Preconcentration of Proteins on Microfluidic Devices Using Porous

Robert S. Foote,* Julia Khandurina,† Stephen C. Jacobson,‡ and J. Michael Ramsey§. Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tenne...
0 downloads 0 Views 265KB Size
Download PDF
Anal. Chem. 2005, 77, 57-63

Preconcentration of Proteins on Microfluidic Devices Using Porous Silica Membranes Robert S. Foote,* Julia Khandurina,† Stephen C. Jacobson,‡ and J. Michael Ramsey§

Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennessee 37831-6142

Fluorescently labeled proteins were electrophoretically concentrated on microfabricated devices prior to separation and laser-induced fluorescence detection on the same device. The proteins were concentrated using a porous silica membrane between adjacent microchannels that allowed the passage of buffer ions but excluded larger migrating molecules. Concentrated analytes were then injected into the separation column for analysis. Two basic microchip designs were tested that allowed sample concentration either directly in the sample injector loop or within the microchannel leading from the sample reservoir to the injector. Signal enhancements of ∼600-fold were achieved by on-chip preconcentration followed by SDSCGE separation. Preconcentration for CE analysis in both coated and uncoated open channels was also demonstrated. Fluorescently labeled ovalbumin could be detected at initial concentrations as low as 100 fM by using a combination of field-amplified injection and preconcentration at a membrane prior to CE in coated channels. Preconcentration has long been used in capillary electrophoresis and electrochromatography to enhance the detection of analytes. Methods for on-line preconcentration include fieldamplified stacking,1-3 sweeping with ionic detergent micelles,4,5 and solid-phase extraction (SPE) into immobilized phases attached either to the walls of open channels6 or to porous monoliths polymerized within the channel.7 Following SPE in sample buffer, the concentrated analytes are eluted from the immobilized phase by changing the buffer or solvent composition. Porous monoliths have been used to preconcentrate peptides and proteins by onchip SPE by factors as high as 1000.7,8 The degree of on-line concentration achievable by these methods is limited by the difference in the conductivities of sample and run buffers (field* To whom correspondence should be addressed: (phone) (865)-576-2032; (fax) (865) 574-8363; (e-mail) [email protected]. † Present address: Diversa Corp., 4955 Directors Place, San Diego, CA 92121. ‡ Present address: Department of Chemistry, Indiana University, Bloomington, IN 47405. § Present address: Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290. (1) Jacobson, S. C.; Ramsey, J. M. Electrophoresis 1995, 16, 481-486. (2) Yang, H.; Chien, R. J. Chromatogr., A 2001, 924, 155-163. (3) Jung, B.; Bharadwaj, R.; Santiago, J. Electrophoresis 2003, 24, 3476-3483. (4) Quirino, J.; Kim, J.; Terabe, S. J. Chromatogr., A 2002, 965, 357-373. (5) Kim, J.; Terabe, S. Pharm. Biomed. Anal. 2003, 30, 1625-1643. (6) Kutter, J. P.; Jacobson, S. C.; Ramsey, J. M. J. Microcolumn Sep. 2000, 12, 93-97. (7) Yu, C.; Davey, M. H.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2001, 73, 5088-5096. 10.1021/ac049136w CCC: $30.25 Published on Web 11/23/2004

© 2005 American Chemical Society

amplified stacking), the length of the injected sample plug (sweeping), or the adsorptive capacity of the immobilized phase (SPE). Proteins have also been preconcentrated by up to 40-fold in capillaries by using an electric field to oppose the flow of charged proteins within a short section of capillary through which the sample is hydraulically pumped.9 We have previously described the concentration of DNA fragments on a microchip prior to their separation on the same device by capillary gel electrophoresis (CGE).10,11 The microchips incorporated a porous membrane structure consisting of two parallel channels separated by 4-6 µm with a silicate bonding layer between the substrate and cover plate (Figure 1). The porous bonding layer12 allowed ionic current to pass but trapped migrating DNA molecules. The concentrated DNA was then injected into a separation channel containing sieving matrix for size analysis. Signal enhancements of 100-fold could be obtained with less than 5 min of preconcentration. This preconcentration method has been extended to proteins. In addition to the original microchip design, in which concentration occurred directly in the injector channel, concentration in a side channel followed by electrokinetic loading of the concentrated sample into the injector has been examined. In theory, the degree of concentration achievable by trapping at a porous membrane is limited only by the ratio of the initial sample volume to the volume of the preconcentration channel and the solubility limit of the protein(s). Microdevices used in this study contained sample reservoirs and preconcentration channels with volume ratios of up to ∼106. Use of low-conductivity sample buffer, as in field-amplified injection, further enhanced the detection sensitivity achieved by concentration at the membrane. EXPERIMENTAL SECTION Reagents. Fluorescein-labeled proteins and protein mixtures for CGE were obtained from Sigma Chemicals (St. Louis, MO), except as noted below, and stock solutions were prepared according to the vendor’s instructions prior to dilution in sample buffer for electrophoresis. Fluorescein-labeled ovalbumin was obtained from Molecular Probes (Eugene, OR). CE-SDS protein run buffer and sample buffer were obtained from Bio-Rad (8) Barrett, L. M.; Svec, F.; Fintschenko, Y. In Proc. 7th Int. Conf. Miniaturized Chem. Biochem. Anal. Syst. 2003, 1077-1080. (9) Astorga-Wells, J.; Swerdlow, H. Anal. Chem. 2003, 75, 5207-5212. (10) Khandurina, J.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1999, 71, 1815-1819. (11) Khandurina, J.; McKnight, T. E.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 2995-3000. (12) Wang, H. Y.; Foote, R. S.; Jacobson, S. C.; Schneibel, J. H.; Ramsey, J. M. Sens. Actuators, B 1997, 45, 199-207.

Analytical Chemistry, Vol. 77, No. 1, January 1, 2005 57

Figure 1. (a) Schematic of microchip layout for preconcentration in the sample injector; (b) microscopic image of preconcentrator-injector channels; (c) schematic cross section through injector and preconcentrator channels (thickness of the silicate layer greatly exaggerated relative to the channel dimensions).

Laboratories (Hercules, CA). Tris-borate buffer, pH 8.9 (50 mM), contained 50 mM Trizma base and 10 mM boric acid. Microchip Fabrication. Microchannel designs were etched in glass substrates (White Crown B-270; Telic, Santa Monica, CA) by photolithography and chemical wet etching, as previously described.13,14 The channels were then enclosed by bonding the substrate to a glass cover plate using a silicate adhesive layer.10,12 Briefly, a 30 wt % solution of potassium silicate (KASIL 2130; PQ Corp., Valley Forge, PA) was diluted 1:10 with deionized water (final concentration ∼2.7%) and then spin-coated (3500 rpm for ∼8 s) onto one surface of the cover plate. The coated surface was then immediately brought into contact with the clean, etched substrate surface, and the assembly was heated at 95 °C for 30 min followed by ramping to 200 °C at 0.3 °C/min and continued heating at 200 °C for 10 h. The thickness of the silicate layer was not determined for these devices; previous ellipsometry measurements of the silicate layers produced by similar spin-coating of 2 and 5 wt % sodium silicate solutions gave thicknesses of approximately 10 and 20 nm, respectively.12 The microfluidic channels were typically 10 µm deep and 50 µm wide at half-depth, except where otherwise noted. The membrane width, i.e., the distance between parallel channels at the substrate surface (see Figure 1), was 4-6 µm. These dimensions were measured using a stylus-based surface profiler (Tencor P-10; Tencor, Mountain View, CA). The 2-mm-diameter holes were drilled vertically through the substrate at the channel termini prior to cover plate bonding. After bonding the cover plate, cylindrical glass reservoirs (∼100 µL capacity) were bonded concentrically over the holes using epoxy. Where noted, microchannels were coated with poly(dimethylacrylamide) (PDMA), as previously described,15 to suppress electroosmotic flow.16 Instrumentation. The electrokinetic concentration and separation of samples were controlled using a custom-built high-voltage

power supply with five individually controlled outputs and relays. Voltages were applied to solutions in the microchip reservoirs via platinum electrodes. Separations were monitored by single-point laser-induced fluorescence detection, as previously described,11,17 using an argon ion laser (488 nm, 543-AP, Omnichrome, Chino, CA). Computer programs written in LabView (National Instruments, Austin, TX) were used for control of voltage outputs and relays and for data acquisition. Imaging was performed using a Nikon TE300 microscope equipped with a CCD camera (model TE/CCD, Princeton Instruments, Trenton, NJ). IPLab Spectrum software (Signal Analytics Corp., Fairfax, VA) was used for camera control and image analysis.

(13) Jacobson, S. C.; Hergenro¨der, R.; Koutny, L. B.; Ramsey, J. M. Anal. Chem. 1994, 66, 1114-1118. (14) Ko, W. H.; Suminto, J. T. In Sensors; Gopel, W., Hesse, J., Zemel, J. N., Eds.; VCH: Weinheim, 1989; Vol. 1, pp 107-168.

(15) McClain, M. A.; Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2001, 73, 5334-5338. (16) Hjerten, S. J. Chromatogr. 1985, 347, 191. (17) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723.

58

Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

RESULTS AND DISCUSSION Microchip Designs for Preconcentration. Two general designs were tested for preconcentration of proteins on microchips. The microchip design shown in Figure 1a was used to concentrate protein samples within a 100- or 150-µm-long injector channel at the top of the separation column. This design is identical to that previously described for preconcentration of DNA samples.10,11 The injector channel was separated from a parallel region of the preconcentrator channel by a distance of 4-6 µm, measured at the top of the substrate (Figure 1b and c). The porous silicate-bonding layer between the substrate and cover plate (Figure 1c) acted as a membrane filter between the two channels. The electrical resistance across the membrane was determined by measuring the resistance between the sample reservoir and a preconcentrator reservoir and subtracting the resistance due to the open channel segments. The latter resistances were calculated from the resistance per centimeter measured in an open channel, such as that between the sample and sample waste reservoirs, that was filled with the same buffer. The resistance of the membrane varied depending on its width and integrity. A

Figure 2. Fluorescence images of fluorescein-labeled ricin injected (a) without preconcentration or (b) with preconcentration for 1 min. Exposure time, 1 s.

resistance of ∼280 MΩ was measured for a 4-µm-wide membrane when the channels were filled with CE-SDS protein run buffer (Bio-Rad) and allowed a current of ∼1.2 µA when a voltage of 1 kV was applied between preconcentration reservoir 1 and the sample reservoir. Leakage of analytes through the membrane can result from imperfect bonding or breakdown of the silicate structure at high applied voltages (>2-3 kV). Individual microchips were used for up to >200 preconcentration cycles using an applied voltage of 1 kV and an average preconcentration time of ∼1 min. For the microchip layout shown in Figure 1a, sample preconcentration was performed by applying a voltage difference between the sample reservoir and the preconcentrator reservoirs. Preconcentrator reservoirs 1 and 2 were typically bridged with platinum wire, and the positive voltage electrode was placed in reservoir 1. Samples could also be loaded into the injector without preconcentration, by applying a voltage difference between the sample and sample waste reservoirs, with no voltage applied to the preconcentrator reservoirs. Samples injected without preconcentration were used to establish base peak heights for calculating the signal enhancement due to preconcentration. Figure 2 shows fluorescence images of fluorescein-labeled Ricinus communis agglutinin (RCA120, FITC labeled, Sigma) loaded into the injector with and without preconcentration. (Note: agglutinin RCA120 is highly toxic; be aware of the risks and familiar with safety procedures before using.) The channels were filled with CE-SDS protein run buffer (Bio-Rad), and the protein (150 µg/mL in BioRad CE-SDS sample buffer) was added to the sample reservoir. All other reservoirs were filled with run buffer. Loading without preconcentration (Figure 2a) was performed by applying 0 kV to the sample reservoir and 1 kV to the sample waste reservoir. The image shown in Figure 2b shows the concentration of protein in the injector channel after applying 0 kV to the sample reservoir and 1 kV to preconcentrator reservoir 1 for 1 min. Ground potential was also applied to the buffer and analyte waste reservoirs during both normal loading and preconcentration. In this microchip, a small amount of protein leaked through the membrane into the preconcentration channel. An advantage of preconcentrating the sample directly in the injector is that all of the concentrated sample can be subsequently injected into the separation channel for analysis. However, we have observed distortions of peak shape and loss of resolution of some sample components in CGE separations following long preconcentration times using this method. These problems may result from slow desorption of sample from the membrane surface

following the injection or changes in buffer composition in the injection channel and adjoining portions of the separation channel during preconcentration. Using fluorescence microscopy, we have also occasionally observed dispersion of the sample away from the membrane surface during the preconcentration step. This dispersion is apparently due to the generation of electroosmotic flow through enlarged pores in defective membranes and can result in spreading of the sample into the buffer and separation channels, even in the absence of an electric field in those channels. To mitigate these problems, we have investigated the use of the alternative microchip design shown in Figure 3. Devices based on this design allow preconcentration in a region of the sample channel separated from the injector. No electric field is applied to the injector or separation channel during the preconcentration step, so that any buffer changes that may occur in the region of the concentrated sample should not extend into those channels and affect the subsequent separation. Following the preconcentration step, the sample is loaded normally through the doubletee injector by applying high voltage (1 kV) at the sample waste and ground potential at the sample reservoir. Injection into the separation channel is timed to occur before all of the concentrated sample has passed through the injector. Partial voltage is applied at the sample reservoir during separation to prevent continual leakage of sample into the separation channel. Figure 4 shows fluorescent images of the preconcentration, loading, and injection of fluorescein-labeled ovalbumin in one variation of this basic microchip design. Preconcentration for CGE Protein Separations. Figure 5 shows the effect of preconcentration time on the CGE analysis of a mixture of three proteins using a device similar to that of Figure 1. Increases in peak height of up to ∼90-fold were achieved after 4 min of concentration with an applied voltage of 1 kV between the sample and preconcentrator reservoirs (Figure 6). As in the previously reported concentration and analysis of nucleic acids,10 the peak heights increased nonlinearly with preconcentration time and variations occurred in the relative peak heights, areas, and migration times of the sample components. The nonlinear response is presumably due to the lag time for migration from the sample reservoir to the preconcentrator through the sieving gel, and the peak variations are due in part to the differences in the migration rates for the different proteins. In addition, a stacking effect was observed in the leading peak, which resulted in an increase in its relative height, and the peaks became compressed with longer preconcentration times. As indicated above, this effect may be due to progressive changes in buffer concentration in the region of the preconcentrated sample and can result in a loss of resolution of early peaks at longer preconcentration times. In an attempt to avoid the potential problems of buffer changes or sample dispersion in the separation channel, we have tested preconcentration in a device based on the design shown in Figure 3a. In the design variant used for these experiments (image not shown), the channels were of uniform width (47 µm at half-depth). The preconcentrator was 2 mm in length and was separated from the injector by 1 mm at its nearest end. These dimensions were designed to provide a relatively long concentrated sample plug that minimizes the possibility of mistiming the loading and injection steps. Preconcentration in this device was tested using Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

59

Figure 3. (a) Schematic of microchip layout for side-channel preconcentration; (b) microscopic image of preconcentrator and injector for one variant of this basic design. Channel width at half-depth, wide channels ∼220 µm, narrow channels ∼50 µm; injector length, 100 µm; preconcentrator length, 0.5 mm; length of narrow channel between preconcentrator and injector, 1.4 mm.

Figure 4. Fluorescence images of (a) preconcentration, (b) sample loading, and (c) injection of fluorescein-labeled ovalbumin (50 µM) into the separation channel in the device shown in Figure 3b. Channels were coated with linear poly(dimethylacrylamide). 50 mM Tris-borate (pH 8.9) was used as both run and sample buffers.

a mixture of six fluorescein isothiocyanate (FITC)-labeled proteins ranging in molecular weight from 20 100 to 205 000 (Fluorescent High Molecular Weight Standard; Sigma). A stock solution of the proteins was diluted 100-fold with sample buffer and initially analyzed on the microchip without preconcentration (Figure 7, top electropherogram). At this dilution, signal-to-noise ratios for the six proteins ranged from approximately 1.8 to 17. The proteins were then preconcentrated for various times from the same sample by applying 1 kV between the preconcentrator and sample reservoirs. The sample injector was loaded immediately after each preconcentration period by applying 1 kV between the sample and sample waste reservoirs for 5 s prior to injecting into the separation channel. The relative increase in height for the largest peak (peak 5, β-galactosidase) is shown as a function of preconcentration time in Figure 8. Following a gradual increase in peak height at preconcentration times of up to 2 min, the rate of increase became much more rapid for longer times. In this experiment, a signal enhancement of 590-fold was obtained for peak 5 after 8 min of preconcentration. At 10 min of preconcentration (not shown), the peak height declined by ∼16%, possibly due to exhaustion of the sample. Interestingly, the increase in peak height observed at a preconcentration time of 4 min was similar to that obtained by direct preconcentration in the injector (Figure 6), even though only a fraction of the protein concentrated in the sample channel was injected. 60 Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

Figure 5. CGE separations of R-lactalbumin (1), trypsin inhibitor (2), and carbonic anhydrase (3), following preconcentration in the sample injector. Starting with the top electropherogram, preconcentration times were 0, 1, 2, 3, and 4 min. Buffers, CE-SDS protein sample and run buffer (Bio-Rad); separation length, 7 cm; separation field strength, 300 V/cm.

The migration times for all peaks in Figure 7 remained relatively constant for up to 4 min of preconcentration, but progressively shifted to longer times with increased concentration times. The resolution of peaks 5 and 6 improved at the longer times and peak 4 was resolved into two separate peaks. However, significant compression and loss of resolution of the earlier peaks occurred at 8 min. Optimal preconcentration times therefore varied for different proteins, as a function of both resolution and peak

Figure 6. Relative peak height versus preconcentration time from the data of Figure 5: (O) R-lactalbumin, (4) trypsin inhibitor, and (b) carbonic anhydrase. Peak height without preconcentration, 1.

Figure 7. CGE separations of fluorescent high molecular weight protein standards (Sigma) following preconcentration in the sample channel. Starting with the top electropherogram, preconcentration times were 0, 1, 2, 4, 6, and 8 min. Peak numbers: (1) trypsin inhibitor, (2) carbonic anhydrase, (3) alcohol dehydrogenase, (4) bovine serum albumin, (5) β-galactosidase, and (7) myosin. Buffers and separation conditions as in Figure 5.

height. Although these effects are not fully understood, they are indicative of time-dependent buffer changes in the sample plug or portions of the separation column. In general, however, higher preconcentration levels could be achieved in side channels without loss of resolution in CGE analysis than by preconcentration in the injector. Preconcentration and CE in Open Channels. Preconcentration on microchips by electrophoretic concentration at a

Figure 8. Relative peak height of β-galactosidase (peak 5) versus preconcentration time from data of Figure 7. Peak height without preconcentration, 1.

membrane requires the suppression of electroosmotic flow (EOF) between the membrane and sample reservoir. EOF is normally suppressed in CGE analyses by dynamic coating of the channel walls by components of the run buffer.18 To perform preconcentration in open channels, the EOF was suppressed by covalently coating the walls with linear poly(dimethylacrylamide), using a procedure similar to that of Hjerten.16 Coating presumably also occurred on the porous silica membrane but did not suppress the flow of ionic current across the membrane. The preconcentration efficiency was initially tested in microchips of the design shown in Figure 1, in which all channels, including the separation channel, were coated with the polymer. The voltage configurations used for the preconcentration and run steps were similar to those used for the CGE analyses described above. The suppression of EOF was confirmed by the absence of migration of a neutral dye (Rhodamine B) in channels subjected to an electric field, as observed by fluorescence microscopy. Figure 9 shows the effect of a 20-s preconcentration of FITC-labeled ovalbumin (10 nM) on CE analysis in 50 mM Tris-borate buffer. The protein could not be detected in this buffer system without preconcentration. (The inset in Figure 9 shows the detection of nonpreconcentrated protein using 5 mM Tris-borate as sample buffer; preconcentration of 10 nM protein using this dilute sample buffer resulted in off-scale peaks.) The signal-to-noise ratio (S/N) for the preconcentrated peak in Figure 9 was ∼820. Assuming a maximum S/N of 3 for undetected peaks in analyses without preconcentration (in 50 mM sample buffer), the 20-s preconcentration would have resulted in at least a 270-fold increase in peak height. The preconcentration also resulted in an ∼54-fold increase in peak height relative to the analysis performed in 5 mM sample buffer without preconcentration (Figure 9 inset). The increased sensitivity obtained using dilute sample buffer is due to field-amplified injection, in which the protein is concentrated at the interface between high-conductivity run buffer and low-conductivity sample buffer, as it migrates to the injector. As in the case of CGE, high levels of preconcentration resulted in increased migration times for CE in coated channels. In many cases, we also observed anomalous peaks that terminated in a sharp spike on the trailing edge. This effect could possibly be (18) Chu, B.; Liang, D. J. Chromatogr., A 2002, 966, 1-13.

Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

61

Figure 9. CE analysis of 10 nM ovalbumin in PDMA-coated channels following 20-s preconcentration in 50 mM Tris-borate sample buffer, pH 8.9. Inset: analysis of 10 nM ovalbumin without preconcentration, but using 5 mM Tris-borate sample buffer. Separation conditions for both analyses: separation buffer, 50 mM Trisborate, pH 8.9; separation length, 2.2 cm; field strength, ∼340 V/cm; PDMA-coated channels.

due to heterogeneity in the concentrated sample plug that results in initial injection of nonadsorbed sample molecules followed by rapid desorption of more concentrated sample from the membrane surface. However, the abrupt termination of the trailing edge suggested that the peaks were partially stacked by the presence of a discontinuous buffer gradient. This effect was observed both when the run and sample buffers were identical (50 mM Trisborate), and when dilute (5 mM Tris-borate) sample buffer was used. Changes in buffer concentration, pH, or both could potentially occur at the preconcentrator as a result of differences in the relative rates of transport of buffer cations and anions through the membrane, compared to their rates in open channels.19 Such transport differences could be due to differences in ion size or the presence of fixed negative charges in the nanopores of the silica membrane that result in electrostatic repulsion of the buffer anion.20 Regardless of the explanation for the observed stacking effect resulting from preconcentration in these chips, it allowed the detection of very low concentrations of proteins. Stacking was more complete at lower initial concentrations and longer preconcentration times. Figure 10 shows three successive analyses of FITC-labeled ovalbumin at an initial concentration of 10 pM with 20-s preconcentration steps. Figure 11 shows peaks obtained from three successive analyses of the fluorescent protein by 120-s preconcentration from an initial sample concentration of 100 fM. No peaks could be detected using 50 mM Tris-borate sample buffer at this concentration, and 5 mM sample buffer was therefore used to enhance the detection. The average height of the peaks shown in Figure 11 (0.031 ( 0.008) is similar to that of the peaks in Figure 10 (0.035 ( 0.005), indicating that the combination of field-amplified injection and 100-s longer preconcentration times gave an ∼100-fold improvement in the detection limit. (19) Shaposhnik, V. A.; Kesore, K. J. Membr. Sci. 1997, 136, 35-39. (20) Schaep, J.; Van der Bruggen, B.; Vandecasteele, C.; Wilms, D. Sep. Purif. Technol. 1998, 14, 155-162.

62 Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

Figure 10. Three successive analyses of 10 pM ovalbumin by CE in coated channels with 20-s preconcentration steps and 50 mM Trisborate sample buffer. Mean peak height ( 1σ was 0.035 ( 0.005. Traces are offset for clarity; bottom trace shows analysis of pure buffer. Separation conditions as in Figure 9.

Figure 11. Three successive analyses of 100 fM ovalbumin by CE in coated channels with 120-s preconcentration steps and 5 mM Trisborate sample buffer. Mean peak height ( 1σ was 0.031 ( 0.008. Traces are offset for clarity; bottom trace shows analysis of pure buffer. Separation conditions as in Figure 9.

The maximum signal enhancement that could theoretically be obtained by preconcentration in these devices is given by the ratio of the sample volume to the preconcentrated volume. In these experiments, the sample reservoir contained 60 µL of solution and the volume of the preconcentration channel was ∼50 pL, giving a theoretical maximum enhancement of 6 orders of magnitude. Using 5 mM sample buffer, the limit of detection (S/N ) 3) for FITC-labeled ovalbumin was ∼3 nM without preconcentration at the membrane (Figure 9, inset), suggesting that an LOD of ∼3 fM could potentially be achieved. However, no peaks could be detected from a 10 fM solution using preconcentration times of up to 10 min. Detection at this concentration would have required the transfer of at least 30% of the sample molecules to the preconcentration volume, without losses due to adsorption to the channel walls or membrane. Preconcentration coupled with CE in uncoated channels was also demonstrated, in a device similar to that of Figure 3, by

selectively coating only the walls of the preconcentration chamber and sample channel with PDMA. (This required the addition of a reservoir and channel that allowed coating reagents from the sample reservoir to be drawn through the preconcentration channel with vacuum, without entering other microchip channels which therefore remained uncoated.) The absence of EOF in the coated sample channel allowed migration of protein to the preconcentration membrane, as in fully coated chips. The concentrated sample could then be loaded by applying positive voltage to the sample reservoir with ground potential at sample waste; EOF generated in the uncoated channels between the end of the preconcentration chamber and sample waste reservoir drew sample from the chamber and through the injection valve. The loaded sample was then injected into the separation channel for normal CE analysis by applying positive voltage at the buffer reservoir with ground potential at analyte waste; partial voltages were applied at the sample and sample waste reservoirs to offset buffer flow into the side channels. Preliminary experiments using this device demonstrated a 32-fold increase in peak height for a 60-s preconcentration of 1 µM fluorescein-labeled ovalbumin in 50 mM Tris-borate buffer (data not shown). There was no evidence of sample stacking in this experiment. Lower precon-

centration could also be due in part to countermigration of analyte during the sample loading step, as well as to adsorption of protein to the uncoated channel walls, which resulted in broader peaks than seen for CE in coated separation channels. These experiments, along with earlier studies of DNA preconcentration,10,11 demonstrate the utility of this method for significantly improving the limits of detection of biomolecules in microchip analysis. Combining preconcentration with on-chip labeling of proteins21,22 would provide a fully integrated lab-on-achip device for protein analysis.

(21) Liu, Y.; Foote, R. S.; Jacobson, S. C.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 4608.4613. (22) Gottschlich, N.; Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. J. Chromatogr., B 2000, 745, 243-249.

Received for review June 11, 2004. Accepted October 4, 2004.

ACKNOWLEDGMENT This research was sponsored by the U.S. Department of Energy Office of Science and the U.S. Department of Homeland Security. Oak Ridge National Laboratory is managed and operated by UT-Battelle, LLC, under Contract DE-AC05-00OR22725 with the U.S. Department of Energy. J.K. was supported by the Oak Ridge National Laboratory (ORNL) Postdoctoral Research Associates Program, administered jointly by ORNL and the Oak Ridge Institute for Science and Education. The authors thank Christopher D. Thomas and Leslie Wilson for microchip fabrication.

AC049136W

Analytical Chemistry, Vol. 77, No. 1, January 1, 2005

63