Anal. Chem. 2000, 72, 1704-1706
Technical Notes
Effects of Alkaline Hydrolysis and Dynamic Coating on the Electroosmotic Flow in Polymeric Microfabricated Channels Shau-Chun Wang, Catherine E. Perso, and Michael D. Morris*
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055
Protocols are described for control of the electroosmotic flow in microfabricated channels in Vivak copolyester. Alkaline hydrolysis of surface ionizable groups alone or such hydrolysis in combination with dynamic coating with cetyltrimethylammonium bromide (CTAB) is shown to provide reproducible electroosmotic flows. Dynamic coating with CTAB can be used to eliminate electroosmosis or to reverse its direction, depending on the concentration employed. Thermoplastic polymers are attractive materials for fabrication of microfluidic instrumentation. The polymers are easier to process than silica, glass, or silicon. Fabrication techniques appropriate to several important polymers have been described. These include the use of poly(dimethylsiloxane) (PDMS),1-3 epoxy-based photopolymer,4 polycarbonate,5,6 poly(methyl methacrylate (PMMA),5,7-9 cellulose acetate,6 poly(ethylene terephthalate),6 acrylic copolymer,10,11 polystyrene,6,11 copolyester,11 and poly(methyl methacrylate) substrates.12 Electrophoresis in these devices has been demonstrated. (1) Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A. Anal. Chem. 1997, 69, 34513457. (2) Folch, A.; Ayon, A.; Hurtado, O.; Schmidt, M. A.; Toner, M. J. Biomech. Eng. 1999, 121, 28-34. (3) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984. (4) Renaud, P.; van Lintel, H.; Heuschkel, M.; Guerin, L. In Micro Total Analysis Systems ’98; Harrison, D. J., van den Berg, A., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998; pp 17-22. (5) Becker, H.; Dietz, W.; Dannberg, P. In Micro Total Analysis Systems ’98; Harrison, D. J., van den Berg, A., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998; pp 253-256. (6) Roberts, M. A.; Rossier, J. S.; Bercier, P.; Giralt, H. Anal. Chem. 1997, 69, 2035-2042. (7) Ford, S. M.; Davis, J.; Kar, B.; Qi, S. D.; McWhorter, S.; Soper, S. A.; Malek, C. K. J. Biomech. Eng. 1999, 121, 13-21. (8) Boone, T. D.; Hooper, H. H. In Micro Total Analysis Systems ’98; Harrison, D. J., van den Berg, A., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998; pp 257-260. (9) Ford, S. M.; Kar, B.; McWhorter, S.; Davis, J.; Soper, S. A.; Klopt, M.; Calderon, G.; Saile, V. J. Microcol. Sep. 1998, 10, 413-422. (10) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626-2630. (11) Locascio, L. E.; Perso, C. E.; Cheng, S. L. J. Chromatogr., A 1999, 857, 275-284. (12) Martynova, L.; Locascio, L. E.; Gaitan, M.; Kramer, G. W.; Christensen, R. G.; MacCrehan, W. A. Anal. Chem. 1997, 69, 4783-4789.
1704 Analytical Chemistry, Vol. 72, No. 7, April 1, 2000
As do those of glass and silica, the surfaces of many polymers contain ionizable functional groups. Consequently, electroosmosis is observed in channels fabricated in these surfaces. The electroosmotic properties of several polymers including polystyrene, copolyester (Vivak), and acrylic copolymer (Lucite CP) have been described by Locascio and co-workers.11 The surfaces contain carboxylate ions. Electroosmotic flow is directed toward the more negative electrode, and electroosmotic mobilities are similar to those observed in glass or silica. Although control of surface charge and electroosmotic flow is as important in electrophoresis in plastic microchannels as it is in glass or silica, little has been reported on this topic. Oxidation of poly(dimethylsiloxane) (PDMS) in an oxygen plasma has been shown to increase the number anionic sites on the microchannel surface.3 UV laser machining of microchannels on polystyrene, polycarbonate, cellulose acetate, and poly(ethylene terephthalate) slides produces surfaces with high densities of carboxylic acid sites.6 On the other hand, electroosmotic flow can be reduced or suppressed by working at high ionic strength6 or by coating surfaces with proteins such as Eosin B6 or antibodies.11 There remains a need for surface charge control methods that do not require high ionic strength solutions with consequent Joule heating or the use of proteins that themselves may be a source of instability or interference. In this paper, we show that electroosmotic flow can be controlled by the same techniques that have proven successful in glass and silica: pretreatment with strongly alkaline solutions and dynamic coating with small organic cations.13,14 EXPERIMENTAL SECTION Chemicals. Copolyester plastic slides were cut from Vivak plastic sheets (DSM Engineering Plastic Products, Sheffield, MA). Chromel wire (50 µm in diameter) was obtained from Omega (Stamford, CT). ACS grade reagents, including Tris, boric acids, and disodium ethylenediaminetetraacetate (EDTA), were used for the preparation of TBE buffers. Cetyltrimethylammonium bromide (CTAB) was obtained from Aldrich Chemical Co. (Milwaukee, WI). (13) Tavares, M. F. M.; Colombara, R.; Massaro, S. J. Chromatogr., A 1997, 722, 171-178. (14) Cifuentes, A.; Rodriguez, M. A.; Garcia-Montelongo, F. J. J. Chromatogr., A 1996, 742, 257-266. 10.1021/ac9909148 CCC: $19.00
© 2000 American Chemical Society Published on Web 02/23/2000
Channel Fabrication Protocol. Channels were fabricated in Vivak (DSM Plastics) using the hot wire imprinting method.11,15 Briefly, 50 µm Nichrome wire was stretched lengthwise over a clean piece of plastic (2.5 cm × 7.6 cm × 0.16 cm). To fabricate a channel, the wire and plastic were clamped between two glass microscope slides and placed in a convection oven for 15 min at 80 °C. For electroosmotic studies, only a single channel was fabricated in each device. A second piece of Vivak of the same dimensions was used to cover and seal the channel. Prior to sealing, 2 mm holes for buffer wells were drilled approximately 2 cm apart through the cover piece. The two plastic pieces were clamped together and placed in the oven at 75 °C for 25 min to seal them together. The channels of the completed devices were kept filled with water during storage. Apparatus. Equipment used was similar to that used in the previous paper.11 Briefly, a personal computer with an attached data acquisition board was used to drive a high-voltage amplifier (model 20/20, Trek, Inc., Medina, NY) and monitor the IR drop across a 100 kΩ load resistor in series with the microchannel. Driving potentials between 600 and 2000 V were used. Flow was measured in TBE (Tris, borate, EDTA) buffers. Measurement of Electroosmotic Flow. The electroosmotic flow in the microchannel was measured by current monitoring.11,15 In a typical measurement sequence, the channel was filled under vacuum with a solution of 1X TBE, pH 8.2. Platinum electrodes were placed in each well. The buffer was next removed from the wells and replaced with equal aliquots of 0.5X TBE, and the IR drop was measured. This measurement provided a baseline current. After completion of this measurement, the buffer was removed from both wells. An aliquot of 0.5X TBE was then placed in one well, and an aliquot of 1X TBE was placed in the other. The amplifier was turned on, and the current decrease was monitored until steady state was reached, when 0.5X TBE completely displaced 1X TBE in the channel. Measurements were made in 150 V/cm increments at field strengths from 150 to 900 V/cm. Four replicate measurements were made at each field strength. The average of these 20 measurements is reported. The electroosmotic mobility, µeo, was calculated according to eq 1,
µeo ) (L/t)E-1
(1)
where L is the length of the microchannel (2 cm), t is the time for 0.5X TBE to completely displace 1X TBE in the channels, and E is the electric field strength (V/cm). Alkaline Hydroylsis. Using a vacuum technique, 0.1 M NaOH was continuously pumped for 5 min through a clean microchannel. After 5 min, the NaOH solution was replaced with 5X TBE, which was pumped through the channel for another 5 min. Equal aliquots of fresh 5X TBE buffer were then placed in both wells. The device was stored in a closed container for 40 min to allow time to reach a steady-state surface charge density. After 40 min, the 5X TBE was removed from the wells and equal aliquots of fresh 1X TBE buffer were placed in both wells and the channel was filled with 1X TBE by electroosmotic pumping. The liquid was again removed from the wells, and equal aliquots of fresh 1X TBE buffer were placed in both wells. The electroosmotic mobility was then measured, as described above. (15) Huang, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 1837-1838.
Table 1. Enhancement of EO Mobilities in the Microchannels after Pretreatment with Alkaline Solution EO Mobility in Microchannels without Alkaline Hydrolysis EO mobility (cm2 V-1 s-1 device 1: (5.4 ( 0.4) × 10-5 device 2: (3.0 ( 0.4) × 10-5 device 3: (7.0 ( 0.6) × 10-5 av EO mobility (cm2 V-1 s-1) (5.1 ( 0.5) × 10-5 chip-to-chip rel error (%), 78% (range/av) × 100% EO Mobility in Microchannels without Alkaline Hydrolysis EO mobility (cm2 V-1 s-1) device 1: (3.46 ( 0.22) × 10-4 device 2: (3.26 ( 0.16) × 10-4 device 3: (3.42 ( 0.24) × 10-4 av EO mobility (cm2 V-1 s-1) (3.38 ( 0.21) × 10-4 chip-to-chip rel error (%), 6.0% (range/av) × 100%
Additionally, some measurements were repeated using 1X TBE and 0.95X to validate the more extensive 1X TBE/ 0.5X TBE electroosmotic flow data. Dynamic Coating with Cetyltrimethylammonium Bromide (CTAB). In different experiments, CTAB concentrations between 0 and 8.0 × 10-5 M were used to coat microchannels. All CTAB solutions were prepared in 1X TBE. Through a clean microchannel 0.1 M NaOH was continuously pumped for 5 min, using the vacuum technique. After 5 min, the CTAB solution being tested was pumped through the channel. The CTAB solutions were removed from the wells and replaced with fresh aliquots. The plastic chip assembly was placed in a closed container for 40 min to allow time to reach a steady-state surface charge density. After 40 min, the CTAB was replaced by fresh aliquots (CTAB in TBE buffers) and the electroosmotic mobility was measured. RESULTS AND DISCUSSION The electroosmotic mobility in an untreated channel containing 1X TBE was variable. In three chips prepared from the same sheet of Vivak, we observed a µeo range from (3.0 ( 0.4) × 10-5 to (7.0 ( 0.6) × 10-5 cm2 V-1 s-1. Within each chip, mobility measurements were reproducible to about 10% (Table 1). The alkaline hydrolysis procedure increased the number of surface anionic sites, so that µeo increased to (3.38 ( 0.21) × 10-4 cm2 V-1 s-1. The chip to chip variation of electroosmotic mobility in treated chips was reduced to about 6% (Table 1). Prolonged alkaline hydrolysis causes ionization of nearly all of the potentially anionic sites. The observed µeo is close to that typically reported for a fused-silica capillary under similar conditions,15 5.0 × 10-4 cm2 V-1 s-1. The resulting electroosmotic flow should allow transfer of zone electrophoresis procedures from silica to Vivak with minimal changes. These results were confirmed by measurements of displacement of 1X TBE by 0.95X TBE. Very similar results were obtained (Table 2). Alkaline hydrolysis is a well-known phenomenon in the degradation of polymers that contain ester structures.16,17 Conversion of ester moieties to carboxylates occurs. These carboxylate moieties are negatively charged under basic conditions. Conver(16) Belan, F.; Bellenger, V.; Mortaingne, B.; Verdu, J. Polym. Degrad. Stab. 1997, 56, 301-309. (17) Zeronian, S. H.; Buschlediller, G.; Inglesby, M. K. Polymer 1994, 35, 25872590.
Analytical Chemistry, Vol. 72, No. 7, April 1, 2000
1705
Table 2. Validation of EO Mobility Measurementsa buffer solns 1X TBE and 0.5X TBE 1X TBE and 0.95X TBE a
no. of replica 4 5
av EO mobility (cm2 V-1 s-1) 10-4
(3.41 ( 0.05) × (3.33 ( 0.08) × 10-4
Relative error: 5%.
Table 3. EO Mobility Dependence on CTAB Concentrationa CTAB concn (M)
EO mobility (cm2 V-1 s-1)
CTAB concn (M)
EO mobility (cm2 V-1 s-1)
0.0 3.0 × 10-5 5.0 × 10-5 1.1 × 10-4
(2.3 ( 0.0) × 10-4 (8.8 ( 0.7) × 10-5 (2.1 ( 0.2) × 10-5 (-7.4 ( 0.2) × 10-5
1.9 × 10-4 3.8 × 10-4 7.6 × 10-4
(-7.3 ( 0.4) × 10-5 (-6.7 ( 0.4) × 10-5 (-6.7 ( 0.2) × 10-5
av reverse EO mobility: (-7.0 ( 0.2) × 10-5 a
The sign of reverse EO mobility is designated as negative.
× 10-4 to 7.6 × 10-4 M is (7.0 ( 0.32) × 10-5 cm2 V-1 s-1. Similar results have been reported for fused-silica capillaries.13,14 A coating time of 40 min was sufficient to achieve equilibrium because microchannels did not have greater EO mobility and it did not change, even after equilibration. CTAB-coated microchannels had stable EO mobility, lasting for 3-4 h. The same chip generated a similar EO mobility (less than 5% difference) after storage in deionized water for 3 days and recoating. The lifetime of a laboratory-fabricated chip is usually only 3-5 days.
Figure 1. Dependence of electroosmotic mobility (µeo) in Vivak, 1X TBE, on concentration of cetyltrimethylammonium bromide (CTAB). Averages of measurements over the range 150-900 V/cm are shown.
sion can be achieved with any alkaline buffer. In this work, TBE was used because of its widespread application in the electrophoretic separation of biopolymers such as nucleic acids and proteins. Quaternary ammonium ions containing long-chain alkyl groups are strongly adsorbed on polyanions. These ions can be used to reduce or even reverse the direction of electroosmotic flow. The commonly available CTAB serves this function, as shown in Figure 1 and Table 3. As the CTAB concentration increases, µeo drops rapidly. The curve crosses zero at about 8.0 × 10-5 M. Neutralization of surface charges occurs at concentrations close to that observed in quartz capillaries,13 9.0 × 10-5 M. At higher CTAB concentrations, the surface becomes cationic and the direction of electroosmotic flow is reversed. The reversed electroosmotic mobility is only weakly dependent on concentration, to the highest concentration in our measurement series, near the critical micelle concentration (cmc) of 9.0 × 10-4 M. The average value of µeo over the CTAB concentration range from 1.2
1706 Analytical Chemistry, Vol. 72, No. 7, April 1, 2000
CONCLUSIONS Two important techniques for controlling electroosmotic flow in quartz capillaries and microchannels are shown to work equally well in Vivak microchannels. Both techniques are based on complete or nearly complete hydrolysis of ionizable surface moieties. In the case of CTAB solutions, both physical absorption and ion-pair formation are used to neutralize some of the surface charges of these carboxylate moieties. Many other techniques for electroosmotic flow control have been developed for silica surfaces. Unless they employ silica-specific chemistry, most of these techniques should also be transferrable to plastics with easily ionizable surfaces. ACKNOWLEDGMENT This study was supported by the National Institutes of Health through Grant 1R01 GM37006. We also thank Laurie E. Locascio, National Institute of Standards and Technology, for assistance with this work. Received for review August 11, 1999. Accepted January 4, 2000. AC9909148
