Anal. Chem. 1997, 69, 253-258
Micromachining Sensors for Electrochemical Measurement in Subnanoliter Volumes Craig D. T. Bratten,†,‡ Peter H. Cobbold,‡ and Jonathan M. Cooper*,†
Bioelectronics Group, Department of Electronics and Electrical Engineering, University of Glasgow, G12 8QQ, UK, and Department of Human Anatomy and Cell Biology, University of Liverpool, Liverpool, L69 3BX, UK.
A miniaturized electrochemical cell was produced using lithographic patterning of metals and photopolymerizable polyimide to give a high aspect ratio circular well with clearly defined, clean gold microelectrodes at its bottom. The device, which consisted of a gold working and counter electrode and a Ag|AgI pseudoreference, located in a microchamber of diameter 200 µm and depth 20 µm (0.6 nL), was fabricated in order to analyze very small volumes of solution. An experimental procedure for routinely conducting assays in subnanoliter volumes, involving micropipetting of small quantities of reactants and solvents, was developed in order that the electrochemical characteristics of the device could be defined using the model redox compound ferrocene monocarboxylic acid. The motivation for developing this technology has been to establish a protocol that can be used to make measurements of the responses of single living cells to hormones or metabolic insult, although it is predicted that these general methods may also be of value to those interested in a variety of other fields, including technologies associated with fast-throughput diagnostics. As a consequence, issues concerning solvent evaporation and biocompatibility have also been addressed. The electrochemical characteristics of microelectrodes have been the subject of intense investigation for a decade and their behavior is now well appreciated and understood.1,2 Recently, they have found various applications in bioanalytical science, not least because their small physical dimensions have enabled their placement in precisely defined locations.3 Microelectrodes have also been used in order to analyze progressively smaller volumes of solution: for example, a three-dimensional flow cell has been used to measure glucose in 2-µL volumes;4 band electrodes constructed from metal foil sandwiched between layers of Tefzel film have been used to perform voltammetry on volumes as small as 0.05 µL5,6 or within a ∼2-µL volume of solution placed directly on electrodes within a lithographically defined electrochemical †
University of Glasgow. University of Liverpool. (1) Wrightman, R. M.; Wipf, D. O. In Electroanalytical Chemistry; Bard, A. J., Ed.: Marcel Dekker: New York, 1989; Vol. 15, pp 267-353. (2) Heinze, J. Angew. Chem., Int. Eng. Engl. 1993, 32, 1268-1288. (3) Arbault, S.; Pantano, P.; Jankowski, J. A.; Vuillaume, M.; Amatore, C. Anal. Chem. 1995, 67, 3382-3390. (4) Murakami, Y.; Uchida, T.; Takeuchi, T.; Tamiya, E.; Karube, I.; Suda, M. Electroanalysis 1994, 6, 735-739. (5) Bowyer, W. J.; Clark, M. E.; Ingram, J. L. Anal. Chem. 1992, 64, 459-462. (6) Clark, M. E.; Ingram, J. L.; Blakely, E. E.; Bowyer, W. J. J. Electroanal. Chem. 1995, 385, 157-162. ‡
S0003-2700(96)00743-3 CCC: $14.00
© 1997 American Chemical Society
cell,7 (the latter case illustrating the potential in using semiconductor fabrication methods in this technology). However, equally important to these considerations, and as a direct consequence of their reduced geometries, microelectrodes have also been shown to have an enhanced analytical performance,1,2 for example, through more efficient diffusion of analytes to the electrochemical surface. In the case of the design that we propose in this paper, where the microelectrode is fabricated within a chamber, potentially, there is an additional advantage; for if the volume surrounding the sensor is confined, when compounds are produced locally within the well, their mean diffusion path length to the electrode surface is greatly reduced when compared with the situation for the same electrode geometry in bulk solution. In the light of recent developments in micromachining,7,8 we have been investigating the application of semiconductor processing techniques in making devices for analyzing responses from single cells in vitro. To this end, we have fabricated an electrochemical device by photolithography, with gold electrodes patterned onto a quartz slide. The microstructures were covered with a sacrificial layer of nickel-chromium, which could be easily removed after the polymer processing. The microchamber was formed from a thin layer of polyimide that was selectively photopolymerized and developed, so exposing the electrodes and defining a chamber of 200-µm diameter and 20-µm depth, with a volume of 600 pL (the polyimide layer also serving to insulate the metal tracking leading to exposed bonding pads). At the end of this procedure, removal of the nichrome layer with a wet etch resulted in three individually addressed clean microelectrodes, one of which was subsequently coated with silver and converted to a silver|silver halide reference (the other two were used as the working and the counter electrodes). Having developed this miniaturized device, we have also optimized a pipetting protocol that could be used in order to perform electrochemical experiments using subnanoliter volumes, while simultaneously negating problems of evaporation by using mineral oil. The technique, which relies upon being able to maintain the hydrophobicity of the polyimide after polymer processing, can be used routinely and at minimal cost. As a consequence, we have been able to characterize the responses of a microelectrochemical cell to the model redox compound, ferrocene monocarboxylic acid (FMCA), paying particular attention to the reproducibility of the response with different integrated silver|silver halide pseudoreference electrodes. (7) Lambrechts, M.; Sansen, W. Biosensors: Microelectrochemical Devices; Institute of Physics Pub.: New York, 1992; Chapter 5. (8) Kovacs, G. T. A.; Petersen, K.; Albin, M. Anal. Chem. 1996, 68, 407A412A.
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Figure 1. Schematic diagram of the major fabrication steps (in transverse section). Key: (1) The cleaned coverslip was coated with a positive photoresist; (2) the resist was patterned, and the sample was vacuum coated with Ti/Pd/Au/NiCr; (3) the resist was lifted off, leaving a microelectrode pattern; (4) the sample was coated with a photocurable polyimide; (5) the polyimide was patterned and developedsa slight residue remained; (6) the sacrificial nichrome layer was removed leaving clean microelectrodes at bottom of 0.6-nL chamber.
The device has been designed with the aim of monitoring the time course of metabolites released from a single mammalian cell in vitro, and the ring geometry of the working and counter electrodes has been fabricated so that the cell can be placed in the center of the microstructure. Clearly, however, lithography provides sufficient flexibility that other designs could be used in future, not least through further geometric reduction of the chamber size. For the present, however, by developing a method for making amperometric measurement in a subnanoliter volume, we are now in a position where we can routinely detect less than 1 pmol of analyte electrochemically (with the realistic prospect of the measurement of femtomoles of material). It is therefore expected that the general methodology will be of interest to readers in other analytical fields. EXPERIMENTAL SECTION Microelectrochemical Devices. The major steps involved in fabricating the device are illustrated schematically in Figure 1. Whereas some of the procedures followed standard lithographic methods, in common use in other laboratories,7,9 many of the steps were optimized and modified for this particular application. Those processes, which were essential for the reproducible machining of the microchamber devices, are detailed below. Electrode Patterning. Glass coverslips (18 mm) were cleaned and spin-coated (at 4000 rpm) with Shipley 1400-31 positive photoresist, which was baked for 15 min at 90 °C, soaked for 10 min in chlorobenzene in order to define electrodes with clean, sharp edges,10,11 and then rebaked for 15 min, again at 90 (9) Morita, M.; Longmire, M. L.; Murray, R. W. Anal. Chem. 1988, 60, 27702775. (10) Fathimulla, A. J. Vac. Sci. Technol. B 1985, 3, 25-27. (11) Mimura, Y. J. Vac. Sci. Technol. B 1986, 4, 15-21.
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°C. The resist was patterned by UV exposure through a purpose made ferric oxide photomask and was subsequently developed. Two aspects of the microelectrode formation were recognized as paramount: adequate adhesion of the gold electrodes to the photoresist patterned glass substrate and cleanliness of the final electrode surfaces. In order to achieve these objectives, after lithographic patterning, all samples were recleaned prior to metal evaporation using a low-power O2 plasma etch (PlasmaFab ET340 ∼70 W, 1 min), in order to remove partially developed resist or resist residue. Metals were deposited by electron beam evaporation of a Ti/Pd/Au (10:10:100 nm) multilayer structure, providing excellent adhesion of the metal to the glass substrate, and good electrochemical stability, even at high oxidative potentials in aqueous solutions.7 Finally, 50 nm of nickel-chromium (60:40), also known as nichrome, was evaporated as a sacrificial protective mask in order to stop the gold electrodes becoming fouled by a polyimide residue which persisted after the microchamber had been formed (see later). Having deposited these metals, the removal of the undeveloped photoresist left a pattern of nichromecoated gold microelectrodes in the center of the slide, connected by tracks to bonding pads at one edge. Microchamber Formation. Samples were then baked at 180 °C for 10 min to drive off surface moisture and were spin-coated (2000 rpm for 9 s) with photosensitive polyimide (Probimide 7020) to a thickness of 40 µm. The layer that formed was slightly thicker at the edges of the samplessalthough after letting the samples stand for 5 min, the polymer thickness was found to become uniform (this latter detail being essential in order to ensure a close and even photomask contact in later processing). The polyimide was finally soft-baked in two stages: the samples were placed in a covered dish onto a 5-mm-thick aluminium grating, which was placed for 10 min onto a hot-plate at 90 °C for 10 min, and were then placed directly onto the hot-plate for a further 10 min. Following soft-baking, the polyimide was photopolymerized and developed to expose a circular micrometer-scale titer well over the microelectrode area, with large rectangles over the bonding pads. The particular brand of photosensitive polyimide has a peak light sensitivity at λ ) 436 nm and was therefore patterned using a chrome photomask: ferric oxide masks gave inferior resolution, possibly as they permitted too much blue light through the masked areas. Development was carried with OCG developer for ∼4 min. In order to remove as much polyimide as possible at this stage, the developer was agitated, if necessary using a small plastic scribe to carefully break the surface of the polymer over the chamber, taking care not to scratch the UV-exposed areas of polyimide surrounding the chamber. The devices were rinsed for 10 s in OCG rinse and were examined visually under a microscope. Usefully, it was found that any device that had not been adequately developed could be reimmersed in developer and the process repeated in an iterative fashion without any apparent detriment to the surrounding polyimide. The samples were blow-dried and hard-baked at 300 °C for 1 h. This second baking resulted in a ∼50% shrinkage in the polyimide thickness without affecting the diameter of the chamber. Dimensions were measured with a DekTak surface profiler, enabling accurate calculation of the volume. The examples shown in Figure 2 are scanning electron micrographs (SEMs) of the finished device, of diameter 200 µm with a polyimide thickness (z) of 20 µm.
Figure 2. Scanning electron micrographs showing (a, top) the chamber and (b, bottom) detail of the gold microelectrode. The reference electrode is subsequently coated with silver and converted to a silver|silver halide reference. The scale bar represents 100 µm. Key: (1) counter electrode; (2) reference electrode; (3) working electrode. The base of the chamber is transparent glass while the walls are photopolymerized polyimide, 20 µm deep. The structures was coated with Au/Pd (particle size nominally 20 nm) prior to being imaged using a Hitachi S800 scanning electron microscope (Hitachi, UK) at 30 kV.
Polyimide Residue Removal. Despite efforts to optimize the above procedure and remove all the unwanted polyimide from the microchamber, a residue always persisted on the electrode surfaces. This thin layer, which bound strongly to the metals but not the glass substrate, was only visible under the microscope after the hard-bake stage. To remove the polymer, the nichrome sacrificial layer was etched by placing a drop of 0.6 M acetic acid and 0.37 M ammonium hexanitrocerate on the microelectrode area for 1 min, prior to being rinsed off with reverse osmosis (RO) water. The devices were then ultrasonicated for 15 min in 1:1
water/Decon 90 and, again, were rinsed with water. Etching of the protective nichrome overlayer created pin holes and undercut the residue layer, weakening its adhesion to the underlying metal. Periodic visual examination of the device during the ultrasonication revealed that the residue layer was removed from the electrodes. Under the action of the acid in the nichrome etch, the polyimide first became hydrophobic, as determined by contact angle measurement, although subsequent exposure to strong alkali12 (e.g., Decon 90, used in the cleaning process) left an essentially hydrophilic surface. It is noteworthy that this (hydrophilic) surface made filling the microchamber with aqueous solution very difficult, although it was found that the hydrophobicity could readily be regenerated by dipping the devices in acid. Consequently the microchamber was immersed in 2.0 M HCl for 1 min, before being rinsed in RO water, and then placed directly on a 200 °C hot plate for 10 min. As stated, examples of the fabricated structures are shown as SEMs in Figure 2, with three gold microelectrodes situated at the bottom of a 0.6-nL-capacity microchamber. The microring working electrode has a diameter of 100 µm, with a width of 10 µm, and is surrounded by a larger horseshoe (counter) electrode. The working, reference, and counter electrodes were all individually addressed. Reference Electrodes. A layer of silver was electrodeposited on the smallest of the gold electrodes, Figure 2 (no. 2), from an aqueous plating solution of 0.2 M AgNO3/2 M KI/0.5 mM Na2S2O3, containing the complex ion [AgI2]- K+. The sodium thiosulfate was added to reduce the size of nucleation of the deposited silver crystals.13 A current of -0.5 µA was passed for 1 min through the electrode, using a coil of platinum wire as a counter electrode. Although the deposition time was not critical, care had to be taken not to pass too much charge, otherwise the silver layer grew to such an extent that it “shorted” to the working electrode. Finally, the silver halide reference electrode was completed by passing +0.5 µA through the electrode in a solution of either 0.1 HCl or 0.1 KI and allowing the current to flow for 30 s, using the same platinum wire as a counter. During this period the current decayed slowly as the halide layer was deposited on the silver surface. Originally, the silvered gold layer was a shiny, yet rough, continuous layer: the deposited AgCl was purple/ brown while the AgI was yellow/white. Finished devices were stored between tests, dry and covered, but were rinsed in double-distilled water and blow-dried before reuse. It may be helpful to note that, if necessary, devices can be stored before the nichrome etch, with the later steps performed immediately prior to the experiments. Connections to the reference and working microelectrodes were made by attaching multicore wire to the bonding pads with silver paint. The wires were secured in place with epoxy resin. An epoxy rings1 mm wide, between 3 and 5 mm diameter, and 1 mm deepswas adhered around the polyimide microchamber, a process that was essential for the handling of ultralow volumes of solution, as described below. Nanoliter Electrochemical Experiments. The electrochemical performance of the device was investigated using an aqueous solution of 0.5 mM monocarboxylic acid FMCA, with 50 mM (12) Lee, K.-W. J. Adhes. Sci. Technol. 1994, 8, 1077-1092. Lee, K.-W.; Viehbeck, A. IBM J. Res. Dev. 1994, 38, 457-474. (13) Brimi, M. A.; Luck, J. R. Electrofinishing; Am. Elsevier Pub. Co.: New York, 1965; Chapter 10.
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LiClO4 as the supporting electrolyte. FMCA was dissolved in 3% methanol and diluted in the supporting electrolyte, as appropriate. All solutions were made immediately prior to each experiment, not being stored for longer than 30 min. A ∼0.5 µL droplet of FMCA (0.5 mM) was placed directly onto the microchamber area of the device and immediately covered with mineral oil (Sigma, Poole, UK). The aqueous droplet formed as a hemisphere due to the hydrophobicity of the polyimide surface, with the mineral oil spreading outward. Importantly, the epoxy washer acted to prevent the oil layer spreading too thinly and allowing the droplet to come into contact with the air and start to evaporate. The device was observed under a Nikon MZ6 stereoscopic microscope at a magnification of ×100, the droplet being best viewed when both transmitted light and independent side illumination (from a fiber optic) were used simultaneously. A pulled glass micropipet, with a diameter of between 10 and 20 µm, was attached to a suction ball and was filled with the mineral oil until the entire tapered region was full. When placed in the aqueous solution within the microchamber, the pipet filled by capillary action. Providing the tip diameter was small enough, the rate of capillary filling was slow. As the droplet volume reduced, viewed under the microscope, it maintained its hemispherical shape due to the hydrophobicity of the surface. When the droplet came to the required volume, the pipet was removed. This procedure proved to be both a quick and a reproducible way of restricting the volume of the droplet to the microchamber alone. The lack of a lens effect was taken as an indication that the oil droplet meniscus was horizontal with the lip of the cell and that the volume was therefore close to its geometric dimension, e.g., ∼0.6 nL. After use, the cell could be washed with diethyl ether in order to remove the mineral oil, without damaging the polymerized polyimide. If necessary, the gold was recleaned using a low-power oxygen plasma etch, although this was found to reduce the height of chamber (as a function of the length of the etch and its power). Cyclic Voltammetry. A low-current potentiostat (BAS CV37, Stockport, UK) was used to perform the cyclic voltammetry, with data collected on a personal computer. Scan rates (ν) were varied from 1 to 1000 mV s-1, with a filter time constant of 0.1 s. Low-current, low-volume measurements were made inside a Faraday cage. The response of devices was investigated for a variety of reference electrode configurations: (a) an external Ag|AgCl microreference inserted into a large droplet placed on the device; (b) the silvered microreference electrode as a silver pseudoreference; (c) an in situ Ag|AgCl reference; and (d) a pseudo-Ag|AgI reference. Individual cyclic voltammograms could be analyzed without any postprocessing of the data. To compare two or more curves, especially of similar scan rate, the data obtained for each sweep were digitally filtered using simple lowpass FIR filters of appropriate bandwidth with 15 coefficients. The filter coefficients were calculated using the FGEN digital filter design program, obtained from Microstar Laboratories. The filtered wave form was compared with the unfiltered version to ensure no scale shifting had taken place. Reagents. Reagent grade hydrochloric acid, chlorobenzene, diethyl ether, acetic acid, and acetone were from Aldrich, (Gillingham, UK); silver nitrate, potassium iodide, lithium perchlorate, sodium thiosulfate, glacial acetic acid, Decon 90, and ferrocenemonocarboxylic acid were from Sigma; ammonium hexanitrocerate was from Fluka, UK; Probimide 7020 photopatternable polyimide and OCG developer and rinse were from OCG, London, 256
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UK; 1400-31 photoresist was from Shipley, UK; metals, including Au, Cr, Ni, Ti, and Pd, were from Goodfellows (Cambridge, UK). All chemicals were used as received. Safety: Care was taken when handling silver nitrate (toxic) and potassium iodide (teratogen), especially in the powdered form. RESULTS AND DISCUSSION Device Fabrication. The procedure described above, and illustrated schematically in Figure 1, has proved to be a reliable and reproducible method for producing subnanovolume electrochemical cells, typified by the SEMs (Figure 2). Devices were generally made in batches of between 9 and 18, and no significant inter- or intrabatch variation was detected for electrochemical measurements made with freshly cleaned devices. The only problem encountered in the process involved the degradation of soft-baked polyimide adhesion when the relative humidity of the clean room was above ∼40%, which manifested itself during the development of exposed samples by the edges of the insulator rolling back. Regardless of this latter problem, there are a number of important criteria for choosing this particular polyimde: first, the polymer is not only appropriate for photopatterning but can also be deposited to a greater thickness than is the case for a variety of other insulators, e.g., silicon nitride or photoresist; second, the photopatterning process is the equivalent of an anisotropic etch, giving high aspect ratio walls (most etching of thick materials requires an isotropic wet etch, preventing material redeposition but giving shallow sides); third, the matrix can be readily spun onto lithographically defined electrodes, enabling the relative positions and sizes of the microstructures to be easily defined and reproduced (and if necessary, redefined), a situation that is in contrast to the technology associated with manually inserted probe-type electrodes;3 and finally, the thermally cross-linked polyimide does not leach toxic compounds, e.g., into cell culture (see later). Electrochemical Characterization. FMCA was used as a model redox compound in order to investigate the performance of the device. In initial experiments, a small standard Ag|AgCl electrode, made from a choridated silver microwire (Goodfellows) was inserted from above, in order to act as a reference, from which comparisons of the behavior of the integrated silver halide reference electrodes, fabricated in situ, could be made. The response of the ferrocene compound showed appropriate Fe2+|Fe3+ redox behavior (data not shown), with an experimentally measured E′1/2 for the redox couple of 0.31 V (∆Ep ) 80 mV) which compared favorably with the literature value of ∼0.30 mV (vs Ag|AgCl) previously measured using a carbon electrode.14 The deposition of chloride, to give an integrated (in situ) Ag|AgCl microreference, produced an electrode that performed well over short periods of time, with FMCA showing redox characteristics similar to that for the probe Ag|AgCl reference. However, repeated voltammograms with identical solutions, either in the presence of Cl-, or without added electrolyte, displayed increasing hysterisis with ∆Ep becoming larger on successive scans. In particular, in the absence of Cl-, with the Ag|AgCl acting as a pseudoreference, distortion of the i-v curve at ∼+65 and at -60 mV was assumed to be indicative of a Ag|Ag+ redox couple, suggesting dissolution of the reference electrode. This could be readily confirmed by rechloridating the reference after the response had begun to drift and finding that the original performance was regained.
Figure 4. Plot of the log of oxidation charge (Q) against the log of scan rate (v) for 0.5 mM FMCA at the microring electrode (A ) 40 µm2) within a 0.6-nL electrochemical cell. The regression is shown for the points v ) 1-100 mV s-1, with obvious deviation from linearity for the scan at both 1000 and 500 mV s-1.
Figure 3. Steady state cyclic voltammetric responses of ∼0.6 nL of 0.5 mM FMCA in 50 mM LiClO4, measured at a 40-µm2 microelectrode in the chamber using an integrated Ag|AgI pseudoreference. Examples that are shown lie within the range 1-500 mV s-1.
As an alternative, a Ag|AgI reference electrode was used, for not only is I- less soluble than Cl-, Ksp for I- ) 8 × 10-17 and for Cl- ) 2 × 10-10 (The Merck Index; Merck & Co.: Rahway, NJ, 1996), but X-ray photoelectron spectroscopic studies have shown that AgI is a particularly stable reference material when exposed to aqueous electrolytes.15 Ag|AgI was therefore deposited onto the silvered Au microelectrode with ease, giving a device that performed without degradation of response over periods of several hours, even when the experimental protocol involved repeated rinses. Cyclic voltammograms of FMCA using a pseudo-Ag|AgI microreference are shown in Figure 3 for scan rates in the range 1-500 mV s-1, with the redox characteristics of the couple estimated as E′1/2 ) 430 mV, ∆Ep) 102 mV at v ) 500 mV s-1. Iodide ions were not added to the working solution, as it was decided to characterize the stability of the reference electrode, and the response of the working electrod,e under conditions similar to that which would be used in its future biological application. It should however be noted that, for experiments either involving a pseudo-Ag|AgI or for those with a standard Ag|AgCl electrode, the background voltammogram, in the absence of FMCA, was essentially flat, with no contribution visible from either Ni or Cr ions, previously present in the protecting alloy layer. The limiting current generated at the working electrode was estimated from the i-t curves “contained” within the voltammo(14) Cass, A. E. G.; Davis G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667-671. (15) Strydom, C. A.; Van Staden, J. A.; Strydom, H. J. Electroanalysis 1992, 4, 969-973.
grams in Figure 3. Linear regression of a plot of t-1 (s-1) against the steady state diffusion current (nA) at 600 mV gave a steady state approximation of 1.30 nA, which, notwithstanding the nonideal geometry of the microring due to the connecting tracking, Figure 2a, was within 1 order of magnitude of the value predicted for the electrode microring structure in a bulk solution.16 In this respect, it is interesting to note that when electrochemical measurements are performed in such a small volume, the total charge that could be generated by oxidation or reduction of all of the ferrocene in the microchamber is very small, in this case only ∼30 nC. Although a plot of the reciprocal of scan rate against the oxidation charge is linear between 1 and 100 mV s-1 (Figure 4), as might be expected for a microelectrode in bulk solution,1,2 it is evident that redox recycling is occurring. The process is, however, sufficiently fast that it does not limit the electrochemical processes (the diffusion coefficient for FMCA of 4 × 10-6 cm2 s-1).14 Finally, it is also clear from Figure 4 that there is a deviation from linearity at the fastest scan rates (i.e., at 1000 and 500 mV s-1), an observation that is in accord with a change in the diffusion mechanism that is occurring at increased scan rates (see Figure 3). Device Applications. Consistent with the aim of using the device to monitor substance released from single mammalian cells, it has been found possible to culture both baby hamster kidney (BHK) and rat neonatal fibroblasts over the device and in the chamber, using standard procedures,17 with the particular polyimide used showing no apparent signs of toxicity, as determined by cell longevity. The base of the chamber is transparent glass and is suitable for use with light microscopy, which will, in future studies, enable the cell to be placed appropriately within the chamber. Many of the materials used in the fabrication process are in common use in the semiconductor industry, and it would be feasible to produce such devices in large numbers, with a commensurate reduction in the cost of each sample. Indeed, since (16) Fleischmann, M.; Pons, S. J. Electroanal. Chem. 1987, 222, 107-115. (17) Freshney, R. I. Culture of Animal Cells: A Manual of Basic Technique. 3rd ed.; Wiley: New York, 1994.
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at no stage during the procedure were the limits of lithographic resolution reached, structures could be readily fabricated in very high yield, thus offering the prospect for developments such as the production of arrays of microchamber sensors on one substrate, even smaller electrochemical cells, or devices with more than one working electrode. Among the possible applications of such devices would be monitoring the release of various compounds from single mammalian cells, with the prospect of discovering patterns of cell signaling or, in the longer term, developing methods for low-cost, high-throughput diagnostics. In this context, two important conclusions can be drawn from this initial study: (i) if the device is to be used to obtain an absolute measurement of amounts of material within the cell, then not withstanding possible problems regarding capacitive or inductive coupling between the electrodes, it may be preferable to use a short pulsed electroanalytical technique, so that quantification at the working electrode is not complicated by the regeneration of species that have diffused from the counter electrode; (ii) given (18) Paeschke, M.; Hintsche, M.; Wollenberger, U; Jin, W.; Scheller, F. J. Electroanal. Chem. 1995, 393, 131-135.
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the demonstrated ability of the device to recycle redox mediators, there is the possibility of using the chamber as a miniature amplifier, thus regenerating a signal without losing species to bulk (a phenomenon previously observed at proximal microband electrodes18). Although both of these latter strategies offer exciting future prospects in the monitoring of the biology of cells in picoliter volumes, care will be needed in future designs of the electrochemical cell to ensure that potential biological toxins, including H+, OH- and Cl2, do not accumulate at the counter or working electrode (e.g., it may be necessary to use a polymermodified electrochemical surface). ACKNOWLEDGMENT The authors thank the Wellcome Trust and Glaxo-Wellcome for supporting this work. Received for review July 24, 1996. Accepted October 18, 1996.X AC960743W X
Abstract published in Advance ACS Abstracts, December 1, 1996.
