Anal. Chem. 1997, 69, 259-263
Electrochemical Analysis in Picoliter Microvials Rose A. Clark, Paula Beyer Hietpas, and Andrew G. Ewing*
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802
Lithographically fabricated templates have been used to imprint picoliter to femtoliter microvials in polystyrene. The clear microvials produced in conjunction with a transmission optical microscope and micromanipulators make it possible to handle volumes as small as 1 pL. Voltammetry has been carried out in these well-defined microenvironments with restriction of reagents near the electrode surface. Ferrocenecarboxylic acid has been utilized to characterize the electrochemical response in these ultrasmall volumes. Sigmoidal voltammograms (0.4-2 V/s) are obtained for vial sizes down to 1 pL. Important problems in biology and medicine continue to benefit from the ability to perform analytical measurements on minute quantities. For example, the information gained by chemical analysis of nerve cells can provide better models of the cellular neurotransmission process, which can lead to a better understanding of neurodegenerative diseases such as epilepsy, Alzheimer’s disease, and Parkinson’s disease.1 One analytical method that is emerging as an important technique for selective and sensitive detection in ultrasmall environments is electrochemistry. The small size of microelectrodes has facilitated this growth in the last decade.2 Cellular investigations in vitro3-6 and in vivo7,8 that require detection of minute amounts of material are being conducted with carbon fiber microelectrodes. However, analyzing a sample on the order of a single cell is not easy due to the small volume released upon stimulation of a cell and the femtomole to zeptomole levels of analyte present. To realize the full potential of electrochemical techniques, further developments in smallvolume sample handling are needed to prevent dilution by restricting the low analyte levels present in single cells to extremely small volumes. Achieving low sample volume handling techniques is of current interest in separations, mass spectrometry, and other areas where small volumes are required for the analysis. Picoliter to femtoliter volume methods have been explored by Graztl and co-workers9-11 for microscopic diffusional titration which involve isolating small aqueous drops in organic media. Small samples have also been (1) Shepherd, G. M. Neurobiology, 2nd ed.; Oxford: New York, 1988; pp 3-9. (2) O’Neill, R. D. Analyst 1994, 119, 767-779. (3) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, E. J., Jr.; Viveros, O. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10754-10758. (4) Chow, R. H.; von Ru ¨ den, L.; Neher, E. Nature 1992, 356, 60-63. (5) Chen, T. K.; Lou, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (6) Zerby, S. E.; Ewing, A. G. J. Neurochem. 1996, 66, 651-657. (7) Garris, P. A.; Ciolkowski, E. L.; Pastore, P.; Wightman, R. M. J. Neurosci. 1994, 14, 6084-6093. (8) Chen, G.; Gavin, P. F.; Lou, G.; Ewing, A. G. J. Neurosci. 1995, 15, 77477755. (9) Gratzl, M.; Yi, C. Anal. Chem. 1993, 65, 2085-2088. (10) Yi, C.; Gratzl, M. Anal. Chem. 1994, 66, 1976-1982. S0003-2700(96)00559-8 CCC: $14.00
© 1997 American Chemical Society
isolated directly in micrometer-sized containers. Vials capable of handling nanoliter volumes have been developed for analysis of single cells in microcolumn liquid chromatography.12,13 Another rendition of nanoliter-sized vials has been demonstrated by Roeraade et al.14 for capillary electrophoresis (CE). Even though these microvials restrict the sample volume, only a small fraction of the total volume was actually analyzed. In efforts to further minimize the sample to picoliter volumes, ultrasmall vials have been fabricated on silicon wafers for CE applications15 and for matrix-assisted laser desorption/ionization mass spectrometry.16 The vials allow the isolation of an analyte, in this case bradykinin, cytochrome c,16 and neurotransmitters,15 to picoliter volumes. Combining these microvial technologies with electrochemistry will provide substantial improvements in sample handling by decreasing dilution for single-cell analysis as well as small-volume samples. Thus, opportunities will be created for biological and chemical analysis previously unexplored in the solution phase. In this article, the fabrication of arrays of picoliter vials in polystyrene and their utility for electrochemical investigations are demonstrated. Vials ranging in volume from 0.4 to 300 pL solve sample handling problems by allowing for localization of small volumes with high spatial resolution. In contrast to microvials fabricated in silicon, polystyrene vials are transparent, which is advantageous for transmission optical microscopy. The microvials are characterized both structurally with scanning electron microscopy and electrochemically with cyclic voltammetry of ferrocenecarboxylic acid solutions. EXPERIMENTAL SECTION Microvial templates were fabricated at the National Nanofabrication Facility, Cornell University, using standard photolithographic and wet chemical etching techniques. The computeraided design was transferred to a 4-in. chrome photomask using a GCA PG3600F optical pattern generator. Three-inch singlecrystal 〈100〉 silicon wafers were coated with 1500-Å silicon nitride (Ipe Model 1000 plasma-enhanced chemical vapor deposition system), followed by spin-coating with P-20 primer and Shipley 1400-27 photoresist. The photomask was then utilized to transfer (11) Yi, C.; Haung, D.; Gratzl, M. Anal. Chem. 1996, 68, 1580-1584. (12) Holland, L. A.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3275-3283. (13) Oates, M. D.; Cooper, B. R.; Jorgenson, J. W. Anal. Chem. 1990, 62, 15731577. (14) Jansson, M.; Emmer, A° .; Roeraade, J.; Lindberg, U.; Ho ¨k, B. J. Chromatogr. 1992, 626, 310-314. (15) Beyer Hietpas, P.; Ewing, A. G. J. Liq. Chromatogr. 1995, 18, 3557-3576. (16) Jespersen, S.; Niessen, W. M. A.; Tjaden, U. R.; van der Greef, J.; Litborn, E.; Linberg, U.; Roeraade, J. Rapid Commun. Mass. Spectrom 1994, 8, 581584.
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Figure 1. Schematic of polystyrene microvial fabrication procedure. See text for details.
the pattern to the silicon wafer using an HTG System 3HR contact/proximity aligner (UV 405 nm, 2.4 s). The UV-decomposed resist was removed by a chemical etch, leaving the pattern in the resist (Figure 1A). The exposed underlayer of silicon nitride was removed using a 3-min CF4 plasma etch (Applied Materials reactive ion etching (RIE) chamber), followed by removal of the remaining resist with a 4-min O2 plasma (RIE), Figure 1B. Wet etching was then performed in 28% (w/w) KOH (80-90 °C) solution for varying times, depending on the desired depth of the vials. KOH etched the silicon anisotropically, leaving square-pyramidal structures protruding from the surface (Figure 1C). A CHF3, O2 plasma treatment in the RIE was then used to strip the silicon nitride mask. The pattern on the completed silicon wafer template (Figure 1C) was then transferred into polystyrene (Figure 1D) using a hot press method. A 2-cm2 piece of polystyrene was placed onto the silicon wafer template, which was lubricated with Vaseline (Chesebrough Ponds, Inc. Greenwich, CT). The polystyrene and silicon wafer were sandwiched between two glass sheets (8.9 cm2), and pressure was applied while the assembly was heated to 180 °C for 15 min. After cooling, the polystyrene microvials (Figure 1E) were removed from the template with tweezers. Scanning electron microscopy images were collected using a Jeol Model JSM 5400 (Peabody, MA). All voltammetry was performed using an Ensman Instruments EI400 microelectrode potentiostat (Bloomington, IN) in the two-electrode mode. A Gateway 2000 486/33 PC computer and interface (Labmaster, Scientific Solutions, Solon, OH) with local software5 were used for data acquisition and analysis. Experiments were performed on the stage of an inverted microscope (IM-35, Carl Zeiss, Thornwood, NY) equipped with three micromanipulators (one PCS-750/100, Burleigh Instruments, Fishers, NY, and two Mertzhouser, Zeiss, Thornwood, NY) for positioning electrodes and injectors. Microelectrodes were fabricated by aspiration of a 5-µm carbon fiber (Amoco, Greenville, SC) into a 1.2-mm × 0.68-mm glass capillary (A-M Systems, Everett, WA). The fiber was sealed in the glass capillary using a vertical capillary puller (Ealing, Harvard 260
Analytical Chemistry, Vol. 69, No. 2, January 15, 1997
Apparatus, Edenbridge, KY). The electrodes were back-filled with colloidal graphite (Polysciences, Inc., Warrington, PA), and a nichrome wire was inserted for contact. The carbon fiber was then coated with phenol-allylphenol copolymer with slight modification of a previously described procedure.17 Briefly, 150 µL of phenol and 300 µL of allylphenol were added to a 2% by (w/w) solution of butyl cellosolve in 25.0 mL of a 1:1 methanol/ water mixture. The electrodes were coated with the copolymer electrochemically by applying 2 V vs a platinum wire counter/ reference electrode for 12 min. Polymer curing was then accomplished by baking at 150 °C for 30 min. Electrodes were used after cleaving the tip to expose a carbon disk (∼5 µm) and could be reused by repeating the procedure. A miniature, tip ∼1 µm, Ag/AgCl (1 M KCl) reference was constructed in-house for the electrochemical measurements. The half-wave potentials reported were estimated from the steady state cyclic voltammograms as the potential where the current is half the maximum value. Injectors are prepared as previously described15 by HF etching a CE capillary with external dimensions of 150 µm down to values close to the internal dimensions, which ranged from 10 to 2 µm. The etched capillary tip was epoxied into a larger glass capillary tube for support and used for injection of samples. Injections were carried out with a Picospritzer II (General Value, Fairfield, NJ). Microvials were cleaned by consecutive 3-min sonications in ethanol, 1 M sulfuric acid, and doubly distilled water. Vials were cleaned immediately before use and were, in some cases, reused after repeating the cleaning procedure. All solutions were prepared in doubly distilled water (Corning Mega-Pure MP-3A, Corning, NY). Glycerol was added at 35% (w/w) to the 0.1 M phosphate buffers (pH 7.4) utilized for preparation of the ferrocenecarboxylic acid solutions to control evaporation. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. RESULTS AND DISCUSSION In Figure 1, fabrication procedures are summarized for the polystyrene microvials. This lithographic procedure creates arrays of microvials spaced 1 mm apart. The vial size can be easily manipulated by changing the photomask or by controlling the KOH etch depth. Five sizes have been fabricated in polystyrene, with sizes ranging from 300 down to 0.4 pL. These transparent microvials are easily viewed with transmission optical microscopy, which is required for manipulation of extremely small volumes and single cells. Microvials fabricated in silicon14,15 are perfectly symmetrical inverted square pyramids, as depicted for an ideal microvial in Figure 1F; in contrast, the polystyrene microvials show some nonuniformities. Scanning electron microscopy (SEM) reveals the actual structural features observed for 75-, 20-, and 0.4-pL vials (Figure 2). Microvial volumes have been estimated from the measured dimensions, and these values are given in Table 1. Inner and outer diameters have been measured employing optical microscopy and SEM. Microvial depths are determined using a surface profiler (Tencor Alpha-Step 200). Surface roughness contributions have not been considered in these calculations; however, calibration of the exact volume by injection of known volumes is planned. The values reported in Table 1 are consistent over a single silicon wafer template. Vial sizes can vary from wafer to wafer since the depth and, therefore, the outer diameter and volume are controlled by the KOH etch (17) Strein, T. G.; Ewing, A. G. Anal. Chem. 1992, 64, 1368-1373.
Figure 3. Bright field photomicrographs of a 75-pL polystyrene microvial (A) empty and (B) filled. The injection pipet is shown in the lower left-hand corner. The reference and working electrodes are on the bottom right and top right, respectively.
Figure 2. Scanning electron micrographs of polystyrene microvials: (A) 75, (B) 20, and (C) 0.4 pL. The microvials were sputtercoated with Pd/Au mixture before imaging to make them conducting. Table 1. Physical Characteristics of Polystyrene Microvials volume (pL) top length (µm)a bottom length (µm)a depth (µm)b 300 75 20 14 1 0.4
120 75 40 30 14 10
70 20 20 20 9 4
33.5 36.0 20.4 16.5 8.4 8.4
a Lengths were determined using optical microscopy and SEM. Depth was measured using an R step depth profiler. Volumes were calculated on the basis of square pyramids for 300 down to 14 pL and conical shapes for 1- and 0.4-pL vials. b
rate. The rounding observed in 300-pL vials (not shown) is a small fraction of the total volume and should not be a factor in the estimated volume. The rounding is more pronounced for
