Anal. Chem. 1999, 71, 678-686
Adaptation of Capillary Isoelectric Focusing to Microchannels on a Glass Chip Oliver Hofmann,*,† Diping Che, Kenneth A. Cruickshank, and Uwe R. Mu 1 ller
Vysis, Inc., 3100 Woodcreek Drive, Downers Grove, Illinois 60515
As a first step toward adaptation of capillary isoelectric focusing (cIEF) to microchannels on a glass chip, we have compared the three most common mobilization methods: chemical, hydrodynamic, and electroosmotic flow (EOF)-driven mobilization. Using a commercial cIEF apparatus with coated or uncoated fused-silica capillaries, both chemical and hydrodynamic mobilization gave superior separation efficiency and reproducibility. However, EOF-driven mobilization, which occurs simultaneously with focusing, proved most suitable for miniaturization because of high speed, EOF compatibility and low instrumentation requirements. When this method was tested in a 200-µm-wide, 10-µm-deep, and 7-cm-long channel etched into planar glass, a mixture of Cy5-labeled peptides could be focused in less than 30 s, with plate heights of 0.4 µm (410 plates/s) upon optimization. For a total analysis time of less than 5 min, we estimate a maximum peak capacity of approximately 30-40. Interestingly, the order of migration was found to be reversed compared to capillary-based focusing. Isoelectric focusing (IEF) is a powerful separation technique to resolve amphoteric molecules based on their isoelectric points (pI’s). Most commonly, IEF is applied to peptide and protein characterization. In 1985, Hjerten and Zhu transferred IEF from the conventional slab gel format to capillary electrophoresis (CE) equipment, thereby combining high resolving power with ease and speed of separation.1 In capillary isoelectric focusing (cIEF), the capillary is filled with a mixture of ampholytes and analyte, whereby the ends are immersed in acidic buffer (anolyte) and basic buffer (catholyte). Upon application of an electric field, a pH gradient is formed in the capillary and analyte molecules migrate to the position within the gradient where the pH equals their pI, thus losing their net charge. Typically, the focused zones are mobilized for detection. Chemical mobilization can be achieved by replacing anolyte with a base (anodic mobilization), by replacing catholyte with an acid (cathodic mobilization), or by adding zwitterions to anolyte or catholyte.1-7 In cathodic mobilization, analyte molecules previ† Present address: Department of Chemistry, Imperial College, London SW7 2AY, England; (e-mail)
[email protected]. (1) Hjerten, S.; Zhu, M.-D. J. Chromatogr. 1985, 346, 265-270. (2) Hjerten, S. J. Chromatogr. 1985, 347, 191-198. (3) Hjerten, S.; Liao, J.-L.; Yao, K. J. Chromatogr. 1987, 387, 127-138. (4) Hjerten, S.; Elenbring, K.; Kilar, F.; Liao, J.-L.; Chen, J. C.; Siebert, C.; Zhu, M.-D. J. Chromatogr. 1987, 403, 47-61. (5) Kilar, F.; Hjerten, S. J. Chromatogr. 1989, 480, 351-357.
678 Analytical Chemistry, Vol. 71, No. 3, February 1, 1999
ously focused at their pI values become positively charged and migrate toward the cathodic end of the capillary where they can be detected. The resulting pH gradient linearity is limited by the pH shift employed for mobilization and the presence of electroosmotic flow (EOF), which exhibits stronger mobilization at the basic end. Hjerten et al. have shown that coating of the capillary wall is required to minimize EOF and to reduce analyte adsorption.1-6 However, covalent coatings suffer from poor hydrolytic stability, especially at alkaline pH, and the related cost represents a major disadvantage.8 Hydrodynamic mobilization can be achieved by applying pressure or vacuum to the capillary while maintaining an electric field to reduce band dispersion.9-12 This is the only mobilization method that guarantees a constant flow rate and, as a consequence, provides a linear correlation between retention time and analyte pI.9 However, the need for coating capillaries combined with relatively high instrumentation costs present major drawbacks.9,10 In an effort to both simplify cIEF and circumvent the use of coated capillaries, a one-step cIEF method with simultaneous focusing and mobilization was developed by Mazzeo et al.13-15 and Thormann et al.16 By employing uncoated capillaries, a strong EOF is generated once the voltage is applied, resulting in continuous migration of the fluid column toward the cathode. Dynamic coating additives such as methylcellulose13-15 and hydroxypropylmethylcellulose16 are added to the ampholyte/analyte mixture to control the EOF and to minimize adsorption of analyte to the capillary wall. Computer models17,18 predict that due to its pH dependence the EOF may vary along the mobilized pH gradient by a factor of (6) Kilar, F.; Hjerten, S. Electrophoresis 1989, 10, 23-29. (7) Capillary Electrophoresis Application Note 31, Bio-Rad Laboratories, Hercules, CA. (8) Cobb, K.; Dolnik, V.; Novotny, M. Anal. Chem. 1990, 62, 2478-2483. (9) Chen, S.-M.; Wiktorowicz, J. E. Anal. Biochem. 1992, 206, 84-90. (10) Huang, T.-L.; Shieh, P. C. H.; Cooke, N. Chromatographia 1994, 39, 543548. (11) Instructions 015-726466-AA, Beckman Instruments, Fullerton, CA. (12) Minarik, M.; Groiss, F.; Gas, B.; Blaas, D.; Kenndler, E. J. Chromatogr., A 1996, 738, 123-128. (13) Mazzeo, J. R.; Krull, I. S. Anal. Chem. 1991, 63, 2852-2857. (14) Mazzeo, J. R.; Krull, I. S. J. Chromatogr. 1992, 606, 291-296. (15) Mazzeo, J. R.; Martineau, J. A.; Krull, I. S. Anal. Biochem. 1993, 208, 323329. (16) Thormann, W.; Caslavska, J.; Molteni, S.; Chmelik, J. J. Chromatogr. 1992, 589, 321-327. (17) Thormann, W.; Molteni, S.; Stoffel, E.; Mosher, R. A.; Chmelik, J. Anal. Methods Instrum. 1993, 1, 177-184. (18) Steinmann, L.; Mosher, R. A.; Thormann, W. J. Chromatogr., A 1996, 756, 219-232. 10.1021/ac9806660 CCC: $18.00
© 1999 American Chemical Society Published on Web 12/29/1998
4-6, resulting in considerable nonlinearity of the pH gradient15 and somewhat irreproducible migration times.18 The fact that one-step cIEF is typically the fastest cIEF method is not only due to EOF being a strong mobilization force but also due to the short separation distances employed.14,19 In the reversed polarity mode reported by Moorhouse et al.,19 anolyte and positive potential are assigned to the short end of the capillary and catholyte and ground to the long end. Since EOF is generated in the direction of positive potential to ground, only the short capillary end is accessed as separation distance prior to detection. For a 27-cm capillary, this reduces the separation distance from 20 to 7 cm, leading to significantly shorter run times. In this configuration, even coated capillaries can be used, with the residual EOF still being strong enough to allow for run times of less than 10 min.19 Over the last 10 years, intensive work has been directed toward miniaturization of analytical methods most notably in the field of CE.20 By using conventional photolithography and microfabrication technology, channel structures and even integrated devices can be micromachined on chips made of silicon or glass.21 Gains include low cost, speed, and portability. We have developed a novel generic multiplex diagnostic assay system that is equally applicable to genetic assays as well as immunoassays. Multiplexing is achieved through a new set of fluorescent detection moieties, which are differentiated from each other by their pI.22 This allows the development of sandwich assays with simplified detection (single excitation and emission wavelength), whereby moieties are released from their detection probe (or antibody) in the final step of the assay and resolved by cIEF. The goal is to perform the complete sandwich assay, including cIEF, in a single integrated device, through miniaturization of all assay and detection steps. In the course of this development, it was critical to determine whether cIEF could be performed in a microchannel, and which mobilization method would be most suitable. While a cIEF method comparison is described by Schwer,23 it is based on experiments conducted at different field strengths using different coated capillaries. Since uncoated fused-silica capillaries were not employed in that study, suitability for miniaturization onto a glass chip cannot be deduced. We have therefore conducted a comparison of different cIEF methods, with subsequent transitions to uncoated fused-silica capillaries and to shorter separation distances in order to mimic the conditions in microchannels more closely. The feasibility of chip-based cIEF with laser-induced fluorescence (LIF) detection was then demonstrated by separating Cy-5 labeled peptides. EXPERIMENTAL SECTION Material and Reagents. For the cIEF method comparison, an eCAP neutral capillary and a P/ACE 5510 system with absorbance detection at 280 nm or LIF detection (excitation at (19) Moorhouse, K. G.; Eusebio, C. A.; Hunt, G.; Chen, A. B. J. Chromatogr., A 1995, 717, 61-69. (20) Effenhauser, C. S. In Microsystem technology in chemistry and life science; Becker, H., Manz, A., Eds.; Current Topics in Chemistry, Special Volume; Springer-Verlag: Heidelberg, Germany, 1998; pp 51-82. (21) Van den Berg, A., Bergveld, P., Eds. Micro Total Analysis Systems; Kluwer Academic Publisher: Boston, 1995. (22) Cruickshank, K. A.; Olvera, J.; Mu ¨ ller, U. R. J. Chromatogr., A 1998, 817, 41-47. (23) Schwer, C. Electrophoresis 1995, 16, 2121-2126.
Table 1. Protein pI Markers Used To Determine pH Gradient Linearity of cIEF Methods no. 1 2 3a-c 4a,b 5 6 7 8 9
protein hearta
cytochrome c from bovine ribonuclease A trypsinogen from bovine pancreasb lentil lectin from lens culinaris myoglobin from horse heart carbonic anhydrase I from human erythrocytes carbonic anhydrase II from bovine erythrocytes β-lactoglobulin A from bovine milk trypsin inhibitor from soya bean glucose oxidase from Aspergillus nigerb amyloglucosidase from A. niger pepsinogen from porcine stomacha,b
concn (mg/mL)
pI
0.09 0.28 0.10 0.35-0.36 0.07-0.09 0.06-0.14
10.25 9.45 9.3 8.8/8.6/8.2 7.2/6.8 6.6
0.03-0.09
5.9
0.07-0.17 0.03-0.13 0.20 0.03-0.21 0.14-0.18
5.1 4.6 4.2 3.6 2.8
a Purified proteins, not specified as pI markers. b Initially tested as pI markers; not used for shown results due to bad focusing performance.
488 nm) were used (Beckman, Fullerton, CA). The He-Ne laser (excitation at 632.8 nm), which was used for the chip-based experiments, was not commercially available at the time for the Beckman system. The internal diameter of the capillary was 50 µm, and the distances from inlet and outlet to the detection window were 20 and 7 cm, respectively. An uncoated fused-silica capillary of similar dimensions was employed for one-step cIEF modifications. All protein pI markers for the method comparison (see Table 1) were purchased from Sigma (St. Louis, MO). BioLyte ampholytes pH 3-10, anolyte, catholyte, and cathodic mobilizer used for cIEF with chemical mobilization were purchased from Bio-Rad (Hercules, CA).7 For cIEF with hydrodynamic mobilization, a commercially available kit from Beckman was used, including Beckman ampholytes pH 3-10 in cIEF gel, anolyte, and catholyte.11 For one-step cIEF, Pharmalyte ampholytes pH 3-10 were obtained from Pharmacia LKB (Piscataway, NJ), 99% glycerol was from Sigma, and N,N,N′,N′tetramethylethylenediamine (TEMED) was from Bio-Rad. Using standard protocols, synthesized peptides were labeled with Fluorolink monofunctional Cy5 dye purchased from Amersham (Pittsburgh, PA). Capillary Conditioning. Before each run, the capillary was conditioned with a series of high-pressure rinses. Coated capillaries were rinsed with 0.1 M HCl, 10 mM H3PO4, and water for 2 min each. Uncoated fused-silica capillaries were rinsed with 0.1 M NaOH for 2 min followed by water for 2 min. cIEF with Chemical Mobilization. The experimental conditions described in a Bio-Rad application note7 were modified as follows. Focusing and mobilization voltage were reduced to 13.5 kV, corresponding to an electric field strength of 0.5 kV/cm, while focusing and mobilization time were extended to 10 and 40 min, respectively. All runs were performed in the normal polarity mode (see Figure 1A). cIEF with Hydrodynamic Mobilization. No changes were made to the experimental conditions recommended by the Beckman kit.11 Focusing voltage was kept at 10.8 kV (0.4 kV/ cm) and maintained during pressure-induced mobilization. Focusing and mobilization times were set to 5 and 30 min, respectively. All runs were performed in the normal polarity mode (see Figure 1A). Analytical Chemistry, Vol. 71, No. 3, February 1, 1999
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Figure 1. Polarity modes used for cIEF. (A) Normal polarity mode for capillaries, separation distance 20 cm. (B) Reversed polarity mode for capillaries, separation distance 7 cm.19 Due to instrumental constraints the point of detection is fixed. (C) Polarity mode for microchannels on a chip. The point of detection can be moved. Separation distances of 0.5, 3.5, and 6.5 cm were chosen. D represents the point of detection. aBased on mechanistic hypothesis (see Results and Discussion).
One-Step cIEF. Minor changes were made to the experimental conditions given by Moorhouse et al.19 As problems with analyte solubility were not observed, urea was omitted from the sample/ampholyte mixture. Furthermore, only wide range Pharmalyte ampholytes pH 3-10 (3 w/v %) were used. A voltage of 13.5 kV (0.5 kV/cm) was applied, causing simultaneous focusing and mobilization. Runs for the method comparison were performed in a coated capillary in the reversed polarity mode (see Figure 1B). In the course of modifying one-step cIEF, uncoated fused-silica capillaries in both the normal polarity mode (see Figure 1A) and the reversed polarity mode were also employed. Microchannel Layout for Chip-Based One-Step cIEF. The aim of this research was to evaluate changes associated specifically with the transfer from a capillary to a chip format rather than studying the effects of specific channel configurations. Thus, we have designed the microchannels in planar glass to mimic the short end of the 50-µm-i.d. capillary that was used for the reversed polarity experiments (see Figure 1B). At identical volume (140 nL) and length (7 cm), the 200-µm-wide and 10-µm-deep plain microchannels exhibit a 3-fold higher surface-to-volume ratio which should yield not only stronger EOF and consequently shortened separation times but also reduced Joule heating allowing for higher field strength to be applied. 680 Analytical Chemistry, Vol. 71, No. 3, February 1, 1999
Fabrication of the Glass Chip Devices. The microchannels were fabricated in 2-mm-thick soda lime glass (Nanofilm, Westlake Village, CA) using standard photolithographic techniques24 and chemical wet etching. For isotropic etching, a 1:10 dilution of buffered oxide etch (BOE) with surfactant was employed (Hawkins Chemical, Minneapolis, MN). Addition of the surfactant yielded better dimensional consistency and smoothness of the channel edges. Etching was performed at room temperature with ultrasonication used for agitation. Glass cover plates with holes coinciding with the channel ends were fabricated by means of water-cooled drilling with a diamond core drill bit (Diamond Industrial Tools, Lincolnwood, IL). Prior to thermal bonding of a cover plate onto the top of a structured plate, a standard cleaning procedure was performed including wiping the surface with a cleaning solution, water rinse, and ultrasonication in acetone for 10 min, followed by a second water rinse and blow drying with a stream of dry nitrogen. The two aligned plates were then heated to 665 °C (1 h), kept at this temperature for 4 h, and allowed to cool slowly (5 h). Bonding of ∼80% of the area was routinely achieved. (24) Effenhauser, C. S.; Manz, A.; Widmer, H. M. Anal. Chem. 1993, 65, 26372642.
Chip Preparation and Operation. Pipet tips were inserted into the holes of the cover plate to serve as fluid reservoirs. Prior to sample loading, the microchannel was first cleaned and rinsed using the identical procedure in each case in order to preserve reproducibility. Rinse liquids (prefiltered with a 0.22-µm-pore size syringe filter) were added to the reservoirs and forced through the capillary using a nitrogen stream at 60 psi (1 psi ) 6894.76 Pa). NaOH (0.1 M) was rinsed for 5 min in one direction followed by a second 5-min rinse from the second direction. This process was followed by water rinses (2 min each direction). The microchannel was then filled with the sample/ampholyte mixture using the same process. By rinsing for 5 min, filling of the entire microchannel was achieved. Excess sample/ampholyte was then removed from the reservoirs and replaced by 300 µL of 20 mM NaOH (catholyte) and 10 mM H3PO4 (anolyte). Potential mixing of sample/ampholyte in the microchannel with catholyte or anolyte in the reservoirs by hydrodynamically induced flow was minimized by filling both reservoirs simultaneously. The loaded microchannel was then subjected to voltage application with minimal delay. Setup for Chip-Based One-Step cIEF. The chip was positioned using an x, y, z stage, and platinum wires serving as electrodes were inserted into the reservoirs. Power was supplied by a high-voltage power supply model HCE 7-12500 (Fug Electronic, Rosenheim, Germany). Electrophoretic current was measured with an electrometer (model 617, Keithley, Cleveland, OH). Epi-illumination configuration was used for LIF detection. The excitation source was a 50-mW, 632.8-nm He-Ne laser (model 05LHR831, Melles Griot, Irvine, CA). The laser light was reflected by a dichroic beam splitter (Chroma Technology, Brattleboro, VT) and focused onto the microchannel by a 20×, NA 0.4 objective (Olympus, Melville, NY). The objective also collected the fluorescent emission from the microchannel, which was then focused through a long-pass interference filter (HQ650LP, Chroma Technology; cutoff at 650 nm) and a 1-mm-diameter pinhole onto the PMT (model R298P, Hamamatsu, Shizuoka, Japan). By means of the pinhole, a virtual detection window of 50 µm was defined on the microchannel. Laser power was attenuated with a neutral density filter (OD 2.0, Oriel, Stratford, CT) to limit the anode current below the PMT rating. A photodetector amplifier (model PDA 1, World Precision Instruments, Sarasota, FA) coupled with a data acquisition board (model PCI-MIO-16XE-50, National Instruments, Austin, TX) was used to record the PMT output. To suppress electronic noise, the signal was filtered with a low-pass filter at a time constant of 0.1 s. Typically a sampling rate of 5 Hz was used. System control and data acquisition were accomplished through a Power Mac. While one DAC output was used to control the HV power supply, two ADC inputs were employed to record the signals from the electrometer and PMT. The user interface consisted of an application window generated with LabVIEW software. Voltage was adjusted by means of a dial and both the electropherogram (PMT signal) and electrophoretic current were displayed in real time. Chip-Based One-Step cIEF. Cy5-labeled peptides were directly dissolved in 40 w/w % aqueous glycerol containing 1.85 w/v % Pharmalyte ampholytes (optimized value). Addition of 7.5
v/v % TEMED, intended to be used as a basic blocker, resulted in broad peaks of low intensity and was therefore discontinued. The sample/ampholyte mixture was introduced into the microchannel using a standardized filling procedure (see Chip Preparation and Operation). Positive high voltages of 4-10.5 kV (0.61.5 kV/cm) were then applied to the anolyte while the catholyte was connected to ground (see Figure 1C). The detection window was placed 0.5, 3.5, and 6.5 cm, respectively, away from the cathodic end. Calculations To Determine Separation Performance. Resolution, R, in terms of absolute value25 was calculated for two lentil lectin peaks (pI 8.2/8.6, capillary-based results) to determine the degree of baseline resolution. Plate number, N, plates per second, N/s, and plate height, h, were all determined for representative peaks at the center of the pH gradient using standard equations.25 For carbonic anhydrase II (pI 5.9, capillary-based results) and Cy5GHK (centered between Cy5-VESSK and Cy5-VHLTPVQK, chipbased results), the velocity-induced differences in peak width at the pH extremes ought not to be considered. RESULTS AND DISCUSSION We have compared several cIEF methods in terms of separation efficiency, pH gradient linearity, speed, reproducibility, robustness, EOF compatibility, and instrumentation requirements in order to determine their suitability for miniaturization. Each method was tested with multiple runs of a mixture of protein pI markers (Table 1), and a representative electropherogram, as well as a plot of mean migration time (n ) 3-5) versus pI, is shown in Figure 2. Additional performance data for each method are compared in Table 2. To achieve chemical mobilization, the catholyte was exchanged against a zwitterionic solution serving as a mobilizer as soon as the current dropped to a minimum (not shown).7 The total time for focusing and mobilization of the complete gradient was typically ∼50 min (Figure 2A). As a result, resolution of better than 0.1 pI unit was achieved as determined by the baseline resolution of lentil lectin peaks 3a and 3b, which differ by 0.2 pI unit. This value for nonoptimized conditions compares to 0.05 pI unit resolution for optimized conditions reported previously for this method.7 In addition to high resolution, this method yielded submicrometer plate heights with plate numbers reaching 600 000 and a time-based separation efficiency of almost 300 plates/s (Table 2). Reproducibility proved to be good for migration time and excellent for peak height. The low reproducibility of the peak area for the sharp peaks obtained might be attributed to a nonoptimized integration protocol. The plot of migration time versus pI of focused proteins (Figure 2A) suggests that a fairly linear pH gradient is established between pH 4.6 and 8.2 with a correlation coefficient of 0.991 for the linear regression. On both ends of the gradient, however, this linearity is lost. In addition, the electropherogram reveals that early (basic) peaks are generally broader than late (acidic) peaks. Because of the results achieved with hydrodynamic mobilization (discussed below), we believe that this is due to relative peak velocities rather than the physical distribution of the pH gradient. In other words, because of pH effects on the zwitterionic mobilizer, its mobilization force changes such that early proteins with basic pI migrate past (25) Giddings, J. C. Unified Separation Science; John Wiley: New York, 1991.
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Figure 2. Electropherograms (left) and pH gradient linearity plots (right) for cIEF methods. (A) cIEF with chemical mobilization. (B) cIEF with hydrodynamic mobilization. (C) One-step cIEF. Experimental conditions were as described in the Experimental Section. Key: (1) cytochrome c, (2) ribonuclease A, (3a-c) lentil lectin I-III, (4a,b) myoglobin I-II, (5) carbonic anhydrase I, (6) carbonic anhydrase II, (7) β-lactoglobulin, (8) trypsin inhibitor, and (9) amyloglucosidase. Proteins 1 and 3-9 injected for (A) (no peak obtained for 9); proteins 3-9 injected for (B); proteins 2, 4, 6, and 8 injected for (C). Concentrations, see Table 1.
the detection window relatively slowly compared to the more acidic proteins, making their peaks appear broader than they really are. At the acidic end of the gradient peaks appear extremely sharp, and peak 9 is missing. Presumably this very acidic protein is focused in a stationary steady state just before the detection window.3 For demonstration we chose hydrodynamic mobilization to be faster than chemical mobilization, reducing average run times to 682
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35 min (Figure 2B). For the sake of clarity, protein 1 was not injected. A significantly different profile for peak separation was obtained, whereby relatively poor resolution was achieved at the basic end (incomplete separation of peaks 3a-4a), an equal resolution in the center of the gradient (baseline separation of peaks 4b-7; peaks 4b and 5 separated by 0.2 pI unit), and an increased resolution at the acidic end. Peak 8 now appears to be separated into multiple smaller peaks, and a well-separated peak
Table 2. Comparison of Different cIEF Methods cIEF property
chemical mobilizationa
hydrodynamic mobilizationb
one-stepc
time windowd (min) repr migr time (% RSD)d repr peak height (% RSD)e repr peak area (% RSD)e linearity pH gradient (r) resolutionf plate no.e plates/se (1/s) plate heighte (µm) robustness instrumentation requirements
10-42 2.0 0.6 11.0 0.991 (pI 4.6-8.2) 4.5 592000 263 0.35 excellent moderate
15-33 1.9 2.5 4.7 0.994 (pI 5.1-7.2) 0.7 135000 108 1.50 good high
9-13 4.0 3.2 1.8 0.997 (pI 5.9-9.5) ndg 27000 41 2.64 ndg none
a E ) 0.5 kV/cm, n ) 5. b E ) 0.4 kV/cm, n ) 3. c E ) 0.5 kV/cm, n ) 5. d Based on migration times for protein pI markers injected; see caption to Figure 2. e Values determined for carbonic anhydrase II peak. f Values determined for lentil lectin peaks 2 and 3. g Not determined.
Figure 3. One-step cIEF in a fused-silica capillary using the reversed polarity mode, E ) 0.5 kV/cm. (A) Electropherogram; (B) pH gradient linearity plot. Peak identification and concentrations as for Figure 2C.
9, followed by an additional smaller peak, becomes visible. We have eliminated salt-related protein precipitation as a possible explanation for these new peaks since they also appear with oncapillary desalting11 (results not shown). The compression of peaks at the basic end may be due to the additional mobilization caused by not totally suppressed EOF, which is considerable at the basic end of the pH gradient. Consequently, basic peaks may be swept past the detection window prior to complete focusing, resulting in poor resolution.
Overall, at moderate speeds, this method produces good separation efficiency for the lower pH range with plate heights of ∼1.5 µm, plate numbers topping 100 000, and a relatively high time-based separation efficiency of ∼110 plates/s (Table 2). Reproducibility was found to be good for migration time and peak height and moderate for peak area. The third method tested uses EOF as the only force to move the ampholyte solution toward the cathode. Even with a neutral capillary coating, a sufficiently strong EOF is generated.19 To reduce run times, the approach of Mazzeo and Krull14 and Moorhouse et al.19 was used which allows for shortened separation distance without changing the capillary. In the reversed polarity mode, only 7 out of 27 cm separation distance was accessed prior to detection (Figure 1B). When applied to a neutraly coated capillary this results in low run times of less than 15 min (Figure 2C). Based on the anticipated resolution, only proteins 2, 4, 6, and 8 were injected. A near-baseline separation of myoglobin peaks 4a and 4b suggests a resolution of ∼0.4 pI unit. The whole pH gradient appears to be confined to a 5-min window, which is preceded by a low absorbance plateau and followed by a high absorbance plateau (similar window reported in ref 18). This baseline profile was found for all EOF-mobilized cIEF runs, including those with an uncoated fused-silica capillary (Figure 3) or uncoated microchannels in glass (fluorescence profile in reversed order, Figures 4-6). For the runs in a capillary with UV detection we attribute this to changes of the liquid phase, from TEMED/catholyte to focused sample/ ampholytes and then to anolyte. Ribonuclease A with a pI of 9.5 seems to coincide with the basic edge of the pH gradient (ampholytes pH range 3-10), while trypsin inhibitor (pI of 4.6) is distinctly focused away from the acidic edge. The plot of migration times versus pI’s (Figure 2C) suggests linearity in the pH range of 5.9-9.5. At the acidic end, the linearity plot gets steeper, possibly indicating that the acidic proteins are relatively retarded. Since EOF is the driving force for mobilization, a weaker flow is expected at acidic pH.18 Additionally, migration of the anodic end of the ampholyte gradient toward the anode may contribute to this retardation (anodic drift26). This effect can be minimized by increasing the anolyte concentration.14,26 Analytical Chemistry, Vol. 71, No. 3, February 1, 1999
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Table 3. Comparison of Different One-Step cIEF Modifications
property
coated capillary, normal polaritya
time windowc (min) linearity pH gradient (r) plate no.d plates/sd (1/s) plate heightd (µm)
59-89 0.999 (pI 5.9-9.5) 43000 9 6.3
fused-silica capillary normal polaritya reversed polarityb 35-50 0.999 (pI 5.9-9.5) 705000 279 1.5
3-5 0.982 (pI 4.6-9.5) 210000 816 0.3
a E ) 0.5 kV/cm, distance from anodic capillary end to point of detection 20 cm. b E ) 0.5 kV/cm, distance from anodic capillary end to point of detection 7 cm. c Based on migration times for protein pI markers injected; see caption to Figure 2C. d Values determined for carbonic anhydrase II peak.
The overall performance characteristics for this high-speed method are listed in Table 2. A moderate separation efficiency is documented by a plate height of 2.6 µm, plate numbers of ∼30 000 with a time-based separation efficiency of ∼40 plates/s. Reproducibility of migration time is only half as good as for the other methods, while peak height is moderately and peak area highly reproducible. Overall, chemical mobilization appeared to produce the best separation characteristics for cIEF while hydrodynamic mobilization allows for faster run times with somewhat decreased separation efficiency (Table 2). Though hydrodynamic mobilization could be made even faster (at the expense of reduced separation efficiency12), it does not lend itself to miniaturization. The onestep cIEF with shortened separation distance and EOF as the only mobilization force is much more suitable in that regard, albeit at the cost of further reductions in separation efficiency and reproducibility. It is important to realize that adaptation of cIEF to microchannels on a glass chip as part of an integrated device requires that the method be compatible with EOF, which is intended to be used for liquid handling, and should be as simple as possible to ease automation. Both issues are well addressed by the one-step cIEF method, which has the additional advantage that the feasibility of this method on fused-silica capillaries has already been demonstrated,13-16 suggesting that a transfer to microchannels on a glass chip should present few technical hurdles. To mimic the conditions in microchannels on planar glass more closely, the one-step cIEF method was transferred to an uncoated fused-silica capillary (Figure 3). In the reversed polarity mode (Figure 1B), exceptionally low run times of less than 5 min were achieved corresponding to a 3-fold reduction in run times compared to a coated capillary (Figure 2C). While a slight loss of peak resolution was observed, possibly due to the reduced focusing time, plate height and plate numbers were significantly improved (Table 3). The effect of focusing time on separation efficiency was tested with both uncoated and coated fused-silica capillaries in the normal polarity mode (electropherograms not shown). As shown in Table 3, excellent separation efficiencies were obtained with the uncoated fused-silica capillary, with typical run times of up to 50 min. In contrast, the separation efficiency for the coated capillary was markedly deteriorated, presumably due to low and varying residual EOF. A 200-µm-wide, 10-µm-deep, and 7-cm-long plain channel etched into planar glass was used to investigate potential changes
associated with the transfer of the one-step cIEF method to the chip format. Protein pI markers were replaced by Cy5-labeled peptides to allow detection by laser excitation at 632 nm (see Experimental Section). All other experimental conditions remained unchanged. Several problems were encountered in initial experiments including lower than expected peak intensities, a sample concentration-related baseline fluorescence plateau at the start, and a broad peak at the end of the gradient. We attributed the fluorescence plateau and sample loss to early EOF-driven carryout of sample prior to focusing, after determining that this was not due to sample degradation upon contact with anolyte and/or catholyte. This was tested by stabilizing the sample/ampholyte mixture in the channel with aqueous glycerol (40 w/w %) and introducing a density gradient between the ends of the channel and the anolyte and catholyte in the reservoirs. Since only minor improvements were achieved with these modifications, their use was discontinued. From this point on, however, we substituted HPMC with glycerol as the EOF suppression agent. Subsequent experiments revealed that at least some of the above-mentioned problems were related to TEMED, which extends the pH gradient to pH 12 and serves as a cathodic blocker.27 A representative electropherogram of a chip-based run without TEMED in the sample/ampholyte mixture is depicted in Figure 4. Good run-to-run reproducibility was achieved with typical
(26) Mosher, R. A.; Thormann, W. Electrophoresis 1991, 11, 717-723.
(27) Guo, Y.-J.; Bishop, R. J. Chromatogr. 1982, 234, 459-462.
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Figure 4. One-step cIEF in a microchannel on a glass chip, E ) 0.6 kV/cm, detection at l ) 0.5 cm (cathodic channel end). Peptides in 40 w/w % aqueous glycerol/1.85 w/v % Pharmalytes. Key: (1) Cy5lysine, (2) Cy5-VHLTPVEK, and (3) Cy5-VHLTPVQK (900, 600, and 150 nM, respectively).
Table 4. Optimization of Separation Efficiency for One-Step cIEF in a Microchannel on a Glass Chip detector position, la property time windowd (min) plate no.e plates/se (1/s) plate heighte (µm)
0.5 cm
3.5 cm
0.3-0.5 5-7 8000 342 0.6
sample mediumb dd H 2O 1.5-2.5
44000 15000 120 108 0.8 2.3
electric field strength, Ec
40 w/w % 1 aq glycerol kV/cm
1.5 kV/cm
5-7
2.5-3.5 1.5-2.5
44000 120 0.8
83000 411 0.4
42000 319 0.8
a Distance from cathodic channel end to point of detection; results for analytes in 40 w/w % aqueous glycerol/1.85 w/v % Pharmalytes, E ) 0.6 kV/cm. b Medium in which analytes and 1.85 w/v % Pharmalytes are dissolved; results for point of detection at 3.5 cm, E ) 0.6 kV/cm. c Results for point of detection at 3.5 cm; analytes in 40 w/w % aqueous glycerol/1.85 w/v % Pharmalytes. d Based on migration times for peptides injected: Cy5-VESSK, Cy5-TSK, Cy5-VTKG, Cy5-GHK (300 nM each). e Values determined for Cy5-GHK.
RSD values of below 2% for migration time (n ) 3). Peak intensities are as expected and the late broad peak is gone, suggesting that TEMED may have been responsible for peptide breakdown in previous runs. Two additional features of the electropherogram are noteworthy: (i) considering the expected separation distance of 6.5 cm, the extremely short migration times are surprising, and (ii) the baseline profile is reversed compared to the results obtained with a fused-silica capillary (Figure 3). Peak identification (by varying input concentrations; data not shown) revealed that the migration order is reversed also, being from low to high pI. However, it has to be noted that the expected pI values of our peptide markers were estimates solely based on the functional groups of the peptide sequences (experimentally determined pI values not available). The current profiles recorded for the chip-based runs (drop to a minimum followed by a slight increase; data not shown) strongly suggest that a typical focusing process occurred, despite the migration order reversal. Moreover, only focusing allows for peak formation without a proper injection step (entire microchannel filled with sample; see Experimental Section). On the basis of these considerations, we propose the following mechanism: At the start of the run, the faster migrating ampholytes begin to focus at the cathodic end of the channel and are then mobilized toward the anodic end. While the ampholyte gradient is mobilized the Cy5-peptides complete focusing, resulting in an increase of peak intensities. Plausible explanations for the initial zone formation at the cathodic end are the absence of TEMED (used as a cathodic blocker in the capillary-based experiments) and the presence of difficult-to-control hydrostatic pressure originating from the use of pipet tips as fluid reservoirs and potentially leading to displacement of the sample/ampholyte mixture within or out of the channel. Why all focusing zones are mobilized toward the anode is still unclear. To test this hypothesis, the detector was moved from the cathodic end toward the anode (see Figure 1C). Figure 5 shows the changes associated with moving the detector from 0.5 to 3.5 cm away from the cathodic end. As anticipated by our hypothesis, peak intensities more than doubled and peak resolution improved to baseline separation. A subsequent run with detection at 6.5cm distance from the cathodic end (i.e., 0.5 cm from the anodic
Figure 5. One-step cIEF in a microchannel on a glass chip, E ) 0.6 kV/cm. (A) Detection at l ) 0.5 cm (cathodic channel end); (B) detection at l ) 3.5 cm (half channel length). Peptides in 40 w/w % aqueous glycerol/1.85 w/v % Pharmalytes. Key: (1) Cy5-VESSK, (2) Cy5-TSK, (3) Cy5-VTKG, and (4) Cy5-GHK (300 nM each).
end) yielded no peaks and only broad baseline features, suggesting that focused zones become stationary prior to reaching the anodic end. This is not surprising in that a stationary steady state for acidic analytes was predicted for capillary-based experiments.18 For subsequent runs aimed to optimize the separation efficiency, the detector position was fixed at 3.5 cm. Parameters investigated included the medium used for the sample/ampholyte mixture and the effect of field strength (Table 4). Best results were obtained with 40 w/w % glycerol and 1 kV/cm, resulting in plate heights of 0.4 µm and more than 400 plates/s. This performance is at least equal, if not superior to what was achieved with cIEF and chemical mobilization in a capillary (Table 2). Note that a higher field strength was used for the optimized chip-based one-step cIEF results. The total peak-resolving capacity of cIEF under our optimized conditions was evaluated with a mixture of either six Cy5-peptides (Figure 6) or seven Cy5-peptides (including Cy5-lysine; not shown). We estimate that with analytes covering the whole pI range of 3-10 up to 40 peptides could be resolved. Analytical Chemistry, Vol. 71, No. 3, February 1, 1999
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namic mobilization gave superior separation efficiency and reproducibility, this one-step cIEF mode (simultaneous focusing and mobilization) combined high speed with EOF compatibility and low instrumentation requirements. When TEMED, typically used as a cathodic blocker, is deleted from the sample/ampholyte mixture, chip-based focusing can be achieved in less than 30 s. For total run times of less than 5 min, a peak capacity of 30-40 may be achievable. ACKNOWLEDGMENT
Figure 6. Optimized one-step cIEF in a microchannel on a glass chip, E ) 1 kV/cm, detection at l ) 3.5 cm (half channel length). Peptides in 40 w/w % aqueous glycerol/1.85 w/v % Pharmalytes. Key: (1) Cy5-VESSK, (2) Cy5-TSK, (3) Cy5-VTKG, (4) Cy5-VHLTPVEK, (5) Cy5-GHK, and (6) Cy5-VHLTPVQK (400, 300, 250, 150, 100, and 15 nM, respectively).
CONCLUSIONS On the basis of a comparison of several cIEF mobilization methods, we determined EOF-driven mobilization to be most suitable for miniaturization. While both chemical and hydrody-
686 Analytical Chemistry, Vol. 71, No. 3, February 1, 1999
The authors gratefully acknowledge Dr. D. Naylor, Mr. T. Cocco, and Mr. D. Gielow, of the University of Illinois at Chicago, for assistance with the microfabrication of the glass structures. We also thank Dr. Paul Bao and Mr. Joe Olvera (Vysis Inc.) for useful advice on the peptide moieties. This research was supported by the National Institute of Standards and Technology (ATP Award 95-08-0012).
Received for review June 18, 1998. Accepted November 8, 1998. AC9806660
