1880
Anal. Chem. 1984, 56,1880-1884
(10) Mizutani, F.; Sasaki, K.; Shimura, Y. Anal. Chem. 1983, 55, 35-38. (11) Hlrose, S.; Yasukawa, E.; Nose, T. J . Appl. Po/ym. Sci. 1981, 26, 1039- 1046. (12) Hlrose, S.; Yasukawa, E.; Hayashl, M.; Vleth, W. R. J . Membr. Sci. 1982, 11, 177-105. (13) Harger, L. P.; Geller, D. M.; Llpmann, F. Fed. Proc., Fed. Am. Soc. Exp. Biol. 1954, 13, 11-15.
(14) Hotion, A. A.; Kornberg, H. L. Biochim. Biophys. Acta 1984, 89, 381-383.
for review January 277 1984. Accepted April 23, 1984.
Externally Buffered Enzyme Electrode for Determination of Glucose Neil Cleland and Sven-Olof Enfors*
Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, Sweden
A new type of electrode for In situ analysis, the externally buffered enzyme electrode, is presented. I n this system an lmmobllized enzyme Is Immersed In a buffer flow in such a way that the enzyme Is confined to a chamber, with an electrochemical sensor to one side and a dlalysls membrane facing the sample solutlon to the other. While constant chemical conditions are maintained lnslde the enzyme chamber, the buffer flow allows the electrode’s measuring range to be varied through aiteratlons in the buffer flow rate. The system has been applled to glucose determination by uslng glutaraldehyde-Immobilizingglucose oxidase and an amperometric oxygen electrode. Llnear response has been extended from 5 g/L to 150 g/L in phosphate buffer. Havlng an oxygen stablllzation system, the electrode can be used in completely anaeroMc medla. I n this case it has been used in 8 cell-free medium from acetone-butanol fermentation and In corn steep ilquor-based penicillin medium. The electrode Is characterlzed with respect to several Important parameters and the conditlons lnslde the enzyme chamber are dlscussed.
Ever since Clark and Lyons presented the first enzyme electrode in 1962 (I)many workers have been occupied with the development of enzyme electrodes in general (2, 3). Glucose electrodes, due to their great potential applicability, have been subject to particular attention ( 4 , 5 ) . Since there is a strong need for a rapid continuous monitoring of blood glucose in diabetic patients, much work has been focused on development of clinical glucose electrodes for in vivo use (6,
7). For fermentation applications, however, only a few reports on sugar sensors have appeared to our knowledge. One describes an enzyme thermistor device for sucrose analysis (8) and another an enzyme electrode of the self-contained type for glucose analysis (9). The sensors mentioned represent two different concepts: ( a ) The analytical enzyme reactor in which sample is withdrawn from the medium and pumped to the sensor stie which is outside the fermentation vessel. The sample can be diluted or treated in different ways prior to contact with the sensor. There is a drawback in that the sample must be withdrawn from the fermentation medium, since when the microorganisms grow, they consume substrate fairly rapidly and a t a varying rate during the course of operation. TO prevent alterations in substrate concentration during trans0003-2700/84/0356-1880$01 S O / O
port, continuous dialysis or filtration of the sample is necessary. ( b ) The enzyme electrode, which can be immersed in the sample and thus has the advantage of being able to measure in situ provided that it is sterilizable. Since in this case the sample is not diluted, the enzyme electrode is severely restricted upward in its linear measuring range which is determined by the intrinsic enzymatic properties, i.e., the apparent Michaelis K,,, of the immobilized enzyme preparation. Also, reaction products may in time build up to detrimental levels inside the enzyme membrane. The enzyme electrode can be said to be sample buffered, since the only buffer capacity available is that of the sample. In this study, a new type of enzyme electrode has been developed, in which some of the advantages of the two aforementioned types are combined: the externally buffered enzyme electrode. I t is of type b but incorporates a flowthrough system so that the enzyme chamber is continuously washed with a buffer solution. Another feature of the enzyme electrode is that is contains a Pt anode for electrolytic oxygen production. This system has been described earlier (9), as well as the use of a Pt anode (though not for O2 production) in clinical p 0 2 and pC02 measurement (10). Though demonstrated here for the case of a glucose electrode, the external buffer concept should be generally applicable to enzyme electrodes.
EXPERIMENTAL SECTION Solutions and Reagents. Unless stated otherwise, the buffer used both for samples and flow-through buffer was 0.025 M Na phosphate buffer of pH 6.0. The penicillin medium had the following composition (g/L): lactose, 10; corn steep liquor (Fermenta AB, Strangnas, Sweden) 30; (NH4)2S04,2; CaC03,5; KH2P04,0.5. The butanol medium contained originally the following compounds: glucose, 40 g/L; yeast extract (Difco Lab., Detroit, MI), 2 g/L; tryptone (Difco Lab., Detroit, MI), 3 g/L; (NH4)S04,2 g/L; KH2P04,2 g/L; K2HP04,2; CoCl,, 1.3 mg/L; Na2Se0,, 90 wg/L; MgSO4.7Hz0,0.1 g/L; CaCl,, 10 mg/L; FeS04.7H2010 mg/L; NazMoO4.2H20,2 mg/L; MnS04.H20,2 mg/L. The medium was kept anaerobic during measurement wth the glucose electrode by bubbling with nitrogen and as at the end of the fermentation with Clostridium acetobutylicum (when all glucose had been consumed) found to contain (g/L): glucose, 0.0; ethanol, 0.61; acetone, 1.84; butanol, 8.33; acetate, 0.54. Stock glucose solution used for additions was 200 g/L. All chemicals were analytical grade. Enzymes and Immobilization. The enzymes and amounts used were 2.5 mg of glucose oxidase (glucose, oxygen oxidoreductase, EC 1.1.3.4) from Aspergillus niger (Worthington, 0 1984 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
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Millipore Corp., Freehold, NJ) containing 157 U/mg, 2.5 mg of catalase (hydrogen peroxide hydrogen peroxide oxidoreductase, EC 1.11.1.6) from beef liver (Worthington, Millipore Corp., Freehold, NJ) containing 12925 U/mg, and 5 mg of bovine serum albumin (Sigma Chemical Co., St. Louis, MO). Immobilization with glutaraldehyde was performed as previously described (9). Approximately 0.4 mg of glucose oxidase was immobilized on each net. Assays for Glucose, Gluconic Acid, and Solvents. Enzymatic methods were used to measure glucose and gluconic acid contents of the samples. Glucose was determined by means of GLOX (Kabi AB, Uppsala, Sweden). Gluconic acid was assayed by using the corresponding enzymatic test kit (no 428 191) from Boehringer Mannheim (measurements were made at 340 nm). Solvents in the butanol medium were determined by means of a Varian Vista gas chromatograph. Instrumentation, Figure 1 shows schematically the construction of the glucose electrode. Onto the membrane of an oxygen electrode constructed according to Johnson et al. (11)was placed a platinum net with immobilized glucose oxidase and catalase. A dialysis membrane separated the enzyme chamber from the outer solution. A 20-mm Pt wire (diameter 2 mm) immersed in the sample solution served as cathode of the electrolytic circuit. Between the enzyme and the membrane and between the enzyme and the oxygen sensor were inserted nylon net (15 mesh, Monyl HD, ZBF, Zurich, Switzerland) spacers to ensure good flow characteristics of the external buffer. This flow was directed to and from the measuring enzyme chamber by means of 1mm i.d. metal syringes connected to Teflon tubings. Outside of the electrode the entire flow system was made up of Tygon tubing. A piston pump (Lab-pump JR, Fluid Metering, Inc., New York) was used to create the buffer flow through the system. The flow was divided prior to entry in the enzyme chamber to achieve a fine adjustment of the flow rate. Glucose and other low molecular weight compounds diffuse into the buffer of the enzyme chamber but only glucose reacts with the glucose oxidase and causes a reduction of the oxygen tension of the buffer which is sensed by the oxygen electrode. The oxygen consumed by the enzymatic reaction is replaced by electrolytic decomposition of water and the electrolysis current is used as the enzyme electrode signal output. The details of the electronic instrumentation and mode of operation have been previously described (9, 12). The oxygen-stabilized glucose electrode without the buffer flow system (sample buffered) was built as described elsewhere (9). Procedure. The external buffer is pumped through the measuring chamber, to which the sample has access by means of a dialysis membrane. The flow rate of external buffer is chosen so that one gets good response characteristics in the substrate concentration range of interest. It is of utmost importance that the external buffer does not contain dissolved gases to such an extent that they form bubbles in the measuring chamber. The
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Figure 1. Main parts of the externally buffered enzyme electrode: a, oxygen electrode; b, ff gauze with immobilized enzymes; c, Pt coil (cathode); d, nylon nets; e, dlalysis membrane; f, in going buffer stream; g, buffer effluent; h, buffer reservoir; iI PID controller; j, reference potential; k, recorder; I, electrolysis current; F, buffer flow.
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