Characterization of Electrochemical Responses in Picoliter Volumes

conditions such as scan rate, vial size, and concentration. The voltammetric properties have been shown to be dependent on all three parameters. The p...
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Anal. Chem. 1998, 70, 1119-1125

Characterization of Electrochemical Responses in Picoliter Volumes Rose A. Clark and Andrew G. Ewing*

Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802

Cyclic voltammetry is conducted in picoliter microvials to investigate the influence of small volumes on the electrochemical response. Electrochemical experiments using a standard reduction-oxidation couple, ferrocenecarboxylic acid, have been performed in volumes as small as 1 pL. Peak-shaped voltammetry and an increase in the current on the reverse wave of the cyclic voltammogram are observed in the voltammetric response when ultrasmall volumes (16 pL or less) are used. This deviation from bulk microelectrode behavior is observed only at slower scan rates in the smaller microvials. The origins of the voltammetric behavior in the small-volume experiments have been probed by varying experimental conditions such as scan rate, vial size, and concentration. The voltammetric properties have been shown to be dependent on all three parameters. The peak-shaped voltammetry and increases in the current on the reverse wave are attributed to depletion of oxidizable species on the forward scan and reduction of the analyte restricted at the electrode surface, respectively. A physical model based on restriction of analyte in these well-defined microenvironments is proposed to explain the differences in current compared to that predicted by microelectrode theory in bulk solutions. Small-volume chemistry applications are being advanced as the demands for analysis of precious samples, sampling from biological microenvironments, and analysis of fewer molecules grow in the scientific community. Small-volume techniques are being developed simultaneously by many groups for different purposes. Several reports have emphasized the importance for minimization of detection volumes. One detection method amenable to smallvolume analysis (nano- to picoliter volumes)1-4 is fluorescence. Detection of fluorescent analytes has been demonstrated in small droplets on the nanoliter scale1 as well as in nano- to picoliter volumes at the end of a capillary electrophoresis system.2-4 Another detection method, NMR, traditionally a large-volume technique, is now being developed for analysis of samples in the * To whom correspondence should be addressed. Phone: (814) 863-4653. Fax: (814) 863-8081. (1) Mahoney, P. P.; Hieftje, G. M. Appl. Spectrosc. 1994, 48, 956-958. (2) Holland, L. A.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3275-3283. (3) Lippard, S. J.; Yeung, E. S.; McCloskey, M. A. Anal. Chem. 1996, 68, 28972904. (4) Larson, A. P.; Ahlberg, H.; Folestad, S. Appl. Opt. 1993, 32, 794-797. S0003-2700(97)00649-5 CCC: $15.00 Published on Web 02/07/1998

© 1998 American Chemical Society

nanoliter range.5,6 Small-volume detection has been possible by electrochemical methods since the advent of microelectrodes in the mid-1970s. Microelectrodes are currently being used extensively as nanoliter to picoliter detectors for microbore liquid chromatography7 and capillary electrophoresis.8 Detection in subfemtoliter volumes with microelectrodes has also been reported to measure the activity of single molecules trapped at the tip of a scanning electrochemical microscope probe.9 In addition to small-volume detection techniques, methodology to handle small volumes is gaining interest for sample isolation and introduction. Methods have been developed to isolate microliter to femtoliter aqueous drops in an organic matrix.10-12 In these isolated drops, diffusional titration of acid/bases10 and metal ions11 has been performed with various detection schemes.12 Another technology developed to handle small volumes is lithographically fabricated microvials. Arrays of vials etched in silicon produce nanoliter13 and picoliter14 sample wells useful for sample introduction in capillary electrophoresis and in mass spectrometry.15 This microvial technology provides a means for sample handling that previously was unavailable. The ability to isolate samples into nanoliter to picoliter microvials is potentially beneficial for other applications where silicon wafers are not ideal substrates due to their opaque nature. Vials (or wells) are now being produced at the end of a bundle of optical fibers by etching back the fiber. The transparent vials produced are useful in spectroscopic applications where light must pass through the structure.16 Vials with micrometer to nanometer dimensions have been produced, which corresponds to volumes from picoliters to subfemtoliters. Transparent picoliter to femtoliter microvials fabricated in polystyrene have also been reported. A photolithographically designed silicon template is used to (5) Olson, D. L.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Science 1995, 270, 1967-1970. (6) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1995, 22, 3849-3857. (7) Cooper, B. R.; Wightman, R. M.; Jorgenson, J. W. J. Chromatogr. B 1994, 653, 25-34. (8) Mesaros, J. M.; Gavin, P. F.; Ewing, A. G. Anal. Chem. 1994, 66, 527A537A. (9) Fan, F. F.; Kwak, J.; Bard, A. J. J. Am. Chem. Soc. 1996, 118, 9669-9675. (10) Gratzl, M.; Yi, C. Anal. Chem. 1993, 65, 2085-2088. (11) Xie, H.; Gratzl, M. Anal. Chem. 1996, 68, 3665-3669. (12) Yi, C.; Huang, D.; Gratzl, M. Anal. Chem. 1996, 68, 1580-1584. (13) Jansson, M.; Emmer, Å.; Roeraade, J.; Lindberg, U.; Ho¨k, B. J. Chromatogr. 1992, 626, 310-314. (14) Beyer Hietpas, P.; Ewing, A. G. J. Liq. Chromatogr. 1995, 18, 3557-3576. (15) 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. (16) Pantano, P.; Walt, D. R. Chem. Mater. 1996, 8, 2832-2835.

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produce microvials similar to those described above in silicon, except these are transparent.17 Combining the advantages of the transparent microvials with the small-volume detection ability of microelectrodes has been the focus of our research. Electrochemistry in these vials has applications for analysis of extremely low sample volumes and possibly controlled immobilization of specimens such as single cells. It is essential to understand the basic electrochemical responses in the microvials so that further studies of small-volume samples and isolated cells can be fully understood. The first report of electrochemical investigations in picoliter volumes demonstrated the feasibility of combining microvial technology and electrochemistry. However, in the initial report, only a limited range of parameters was investigated. In this report, the results of experiments aimed at gaining further insight into the electrochemical response in picoliter volumes are described. A more detailed investigation of the cyclic voltammetric response over a wide range of conditions is performed. The focus of this article is to investigate the voltammetric behavior in the smallest microvials compared to the currents predicted from microelectrode theory for bulk solutions. Peak-shaped voltammograms and an increase in current on the reverse wave for cyclic voltammetry are observed at slow scan rates in 16-pL vials or less. It is proposed that this behavior is due to the analyte being restricted near the electrode surface, similar to a thin-layer cell. Analyte restriction leads to depletion of electroactive species near the electrode, peak-shaped voltammetry, and an increase in the current on the reverse wave of the CV. The results with changing scan rate, microvial size, and concentration all support the proposed quasi-thin-layer model. EXPERIMENTAL SECTION Microvial Fabrication. The silicon wafer microvial templates were fabricated at the Cornell Nanofabrication Facility, Cornell University (Ithaca, NY), using standard photolithographic and wet chemical etching techniques. The template pattern is then transferred using a hot press method into polystyrene to form an array of transparent microvials.17 Microvial volumes were determined using an Eppendorf microinjector 5242 (Eppendorf North America, Inc., Madison, WI), calibrated such that the delivery rate was known. Solutions containing 58% glycerol were used during the calibration process to ensure minimal evaporation. The vial sizes reported were determined by timing the filling process and calculating the volume from the calibration curve. The range of microvials employed in these investigations was 390 ( 19, 81 ( 2, 16 ( 1, and 7 ( 1 pL. These numbers represent the volumes of the microvial, plus or minus the standard deviation for n ) 6 trials. These values match up extremely well with those predicted from calculations based on the actual microvial dimensions.17 The 1-pL vial was not calibrated since it was difficult to calibrate the Eppendorf for such small volumes. The 1-pL value used is based solely on a calculation using the dimensions of the vial measured with scanning electron microscopy and surface profilometry as previously described.17 Microvials were cleaned by consecutive 3-min sonications in ethanol, 1 M sulfuric acid, and doubly distilled water. Vials were (17) Clark, R. A.; Beyer Hieptas, P.; Ewing, A. G. Anal. Chem. 1997, 69, 259263.

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cleaned immediately before use and were reused after repeating the cleaning procedure. Electrochemical Instrumentation. All voltammetric data were collected using an Ensman Instruments EI400 microelectrode potentiostat (Bloomington, IN) in the two-electrode mode. A Gateway 2000 486/33 PC (interface, Labmaster, Scientific Solutions, Solon, OH) with local software18 was 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, Fisher, NY, and two Mertzhouser, Zeiss, NY) for positioning of the electrodes and an injector. Electrodes. 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).20 The fiber was sealed in the glass capillary using a vertical capillary puller (Ealing, Harvard Apparatus, Edenbridge, KY). The electrodes were back-filled with colloidal graphite (Polysciences, Inc., Warrington, PA) and a Nichrome wire inserted for contact. The carbon fiber was then insulated by electrodepositing a phenolallylphenol copolymer, followed by tip exposure using a scalpel.19 A miniature (tip ∼1 µm) Ag/AgCl (1 M KCl) reference was constructed by pulling a capillary tube to a fine tip and trimming to provide a small opening for solution contact. References were prepared immediately before use and kept in solution at all times. The references did, at times, especially if the tip was broken, change the volume of the solution in the vial. In these cases, new references were prepared. Additionally, the reference solution contained a dye that could be observed if mixing were to occur. Injectors were prepared as previously described14 by HF etching a CE capillary with external dimensions of 150 µm down to values close to the internal dimensions, which range from 10 to 2 µm. The etched capillary tip is epoxied into a larger glass capillary tube for support and used for injection of samples. Injections were made using an Eppendorf microinjector or Picospritzer II (General Value, Fairfield, NJ). Chemicals. All solutions were prepared in doubly distilled water (Corning Mega-Pure MP-3A, Corning, NY). To control evaporation, glycerol was added at 35% (w/w) to the 0.1 M phosphate buffers (pH 7.4) used for preparation of the ferrocenecarboxylic acid solutions. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. RESULTS AND DISCUSSION Voltammetry in Picoliter Microvials. Details of polystyrene microvial preparation, structure, and filling procedures have been previously described.17 Figure 1A shows a schematic of the microvial experimental setup. During an experiment, a microvial is first filled with the redox solution using a microinjector, and then the working and reference electrodes are placed into the vial to conduct voltammetric measurements. A photomicrograph of a typical 16-pL microvial is shown in Figure 1B, with the electrodes and injector retracted for clarity. The vial pictured is (18) Chen, T. K.; Lou, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (19) Strein, T. G.; Ewing, A. G. Anal. Chem. 1992, 64, 1368-1373. (20) Wightman, R. M.; Wipf, D. O. Voltammetry at Ultramicroelectrodes In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1989; Vol. 15, pp 267-351.

Figure 2. Cyclic voltammograms of 1.0 mM ferrocenecarboxylic acid in a bulk drop on polystyrene. The scan rates are (A) 0.1, (B) 1.0, and (C) 10 V/s. Carbon fiber working electrodes are 5 µm in diameter. Figure 1. (A) Schematic of the placement of electrodes in a polystyrene microvial for voltammetric measurements and (B) a brightfield photomicrograph of a 16-pL polystyrene microvial: (a) injector, (b) working electrode, and (c) reference electrode. The base of the microvial in panel B is 20 µm.

empty to aid visualization, since the filled vials become mostly clear.17 An important issue in these experiments is the evaporation rate in the microvials. We have previously shown that this rate is negligible during voltammetric experiments when 35% glycerol is added to the analytical solution.17 The cyclic voltammetry responses for fast, intermediate, and slow scan rates in picoliter microvials have been compared to responses for microelectrodes in bulk solutions to investigate microelectrode performance in restricted volumes. Voltammetric measurements have been conducted on a common redox couple, ferrocenecarboxylic acid. Figure 2 shows representative cyclic voltammetric responses for a large drop of ferrocenecarboxylic acid on a polystyrene template used to model bulk solution. Cyclic voltammetric responses follow that expected for a microelectrode: steady-state behavior at slow scan rates (0.1 V/s, Figure 2A), mostly steady state with a small contribution from linear diffusion at intermediate scan rates (1 V/s, Figure 2B), and finally, more peak-shaped behavior when diffusion becomes the limiting step at faster scan rates (10 V/s, Figure 2C). Figure 3C depicts a similar peak-shaped response for fast scan rates in a 16-pL microvial. For the intermediate scan rate (Figure 3B), the response is mostly sigmoidal (similar to Figure 2B), with a slight increase in the current on the reverse wave of the CV. Finally, the response at slow scan rates in the microvial (Figure 3A) is substantially different from the bulk response, Figure 2A. The slower scan rate cyclic voltammograms are peak-shaped, with a large increase in current on the reverse wave. In the preceding sections, the data that follow microelectrode theory will be discussed first, and then the details of the slower scan rate responses (quasi-thin-layer behavior) will be addressed.

Figure 3. Cyclic voltammograms of 1.0 mM ferrocenecarboxylic acid in a 16-pL vial. The scan rates are (A) 0.1, (B) 1.0, and (C) 10 V/s. Carbon fiber working electrodes are 5 µm in diameter.

Typically, microelectrode currents at slower scan rates are dominated by steady-state behavior and are governed by the following equation for hemispherical electrodes in bulk solutions:

il ) 2πrnFDC*

(1)

where il is the limiting current, r the electrode radius, D the diffusion coefficient, and C* the bulk analyte concentration.20 In this work, the limiting current at the unshielded disk (carbon fiber with a thin, insulating phenol/allylphenol copolymer) is approximated by the equation for a hemispherical electrode. When the insulation of a disk electrode is less than twice the radius of Analytical Chemistry, Vol. 70, No. 6, March 15, 1998

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Table 1. Currents at Various Scan Rates scan rates (V/s)

ipa (nA)

isb (nA)

iTc (nA)

iviald (nA)

ibulke (nA)

0.1 0.4 1 4 10

0.042 0.085 0.13 0.27 0.42

0.18 0.18 0.18 0.18 0.18

0.22 0.27 0.31 0.45 0.60

0.28 0.34 0.31 0.41 0.46

0.28 0.30 0.33 0.47 0.53

a Current expected with planar diffusion (i ) n3/2AD 1/2ν1/2C *).22 p o o n, number of electrons ) 1; A, electrode area ) 3.93 × 10-7 cm2; Do, diffusion coefficient ) 1.6 × 10-6 cm2/s; ν, scan rate; Co*, bulk concentration ) 1.0 × 10-6 mol/cm3. This is approximating the unshielded disk as a hemisphere. b Spherical correction to ip (is ) (0.725 × 105)nDoCo2πr). Electrode radius ) 2.5 × 10-4 cm. c Total current, iT ) ip + is. d Current for 1.0 mM ferrocenecarboxylic acid in a 16-pL microvial, Figure 3. e Current for 1.0 mM ferrocenecarboxylic acid in a bulk drop, Figure 2.

the electrode, the limiting current is increased.21 Diffusion from behind the unshielded electrode results in a larger limiting current more closely approximated by a hemisphere. Using eq 1 for steady-state conditions at microelectrodes and the parameters specified at the bottom of Table 1, the limiting current expected is 0.24 nA. With the hemispherical electrode assumption, the current calculated for 1.0 mM ferrocenecarboxylic acid at 0.1 V/s in bulk solution is close to the experimental values (Figure 2A, Table 1). As the scan rate is increased to 10 V/s, the signal is no longer completely sigmoidal, as peaks appear in the voltammograms. Additional terms are needed in the calculation to account for the increased peak currents, since the voltammetry is no longer governed by steady-state conditions. Table 1 summarizes the currents over a range of scan rates that traverse this boundary from mostly spherical diffusion to larger contributions from planar diffusion. In the intermediate scan regions, contributions from both planar diffusion and spherical diffusion are combined to give the total current.22 In our case, the spherical correction term22 (see Table 1) is modified by replacing A/r in Nicholson’s original correction term by 2πr to approximate a hemispherical microelectrode. Howell and Wightman23 have used a similar approach to approximate fast-scan currents at disk microelectrodes by replacing A/r by 4r. It is well-known that scan-rate-dependent planar diffusion dominates the current at faster scan rates, and this can be seen in Table 1; at 10 V/s, ip (planar) ) 0.42 nA and is (spherical) ) 0.18 nA. The calculated currents based on the combined current contributions correspond well with those obtained in the bulk ferrocenecarboxylic acid solutions (Figure 2, Table 1) and in the microvials at faster scan rates (Figure 3B,C, Table 1). The current values reported in Table 1 are calculated with the diffusion coefficient adjusted for the viscosity of a 35% glycerol solution using the Stokes-Einstein equation (diffusion coefficient proportional to inverse viscosity). For a 35% glycerol solution, the diffusion coefficient is 1.6 × 10-6 cm2/s. The slow diffusion of the analyte in the 35% glycerol solutions causes the scan rate dependence of the current to be observed at slower scan rates. The transition from dominant spherical to dominant planar diffusion also shifts to slower scan rates as the electrode area (21) Shoup, D.; Szabo, A. J. Electroanal. Chem. 1984, 160, 27-31. (22) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; John Wiley & Sons: New York, 1980; pp 218-220. (23) Howell, J. O.; Wightman, R. M. Anal. Chem. 1984, 56, 524-529.

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increases (mostly planar diffusion at macroelectrodes), so small variations in electrode size between experiments affect where the transition region occurs. Table 1 displays the data collected on the same day using the same electrode. To report statistics for microvial experiments conducted on different days, the currents are normalized to account for electrode changes. The results for four experiments in the 16-pL microvial yield currents with standard deviations as follows: 0.23 ( 0.04 (0.1), 0.28 ( 0.07 (0.4), 0.33 ( 0.04 (1.0), 0.43 ( 0.06 (4.0), and 0.52 ( 0.06 nA (10.0 V/s). Careful inspection of the microvial currents in Table 1, however, shows a slight increase in current at the slower scan rates and a slight decrease in current at faster scan rates. Evaporation should be minimal.17 A small amount of evaporation is not expected to significantly change the current. Upon evaporation, the diffusion coefficient would decrease due to increases in viscosity, and the concentration of the ferrocenecarboxylic acid would increase. If evaporation of 10% of the vial volume occurs, the diffusion coefficient and concentration would become 1.49 × 10-6 cm2/s and 1.11 × 10-3 M, respectively. The corresponding current at 1 V/s would change from 0.31 nA (iT, Table 1) to 0.33 nA. With 25% evaporation, the diffusion coefficient and concentration would become 1.24 × 10-6 cm2/s and 1.33 × 10-3 M, respectively, and the current at 1 V/s would increase to 0.35 nA. Only small changes are observed in the current for rather substantial changes in volume due to the dual effects of concentration increase and viscosity increases. Hence, combined with our previous data demonstrating the relatively small amounts of evaporation in this system, it appears that this is not a factor in these experiments. Bulk Electrolysis in Picoliter Microvials. The above discussion addresses the response in bulk and in Figure 3B,C; however, a marked difference in the voltammetry between bulk solution and the microvials is seen at slow scan rates (0.1 V/s). In bulk solution (Figure 2A), a sigmoidal voltammetric response is obtained, whereas in the microvial (Figure 3A), a peak-shaped response is observed. Only voltammograms at slower scan rates (in our case ∼0.4 V/s or less) exhibit this peak-shaped response, which resembles a diffusion-limited voltammogram. In addition, these peak-shaped responses are observed only in the smaller microvials, 16, 7, and ∼1 pL. This phenomenon can be understood by considering the diffusion in bulk compared to that in a microvial. General microelectrode theory at these scan rates supports the idea that no reverse wave should be observed for steady-state conditions, since the products diffuse away as rapidly as they diffuse in. In the smaller microvials, diffusion to the microelectrode is bounded by the walls of the microvial, thus altering the diffusion profile. Based on this argument, depletion of electroactive species near small electrodes can be accomplished in the smallest microvials. If complete depletion occurs at extremely slow scan rates, ,0.1 V/s, the small microvials can be considered essentially threedimensional thin-layer cells. To examine one extreme of thinlayer conditions, we assumed at these slow scan rates that the peak current expected for complete electrolysis would be

ip ) n2F2νVC*/4RT

(2)

where V is the volume of the solution.22 The value calculated from

eq 2 is 1.4 nA, which can be compared to a peak current in Figure 3A of 0.28 nA. The peak current observed in the 16-pL vial is produced by 21% of the total analyte available for oxidation in the solution. The measured current represents a large percentage of the total current expected for complete oxidation in the microvial; however, thin-layer behavior is only partially achieved. This can be compared to bulk solution, where the amount of analyte oxidized is negligible relative to the bulk analyte concentration. The voltammogram in Figure 3A, in contrast, does not have the characteristic features of a thin-layer response, where the currents return to the baseline and minimal peak splitting is observed (reversible system). This may be because the 16-pL vial is sufficiently large compared to the size of the electrode for some diffusion of the analyte to occur. Scan rates below 0.1 V/s have been attempted; however, the voltammograms become distorted when 35% glycerol is used. Based on the above arguments, it should be possible to quantitatively oxidize the analyte in the microvial. Bulk electrolysis would alleviate problems due to variations in the scan rate (time scale) and could be easily implemented in the microvials to determine the amount of analyte present. Reverse Cyclic Voltammetric Wave in Microvials. Upon careful inspection of the voltammograms in Figures 2 and 3, another difference in the response is observed at the slower scan rates. An increase in the current on the reverse wave is observed for voltammetry in the microvials. To gain a more detailed understanding of this response, the current increase on the reverse wave of the CV has been investigated quantitatively to determine the differences between the microvial and bulk responses. The background currents in bulk solution vary only slightly between runs due to changes in the electrode area. Comparing the bulk background currents (at -0.1 V, Figure 2) to the responses in the microvials (at -0.1 V, Figure 3) for ferrocenecarboxylic acid solutions shows a large increase in the response at the intermediate and slow scan rates. The current at -0.1 V is referred to as an increase in the current on the reverse wave in the microvials, since it is most likely a combination of “true” background and faradaic currents (vide infra). Current measurements are quantitated at -0.1 V to focus on the increase in current on the reverse wave instead of the large faradaic response for ferrocenecarboxylic acid at the formal potential, as well as to be consistent between the mixed responses observed over the range of conditions studied (e.g., steady-state to diffusioncontrolled voltammetry). The bulk ferrocenecarboxylic acid background currents have been used in the calculation of the normalized currents since they are essentially equivalent to the backgrounds without ferrocenecarboxylic acid present and to emphasize the differences between microvial and bulk responses. Blank measurements have also been conducted on phosphate buffer with 35% glycerol in the microvials, and the background currents do not show any increase over background measurements in bulk solution. To examine any possible effects resulting from analyte interactions with the microvial walls, we have carried out voltammetry in vials fabricated from different materials, and different analytes have been employed. The polystyrene used in these experiments was obtained from cell culture dishes, so the surface has been modified with hydrophilic functional groups to promote cellular

Figure 4. (A) Normalized reduction current as a function of scan rate for representative plots of ferrocenecarboxylic acid (b), ruthenium hexaammine (s), and ferrocenecarboxylic acid in a polycarbonate microvial (4). (B) The average response for ferrocenecarboxylic acid (in a polystyrene microvial). Error bars indicate the mean ( standard deviation (see text for n values). Concentrations of solutions are 1.0 mM. Scan rates range from 0.1 to 6 V/s, and the vial size is 16 pL.

attachment. The ferrocenecarboxylic acid is a net negative redox couple; ferrocene is converted from 1- to 0 (at pH 7, the COOH should only be slightly deprotonated). To investigate the possibility that charge interactions are influencing the response, two tests have been conducted. First, the microvials have been constructed out of polycarbonate, a material without surface modifications. Second, the redox couple has been varied to a net positive couple, ruthenium hexaammine (3+ to 2+). By making these changes, it should be easy to see if charge interactions are playing a role in the increased currents. The results in 16-pL microvials for ferrocenecarboxylic acid in polystyrene and polycarbonate vials as well as for ruthenium hexaammine in polystyrene vials are shown in Figure 4A. Normalized currents are defined here as the ferrocenecarboxylic acid currents in a microvial with redox couple present (measured at - 0.1 V), divided by the “true” background response for ferrocenecarboxylic acid (-0.1 V) in bulk solutions. Therefore, the normalized current represents only the additional current in the microvial response. Figure 4 displays normalized current plotted vs scan rate for voltammograms collected in 16-pL microvials. Figure 4A overlays the normalized current plotted for ferrocenecarboxylic acid and ruthenium hexaammine voltammetry, both in polystyrene microvials, and that for ferrocenecarboxylic acid in polycarbonate Analytical Chemistry, Vol. 70, No. 6, March 15, 1998

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microvials. At slower scan rates, a dramatic increase in the normalized current is seen for all three cases, and this does not appear to be affected by the charge of the redox molecule or microvial substrate. Based on these results, interactions between the analytes and the walls of the microvial do not appear to be significant for the analytes investigated. Figure 4B displays a summary of the normalized ferrocenecarboxylic acid currents as a function of scan rate in a 16-pL polystyrene vial. The error bars represent the standard deviation of n trials, where n ) 3 for scan rates of 6, 2, and 0.6 V/s; n ) 4 for 0.2 V/s; n ) 5 for 4 V/s; n ) 6 for 0.4 V/s; n ) 9 for 1 V/s. At fast scan rates (around 6 V/s), the additional currents are essentially the same as background in bulk solutions (normalized current of 1). Differences come into play as the scan rate is decreased and the additional current continues to increase. As the scan rate is decreased, the time of the scan is increased, thereby creating a larger diffusion layer. With the limited volume of the microvial, this larger diffusion layer could extend to the limit of the vial dimensions (16-pL vial, 20-µm depth, 20-µm bottom, and 40-µm top; 7-pL vial, 16-µm depth, 20-µm bottom, and 30-µmm top). Finite diffusion in small volumes has been encountered in scanning electrochemical microscopy. In the “negative feedback” mode, an electrode (micro- to nanometer dimensions) is moved closer and closer to an insulating substrate. As this electrode approaches the insulating substrate, it reaches a point where the diffusion profile is altered by the proximity of the substrate. Theory of the feedback mode has been described in detail by Bard and co-workers24,25 and a working curve generated. In their results with an electrode radius of 2.5 µm, the current was shown to be affected at electrode-substrate distances of 12.5 µm and continued to decrease as the distance decreased. A similar effect would be expected in the microvial work shown here; however, in the microvial, the solution volume is restricted not only by the substrate (vial bottom) but also by the microvial walls. Based on this argument, the onset of current perturbation would be expected to start earlier than that observed at a scanning electrochemical microscope electrode with the same solution conditions. Even slower scan rates have been attempted (0.05 V/s) to deplete the analyte and create a thin-layer response. The voltammograms in this case become distorted, possibly due to reduction of oxidized species that cannot diffuse away in the restrained environment. At these extremely slow scan rates, the variable nature of the processes occurring makes it difficult to describe the system using conventional voltammetry at this time. The background currents in the microvials also increase with increasing concentration of the analyte. Figure 5 displays data for normalized current vs analyte concentration from voltammograms at 0.2 V/s. Ferrocenecarboxylic acid concentrations have been varied between 0.05 and 1 mM in a 7-pL microvial (the error bars represent standard deviations with n ) 4 at 0.5 mM, 6 at 0.1 mM, 7 at 0.05 and 1 mM, and 8 at 0.25 mM). The largest additional currents are observed for the highest analyte concentrations and decrease as the concentration is decreased. Based on (24) Bard, A. J.; Fan, F.-R. F.; Mirkin, M. V. Scanning Electrochemical Microscopy In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1994; Vol. 18, pp 268-287. (25) Kwak, J.; Bard, A. J. Anal. Chem. 1989, 61, 1221-1227.

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Figure 5. Normalized reduction current as a function of concentration. Concentration range from 0.05 to 1.0 mM. Scan rate is 0.2 V/s, and the vial size is 7 pL. Error bars indicate the mean ( standard deviation (see text for n values).

these data, the additional currents measured at -0.1 V can be attributed to reduction of the oxidized form of the analyte. In contrast to bulk solution, material oxidized in the 16-pL microvial cannot diffuse very far from the electrode surface. Once the analyte is oxidized, the oxidized molecules in the microvials are close enough to the electrode surface to be reduced on the reverse scan (Figure 3A) and continue to be reduced as molecules in the vicinity of the electrode diffuse back, thus contributing to the current on the reverse wave of the CV. Higher concentrations of electroactive species lead to more oxidized analyte isolated near the electrode surface for reduction, thus increasing the current observed on the reverse wave of the CV. Thus, an increase in analyte concentration leads to a corresponding increase in faradaic current that directly leads to an increase in the reduction current on the reverse wave of cyclic voltammetry (-0.1 V). The final experiments performed have been to investigate the effect of microvial size on the voltammetric response. Based on the above argument that the restricted space of the microvial changes the current on the reverse wave for cyclic voltammetry, it is expected that the additional reduction current will increase with decreasing vial size. The background current in a 390-pL vial (Figure 6) shows essentially no increase in additional reduction current and has a normalized current of ∼1. A slight increase in additional reduction current to 2 is observed for the 81-pL vial, and the ratio begins to increase rapidly reaching a value 14 times larger than the “true” background in the ∼1-pL vial. The error bars are based on standard deviation with n ) 3 for microvial volumes of 1, 6.7, and 81 pL, n ) 4 for 390 pL, n ) 5 for 16 pL. The size of the vial has a dramatic effect on the amount of additional reduction current measured in the cyclic voltammetric response. Similar to the concentration effect, it has to do with the amount of oxidized material available at the electrode surface to reduce. As the vial size decreases, the restricted volume prevents species oxidized on the forward voltammetric scan from diffusing from the vicinity of the electrode; thus, they are available for reduction on the reverse scan. This reduction current causes the increase observed on the reverse scan in the voltammograms collected in the microvials. Another piece of supporting evidence has been obtained by decreasing the electrode area (data not shown). Reduction currents are decreased by decreasing the electrode area in the same size microvial.

Figure 6. Normalized reduction current as a function of microvial size. Sizes range from 1 to 390 pL. Scan rate and concentration of analyte are 0.2 V/s and 1.0 mM ferrocenecarboxylic acid, respectively. Error bars indicate the mean ( standard deviation (see text for n values).

A simple physical model to explain the voltammetric response in the microvial centers around restriction of analyte in the microenvironment of the picoliter vials. The oxidized analyte attempts to diffuse away from the microelectrode as in normal bulk solutions, except that in the microvials the distance that the analyte travels is minimal. On the reverse scan, the material that is closest to the electrode surface is reduced, and then additional oxidized material in the restricted environment can diffuse to the electrode to be reduced, thus contributing to the current past the normal reduction peak (peak broadening). All of the voltammetric responses support this model. First, the voltammetry at slow scan rates is peak-shaped with both an oxidation and a reduction wave. Second, the magnitude of the reverse cyclic voltammetric wave decreases with increasing scan rate, which is reasonable since the material that is oxidized has a more compact diffusion layer due to the limited time scale, approaching steady-state conditions. Under steady-state conditions, there is no rereduction of the analyte, because it diffuses out of this thin diffusion layer. Third, at higher concentrations, where more oxidized species are produced, an increase in the additional reduction currents is also observed. Finally, the microvial size, which should be the most telling factor, shows a marked increase in the additional reduction currents observed as the size is decreased. In the 390-pL vials, the currents measured are only 0.2% of the total peak currents expected for total oxidation of ferrocenecarboxylic acid in this volume. The currents measured are 60% of the total current

expected in volumes as low as 1-pL, so variations from bulk microelectrode responses are expected. What does this mean for future experiments in the microvials? In our research, the goal is to isolate single cells in the microvial for analysis of the exocytosis process (fusion of small packets of neurotransmitters to the cell membrane and subsequent release). Once the neurotransmitters have been released, they can be monitored electrochemically. Research in this area has been advanced by Wightman et al.26 and others.27-29 using microelectrodes placed directly on the cell. The disadvantage of this method is that only the neurotransmitter released directly under the electrode is detected. We have been able to transfer single cells into the picoliter volumes. In these experiments, glycerol is not used to prevent evaporation. The evaporation is minimized by placing a layer of mineral oil above the microvial to prevent the buffer solution from contacting air, preventing evaporation. Up to this point, it has not been possible to look at total neurotransmitter release from a cell electrochemically. Electrochemistry in microvials should make this possible. One report has used native fluorescence of histamine to carry out total quantitation from a mast cell in capillary electrophoresis.30 Using microvials, total quantitation of exocytosis from single cells should be possible by several methods. First, evaluation of the concentration of the species released into the microvial can be carried out by fast-scan voltammetry, where current theory can accurately define the concentration. Second, modeling of the quasi-thin-layer response should allow accurate quantitation at slow scan rates, where mixed behavior is observed. Finally, it should be possible is to take advantage of the vial as a three-dimensional thin-layer cell to perform bulk electrolysis in extremely small volumes. ACKNOWLEDGMENT This work was funded by the National Science Foundation. R.A.C. is an NSF postdoctoral fellow. Received for review June 23, 1997. Accepted January 4, 1998. AC970649V (26) 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. (27) Chen, T. K.; Lou, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (28) Chow, R. H.; Klingauf, J.; Neher, E. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12765-12769. (29) Kennedy, R. T.; Huang, L.; Atkinson, M. A.; Dush, P. Anal. Chem. 1993, 65, 1882-1887. (30) Lillard, S. J.; Yeung, E. S.; McCloskey, M. A. Anal. Chem. 1996, 68, 28972904.

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