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Anal. Chem. 1986, 58, 1042-1046
the exchange reaction with the carbonyl sites.
LITERATURE CITED Engstrom, R. C. Anal. Chem. 1982, 5 4 , 2310. Kamau, G. N.; Willis, W. S.; Rusling, J. F. Anal. Chem. 1985, 5 7 , 545. Thornton, D. C.; Corby, K. T.; Spendel, V. A,; Jordan, J.: Robbat, A,, Jr.; Rutstrom, D. J.; Gross, M.; Ritzler, G. Anal. Chem. 1985, 5 7 , 150. Welsshaar, D.; Kuwana, T. Anal. Chem. 1985, 5 7 , 378. Gunasingham, H.; Fleet, B. Analst (London) 1982, 707, 896. Falat, L.; Cheng, H. Y. Anal. Chem. 1982, 5 4 , 2108. Hallum, J. V.; Donshel, H. V. J . Phys. Chem. 1958, 6 2 , 1502. Evans, J. F.; Kuwana, T. Anal. Chem. 1977, 4 9 , 1632. Laser, D.; Ariel, M. J . Electroanal. Chem. 1974, 5 2 , 291. Engstrom, R. C.; Strasser, V. A. Anal. Chem. 1984, 5 6 , 136. Cabaniss, G. E.; Diamantis, A. A,: Murphy, W. R., Jr.; Linton, R. W.; Meyer, T. J. J . Am. Chem. SOC. 1985, 707, 1845. Schreurs, J.; Van den Berg, J.; Wonders, A,; Barendrecht, E . Recl.: J . R . Neth. Chem. SOC. 1984, 703, 251. Hu, I.; Karweik, D. H.; Kuwana, T. J . Nectroanal. Chem. 1985, 788, 59. Noda, T.; Inagaki, M.; Yamada, S. J . Non-Cryst. Sollds 1969, 7 , 285. Noda, T.; Inagaki, M. Bull. Chem. SOC.Jpn. W64, 3 7 , 1534. Mildner, D. F. R.; Carpenter, J. M. J . Non-Cryst. Solids 1982, 4 7 , 391.
(17) Jenkins, G. M.; Kawamura, K. Nature (London) 1971, 2 3 7 , 175. (16) Whittaker, A. G.; Tooper, B. J . Am. Ceram. SOC. 1974, 5 7 , 443. Smith, N. J., Jr.; Tu, K. N. J . Appl. Phys. 1974, 4 5 , (19) Nathan, M. I.: 2370. (20) Rousseaux, F.; Tchoubar, D. Carbon 1977, 75, 63. (21) Wignall, G. D.; Pings, C. J. Carbon 1974, 72,5 1 . (22) Nagaoka, T.; Okazaki, S.J . Phys. Chem. 1985, 8 9 , 2340. (23) Blurton, K. F. Electrochim. Acta 1973, 78,869. (24) Bjelica, L.; Parsons, R.; Reeves, R. M. Croat. Chem. Acta 1980, 5 3 , 211. (25) Mattson. J. S.;Mark, H. B., Jr.; Weber, W. J., Jr. Anal. Chem. 1969, 4 7 , 355. (26) Mattson, J. S . ; Mark, H. B., Jr. J . Colloid Interface Sci. 1969, 3 7 , 131. (27) Nicholson, R. S. Anal. Chem. 1965, 3 7 , 1351. (28) Soffer, A.; Folman, M. J . Necfroanal. Chem. 1972, 3 8 , 25. (29) Koresh, J.; Soffer, A. J . Nectroanal. Chem. 1983, 747, 223. (30) Feldberg, S. W. I n "Electroanalytical Chemistry"; Bard, A. J., Ed.; Marcel Dekker: New York, 1969; p 199. (31) Zak, J.; Kuwana, T. J . Electroanal. Chem. 1983, 750, 645.
RECEIVED for review July 19, 1985. Resubmitted December 10, 1985. Accepted January 13, 1986.
New Enzyme Membrane for Enzyme Electrodes Ruth Tor and Amihay Freeman* Center for Biotechnology, T h e George S. Wise Faculty of Life Sciences, Tel-Auiu Uniuersity, Tel-Auiv 69978, Israel
A new method for the preparatlon of thln, unlform, selfmounted enzyme membranes, dlrectly coatlng the surface of glass pH electrodes, was developed. The enzyme is dissolved in a solutlon contalnlng synthetlc prepolymers. The electrode Is dlpped In the solution, drled, and dralned carefully. The backbone polymer Is then cross-linked under controlled condltlons to generate a thin ( 4 0 - p m ) enzyme membrane. The method was demonstrated and characterized by the determinatlon of acetylcholine by an acetylchollne esterase electrode, urea by urease electrode, and penicillin G by penlcillinase electrode. Linear response In a wide range of substrate concentrations and high storage and operatlonal stablllty were recorded for all the enzymes tested.
The development and use of biosensors and, in particular, enzyme electrodes has gained large attention in recent years (1-4). These devices are based, in principle, on the conjugation of a biocatalyst and an electrochemical sensor; the biocatalyst recognizes the substrate to be determined and specifically converts it into a product that is measured by the adjacent sensor. This combination thus allows for a sensitive and selective determination of the substrates in the presence of complex mixtures. No wonder then that biosensors are found useful, in particular, for the determination of metabolites in body fluids or fermentation broth. The main operational parameters involved in the use of an enzyme electrode are linearity, sensitivity, response time, wash time, and stability. These parameters strongly depend on the immobilization method employed for the construction of the enzyme layer on the surface of the electrochemical sensor (1, 2,4 ) . The construction of an enzyme-containing layer on the sensor is mostly based on the preparation of an enzyme
membrane and its attachment to the sensor surface by means of a physical support-mostly a dialysis membrane held in place by a rubber O-ring (5). The enzyme layer may be simply made of buffered enzyme solution, held in place by the dialysis membrane (6),by physical entrapment of the enzyme within a synthetic gel matrix (acrylamide and bisacrylamide copolymerization (7)), by chemical binding to organic or inorganic support (8),or by cross-linking with an inert protein (such as gelatin treated with glutaraldehyde (9)). In this paper we present a new method for the construction of a self-mounted, uniform enzyme membrane, directly attached to the sensor's surface, without the need for a physical support such as a dialysis membrane or rubber O-ring. The method is an extrapolation of a technique originally developed in our laboratory for the gel entrapment of whole cells. The method was based on the controlled chemical cross-linking of prepolymerized linear chains of polyacrylamide-hydrazide by dialdehydes, such as glyoxal. The resulting gels were found useful for the gel entrapment of microbial and plant cells with very high retention of immobilized activity. Moreover, these gels are highly porous, thus minimizing diffusional limitations (IO). Recently, this technique was adopted for the gel entrapment of enzymes too (11). The resulting gels offered good mechanical and chemical stability, with minor kinetic perturbations. Also, the entrapped enzymes exhibited improved storage and thermal stability, although the enzymes were physically retained (not cross-linked) within this gel system (11). The construction of an enzyme membrane on the surface of the sensor is based on the following three-step procedure: (a) An enzyme solution, in a concentrated buffered aqueous solution of an appropriate prepolymer, is prepared; in parallel, the sensor surface is cleaned and dried. (b) The sensor is dipped in the enzyme-prepolymer solution for a short time and removed; the adsorbed viscous layer is carefully drained
0003-2700/86/0358-1042$01.50/00 1966 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
and the remaining adsorbed film is air dried. (c) The condensed enzyme polymer film is chemically cross-linked by dipping the coated electrode into an ice-cold glyoxal solution. Following appropriate washing to remove nonbound glyoxal, the enzyme electrode is ready for use without the need for any additional physical supports. In the following, this technique is presented in detail and its efficiency demonstrated by the construction and use of three enzyme electrodes, all based on glass pH electrodes, for the determination of acetylcholine, urea, and penicillin G , in buffered solutions as well as in body fluids. EXPERIMENTAL S E C T I O N Enzymes and Substrates. Acetylcholine esterase, EC 3.1.1.7, from electric eel, Catalog No. C-2888; urease, EC 3.5.1.5, from Jack Beans, Catalog No. U-1500; penicillinase (p-lactamase),EC 3.5.2.6, from Bacillus cereus, Catalog No. P-0389; acetylcholine bromide, Catalog No. A-6500; and penicillin G, Catalog No. PEN-K were all purchased from Sigma, St. Louis, MO. Urea, Catalog No. 8487, was purchased from Merck, Darmstadt, FRG. Chemicals. Glyoxal hydrate (trimer), Catalog No. 804192, was purchased from Merck, Darmstadt, FRG. N-(Z-Hydroxyethyl)piperazene-N'-2-ethanesulfonic acid (HEPES), Catalog No. 4839/2, was purchased from Raught, Ilford, England; Lhistidinehydrochloricacid, Catalog No. 24830, was purchased from Serva, Heidelburg, FRG. 3-N-(Morpholino)propanesulfonicacid (MOPS), Catalog No. M9381, was purchased from Sigma, St. Louis, MO. All other chemicals employed were of analytical grade. Prepolymers. (a) Polyacrylamide-hydrazide (PAAH) of @@ 100000, acylhydrazide content of 750 pmol/g, was prepared essentially as previously described (12, 13). (b) Copolymer acrylamide-methacrylamide-hydrazide was synthesized as follows: Into a 1-L round-bottom flask, equipped with a magnetic stirrer and thermostated at 40 "C, 150 mL of water was added. Acrylamide (21 g, 0.30 mol) and methacrylamide (10.8 g, 0.13 mol) were then added. Following the complete dissolution of monomers, 50 mL of 0.092 M N,N,N',N'-tetramethylethylenediamine and 150 mL of 0.0175 M ammonium persulfate were added and the polymerization reaction was allowed to proceed at 40 "C for 1h. The temperature was then adjusted to 50 "C; 400 mL of water was added, followed by 225 mL of hydrazine hydrate (final concentration, 4.75 M). The hydrazinolysis reaction was then allowed to proceed at 50 "C for 3 h. The resulting derivative of the acrylamide-methacrylamide copolymer was precipitated by dropwise addition into ice-cold methanol. The precipitate was separated by filtration, redissolved in 350 mL of water, precipitated again in cold methanol, filtered, and stored overnight, at 4 "C, under methanol. The polymer was further dried on a rotavapor for 2 h and desiccated in vacuo over P205. The dry polymer was stored in a tightly closed vessel at -18 "C. The acylhydrazide content of the polymer thus obtained was 800 pmol/g of dry polymer (determined according to previously described procedure (14)). Pretreatment of Glass Electrodes. New Electrodes. Treatment was according to manufacturers' instructions, followed by thorough washing with distilled water and drying with lens paper. Used Electrodes. The electrode was soaked in 1 N HCl and 1 N NaOH alternately 5 times and then incubated under 0.1 N HC1 for 5 h. The electrode was then thoroughly washed with distilled water and stored overnight in storage buffer, as recommended by the manufacturers. The electrodes were then washed with distilled water and dried with lens paper. Preparation of EnzymePrepolymer Solutions. Copolymer Solutions. A 25% (w/v) solution is made by adding the dry polymer to distilled water and stirring magnetically at room temperature for 2-3 h. Enzyme Solutions. (1) Acetylcholine esterase was made as follows: 6600 EUnits/mL of 0.1 M phosphate, pH 8, containing 0.01% (w/v) gelatin; (2) urease was made as follows: 130 mg of the emyme/mL of 0.01 M phosphate buffer, pH 7; (3) penicillinase was made as follows: 2500 EU/mL of 0.01 M phosphate, pH 7. Enzyme-Copolymer Solutions. These were all freshly prepared
1043
prior to use. Acetylcholine esterase: into 1.63 mL of the 25% (w/v) copolymer solution 100 pL of 2 M phosphate, pH 7.5, was added, followed by 20 pL of 1% gelatin solution, 200 pL of the enzyme stock solution, and 50 pL of distilled water (final copolymer concentration, 20% (w/v); final enzyme concentration, 500 EU/mL). Urease: Into 1.63 mL of the 25% (w/v) copolymer solution 100 WLof 2 M phosphate, pH 7.5, was added, followed by 20 pL of 0.1 M EDTA in water, 50 pL of the enzyme stock solution, and 200 pL of distilled water (final copolymer concentration, 20% (w/v); final enzyme concentration, 2.5-3 mg/mL (12 EU/mL)). Penicillinase: Into 1.5 mL of the 25% (w/v) copolymer solution 100 pL of 2 M phosphate, pH 7, was added, followed by 400 pI, of the enzyme stock solution (final copolymer concentration, 20% (w/v); final enzyme concentration, 500 EU/mL). Construction of the Enzyme Electrode. The combined pH electrode (Radiometer Model GK-2401-C or Metrohm Model E4-147) was cleaned and dried as described above and held fixed in a vertical position. The enzyme-copolymer solution (1-2 mL in a small plastic vial) was raised slowly upward until the electrode bulb was completely soaked in the solution. (Care should be taken to avoid entrapment of air bubbles.) The electrode was then held within the solution for 1min at room temperature. The enzyme copolymer solution was then slowly lowered, leaving the electrode surface covered with an adsorbed layer of the solution. Draining of excess liquids was carried out by rotating the electrode in a vertical position at 60 rpm, with occasional stops and very gentle touch of the drained liquid drop, accumulating at the bottom of the electrode bulb with a clean glass surface. This is repeated until no excess liquid is seen at the bottom electrode. A uniform layer of tightly adsorbed film is thus obtained. The enzyme-containing film is then further dried by rotating the electrode at 60 rpm in a vertical position for 1 h at room temperature. The white opaque film thus obtained is then cross-linked by incubating the electrode in vertical position in cold, nonstirred 1% (w/v) glyoxal solution in 0.1 M phosphate (pH 5-9, according to the enzyme involved) for 1h. The electrode is then transferred into a magnetically stirred buffer medium (exact composition according to the enzyme used) for 10 min at room temperature. The electrode is then stored until used at a fixed vertical position, at 4 "C, in the appropriate buffered medium containing 0.01% sodium azide as preservative. Measurements and Characterization of Enzyme Electrodes. The enzyme electrode was incubated in 4.9 mL of the appropriate buffer in a thermostated 9-mL vessel, equipped with a magnetic stirrer (-400 rpm). The potential base line of the electrode was then recorded with a Radiometer PH M-82 pH meter, connected to a Radiometer REC-80 servograph recorder. Following the establishment of a steady base line, 0.1 mL of substrate solution in the same buffer was added and the potential change was followed until a steady value was obtained. R E S U L T S A N D DISCUSSION Construction of Enzyme Membrane on Electrode Surface. Throughout our experience with polyacrylamide we observed its tendency to adhere strongly to glass surfaces. In view of the successful adaptation of our gel entrapment technique for the immobilization of enzymes (11) we tried the construction of enzyme membranes, according to the guidelines described above. First attempts were carried out with 100 000) as the prepolymer polyacrylamide-hydrazide resulted in a thick layer that cracked throughout the crosslinking stage. We, therefore, tested other derivatives of this prepolymer, prepared by copolymerizing acrylic monomers (up to 30% content monomer basis) with acrylamide, followed by controlled hydrazinolysis. Out of several derivatives tested, the copolymer of methacrylamide (30%) with acrylamide (70%) was found to be most appropriate. Thus, following draining, a thin adsorbed layer was obtained that, upon cross-linking, formed a uniform and transparent membrane of ca. 50 pm (Figure 1). The membrane was stable to changes in the composition of the medium as well as to sheer forces raised by the magnetic stirring. This membrane, however,
(m
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
r pH Electrode
--Enzyme gel layer
Figure 1. Schematic presentation of the enzyme pH electrode. + +
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3
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0
I
+
*
I
+
,
~
8.0.O5M
-t-+-+~++..-t_
+
I
I
I
I
1
1
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0
2
4 6 TIME
8 (mid
1
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Figure 2. Response curves for acetylcholine determlnation by acetylcholine esterase electrode. should not be touched by sharp edges or hit mechanically as these may lead to the formation of cracks that would prevent signal formation. Acetylcholine Esterase Electrode. Acetylcholine esterase requires the presence of salts (20 mM MgC12,0.1 M NaC1) and gelatin (0.01% (w/v)) for the stabilization of this membrane-bound enzyme in its purified form (14))hence they were contained in all working buffers and storage solutions employed in this study. The chemical nature and buffer capacity of the buffer system employed for measurements with such electrodes are critical for obtaining high sensitivity and wide-range linearity. When no buffer is present the measured electrode response is directly proportional to the log of substrate concentration (15). On the other hand, when buffer is present the response is directly proportional to the substrate concentration (16,17). Three buffers were tested for the acetylcholine esterase electrode: phosphate, MOPS, and HEPES within the concentration range of 1-100 mM a t p H 8. At 1 mM buffer concentration the pH of the buffer was changing throughout the measurement; a t 10 mM concentration, linear response, without change in the pH of the buffer, was obtained for MOPS but not for phosphate. At 100 mM concentration low response was recorded. HEPES buffer exhibited the best results: a t 5 mM concentration, pH 8, 25 "C, a linear calibration curve within the concentration range 2 X lO-"l X M was obtained without pH change of the buffer throughout the measurement. The slope recorded for this curve was 29.5 X lo3 mV/M with a correlation coefficient of 0.9997. This buffer was hence selected for routine use. The range of linear response recorded for this electrode was wider than that reported for acetylcholine esterase electrode prepared by the gelatin-glutardialdehyde technique (1-10 X M with 10 mM M without buffer; 1 X 10-l-2 X phosphate (9)). The response curves of the acetylcholine esterase electrode are presented in Figure 2. The data show that short response
6040 -
20 -
0
10
20 TIME
30
40
50
60
(days)
Figure 4. Storage stability of urease electrode stored at 4 O C in 10 mM histidine, 1 mM EDTA, pH 7.6, and 0.01 % NaN, (activity recorded in 10 mM histidine, 1 mM EDTA, pH 7.6, 30 "C). Note that in spite of the decrease of enzyme total activity the linearity of calibration curve is retained (A) 2 days and (B) 57 days. times are obtained: 0.5-1 min for low concentrations and 2-3 min for the high-concentration range. Recorded values wash time for this electrode were similar. The storage stability of the acetylcholine esterase electrode is shown in Figure 3. The data show that during the period of 6 months (!) and 180 measurements there was no change in the response and sensitivity of this enzyme electrode. The longest storage stability period reported in literature for another acetylcholine esterase electrode was 3-4 weeks (9). Urease Electrode. Phosphate, Tris, glycine, imidazole, and histidine buffers, all at 10 mM concentration, pH 7.6, were tested as potential working buffers for the urease electrode. All solutions contained 1mM EDTA. With glycine and imidazole buffers no response could be observed; Tris and phosphate gave nonlinear responses. Histidine, on the other hand, allowed for high sensitivity and linear response through the concentration range: 2 X 10-"3 X lo-* M (pH 7.6,30 O C ) . The slope of this curve was 109.4 X lo3 mV/M with a correlation coefficient of 0.9995. This range is similar to that
ANALYTICAL CHEMISTRY, VOL. 58, NO. 6, MAY 1986
201
10
20
30
40
50
60
70
TIME d a y s
Figure 5. Storage stability of penicillinase electrode stored at 4 "C in 10 mM phosphate, pH 8, and 0.01 % NaN, (actlvity recorded in 10 mM phosphate, pH 8, 25 "C).
reported for other types of urease electrodes (7,18). Response and wash times recorded for this electrode were 3-5 min, similar to those reported for other electrodes (7). The storage stability of the urease electrode is presented in Figure 4. The data show a decline of about 50% of the slope obtained for calibration curves taken by the urease electrode throughout a period of 2 months. Nevertheless, though the slope of the calibration curve declined, linear calibration curves are obtained throughout the whole period, thus allowing for measurements taken within the full initial range of detectable concentrations. Penicillinase Electrode. Phosphate, histidine, and HEPES buffers, at 10 mM concentration, pH 8, were tested as working buffers for this electrode. Phosphate was found to be most adequate allowing for linear calibration curve M (pH 8, within the concentration range 4 X 10-5-1 X 25 "C), with a slope of 14 X lo3 mV/M and a correlation coefficient of 0.9952. This range is wider than those reported for other penicillinase electrodes, mostly within the range 10-2-10-4 M (6, 18, 19). The response and'wash times recorded for this electrode were 2-3 min, similar to other systems (6, 18). The storage stability of the penicillinase electrode is presented in Figure 5. The data show a decline of about 25% of the slope obtained for calibration curves taken by this electrode within a period of 70 days. The calibration curves obtained were linear throughout the whole period, thus allowing for measurements within the full range of substrate concentrations. The stability recorded for this electrode is significantly better than other electrodes (mostly 1-3 weeks (6,18, 19)). Application for Analysis of Body Fluids. The capability of an enzyme electrode, prepared according to our method, to measure substrates in complex mixtures was demonstrated by measuring urea concentrations in urine and serum samples. Three urease electrodes were prepared simultaneously and stored under identical conditions. One served as control: measurements of standard buffered solutions only, thus taking repeated standard calibration curves periodically. The second was used throughout the experiment period for the determination of urea in fresh urine samples, donated by healthy volunteers. Due to the high sensitivity exhibited by the electrode, samples had to be diluted 50X with buffer prior to measurement. Every 3-6 days a calibration curve was recorded by using standard buffered solutions with this electrode. The third electrode was used €or the determination of urea in serum samples without dilution, collected at random from the main analytical laboratory of a large hospital. Every
1045
7-10 days a calibration curve was recorded for the same electrode using standard buffered solutions. Throughout a period of 80 days, 150 measurements were carried out with the first electrode (control),150 measurements (of which 68 were carried out on urine samples) were carried out with the second electrode, and 146 measurements (of which 64 were carried out on serum (undiluted) samples) were carried out with the third electrode. All three exhibited similar pattern of decay in the slope of the recorded calibration curves, resembling the data of Figure 5. No significant difference between the three electrodes could be observed. Values recorded for urea measured in urine samples were all within the range 0.1-0.6 M (normal range). Values recorded for urea measured in serum samples were comparable (within 10%) to the values provided by the hospital laboratory (chemical method). These results indicate that the performance of enzyme electrodes, based on the proposed enzyme membrane, is not affected by complex mixtures such as body fluids present in the medium throughout the assay. Reproducibility. The slope of calibration curves recorded for different enzyme electrodes was within 10% of the values mentioned above (e.g., slopes recorded for six urease electrodes were all within the range 87.6-109 X lo3 mV/M). Reuse and Regeneration. Whenever an enzyme electrode, prepared according to our system, is damaged, cracked, or decayed, the enzyme membrane layer may be wiped off readily from the electrode surface with tissue paper. The pH electrode might then be used as a regular pH electrode or regenerated as described for "used electrodes" in the Experimental Section. It is pertinent to note that the method described may also be applicable for the construction of enzyme membranes on the surface of electrodes made from materials other than glass (e.g., carbon and platinum electrodes, as shown by preliminary experiments).
ACKNOWLEDGMENT We thank I. Granot for carrying out the measurements of urea samples derived from body fluids and D. Hare1 of "Beilinson" Hospital, Petah-Tikva, Israel, for supplying serum samples. Registry No. Acetylcholine, 51-84-3; acetylcholine esterase, 9000-81-1;urea, 57-13-6; urease, 9002-13-5;penicillin G, 61-33-6; penicillinase, 9001-74-5. LITERATURE CITED Carr, P. W.; Bowers, L. D. I n "Chemical Analyses: a Series of Monographs on Analytical Chemistry and its Applications"; Elving, P. J., Winefordner, J. D., Kolthoff, I. M., Eds; Wiiey: New York, 1980; Vol. 56, pp 197-311. Guilbault, G. G. Appl. Biochem. Biotechnol. 1982, 7 ,85-98. Lowe, C. R. Trends Blotechnol. 1984, 2 , 59-65. Bowers, L. D.; Carr, P. W. Adv. Biochem. Eng. 1980, 15, 89-129. Guilbault, G. G. I n "Methods in Enzymology"; Mosbach, K., Ed.; Academic Press: New York, 1976; Vol. 44, pp 579-618. Nillson, H.; Mosbach, K.; Enfors, S. 0.; Molin, N. Blofechnol. Bioeng. 1978, 20, 527-539. Vadgama, P. M.; Alberti, K. G. M. M.; Covington, A. K. Anal. Chim. Acta 1982, 136, 403-406. Guilbault, G. G.; Tarp, M. Anal. Chim. Acta 1974, 73, 355-365. Durand, P.; David, A.; Thomas, D. Biochim. Biophys. Acta 1978, 527, 277-281. Freeman, A. I n "Enzyme Engineering 7"; Laskin, A. I., Tsao, G. T., Wingard, L. B., Jr., Eds.; Annals of the New York Academy of Science: New York, 1984; Vol. 434, pp 419-426. Freeman, A.; Blank, T.; Haimovlck, B. I n "Biochemical Engineering 111"; Venkatasubramanian, K., Constantinides, A., Vieth, W. R., Eds.; Annals of the New York Academy of Science: New York, 1983; Vol. 413, pp 557-559. Freeman, A. I n "Immobilized Enzymes and Cells" Mosbach, K., Ed., in press. Freeman, A,; Aharonowitz, Y. Biotechnol. Bioeng. 1981, 2 3 , 2747-2759. Dudai, Y.; Siiman, I.I n "Methods in Enzymology"; Jakoby, W. B., Wilchek, M., Eds.; Academic Press: New York, 1974; Vol. 34, pp 571-580. Guilbault, G. G. Enz. Microbiol. Techno/. 1980, 2, 258-264.
1046
Anal. Chem. 1986, 58, 1046-1052
(16) Cullen, L. F.; Rusling, J. F.; Schleifer, A.; Papariello, G. J. Anal. Chern. 1974, 4 6 , 1955-1961. (17) Ruzicha, J.; Hansen. E. J.; Ghose, A. K.; Mottola, H. A. Anal. Chern. 1979, 57, 199-203. (18) Nilsson, H.; Akerlund, A. C.; Mosbach, K. Biochim, Siophys, Acta 1973, 320, 529-534.
(19) Papariello. G. J.; Mukherji, A. K.; Shearer, C. M. Anal. Chern. 1973, 4 5 , 790-792.
RECEIVED for review May 20, 1985. Resubmitted September 13, 1985. Accepted November 13, 1985.
Theory and Application of Diffusion-Limited Amperometric Enzyme Electrode Detection in Flow Injection Analysis of Glucose Bo Olsson, Hans Lundback, and Gillis Johansson* Analytical Chemistry, University of Lund, P.O. Box 124, 221 00 Lund, Sweden
Frieder Scheller and Jiirgen Nentwig Akademie der Wissenschaften der DDR, Zentralinstitut fur Molekularbiologie, Bereich Angewandte Enzymologie, DDR-1115 Berlin-Buch, GDR
A mathematical expresslon describing the transient response of a diff usion-limited amperometric enzyme membrane electrode in flow Injection analysls Is derlved. The formula accounts for the injection volume, the dlsperslon In the flow manifold, and for the diffusional characterlstlcs of the enzyme membrane electrode. The maxlmum rate of sample throughput and the peak height sensltlvlty were calculated for a broad range of operatlng conditions, and the design of an optimal system Is discussed. The experimental characterlstlcs of a glucose oxidase membrane electrode In a flow Injection system were investlgated, and the behavior of the system compared well with theory. A system optlmlzed for hlgh sample throughput was operated at 300 sampleslh with a relative standard deviation of 0.5 % and with a carry-over of 1%. The linear range of detectlon was 0.01-100 mM glucose with a 1.5-pL sample volume.
A large number of enzyme membrane electrodes have been developed using various kinds of membranes and sensor arrangements (1, 2). Theoretical work has been aimed a t an understanding of the compounded sensor per se, and several mathematical models with closed-form solutions for the steady-state response have been presented (3-8). The dynamic response has been investigated mainly with numerical simulation methods (9-13), but some explicit solutions have been derived, particularly for potentiometric sensors (14, 15). Membrane diffusion transients have been calculated for time-lag experiments (26) and for potential step amperometry (17). A formula was recently derived for the transient behavior of the concentration profiles in amperometric enzyme membrane electrodes using a reaction-diffusion model (18). Flow injection analysis (FIA) is a dynamic process that produces a well-defined and highly reproducible concentration transient at the detector site. The relation between peak shape and system dimensions has been described theoretically (19-23). The models have been made with the assumption that the detector response is instantaneous, and they are therefore of limited use when the response time is significant (24). There is thus a need for a model that describes the effects of the most important parameters on the overall response of FIA systems containing enzyme membrane elec0003-2700/86/0358-1046$0 1.50/0
trodes. A few experimental results with such systems are already available (25, 26).
EXPERIMENTAL SECTION Amperometric Cell. An amperometric detector for hydrogen peroxide was made from an oxygen electrode (VEB Metra Radebeul, Berlin, GDR) with a 0.5-mm platinum disk indicator electrode. The electrode was covered with a glucose oxidase membrane, and a steel capillary inlet (length 80 mm; i.d. 0.5 mm) was directed toward the membrane in a confined wall-jet arrangement. The distance between the nozzle and the membrane was approximately 1 mm, and the diameter of the exposed membrane surface was 1.4 mm. The outlet was placed concentrically with respect to the inlet nozzle. A potentiostat (PAR Model 174) was used to polarize the indicator electrode at +600 mV vs. the Ag/AgC1/0.1 M KC1 reference electrode. The steel capillary inlet was connected to the ground wire of the potentiostat to reduce the flow-dependent background noise. Enzyme Membrane. The glucose oxidase membrane consisted of two cellulose dialysis membranes (Nephrophan, VEB CKB Bitterfeld, GDR; thickness .02 mm) sandwiched around a polyurethane layer (thickness 0.02 mm) containing the entrapped glucose oxidase (EC 1.1.3.4from Penicillium notatum, 50 IU cm-2). The preparation and properties of the enzyme membrane are given in detail elsewhere (27). The rate of the enzymatic reaction is completely controlled by the rate of the diffusion of glucose in the membrane because of the high glucose oxidase activity (28). Flow Injection System. A diagram of the flow injection manifold is shown in Figure 1. The sample was introduced with a pneumatic injector (Cheminert SVA 8031) with a sample loop (10-320 pL) that was filled by a peristalic pump. The injector was connected to the steel capillary inlet of the electrode assembly with a tubing made of Teflon (length 120 mm; i.d. 0.5 mm). Another slide injector (Cheminert CSVA) with an injection volume of 1.5 fiL was used alternatively. The outlet Teflon tubing (length 80 mm; i.d. 0.8 mm) of this injector was filled with solid glass beads (diameter 0.5 mm) and connected directly to the steel capillary. The carrier was a 0.1 M phosphate buffer, pH 7.0, containing 0.1 M potassium chloride and 1mM sodium azide, and it was pumped by a Gilson Minipuls 2 peristaltic pump. The flow rate was 1.1 mL min-' unless otherwise stated. The sampling and the injection were controlled by a personal computer that was also used for the peak evaluation. THEORY The response curve of a flow injection system with an amperometric enzyme membrane electrode detector can be treated as the result of a series of independent processes acting 0 1986 American
Chemical Society
