Anal. Chem. 1990,62,1631-1636
gene engineering to tailor systems for a specific use in the construction of biosensors. Registry No. EC 1.1.3.4, 9001-37-0; EC 3.2.1.23, 9031-11-2; glutardialdehyde, 111-30-8; lactose, 63-42-3; glucose, 50-99-7; nitrovin, 804-36-4.
LITERATURE CITED (1) Macholn, L. Cdl. Czech. Chem. Commun. 1978, 43, 1811-1817. (2) Matsurnoto, K.; Yarnada, K.; Osajima, Y. Anal. Chem. 1981, 53, 1974-1979. (3) Toul, 2.; Macholln, L. Coll. Czech. Chem. Commun. 1975, 4 0 , 2208-2217. (4) Satoh. 1.; Karube, I.; Suzuki, S. Biotechnd. Bioeng. 1977, 19, 1095-1099. ( 5 ) Nab1 Rahnl. M. A.; Gullbault, G. G. And. Chem. 1986, 58, 523-526. (6) Watanabe, E.;Endo, H.; Hayashl, T.; Toyama, K. Blosensors 1988, 2 , 235-244. (7) Karube, I.; Nakahara, T.; Matsunaga, T.; Suzuki, S. Anal. Chem. 1982, 5 4 , 1725-1727. (8) Karube, I.; Okada, T.; Suzuki. S.;Suzuki, H.; Hikuma, M.; Yasuda, T. Appl. MicroM. Biotechnol. 1982, 15, 127-132. (9) Schubert, F.; Renneberg, R.; Scheller, F. W.; Klrstein, L. Anal. Chem. 1984, 56, 1677-1682. (IO) Machogn. L. Acta Biotechnoi. 1987, 7 , 547-553. (11) Mattiasson, B.; Nilsson, H. FfBS Lett. 1977, 78, 251.
1031
(12) Cordonnler, M.; Lawny, F.; Chapot, D.; Thomas, D. FfBS Lett. 1975. 59,263-267. (13) Pilloton, R.; Mascini, M.; Casella, I. G.; Festa, M. R.; Bottari, E. Anal. Lett. 1987, 2 0 , 1803-1814. (14) Quillardet. Ph.; Huisman, 0.; D’Ari, R.; Hofnung, M. Roc. Net/. Acad. Sci. U . S . A . 1982, 5971-5975. (15) Quillardet, Ph.; Hofnung, M. M a t . Res. 1985, 147, 65-68. (16) Qulllardet, Ph.; de Bellecombe, Ch.; Hofnung. M. M a t . Res. 1985. 147, 79-95.(17) MiirtuS, S.;Svorc, J.; Sturdk, E.; VojtekovB, H. Anal. Chem. 1987, 59, 504-508. (18)Miller, J. H. Experiments in Molecular Gedetix; Cold Springs Harbor Laboratory: Cold Spring Harbor, NY, 1972. (19) Bernfekl, P. Methods fnzymol. 1955, 1 , 149-158. (20) Bailey, M. J. Appl. Microblol. Biotechnol. 1988, 29, 494-496. (21) Toshihiro, 0.; Naoko, N.; Masaaki, M.; Yasuhlko, S.; Tsuneo, K. m t . Res. 1984, 131, 101-109. (22) Dayan, J.; Deguingand, S.;Truzman, C.; Chevron, M. Mutat. Res. 1987, 55-66. (23) Vojtekovl, H.; MiertuS, S. Chem. Listy 1988, 82. 501-524. (24) Walker, G. C. Microbial. Rev. 1984, 48, 60-93. (25) Mell, L. D.; Maloy, J. T. Ana/. Chem. 1975, 47, 299-307. (26) Mell. L. D.;Maloy, J. T. Anal. Chem. 1978, 48. 1597-1601. (27) Svorc, J.; MiertuS, S. Unpublished results, STU Bratislava. 1989.
RECE~VED for review October 27,1989. Accepted April 2,1990.
Microring Electrode/Optical Waveguide: Electrochemical Characterization and Application to Electrogenerated Chemiluminescence Lance S. Kuhn, Anders Weber,’ and Stephen G. Weber* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
A novel optoelectrochemicalmlcroprobe has been developed that allows the combinatlon of optlcai and electrochemlcal methods In a very m a l l system. The key element Is a goldcoated optlcal flber pollshed to a flat surface, such that the gold forms a microrlng electrode surroundlng the optlcal fiber. The electlochemistry of the mlcroring has been characterized; cyclk voltammetrlc and chronoamperometrlcIlmHlng currents agree well wHh publlshed theoretlcal treatments. The probe has also been used for electrogenerated chemiluminescence wHh trls(2,2’4)ypyrMlne)ruthenium( 11) wHh persunate in 5050 MeCNH,O. Detectlon lhHs of 4.3 X lod M Ru(bpy);+ have been obtained.
INTRODUCTION The development of microelectrodes and optical fibers has contributed strongly to the growing movement in analytical chemistry toward microscale analysis. This trend is fueled by the need for remote sensing and for detection of trace amounts of analyte in small volumes in on-line and in situ or in vivo applications. Microelectrodes have spurred great interest in electrochemical detection due to the low iR drop and increased current density afforded by their micrometer dimensions. Mass transport properties of electrodes of various shapes, including microdisk ( I ) , ring (2-5), spherical (6),hemispherical and conical (7), and band electrodes (8, 9), have been del Current address: Department of Meat Technology and Process Engineering, The Royal Veterinary and Agricultural University,
Copenhagen, Denmark.
0003-2700/90/0362-1631$02.50/0
scribed, and several reviews published (8-11). Their use in a wide variety of detection schemes has been demonstrated (12-1 4). Optical fibers are becoming increasingly popular as couriers of information in analytical detection systems. They have been used in fluorescence detection or excitation in a variety of configurations (15-18) and for remote acquisition of spectroelectrochemical data (19). They have also been used in a variety of sensors, including pH sensors (20), luminescence-based biosensors (21, 221, and affinity/fluorescence sensors (23). Recently, they have been used in conjunction with electrodes to develop very small optoelectrochemical sensors (24). The ability to combine intimately optical and electrochemical methods offers many opportunities to develop sensitive and selective analytical detection methods. Aizawa (24) and others (15,16,20-22) have recently described improvements in sensing systems based on the use of optical fibers and microelectrodes. Much of this work has been done with electrogenerated chemiluminescence (ECL). Blum et al. have used ECL for biochemical sensing using enzyme-based systems and luminol as the light-generating species (21). Van Dyke and Cheng (15, 16) have developed combination optical fiber/electrode devices for use with spectroelectrochemistryand ECL. One device, well suited to the measurement of electrochemically modulated optical changes, consists of a pair of optical fibers embedded in a graphite/epoxy composite electrode (16). The other, well suited to the determination of ECL, consists of a fiber bundle inside of a cylindrical Pt mesh or Pt tube electrode (15). ECL has been shown by other investigators to be a very sensitive technique. Ege et al. (25) showed nanomolar or better detection limits of Ru(bpy)32+ using both reductive oxidation and oxidative reduction, and 0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62. NO. 15, AUGUST 1, 1990
Noffsinger and Danielson (26)were able to detect amines at micromolar to nanomolar levels in the presence of aqueous millimolar Ru(hpy),2+. In this paper we describe a prohe that is a combination of optical fiber and microelectrode. The key element is a goldcoated optical fiber, polished to a flat surface, such that the gold forms a microring electrode immediately adjacent to the optical fiber. The advantages are obvious: the optical fiber allows remote sensing, the microelectrode gives enhanced sensitivity and selectivity, and the combination allows sampling of very small volumes and eliminates the need for alignment of excitation and detection systems. The probe described here will serve two main purposes. First, it will make possible a more detailed view of the cooperative interactions between light and electrochemistry in an analytical detection system. Second, it will be useful in at least two different analytical schemes. This paper will describe the general electrochemical behavior of the prohe, as well as that in a system using electrogenerated chemiluminescence. A following paper will describe the probe’s behavior in a photoelectrochemical system.
EXPERIMENTAL SECTION Apparatus. The working electrode in all cases was the gold microring, built as described in the construction subsection. The auxiliary electrode was either a platinum flag electrode or the built-in silver ring of the probe. AU potentials reported are versus a Ag/AgC1(3 M NaC1) reference electrode. Cyclic voltammetry was done with a CV-1B potentiostat (Bioandytical Systems, Inc.) and recorded on a Houston Instruments Model 200 XY recorder. Chronoamperometry was done using a potentiostat built in-house and run with an IBM PC using ASYST scientific software. A Keithley Instruments Model 427 current amplifier was used to collect the signal, which was then plotted and stored with the PC. Electrogenerated chemiluminescence (ECL) was done using the same PC and software. The in-house-built potentiostat was used to apply potentials, with an ITT FW-130 photomultiplier tube (at ambient temperature) collecting light at the distal end of the optical fiber. The signal from the PMT was taken through an Ortec Model 109PC preamplifier to a Hamner NA-11 RC amplifier and then to a Hamner NC-11 analyzer. The signal was then recorded by using a Hamner N-780A LogLin ratemeter and a Heath Model SR-206 strip-chart recorder. A box was designed and built in-house to allow electrode leads and the optical fiber to reach the ECL cell with a minimum of stray light getting to the cell. It was made of aluminum and therefore also served as a shield for the electrochemical experiments. To maintain constant geometry between the working and auxiliary electrodes, a nylon holder was made that held the Pt flag electrode rigid and that allowed the optical fiber connector to screw in and out. Reagents. Ferrocene (98%,Aldrich),potassium chloride (GR, EM Science), and hexaammineruthenium(II1) chloride (99%, Aldrich) were used as obtained for electrochemistry. Acetonitrile (Ominsolve,EM Science) was dried over activated alumina before use. Sodium perchlorate (GFS Chemicals) was recrystallized from methanol and dried at 130 “C under vacuum prior to use. Tris(2,2’-bipyridine)ruthenium(II)chloride hexahydrate (hereafter Ru(bpy)?? from Strem Chemicals and sodium persulfate (98+%, Aldrich) were used as obtained for ECL. Tetraethylammonium perchlorate (TEAP) was made as described by Cox et al. (27) and purified according to Perriu et al. (28). All water was doubly deionized and distilled. Construction of the Probe. Optical fibers (Anhydroguide G ) were purchased from Fiherguide Industries,Stirling, NJ. These fibers have a core diameter of 200 r m , a 10-pm-thick cladding, and a 15-pm-thick gold jacket. Silver tubing (2.0-mm 0.d. 1.6-mm i.d.) was purchased from GoodfellowMetals, Cambridge, England. Epoxy was Epotek (Billerica, MA) type 320, and shrinkable Teflon tubing was from Small Parts, Iuc., Miami, FL. The optical fiber connectors were Ensign Bickford CC-230-3.0 SMA fiber optic Connectors. A short portion of the optical fiber was cut and the gold jacket removed from all hut 1 cm at one end. A piece of 1 W r m copper
is the gold microring, and at the center of the Au ring is the optical fiber. BoItom: Closeup of the Au ring surrounding the optical fiber. Note the slight protrusion of the optical fiber. The bar in each picture represents about half of the staled distance.
wire was soldered to the remaining gold at a point far enough from the end to he sure that no solder covered the tip. The fiber/wire was potted in epoxy and then inserted into the silver tube. A copper wire was soldered to the silver tube, and the whole system potted in epoxy and inserted into a connector. After overnight drying, the ends of the optical fiber were clipped off and both ends of the probe polished. They were hand polished first on a 17-rm particle abrasive sheet (3M Co.) and then successively with 7-pm silicon carhide (Buehler) and 0.3-rm alumina (Leco) using a Leco VP-50 Polishing Wheel and an in-house-made electrode holder. The electrodes were ultrasonicated in water for 2 min after each step of polishing. The final polished face of the prohe is shown in the SEM photo in Figure 1. Procedure. Chronoamperometry was done using 1.0 mM Ru(NH,),“ in aqueous 0.1 M KCI by stepping the potential from +0.1 to -0.3 V. All solutions were deoxygenated for 15 min prior to a trial, due to the interfering reduction of oxygen, which begins at about -0.2 V. Cyclic voltammetry was done with the same couple over a slightly larger potential range, beginning at +0.7 V and scanning in a negative (reducing) direction, and solutions were deoxygenated before use. Electrogenerated chemiluminescence was done by reductive oxidation of Ru(hpy),2t, using S,O,* as shown hy White and Bard (29). Solutious were 18 mM Na2S20,(shown to be at the optimum for luminescence generation (29)),0.1 M TEAP, and 1 X 10-8-1 X lo4 M Ru(bpy),Cl,. A three-electrode system was used for all trials, consisting of the gold ring as working, Pt flag, or Ag ring
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990 5.000E-7
Table I. Experimental Limiting Currents for the Au MicroringO
4.000E-7
i electrode 2 3 5
Y
C
2 L
1833
2.000E-7
3
0
18 s
32 s
ss/(cv)
29 34 b
27 34 b
46 43 35
"The solution was 1 mM RU(NH~)~*+ in aqueous 0.1 M NaC10,. All currents are in nanoamperes. bElectrode5 was not used in the chronoamperometry experiments.
1.000E-7
0.000 2.00
0.00
4.00
6.00
8.00
Table 11. Theoretical Steady-State Limiting Currents for the Au Microring"
Time (sec)
4E-7
7
3E-7
+
1
I
h
