Anal. Chem. 1002, 64, 459-462
one-step fabrication method offers also a fast and sensitive response, associated with the porous microstructure and electrocatalytic rhodium surface. Such fast response would be advantageous for providing information on a real-time basis, for monitoring glucose in dynamic flow systems (e.g. flow injection), or for self-testing purposes. The controllable rhodium/enzyme codeposition scheme should be applicable for the entrapment of other enzymes or the fabrication of microsensors of different geometries and, hence, holds a great promise for the mass production of inexpensive and reproducible biosensing devices.
ACKNOWLEDGMENT This work was supported by the donors of the Petroleum Research Fund, administrated by the American Chemical Society. L.A. acknowledges a fellowship from FundacHo de Amparo a Pequisa do Estado de SBo Paulo (FAPESP). We also acknowledge H. P. Adams (EML, NMSU) for taking the SEM micrographs. REFERENCES (1) Wang. J. Anal. Chem. 1991. 63, 235R-237R. (2) Vadgama, P.; Desal, M.; Crump, P. Electroana&sk 1991,3,597-606.
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(3) B W a , D. S.; Zhang. Y.; Wllson, G. S.; Sternberg, R.; Thevenot, D. R.; Moattl, D.; Reach, (3. Anal. Chem. 1991. 63, 1692-1696. (4) Cronenberg, C.; van Groen. B.; de Beer, D.; van den Heuvel, H. Anal. Chlm. Acta 1901, 242, 275-278. (5) Kimwa. J; Kawana. Y.; Kuriyama, R. Blosensors 1988, 4 , 41-47. (6) Yokoyama, K.; Tamlya, E.; Karube, I. Electroanalysls 1991, 3 , 469-475. (7) Ikarlyama, Y.; Yamauchl, S.; Yuklashl, T.; Ushiodo, H. J . Electrochem. Soc.1989, 736, 702-706. (8) Umana, M.; W a l k , J. Anal. Chem. 1986, 58, 2979-2983. (9) Shinohara, H.; Chlba, T.;’Alzawa, M. Sens. Actuators 1987, 73 (l), 79-84. (10) Marlnclc, L.; Soeldner, J. S.; Colton, C. K.; Qlner, J.; Morris, S. J . Electrochem. Soc. 1979, 726, 43-49. (11) Simon, R. A.; Mallouk, T. E.; Daube, K. A.; Wrighton, M. S. Inorg. Chem. 1985, 24, 3119-3126. (12) Cukman, D.; Vukovlc, M. J . Electroanal. Ct”. Interfaclal Electro&em. 1990. 279, 273-282. (13) Helder, 0. H.; Sasso, S. V.; Huang, K. M.; Yacynych, A. M.; Wieck, H. J. Anal. Chem. 1990, 62, 1106-1110. (14) Wang, J.; U, R.; Lin, M. S. Elecbwenalysis 1989, 7 , 151-154. (15) Gunaslngham, H.; Tan, C. B. Electroanalysis 1989, 7 , 223-227. (16) Harrison. D. J.; Turner, R. F. B.; Baltes, H. P. Anal. Chem. 1989, 60. 2002-2007. (17) Schick, K. 0.; Magearu, V. G.; Huber, C. 0. Clln. Chem. 1978, 2 4 / 3 , 448-450.
RECEIVED for review August 19,1991. Accepted November 6,1991.
Electrochemical Measurements in Submicroliter Volumes Walter J. Bowyer,* Mary Elizabeth Clark, and Jennifer L. Ingram Department of Chemistry, Hobart and William Smith Colleges, Geneva, New York 14456 INTRODUCTION The application of microelectrodes to voltammetry has been reviewed recently.lz The many advantages of using very small working electrodes include faster voltammetry (e.g. see refs 3-6), voltammetry in resistive media,’’* and higher flux.g In vivo measurements are also greatly facilitated by microelectrodes.1° However, small electrodes yield relatively small currents. Band and cylindrical electrodes, which have one small dimension and one large dimension, are used widely as a compromise between macro- and microelectrode^.^^-^^ Multipleband electrodes are used for a variety of interesting experiincluding generator-collector voltammetry similar to that carried out with rotating ring-disk electrodes.1g-22 There has been and continues to be considerable interest in electrochemical analysis of very small volumes of solut i ~ n . ~ The ~ - ” advent of microelectrodes has greatly facilitated this effort. Small electrodes can be inserted into small volumes, and microelectrodes are useful for analyses with high spatial resolution10r2”28including analysis of total solution volumes of less than 30 pL.18,21+33 Also, the current at working microelectrodes is small, so a two-electrode configuration often can be used without significant polarization of the reference/auxiliary e l e ~ t r o d e . ~This ~ * ~is~sometimes >~~ useful for minimizing the volume of solution required for analysis. Baranski and c o - w o r k e r ~have ~ ~ ~been ~ very active in the development of trace stripping analysis in 5- (two-electrode) and 10-pL (three-electrode voltammetry) total solution volume. In their design, the droplet of solution to be analyzed sits on a microdisk working electrode. The other electrode(s) is/(are) inserted into the droplet. Deoxygenation of the droplet is rapidly accomplished by mounting the electrodes in a cell with an argon stream.
* To whom correspondence should be addressed. 0003-2700/92/0364-0459$03.00/0
An alternate design is to contain 10 pL of solution in a stainless steel spinge needle which acts as the reference and auxiliary electrode.32 A graphite fiber inserted into the solution is the working electrode. Of most relevance to our work is a description of a threeelectrode microcell by Morita, Longmire, and Murray.18 Three gold electrodes are applied to a silicon wafer using a lithographic process. One electrode is coated with silver epoxy to form a silver reference electrode. The working electrode is a band (width 11pm) or a square (11pm). A 2-pL droplet contacts the three electrodes, allowing cyclic voltammetry and chronoamperometry. The advantage of this approach is that the three electrodes are fabricated in close proximity to each other and do not need t o be manipulated. Thus, three-electrode voltammetry is possible in a very small volume. Another device containing three electrodes has been constructed by Wang, Creasy, and S h a ~ . ~The * electrodes are sealed in epoxy, and the device may have applications to in vivo studies as well as to the construction of electrochemical sensors. In this paper, we describe a method for performing threeelectrode voltammetry in total solution volumes as small as 0.05 pL. Three parallel band electrodes are embedded in a Tefzel matrix in close proximity to each other. The drop of solution to be analyzed is placed on top of the three electrodes. These microcells are easily fabricated using standard laboratory equipment, and the surfaces can be renewed indefinitely by polishing. Aqueous and nonaqueous solutions can be analyzed.
EXPERIMENTAL SECTION Microcell Construction. The microcell is constructed by including three strips of metal foil in a multidecker sandwich of heat-sealing Tefzel film. After heat sealing and cooling, the assembly is cut to expose the cross section, yielding three band electrodes. Electrical contact is made to the bands at the other side of the foil strip where it extends from the sandwich. 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992 7
Table I. Diameters of Drops of Aqueous 1.0 M KCl on Electrodes, Diameter Calculated for Perfect Hemisphere, and Fraction of Working Electrode Covered vol, pL
1.0 0.5
0.2 0.1 0.05
foil Flgure 1. Diagram of multidecker sandwich after heat sealing and
before cutting. Each electrode is prepared by cutting a strip of metal from foil (Aesar, Ward Hill, MA) approximately 1mm x 25 mm. (For the microcell most used in this study, the reference electrode is from 100-pm silver foil; auxiliary electrode is from 100-pm platinum foil; the working electrode is from 4-pm platinum foil.) Glass microscope slides and Tefzel500 LZ heat sealing film (American Durafilm, Newton Lower Falls, MA, 125 pm thick) are cut into rectangles 12 mm X 20 mm. A multidecker sandwich is built up with one glass slide, four layers of Tefzel, the strip of silver foil, two layers of Tefzel, the strip of 4 pm thick platinum foil, two layers of Tefzel, the strip of 100-pm platinum foil, four layers of Tefzel, and a glass slide. The strips are longer than the glass or Tefzel and extend about 3 mm from each side of the sandwich. The strips are carefully aligned over each other and clamped at the ends. Then the assembly is clamped lightly and heated at 300 "C for 9 min. Figure 1 depicts the assembly after the heat sealing. After cooling, the heat-sealed assembly is cut into two rectangular wafers 10 mm X 12 mm (illustrated by the dotted line in Figure 1) with a glass-cutting saw. This exposes three band electrodes on each wafer. The glass slides are removed (we have recently found that placing 0.5 mL of 5% dichlorodimethylsilane in carbon tetrachloride on one side of each slide and allowing it to evaporate before construction of the sandwich makes removal of the slides much easier). The face of each microcell is polished with increasingly fiie grades of polish to 0.05-pm alumina (Buehler, Lake Bluff, a). Using silver epoxy, one colored lead is attached to each electrode where the foil extends out of the Tefzel matrix. After the silver epoxy is hard, the junction is covered with a thin layer of insulating epoxy (Devcon, Danvers, MA) for rigidity. The leads are threaded through a short length of 6-mm glass tubing, and the wafer is attached to the glass with the Devcon epoxy. The electrode surface is polished with 0.05-pm alumina for 5 min before each experiment. This method appears reliable and general. A total of eight assemblieshave been built by this technique using various working electrode materials and geometries, including gold foil, platinum wire, and silver wire. Of those attempted, only two were unsuccessful-in one case the foil was inadvertently torn after heat sealing so that electrical connection could not be made. In the second case, two electrodes were shorted during the heatsealing step. Microscopic measurements were made with a Bausch and Lomb SV-1070 SteroZoom microscope equipped with a micrometer eyepiece. Differential pulse, normal pulse, and slow cyclic voltammetry were performed with a PARC 174A Polarographic Analyzer. Differential pulse voltammograms were recorded with a time between pulses of 0.5 s, a scan rate of 0.01 V/s, and an amplitude modulation of 50 mV. High-speed cyclic voltammetry was performed with a PARC 175 Universal Programmer, a Nicolet 310 digital oscilloscope with dual disk drive, and a homemade potentiostat with a minimum time constant of 2 ms. All voltammetry was performed with a standard three-electrode configuration. Experiments in 25-mL solution volume were performed in a 50-mL three-necked round-bottom flask. For
diameter measd, mm
diameter calcd, mm
fraction of electrode covered
1.60 1.29 0.96 0.76
1.56 1.24
1.0 1.0
0.91 0.73 0.58
0.87 0.69 0.48
0.53
comparison, some experiments were performed with a silver wire (0.5 mm X 150 mm) reference electrode, a platinum wire (0.5 mm X 150 mm) auxiliary electrode, and the 4-pm platinum band in the microcell. Otherwise, voltammetry was performed with the three band electrodes. Voltammetry in volumes of less than 4 pL was performed by inverting the electrode and placing the drop of solution on the three bands. When nitrogen purging was necessary or when evaporation was significant, a cell similar to that described by B a r a n ~ k iwas ~ ~ used . ~ ~ to hold the electrode and droplet in a nitrogen stream saturated with solvent. When water was the solvent, evaporation from 1 pL was significant after about 60 s (peak currents increased by more than 5%) if the electrode was not in a nitrogen stream saturated with water. NJV-Dimethylformamide is less volatile, and evaporation from 1 pL was significant after 4 min. Drops were dispensed with Hamilton microsyringes. Drops of 1pL or more could be placed on the electrodes reliably without magnification. For drops of 0.5 pL or less, 40X magnification was used for optimum placement. Unless noted, a fresh drop was used for each voltammogram. Capacitance of the bands was measured as described previ0us1y'~by cyclic voltammetry between 1.0 and 1000 V/s. Measurements were made at -0.5 V vs an internal standard of ((dimethylamino)methyl)ferrocene in 1.0 M aqueous potaasium nitrate or 1.0 M potassium chloride. Potassium ferricyanide, ((dimethylamino)methyl)ferrocene, hydroquinone, p-nitrotoluene, anthraquinone, and ferrocene were used as standards and were purchased from either Fisher or Aldrich. NJV-Dimethylformamide (DMF, Aldrich, HPLC grade) was passed over a small column (20 mm X 100 mm) of activated alumina immediately before use. Tetrabutylammonium hexafluorophosphate (TBAHFP, Aldrich) was recrystallized three times from 95% ethanol and dried for 24 h at 100 "C.
RESULTS AND DISCUSSION Microscopic Examination. The individual electrodes were measured using a micrometer eyepiece at 70X. The reference electrode (silver band) and auxiliary electrode (platinum band) are 0.11 mm X 0.93 mm. The working electrode is 1.1mm long, and its width is nominally 4 pm. Thus, the area of the counter electrode is approximately 25 times that of the working electrode. Average separation between the reference and working electrodes is 0.11 mm and between the auxiliary and working electrodes is 0.15 mm. Because of the hydrophobicity of the Tefzel, water droplets are approximately hemispherical when sitting on the electrodes. See Table I for calculated diameters of hemispheres (volume = 2rr3/3) compared to the diameters of the water droplets measured on the electrode surface. As can be seen in Table I, water droplets are large enough to cover the 1.1-mm band working electrode completely if the volume is 0.5 p L or greater. Since DMF has a lower surface tension (and probably a higher attraction for the Tefzel surface than does water), it spreads out over the surface more than water. Thus, 0.2 pL of DMF is sufficient to cover the working electrode fully. For either solvent, 0.05 p L is just sufficient to contact the edge of the reference and auxiliary electrodes and almost half of the working electrode. Capacitance. The capacitance of the working electrode was measured between 1.0 and 1000 V/s in 25 mL and 1.0,
ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
Table 11. Average Capacitance of Platinum Band Working Electrode in Different Volumes Relative to That Measured in 1 p L
vol, p L 1.0
0.5 0.2
av relative capacitance
vol, pL
av relative capacitance
1.00 0.98 0.86
0.1 0.05
0.69 0.52
~
Table 111. Dependence on Solution Volume of Peak Currents of Differential Pulse Voltammograms and Cyclic Voltammograms ( v = 100 V/s) of Ferrocene vol
i, (dpv), nA
% rsda
i, (cv), pA
2.0 mL 1.0 pL 0.5 pL 0.2 pL 0.1 pL 0.05 p L
720 720 690 660 570
1.2 2.6 5.2 6.5
1.05 1.01 1.04 0.89 0.58
Eight trials. 0.5,0.2,0.1, and 0.05 pL of 1.0 M aqueous KC1. The specific capacitance of the working electorde is approximately independent of volume down to 0.5 pL (see Table 11). Below 0.5 pL, the capacitance of the working electrode is approximately proportional to the length which is exposed to the solution (Tables I and 11). Capacitance of the working electrode in 25 mL of solution is independent of whether the band reference and auxiliary electrodes were used or external reference and auxiliary macroelectrodes were used. To test for capacitance due to the relatively large area of coplanar foil strips separated by Tefzel, we recorded voltammograms when the electrode was not immersed in any solution (the leads to the reference and auxiliary electrodes must be connected to each other). In this configuration, there is no significant capacitance (less than 0.01 pF at lo00 V/s). Differential Pulse Voltammetry. Differential pulse voltammograms of the oxidation of ferrocene in DMF/O.l M TBAHFP were recorded with a scan rate of 0.01 V/s and a time between pulses of 0.5 s. Peak shape and peak width at half-height (100-120 mV) are independent of volume. Even with only 0.05 pL no unusual behavior was observed. Peak currents recorded for 1.0 mM ferrocene in 1-pL drops are identical within experimental error to those recorded in larger volumes (see Table 111). However, the peak current recorded in 0.2 pL is about 10% less than the peak current measured in larger volumes. The decrease is surprising since the working electrode is completely covered by 0.2 pL. However, the drop is so thin (0.23 mm) that flux to the electrode surface is decreased on the time scale of the scan.35 At volumes below 0.2 pL, the differential pulse peak current decreases both because the drop is thinner (e.g. 0.090 mm at 0.05 pL) and, more importantly, because the electrode is not completely covered. In 1pL, the reproducibility of the peak current is very good with a relative standard deviation (rsd) of 1.2% for 1.0 mM ferrocene (Table 111). In lower volumes, reproducibility decreases slightly. With 0.05-pL drops, the rsd of the peak current is 6.5%. The lower reproducibility in volumes which do not fully cover the working electrode probably arises from variable effective working electrode length depending on drop placement. A calibration curve for ferrocene in DMF/O.l M TBAHFP was prepared from voltammograms recorded in 1-pL drops. The peak current by differential pulse voltammetry was plotted w the concentration of ferrocene between 5.0 pM and
461
66 mM. Three voltammograms were recorded at each concentration. Between 5.0 and 210 pM the plot is linear (correlation coefficient = 0.9998 with 17 points), and the sensitivity is 0.63 pAfmM. Sensitivity and background currents are the same in 1pL or 2 mL, and we estimate a limit of detection of 2.0 pM in both volumes. Above 1.0 mM, the function of peak current vs concentration is curved downward, and peak width decreases. We attribute this behavior to polarization of the quasireference electrode by diffusion of the ferrocenium from the working electrode to the silver band, shifting the reference point positive. We will describe these results in detail in a future publication. The silver band electrode is only a quasireference electrode, but peak potential reproducibility is good. For example, the Epof 1.0 mM ferri/ferrocyanide in 1.0 M KC1 had a standard deviation of only 7 mV over several hours. The Epof ((dimethy1amino)methyl)ferrocene in 1.0 M KC1 varied by f 1 3 mV. Mild oxidation of the silver band in the presence of chloride to form an Ag/AgCl quasireference electrode did not greatly improve potential stability. In a typical experiment in nonaqueous solvents, the peak potential of the oxidation of ferrocene shifted about 50 mV over the course of 20 scans. The worst shift observed was by 100 mV between scans. During 6 months of experimentation, the peak potential for the oxidation of ferrocene on a newly polished electrode ranged from Ep = +0.37 to +0.64 V. The range of these values is typical for quasireference electrode^^^*^^ and is not a severe limitation in the application of these devices. Cyclic Voltammetry. We recorded cyclic voltammograms of ferrocene in DMFfO.1 M TBAHFP in volumes between 0.05 and 1pL and at scan rates between 0.1 and 500 V/s. At higher scan rates (above 50 V/s), forward and reverse peaks are observed. Peak separations of 1.0 mM ferrocene range from 78 to 140 mV in DMFfO.1 M TBAHFP between 50 and lo00 Vfs. Peak shape and peak separation are independent of volume even down to 0.05 pL. As can be seen in Table 111, a t 100 V/s peak currents are independent of drop volume down to 0.2 NL. Presumably, the peak current measured by high-speed cyclic voltammetry is not diminished in 0.2 pL because the diffusion layer is very small compared to the thickness of the drop. In 0.1 and 0.05 pL, peak currents are less (Table 111) because below 0.2 pL only a fraction of the working electrode is covered by solution. Because of the proximity of the electrodes, we expect that these microcells will be useful for electrochemistry in resistive solutions. Although contamination, particularly with trace water, is probably severe for such small volumes, we recorded voltammograms in DMF with no added electrolyte. At slow scan rates (0.1 V/s), quasi-steady-state currents are independent of whether electrolyte is added. With 1.0 mM ferrocene, the plateau current was 130 nA ( N = 11,ad = 11 nA) with no added electrolyte and 125 nA (N = 6, sd = 7 nA) with 0.1 M TBAHFP. The slope of a plot of E vs log (i/id - i) is 82 mV with no added electrolyte and 57 mV with 0.1 M TBAHFP. To confirm that depletion of the ferrocene in the drop is not a problem with slow-scan experiments, we calculate that to electrolyze 1.0 pL of 1.0 mM ferrocene completely requires approximately 0.1 mC. With a quasi-steady-state current of 130 nA being passed for 10 s, 1.3 pC are consumed. Thus, during a slow scan only about 1% of the analyte is oxidized. To investigate electrochemistry at reducing potentials, we studied the reductions of p-nitrotoluene and anthraquinone by cyclic voltammetry. Approximately 1 min after placing the drop in the nitrogen stream, no voltammetry attributable to oxygen contamination was seen. The f i t reduction of both p-nitrotoluene and anthraquinone is reversible at slow-scan
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 4, FEBRUARY 15, 1992
rates. At higher speeds, cyclic voltammograms are very similar to those for ferrocene. For example, the cyclic voltammogram of the reduction of anthraquinone to ita anion radical appears chemically reversible and has a peak separation of 150 mV at 100 V f s.
CONCLUSIONS Using three band electrodes sealed in a Tefzel matrix, we demonstrate that electrochemistry in volumes as low as 0.05 p L is possible. Measurements are largely independent of volume down to 0.2 pL. Between 0.05 and 0.2 p L currents are smaller than those recorded in larger volumes and are proportional to the fraction of the electrode covered. Precision is less good when the working electrode is not fully covered (volumes less than 0.2 p L with nonaqueous solvents), but is still acceptable. Voltammogram shape is independent of volume even at 0.05 ILL.To our knowledge, this volume is significantly less than any previously reported. Construction of the microcells is simple and requires no specialized equipment. They may be polished and are reusable indefinitely. The proximity of the electrodes makes this design useful for voltammetry in resistive media. Steady-state voltammograms suffer minimal ohmic distortion even at millimolar analyte concentrations. Thus, we plan to develop applications to analysis in flowing streams.
ACKNOWLEDGMENT This research was supported by a grant from Research Corporation and by Hobart and William Smith Colleges. Registry No. TBAHFP, 3109-63-5; Pt, 7440-06-4; KCl, 7447-40-7; ferrocene, 102-54-5;tefzel 500 LZ, 25038-71-5.
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RECEIVED for review July 30, 1991. Accepted November 19, 1991.
