a high potential between reservoirs 2 (buffer) and 5 (waste) drives the injected sample plug into the separation channel and causes electrophoretic separation of the sample components. The T-intersection where the selected sample zones are withdrawn is indicated by the arrow.
urea buffer (PH8.5) according to the method described in ref 18. The injection and separation procedure of the fluorescentlylabeled mixture of phosphorothioate oligonucleotides PS pd010-25 are reported in the same reference. The geometricallydefined length of the injected sample plug is 150 pm (correspondingvolume, 90 pL). The intersection volume at the junction where the fraction collection takes place is 30 pL. The maximum voltage applicable to the separation capillary was limited to a potential of 12 kV due to the breakthrough voltage of the high-voltage relays used for potential switching in the experiment. As a result of the chip layout employed in our experiments (maximum usable length of the separation channel, 7 cm), the separation field strength was limited to 1130 V/cm. This figure is a factor of -2 lower than the field strength used in our earlier experiment.18 The resulting electropherogram recorded at maximum separation length is shown in Figure 2. In order to allow easy referencing of the individual peaks, the nucleotide length has been tentatively assigned. Due to the lower field strength and the longer separation length, the overall analysis time is increased by a factor of -4 compared to our earlier results. The principle of the experiment is schematically shown in Figure 3. Detection was accomplished via laser-induced fluorescence (LIF) with the whole detector unit mounted on a X-Y translational stage,I3so that any point of the channel system can be used as an oncolumn detection volume (length of detection volume, 40 pm). Before the fist run, the LIF detector was adjusted to a spot on the separation channel very close to the intersection. In this way, the arrival time distribution of the
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Figure 3. Schematic representation of the various phases of the withdrawal of a preselected sample zone. (phase 1) Application of a negative high potential at reservoir 2 and ground potential at reservoir 5 (reservoir 6, floating) separates the injected sample plug and drives the sample zones toward reservoir 5. When the sample zones of interest (solid circles) have arrived at the intersection, reservoir 6 is grounded for a few seconds and reservoir 5 left floating, thus switching the direction of the electric field toward reservoir 6 (withdrawal phase 2). The remaining sample zones (shaded circles) are removed from the system by switching back to phase 1 until the separation channel has been completely emptied (phase 3). Finally, the extracted fractions are moved past the detector by grounding reservoir 6 again while leaving reservoir 5 floating (phase 4). During the whole sequence, reservoir 2 is kept at a negative high potential. The location of the LIF detector is indicated by the arrows.
separated sample components at the immediate vicinity of the intersection was determined. Subsequently, the detector was aligned to a spot on the channel connecting the junction with reservoir 6, -2 mm away from the junction (Figure 3). In the following run, the actual fraction manipulation experiment was carried out by applying the separation field for a predetermined period of time until the sample fraction of interest Analytical Chemistry, Vol. 67, No. 13, July 1, 1995
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had arrived at the junction (phase 1 in Figure 3). Then, the direction of the electric field was switched by application of a brief fraction collection field “pulse” (reservoir 6 was set to ground potential while reservoir 5 was left floating). This pulse was applied for a few seconds with a field strength identical to the separation field (1130 V/cm) in order to drive the sample fraction into the left half of the T-bar channel toward reservoir 6 (phase 2). Switching the potentials back to the arrangement of phase 1 by grounding reservoir 5 then removed the remaining sample zones from the separation channel (phase 3). Finally, reservoir 6 was grounded again, and the selected fractions that were “cut out” from the sequence of sample zones were moved past the detector (phase 4). During the whole procedure, reservoir 2 is constantly kept at a potential of -12 kV. The timing protocol of the potential switching was controlled with a Labview I1 program (National Instruments, Austin, TX) running on a Macintosh Quadra 700 personal computer. A National Instruments interface board (NEMIO-16X) was used for data acquisition at a sampling rate of 100 Hz. RESULTS AND DISCUSSION The arrival time distribution of the separated bands at the channel intersection is shown in Figure 2. As has been already mentioned above, due to the lower maximum field strength a p plied and the longer separation length, the analysis time is a factor of -4 longer than our previously published results.1s It is interesting to note that even though the total separation voltage employed is even slightly lower (7.9 vs 8.4 kV in ref 18), the resolution is improved and the bands are almost baseline resolved. We attribute this finding to the fact that the relative band broadening contribution of the sample injection is decreased with increasing separation length (the length of the injected plug (150 pm) is the same in both experiments, whereas the separation length differs by a factor of 2; see discussion of band broadening in ref 18). The separation efficiency o b tained amounts to N = 295 000 theoretical plates for the band assigned as n = 24 with a corresponding plate height of 0.24 pm (separation length, 70 mm). A plot of the migration time (t) of the individual bands vs the nucleotide length (n) proved to be perfectly linear (t = 100.1 s n2.56 s, correlation coefficient r = 0.999 87). Figure 4 depicts the results of two fraction withdrawal experiments. Depending on the length of the collection pulse, a group of three sample zones F i r e 4a) or even a single sample zone (Figure 4b) have been selectively “cut out” of the electropherogram. It is important to note that the time scales indicated in this figure do not correspond to the time scale given in Figure 2, but rather represent the total time scale of the experiment including the manipulation procedure after the separation. The time needed to perform the whole operation was dominated by the relatively “long” analysis time of only little less than 3 min, which itself was caused by electric field strength limitations and the rather long separation length as discussed in the Experimental Section. Nevertheless, given the complexitiy of the procedure, the overall operation time of less than 4 min can be considered fast. The result of the withdrawal of three successive parts of the electropherogram is shown in Figure 5: (a) PS pdO16-18; (b) PS pdOi9-21; (4 PS ~dOzz-24. After the removal of the sample zones, the compounds are available on the chip in a spatially concentrated form that would
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Flgure 4. Automated withdrawal of preselected sample zones. (a, top) Fraction collection of PS pd(T)16-18: potential switching protocol (phase 1) 140 s, (phase 2) 5 s (fraction withdrawal “pulse”), (phase 3) 40 s, (phase 4) 65 s; total acquisition time, 250 s. (b, bottom) Fraction collection of PS pd(T)l8: potential switching protocol (phase 1) 143 s, (phase 2) 2 s (fraction withdrawal “pulse”), (phase 3) 40 s, (phase 4) 65 s; total acquisition time, 250 s. Electric field strength, 1130 Vlcm.
be suitable for performing further analytical operations. Since dead volume interferences have to be carefully avoided when working with such minute sample volumes, these operations should preferably be carried out on the same device. The full width at half-maximum (fwhm), in time units, of the peak shown in Figure 4b is -1 s. The migration velocity of this peak at 1130 V/cm was calculated to be 0.48 mm/s from the data depicted in Figure 2; i.e., the corresponding fwhm in length units amounts to -0.5 mm with a corresponding volume of 300 pL. Compared to the injected sample plug, the average concentration of the zone is reduced by only a factor of 3, with longitudinal diffusion being the dominant (and unavoidable) dispersion mechanism. During the removal of the remaining sample zones (phase 3), the extracted bands are subjected to diffusional band broadening. Furthermore, the intersection itself leads to additional band dispersion as discussed in ref 17 (so-called “comer effects”). For these two reasons, the resolution between individual bands of an extracted group of zones is discemably reduced (see Figure 4a and Figure 5).
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Figure 5. Superposition of three successive portions that have been "cut out" of the electropherogram shown in Figure 2: (a) PS pd(T)te-te; (b) PS pd(T)ie-ni; (c) PS pd(T)22-24. Fraction collection pulse (phase 2) in each experiment, 5 s.
The whole procedure described above was carried out in a completely automated manner under computer control, and in principle, it can be repeated until a sufficient amount of the compound of interest has been collected within the principal limitations of a microanalytical technique like CE. Since more complex manipulation protocols can be easily applied, one of the major advantages of this method is its versatility; it could be easily extended to collect more than one fraction per run,e.g., all oddor even-numbered nucleotides. Furthermore, coupled to other analytical techniques (e.g., two-dimensional or "hyphenated" methods like CE-MS or screening procedures based on bioafh i t y interactions), it would be a valuable tool to increase selectivity and peak capacity. In the current experimental setup, determination of the arrival time of the sample zones at the intersection prior to the actual fraction withdrawal is a necessary prerequisite to establish the timing protocol. The degree of control could be improved by (20) Manz, A; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. Adu. Chromatogr. 1993,33, 1. (21) See, for example, articles in: van den Berg, A, Bergveld, P., Eds. Micro Tofu2 Analysis Sytems; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1995.
installation of a second detector unit at the separation channel, which would allow a computercontrolled prediction of the arrival time of the desired fractions. The quality of the withdrawn zones could then be checked by the second detector unit in the way described above. In conclusion, we would like to emphasize that the excellent reproducibility of the migration time demonstrated in our earlier work**represents an important prerequisite for the experiments presented here. Once the time delay due to the distance between the detector spot and the intersection is known, a single determination of the arrival time distribution is sufiicient to determine the proper timing and pulse width of the collection pulses. In repetitive experiments with gel-filled devices, we have carried out several hundred experiments with the same device without a noticable decrease in operation performance. The results underline the high degree of substance transport control that can be obtained on gel-filled microstructures with sample volumes in the pice to nanoliter range. This capability represents a significant element for the general feasibility of the concept of miniaturized total analysis systems (LTAS)." Even though the sample volumes are small and the corresponding amount of substance in a sample zone is usually in the range of femtomoles and below, highsensitivity detection techniques like LIF will allow for adequate monitoring of the analytical operations on the chip. For special applications, micromachined devices can be operated virtually like a molecular "sorting device" and are of relevance to future developments of microreactors as well as for the micromanipulation and microanalysis of biochemical systems.21 ACKNOWLEDGMENT We thank Dr. Lendell L. C u m " (ISIS Pharmaceuticals, Carlsbad, CA) for the synthesis of the oligonucleotide sample. Furthermore, we thank Dr. A Paulus for assistance with the preparation of the gel-filled devices and Dr. k E. Bruno and Dr. M. A Malone (all Ciba) for reading the manuscript. Received for review January 1995.@
23, 1995. Accepted April 12,
AC9500693 Abstract published in Advance ACS Abstracts, June 1, 1995.
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