Anal. Chem. 1989, 61, 159-162 (2) Williams, D.H.; Bradley, C.; Bojesen. 0.; Santikarn, S.; Taylor, L. C. E. J . Am. Chem. SOC.1981, 103, 5700. (3) Burlingame, A. L.; Balllie, T. A.; Derrick, P. J. Anal. Chem. 1986, 58, 165R. (4) Burlingame, A. L.; Russell, D. H.; Maltby, D.;Holland, P. T. Anal. Chem. 1988, 60. 294R. (5) Tomer. K. 6.; Crow, F. W.; Gross, M. L.; Kopple. K. D. Anal. Chem. 1984, 56, 880. (6) Amster, I. J.; McLafferty, F. W. Anal. Chem. 1985, 57, 1208. (7) Cotter, R. J.; Larsen. E. S.; Heller, D.N.; Campana, J. E.; Fenseiau, C. Anal. Chem. 1985, 57, 1479. (6) Biemann. K.; Martin, S. A. Mass Specfrom. Rev. 1987, 6 , 1. (9) Jensen, N. J.; Gross, M. L. Mass Specfrom. Rev. 1987, 7, 1. (10) Long, 0.L.; Winefordner, J. D. Anal. Chem. 1983, 55, 712A. (11) Caprioll, R. M.; Fan, T.; Cottrell, J. S. Anal. Chem. 1986, 58, 2949. (12) Hunt, D. F.; Shabanowitz, J.; Yates 111, J. R.; Zhu, N. 2.; Russell, D. H.; Castro, M. E. Proc. Nafl. Aced. Sci. U . S . A . 1987, 84, 620. (13) Russell. D. H. Mass Specfrom. Rev. 1988, 5 , 167. (14) Aberth, W.; Straub, K. M.; Burlingame, A. L. Anal. Chem. 1982, 5 4 , 2029. (15) Aberth, W. Anal. Chem. 1986, 58, 1221. (16) Olthoff, J. K.; Cotter, R. J. Nucl. Insfrum. Methods Phys. Res ., Sect. 8 1987, 828. 566. (17) Tecklenburg, R. E., Jr.; Russell, D.H. J. Am. Chem. SOC. 1987. 109, 7654.
150
(18) Tecklenburg, R. E., Jr.; Mlller, M. N.; Russell, D. H. J. Am. Chem. Soc., in press. (19) Tecklenburg, R. E., Jr.; Sellers-Hann, L.; Russell, D. H. Inf. J. Mass Specfrom. Ion Proc ., in press. (20) cooks,R. G. Colhkm Specb.oscopy; Cooks. R. G., Ed.; Plenum Press: New York, 1978; pp 405-406. (21) Ashcroft, A. E.; Brown, R. S.; Coles. A. D.;Evans, S.; Milton, D.J.; Wright. B. Spectroscopy 1987. 3 , 57. (22) Ouwerkerk, C. E. D.; Boerboom, A. J. H.; Matsuo. T.; Sakurai, T. I n t . J. Mass Specfrom. Ion Processes, 1986, 70, 79. (23) Zhaoheng, H.; Chen, H.; Boerboom, A. J. H.; Matsuda, H. Int. J. Mess Specfrom. Ion Processes 1988, 71. 29. (24) Tecklenburg, R. E., Ph. D. Thesis, Texas ABM Unlverslty, 1988.
RECEIVED for review May 23,1988. Accepted October 18,1988. This work was supported by grants from the US.Department of Energy, Office of Basic Energy Sciences (DE-AS0582ER13023) and the National Science Foundation (CHE84 18457).
Microhole Array for Oxygen Electrode Ken-ichi Morita*' and Yoshihiro Shimizu Basic Research Laboratories, Toray Ind., Inc., 1111 Tebiro, Kamakura-shi, Kanagawa-ken, 248 J a p a n
Carbon fiber-epoxy composite ultramicroelectrode arrays were etched to depths of several hundred mlcrometers to create an array of microholes of the diameter of the carbon fiber (4-7 pm). The cylindrical wells that form over active microdisk electrodes (platlnlred carbon fiber) reimpose linear diffusion to each mkroelectrode. A solid-state polarographic oxygen sensor was fabricated by using the microhole array. For the assembled arrays the thickness of the diffusion layer for the reduction of dissolved oxygen was fwnd to be the sum of the depth of the mkrohole and the thickness of the solutlon boundary layer. The ultramkrohde/mkroelrode array has the advantages of being moderately flow-rate-lnsensitlve and dlscrlmlnatlve agalnst macromolecular polsons.
Recently, microelectrodes less than 7 pm in radius have become readily available because of progress in carbon fiber technology (1). They exhibit several striking features that have facilitated their use in electrochemical studies (2-7). The concept of a microhole array with the resulting cylindrical well controlling diffusion has been demonstrated (8,9).The recessed electrode has been examined (10-1 3) for potential use as a polarographic oxygen sensor. Recessed ultramicroelectrode arrays are produced by oxidative etching of polished sections of carbon fiber-epoxy composites, followed by plating with platinum. Scanning electron micrographs of the surface of the microhole electrode and a cross-sectioned view of a microhole showing the platinized carbon fiber are shown in Figure 1. The solution well over a microdisk electrode is protected from convection in the bulk fluid and acts as a diffusion layer in the electrochemical process. Compared to a bare electrode, the electrodes are far 'Present address: Tbin University of Yokohama, 1614 Kurogane-cho, Midori-ku, Yokohama, Kanagawa-ken, 227 Japan. 0003-2700/89/0361-0159$01.50/0
less sensitive to changes in thickness of the boundary layer (outer diffusion layer) on the surface of the probe caused by a flow of liquid. It is also important from a practical viewpoint to note that microholes behave as a filter that prevents electrode contamination and pollution. Dissolved oxygen sensors made from microhole electrode arrays showed several advantages over a conventional Clark-type sensor.
EXPERIMENTAL SECTION Fabrication of Microhole Electrodes. High-tensile-strength carbon fibers were used (heat-treated at 1300-1500 "C, the number of fibers ranging from 1000 to 6000 filaments). High modulus carbon fibers (heat-treated over 2000 "C) resisted oxidative etching. A bundle of 1000 filamenta of high-strength carbon fibers (Torayca T-300,l K, diameter 6.93 pm) was pulled through a resin bath of polymeric binder material consisting of 97 parts of epoxy (Chissonox 221, Yuka Shell, Tokyo, unless otherwise stated) to 3 parts of BF3-monoethylamine,and the resin-impregnated fibers were wound onto a wooden spool. The impregnated yarn was then cured in an air-heated dryer at 130 "C for 30 min. A needle-type composite was obtained. Various types of carbon fibers were treated in the same way (Table I). Diameters of the microelectrode arrays were 0.3 mm for 1000 pieces of fiber and 0.8 mm for 6000 pieces of fiber. The electrode was prepared by placing the composite in a polyethylene tube with an inside diameter of 2 mm and drawning an epoxy resin into the tube. After curing, the rod was removed from the tube and cut into sections about 10 cm long. One end of the rod was ground with sandpaper (2000 grit), and an electrical lead was bonded to it with a conductive silver paste (Dotite D-435, Fuzikura Kasei). The other end was polished to a mirror finish by using a 12-pm lapping film followed by a 0.3-pm lapping film using Handy Fiber Polisher, Type OFL-4 (Seiko Instruments & Electronics, Ltd). After being polished, the rod was rinsed with deionized water and carefully wiped. The mirror section was immersed in a 2 mM sulfuric acid solution containing 0.2 M sodium sulfate and anodically etched at a constant current greater than 800 mA/cm2. The depth of etching was nearly proportional to the amount of charge added, and the limit of etching was about 500 pm. The depths of the microholes were measured by an optical microscope or a scanning electron 0 1989 American Chemical Society
180
ANALYTICAL CHEMISTRY. VOL. 61. NO. 2. JANUARY 15, 1989
100
,
.40 -
~.
..
20 0
0 Figure 1. Scanning eleciron micrographs of a microhole elechode: (a) the surface of a microhole electrode: (b) a Cross-sectioned view of a microhole showing the platinized carbon flber. microscope. After standing at 4 . 6 V (M SCE) for 1 h, the fiber was plated with platinum in the usual manner (plating liquid WBS purchased from Tanaka Kikinzoku, Tokyo). Scanning electron micrographs of a cross-sectioned view of the microhole showing the platinized carbon fiber surface revealed that the thickness of the platinum layer on the etched carbon fibers was about 0.5 pm and the layer was uniform. Electrochemical Measurements. The electrochemical experiments were carried out with a Nikko Keisoku Potentiostat (Model N-POT 2501). The electrwhemical cell consisted of an 100-mL glass beaker with three holes in a silicon rubber lid for the three-electrode system. Potentials were controlled relative io a standard calomel reference electrode. A platinum wire served as the auxiliary electrode. All experiments were done in a 0.9% (w/v) aqueous sodium chloride solution thermcstated at 37 "C. The solution was stirred by a magnetic bar covered with Teflon or by a mechanical stirrer. Potential ( 4 . 6 V) was applied at the microhole electrodes, and the current due to oxygen reduction was measured. For measurements of response times a microhole electrode (poised at 4 . 6 V M Ag/AgCI) wound by Ag/AgCI wire was used. The response time (tm) was defined as the time it took to reach 90 percent of the steady-state current between an air-sparged solution and a nitrogen-sparged solution. Flow dependence of the diffusion current was measured by mechanical stirring (bar, 45 mm long) at various rotation speeds. RESULTS AND DISCUSSION Fabrication of Microhole Arrays. Design and synthesis of microhole arrays for electrochemical studies have been reported. Miller and Majda (8)reported the development of rigid, nonpolymeric electrode films consisting of porous aluminum oxide with the radii of the microholes ranging from 0.01 to 0.2 pm and depths ranging from 0.1 to 10 pm. Hepel and Osteryoung (9) constructed a resin microhole array on a silicon substrate by using electron beam lithography. Each individual member of the array was a disk with radius of 0.375 r m and was connected to the bulk solution by a cylindrical channel through a resist (resin) 1pm long. Penner and Martin (14) reported a Nuclepore-coated platinum electrode. Nuclepore membrane is available in pore diameters ranging from 0.01 to 12 pm and depths ranging from 4 to 10 rm. We prepared a carbon fiber-epoxy composite array (number of fibers ranging from 1000 to 6000 filaments). The carbon fibers of the array were then etched to depths of several hundred micrometers to create an array of microholes of the diameter of the carbon fiber (4-7 pm). The limit of the depth of etching, about 500 pm, probably arises from the limit of the distance water reaches in the microhole capillaries by pressure derived from the surface tension of the etched epoxy wall. Analysis of the surface chemistry of the etched wall by electron spectroscopy for chemical analysis (ESCA) revealed that the oxygen/carbon ratio at the surface of the etched wall
20
40
60 80 100
F l o w v e l o c i t y I c m 5' Figure 2. Flow dependence of me current for oxygen reductlon In a 0.9% ( w h ) aqueous sodium chloride soluticm at 37 'C for microhole ek&cdes pepared from sample D (T-300 (exp)carbon Rbers of 1000 filaments)wllh a deplh of 100 (0) and 200 pm ( 0 ) .
_r
40 50
0
10
20 30 i'IpA'
Flgure 3. Depm of me microhole as a function of the reciprocal of me sleady-stale cmem la oxygen reduction m a 0.9% ( w w aqueous sod um cnlude soluticm at 37 "C In me slattonaly state (01 or on the stirred stale (01. The data are faa mlncimle ektrode hom sample D IT-300 (expl Carbon fibers 01 1000 filamenlsl.
was 0.42, whereas that of the epoxy resin was 0.28. Flow Dependence of Diffusion Currents io Microhole Electrodes. The relationship hetween the flow velwity and the diffusion current that flowed when oxygen was reduced was examined by using the platinized microhole electrodes with depths of 100 pm (the recess depth was 15 times the microhole diameter) and 200 pm (30times). The result is shown in Figure 2. The faster the flow velocity (until 30-50 cmls), the larger was the diffusion current. The current resulting from electrochemical reduction of oxygen in a stirred solution at 37 "C was 74 nA for a 100-pm-depth and 40 nA for a 200-pm-depth electrode array for microhole electrodes made from carbon fiber with loo0 filamenta. The ratio of the current between zero and 50 cmls flow velocity was 1.42 and 1.25 for 100- and 200-pm microhole electrodes. respectively. The ratio is dependent on the depth of the microholes. Diffusion Model of Microhole Electrodes. When the electrochemical reaction is diffusion-controlled. the diffusion.limited current, i. is given by
i = nFDCS/I.
(1)
where n is the stoichiometric number of electrons in the electrode reaction. F is the Faraday constant, D is the diffusion coefficient.C is the concentration of the redox species in water, S is the electrde area. and L is the thickness of the diffusion layer. The depth of the microholes was plotted against I / i where i is the steady-state current resulting from electrochemical reduction of dlasolved oxygen in a 0.9% Iwlv) aqueous pndium
ANALYTICAL CHEMISTRY, VOL. 61,
NO. 2, JANUARY 15, 1989
161
Table I. Values of f and I for Various Electrodes
no. of sample types of carbon fibers filaments A
B C
D E
T-300 T-300 T-300 T-300(exp)b T-300(expIb
f, w PA
diameter, pm
total area, lo4 cm2
for lo
for I ,
calcd"
10
11
6.93 6.93 6.93 6.79 4.96
3.77 11.3 23.0 3.62 5.79
8.1 22.7 45.6 7.9 11.4
8.1 22.7 46.5 7.9 11.5
7.8 23.3 47.5 7.5 12.0
61.5 74.6 129 48.4 54.6
9.2 4.1 12.8 6.8 5.3 7.6'
1000 3000 6000 1000 3000
found
1, pm
mol/cms). bT-300(exp) is basically T-300,but prepared in
"Calculated from RFDCS (n = 4,D = 2.5 x 10" cm2/s (X),C = 2.14 X a laboratory. 'Average.
I
I
a
I
I
E
a .
i 0 0
f
Flgure 4. Schematic illustration of the microhole electrode probe and schematic distribution of the concentration (C) as the function of the distance from the electrode surface inside the diffusion layer in the stirred state (e) and in the stationary state (f): (a)carbon fiber, (b) epoxy resin, (d) microhole. chloride solution at 37 "C in the stationary state or in the stirred state (Figure 3). The current, i, was fit to eq 2 or 3, where io is the current in the stationary state, il is the current in the stirred state, lo and l1 are nonzero intercepts of Figure 3 in the stationary state and in the stirred state (above 50 cm/s flow velocity), respectively, and f is the slope of the line in Figure 3.
io = f / ( h+ 10)
sample
h"pm
A
95 190 270 370 90 180 270 360 80 160
B
O h
+ lo + 11
f = nFDCS
(4)
(5)
(6)
where Lo is the thickness of the diffusion layer in the stationary state and L1is the thickness of the diffusion layer in the stirred state. The thickness of the diffusion layer ( L ) of the microhole electrodes in the electrochemical reaction is determined by the depth of the microholes (h) and the thickness of the boundary layer (1) on the surface of the probe as illustrated in Figure 4. The thickness of the boundary layers on the surface of the probe and f investigated in this work are summarized in Table I. Experimental values o f f agreed reasonably well with the values calculated from nFDCS where S is given as the total area of the carbon fiber cross sections. The area of the carbon fiber cross sections corresponds to that of the microhole's cross section, and not to the area of the assembled electrode surface, which is larger than that of the carbon fiber cross sections as shown in Figure 1; this area is effective since the reaction is controlled by the diffusion of oxygen down the microholes.
5
Table 11. Observed Response Times (tw)
C
Lo = h L1 = h
2 3 4 s " ~ /1 0 - 2 c m
Flgure 5. Boundary layer thickness as a function of the square root of the total area of the carbon fiber cross sections: (0)in the stationary state, ( 0 )in the stirred state.
(2)
We have from eq 1-3
1
t,/s
sample
h" pm
tw/s
5
C
320 400 100 200 270 360 105 180 205 310
50.5 73 5 15.5 28 46 8 19.5 23.5 47
18 35 64 4 15.5 32.5 51 4 12.5
D
E
is depth of microhole.
It was found that lo is proportional to the square root of the total carbon fiber cross sections, and consequently to the diameter of the microhole region (Figure 5 and Table I), while l1 is almost constant (average of 7.6 hm). The observation that the boundary layer thickness (Z1) in the stirred state is nearly constant when the total area of the carbon fiber cross sections is changed is due to the fact that the thickness l1 is determined by the shape of the probe (electrode assembly), and the diameter of the probe is 2 mm in all cases. As expected, the boundary layer thickness in the stationary state (lo)becomes smaller when the total area of the carbon fiber cross sections becomes smaller. Response Times in Microhole Electrodes. The observed response times (tw) of the steady-state current between an air-saturated solution and a nitrogen-sparged solution are tabulated in Table 11. It was found that the observed response time (tw) is proportional to the square of the thickness of the diffusion layer ( L )(Figure 6) and is given by tgO= T(h
+ 1 J 2 + 1.8
(7)
162
ANALYTICAL CHEMISTRY, VOL. 61, NO. 2, JANUARY 15, 1989
0
10
20
i 2 /1O'prn' Flgure 6. Response time ( t W ) as a function of the square of the thickness of the diffusion layer. The data are for the microhole electrode from sample D (T-300(exp) carbon fibers of 1000 filaments).
Table 111. Values of T a n d t ofor Various Electrodes
sample
epoxy resin
T,O lo4 s/rm2
to: s
A B C D E
Chissonox 221 Chissonox 221 Chissonox 221 Toray in-house Chissonox 221
4.4 4.3 4.2 3.4 4.7
1.4 0.5 2.5 1.4 3.1
ntw =
T(h + 1 J 2
+ t,.
where tW is in seconds, h and l1 are in micrometers, and T i s constant. The values of the constants in eq 7 are summarized in Table 111. The value of T seems to depend on the nature of the etched wall and the diameter of the microhole. The value of T for the microhole electrode made of the Toray in house epoxy resin (sample D)is smaller than that of Chissonox epoxy resin. Chissonox epoxy resin is more hydrophobic than the Toray in-house epoxy resin. The microhole electrode with a diameter of 4.96 pm (sample E) has a slightly larger value of T than that of 6.93 pm (samples A, B, and C). Filter Effect of Microholes. A section of an epoxy composite with 1000 carbon fibers was polished and platinized without etching. The electrode was placed in an LB culture (an aqueous solution of a mixture of bactotripton, yeast extracts, sodium chloride, glucose, and antibiotics). A potential of 4 . 6 V was applied a t the electrode, and the current resulting from reduction of dissolved oxygen was measured. The current decreased about 20% after stirring for 48 h and continued to decrease. In the case of the platinized microhole electrode (100-pm depth), on the other hand, the current reduction was about 5% after 48 h and became nearly constant afterward. The decrease of the current undoubtedly resulted from the decrease of the electrode area by contamination with certain components of the LB culture. No current reduction was observed when microhole electrodes were covered by a thin membrane of polyaniline prepared by electropolymerization of aniline (18). The oxygen concentration was measured during fermentation of coliform baccilli by using an oxygen sensor fabricated from microhole electrodes. No baccilli were observed in the microholes when examined by scanning electron microscopy. It is apparent that microholes behave as filters. Applications of Microhole Electrodes to Dissolved Oxygen Sensor and Biosensors. Monopolar and bipolar
polarographic type dissolved oxygen sensors were fabricated by utilizing microhole electrodes (16). Two types of membranes, one hydrophobic and one hydrophilic, have been used to fabricate oxygen sensors. Polymers such as Teflon are permselective to oxygen but virtually impermeable to water and solutes. This type of sensor is referred to as a Clark-type sensor or a bipolar sensor. A hydrophilic membrane such as cellulose is permeable to oxygen and electrolytes but partially impermeable to substances that may poison the electrode. This design is commonly known as a monopolar sensor. It has been reported that the electroinactive polyphenol or polyaniline film deposited on a Pt electrode by electropolymerization of phenol or aniline derivatives possesses selective permeability to dissolved redox species. The swollen hydrophilic membrane had high permeability to hydrogen ion, but inhibited the diffusion of Br-, Cr3+, Eu3+, Fe(CN)63-, and iron(3+)-ethylenediaminetetraacetate complex (17). We found that certain thin membranes coated on the platinized microhole electrodes by electropolymerization had high permeability to dissolved oxygen and electrolytes but inhibited redox species such as vitamin C and the various above-mentioned molecules that may poison the electrode, and we fabricated a solid-state dissolved oxygen sensor. An advantage of the solid-state sensor is its small size, which creates opportunities for in vivo measurements and for multiplexing (combining different sensors on the same probe). Other advantages are the capability of measuring dissolved oxygen at high and low pressure easily and of measuring a concentration of dissolved oxygen instead of measuring a partial pressure of oxygen, as the Clark-type sensor does. Biosensors have also been fabricated by using microhole electrodes (18). A detailed account and other applications will be reported elsewhere.
ACKNOWLEDGMENT We thank S. Asakura for experimental assistance. Registry No. Oxygen, 7782-44-7; platinum, 7440-06-4. LITERATURE CITED (1) Morita, K. Carbon Fibers, Theory and ApplicatJons; Kindai Henshusha: Tokyo, 1984; p 192. (2) Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1980, 5 2 , 946-950. (3) Wightman, R. M. Anal. Chem. 1981, 53, 1125A. (4) Caudiil, W. L.; Howeii, J. 0.; Wightman, R. M. Anal. Chem. 1982, 5 4 , 2532. (5) Howell, J. 0.; Wightman, R. M. Anal. Chem. 1984, 56, 524. (6) Chen, J. W.; George, J. J. Electroanal. Cbem. Interfacial €/echochem. 1988, 210, 205. (7) Michael, A. C.; Justice, J. B., Jr. Anal. Chem. 1987, 59, 405. (8) Mlller, C. J.; Majda, M. J . €lectroanal. Chem. InterfacialElectrochem. 1988, 207, 49. (9) Hepei, T.; Osteryoung, J. J. Electrochem. Chem. 1988, 733, 752. 10) Davies, P. W.; Brink, F., Jr. Fed. f r o c . 1942, 1 , 197; I . fatt. foiarograpbic Oxygen Sensors; E. Krieger Publ. Co.: 1982; p 19. 2nd ed.; Interscience: 11) Kotthoff, I.M.; Lingane, J. J. Polarography, . . New York, 1952; pp 25-27. 12) Uehara, S.; Uchid, A.; Kojima, H. Jpn. Kokai Tokkyo Koho, JP 57 195436 1982. 13) Gnaiger, E.: Forster, H. folarcgraphic Oxygen Sensor; Springer-Verlag: Berlin, Heidelberg, New York, 1983; Chapter 1.4. 14) Penner, R. M.; Martin, C. R. J . Electrochem. SOC. 1086, 133. 2206. 15) Baumgarti, H.; Lubbers, B. W. I n folarographlc Oxygen Sensors; Gnaiger, E., Forster. H., Eds.; Springer-Verlag: Berlin, Heidelberg, New York, 1983; p 57. (16) Morita, K. Jpn. Kokai Tokkyo Koho JP 62 123349 1987. (17) Ohnuki, Y.; Matsuda, H.; Osaka, T.; Oyama, N. J . Electroanal. Chem. Interfacial Electrochem. 1983, 158, 55. (18) Morita, K.; Shimizu, Y. PCT Int. Appi. WO 87 06,701, 1987.
RECEIVED for review December 30,1987. Accepted October 1, 1988.
