Anal. Chem. 1998, 70, 801-806
Pulsed Amperometric Detection of Microdialysates from the Glucose Oxidase Reaction Christine M. Zook and William R. LaCourse*
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, Maryland 21250
Microdialysis is used for in vitro characterization of the glucose oxidase reaction. A microdialysis block, which is designed to accept a wide range of commercially available flat membranes, is mated with high-performance anion-exchange chromatography (HPAEC) followed by pulsed amperometric detection (PAD) to directly and continuously monitor the concentration of glucose over time. Glucose is sampled at 5-min intervals, and the initial velocity is determined from the first 10% of the reaction profile. The Michaelis constant, Km, was determined to be 10 mM. This technique is generally applicable to the study of exo- and endoglycosidases and other enzyme reactions with carbohydrate-based substrates. Microdialysis is a sampling technique that utilizes semipermeable membranes to sample analytes from a surrounding matrix. The membrane separates a flowing perfusion fluid from the matrix, and diffusion across the membrane is driven by a concentration gradient. Small molecules, as defined by the membrane, cross the membrane and are carried by the perfusate for analysis. The membrane excludes large molecules found in the matrix, thus providing samples that require little sample cleanup. In addition, removing the analyte from the matrix also removes it from the reaction, in effect, quenching the reaction at the moment of sampling. This eliminates additional preanalysis steps. Finally, microdialysis is a continuous sampling technique, which makes microdialysis amenable to monitoring reactions in progress to determine kinetics of the reaction. Microdialysis has had much of its development and usage for in vivo applications, particularly in the neurosciences1-4 and pharmacokinetic studies.3,5,6 Here, the microdialysis probes are made small in order to have minimal impact on the surrounding tissues. The characteristics of microdialysis, which make it wellsuited to in vivo applications, also make it a good sampling technique for in vitro situations. Other than characterization studies of in vivo probes external to the biosystem of study, only * Corresponding author: (Phone) 410-455-2105; (fax) 410-455-2608; (e-mail)
[email protected]. (1) Benveniste, H.; Huttemeier, P. C. Prog. Neurobiol. 1990, 35, 195-215. (2) Benveniste, H. J. Neurochem. 1989, 52, 1667-1679. (3) Lunte, C. E.; Scott, D. O.; Kissinger, P. T. Anal. Chem. 1991, 63, 773A779A. (4) Justice, J. B., Jr. J. Neurosci. Methods 1993, 48, 263-276. (5) Stahle, L. Eur. J. Drug Metab. Pharmacokinet. 1993, 18, 89-96. (6) Morrison, P. F.; Bungay, P. M.; Hsiao, J. K.; Ball, B. A.; Mefford, I. N.; Dedrick, R. L. J. Neurochem. 1991, 57, 103-119. S0003-2700(97)01106-2 CCC: $15.00 Published on Web 02/15/1998
© 1998 American Chemical Society
a limited number of in vitro applications have been published. It has previously been used for the measurement of ethylene in apples,7 for monitoring ethanol,8 other alcohols, and carbohydrates9-11 during fermentation, and for monitoring enzymatic mannan nut hydrolysates.12,13 In each of these applications, the microdialysis probes developed for in vivo applications has been used to sample from the fruit or reactors. To implement this methodology for in vitro applications, we have designed a microdialysis block. The block design has the advantage of being more rugged than the probes used in vivo and more versatile in regard to reservoir size, exposed area, and block material for probe inertness. The microdialysis block is also designed to accept any flat membrane, thereby allowing study of a greater variety of membrane materials for dialysis. In addition, system parameters (e.g., temperature and stirring) are controlled precisely, which facilitates their study and optimization. Previous microdialysis membrane studies have concentrated on those membranes in commercially available probes.14-16 Since the membranes in these studies differed in both composition and molecular weight cutoff (MWCO), comparison of membrane properties was inconclusive. In this paper, comparison of different cellulose-based membranes will be made over similar ranges of MWCOs. Glucose oxidase (GOx) is a widely used and well-characterized enzyme. It catalyzes the oxidation of glucose to gluconolactone in the presence of oxygen, with hydrogen peroxide as a byproduct of the reaction. It has been used to monitor glucose on-line in a chromatographic system using a bioreactor17 and has been incorporated into a number of biosensors.18 Typically, the reaction (7) Eklund, L.; Collin, A.-K. J. Plant Physiol. 1991, 137, 375-377. (8) Buttler, T.; Gorton, L.; Jarskog, H.; Marko-Varga, G.; Hahn-Hagerdal, B.; Meinander, N.; Olsson, L. Biotechnol. Bioeng. 1994, 44, 322-328. (9) Buttler, T.; Jarskog, H.; Gorton, L.; Marko-Varga, G.; Ramnemark, L. Am. Lab. 1994, 26 (12), 28I-28M. (10) Marko-Varga, G.; Buttler, T.; Gorton, L.; Gronsterwall, C. Chromatographia 1993, 35 (5/6), 285-289. (11) Buttler, T.; Liden, H.; Jonsson, J. A.; Gorton, L.; Marko-Varga, G.; Jeppsson, H., Anal. Chim. Acta 1996, 324, 103-113. (12) Torto, N.; Buttler, T.; Gorton, L.; Marko-Varga; G., Stalbrand, H.; Tjernel, F. Anal. Chim. Acta 1995, 313, 15-24. (13) Torto, N.; Marko-Varga, G.; Gorton, L.; Stalbrand, H.; Tjerneld, F. J. Chromatogr., A 1996, 725, 165-175. (14) Buttler, T.; Nilsson, C.; Gorton, L.; Marko-Varga, G.; Laurell, T., J. Chromatogr., A 1996, 725, 41-56. (15) Hsiao, J. K.; Ball, B. A.; Morrison, P. F.; Mefford, I. N.; Bungay, P. M. J. Neurochem. 1990, 54, 1449-1452. (16) Stenken, J. A.; Topp, E. M.; Southard, M. Z.; Lunte, C. E. Anal. Chem. 1993, 65, 2324-2328. (17) Huang, T.; Kissinger, P. T. Curr. Sep. 1989, 9 (12), 9-13. (18) Wilson, R.; Turner, A. P. F. Biosen. Bioelectron. 1992, 7, 165-185.
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is monitored nonspecifically either by amperometric detection of hydrogen peroxide18 or by a coupled enzyme reaction with peroxidase and UV detection.18 In this paper, the glucose oxidase reaction will be monitored using microdialysis mated with high-performance anion-exchange chromatography (HPAEC) followed by pulsed amperometric detection (PAD). HPAEC-PAD is used routinely for the separation and direct detection of carbohydrates.19-21 Using commonly available cellulose-based membranes, the loss of glucose will be monitored directly. The Michaelis-Menten constant (Km) is the concentration of substrate at half the maximum velocity (Vmax), and it is used with Vmax to describe empirically the kinetics of an enzyme reaction. These values will be determined for glucose. Membranes are characterized as to selectivity, recovery, and reproducibility for various carbohydrates, and these studies are used to select the appropriate membrane for use in sampling glucose away from the enzyme solution. In addition, microdialysis system parameters (e.g., perfusate composition) and instrumental (i.e., HPAEC-PAD) optimization will be discussed in detail. EXPERIMENTAL SECTION Reagents. All solutions were prepared from reagent-grade chemicals. Sodium hydroxide solutions were diluted from 50% (w/w) stock solution (J. T. Baker Inc., Phillipsburg, NJ.). All mobile phases were filtered with 0.2-µm Nylon-66 filters (Rainin Corp., Woburn, MA) and a solvent filtration apparatus (Microfiltration Systems, Rainin). All mobile phases were deaerated with dispersed N2. Water was purified using a reverse osmosis system coupled with multitank/ultraviolet/ultrafiltration stations (US Filter/IONPURE, Lowell, MA). Glucose oxidase EC 1.1.3.4 from Aspergillus niger was purchased from Sigma (St. Louis, MO) and was used without further purification. Activity for this preparation was listed as 100 000250 000 units/g of solid at pH 5.1, 35 °C. Apparatus. The microdialysis cell was constructed in-house from a Teflon block. Figure 1 shows an expanded view of the cell. The Teflon cap (a), which also serves as a 5-mL sample reservoir, is threaded so that it can be screwed onto the cell bottom. The active dialysis window in the spacer (b) is 40 mm2, which is comparable to microdialysis probe membrane areas available commercially from Bioanalytical Systems, Inc. (West Lafayette, IN). This spacer not only defines the area of the membrane available to the solution but also functions to hold the membrane (c) tightly against the top of the cell bottom, preventing leakage and damage to the membrane from assembly of the cell. All the membranes used were 33-mm membrane disks (Spectrum, Houston, TX). The cell bottom (d) has a 40-µL channel (1.0 mm deep by 3.0 mm wide) machined into its top face, which exposes the membrane to the perfusion medium for collection of the analyte. A 0.0625-in.-diameter Teflon tube connects a Baby Bee syringe pump (Bioanalytical Systems) to the inlet port of the cell, and a PEEK tube (0.0625-in. o.d. and 0.007-in. i.d.) connects the outlet port of the microdialysis cell to the chromatography injection valve. The entire microdialysis cell and injection valve (19) LaCourse, W. R. Pulsed Electrochemical Detection in High Perfromance Liquid Chromatography; John Wiley & Sons: New York, 1997. (20) LaCourse, W. R. Analusis 1993, 21, 181-195. (21) Johnson, D. C.; LaCourse, W. R. Anal. Chem. 1990, 62, 589A-597A.
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Figure 1. Schematic drawing of the microdialysis cell showing (a) threaded cap and sample reservoir, (b) spacer with dialysis window, (c) membrane, and (d) cell bottom with perfusion medium channel.
were placed in an incubator (model 03608-16; Cole Palmer, Vernon Hills, IL) for active temperature control. A rotator fitted with a stirring paddle was used to determine the effects of stirring the solution on the percent recovery of glucose. Since stirring defines a finite diffusion layer, increased recovery was found for any rotation speed over no stirring. In determining the effect on the response of the system to changes in the sample cup, the glucose concentration was incrementally increased and the dialysate analyzed. By stirring the solution, the concentration at the membrane surface is maintained at that of the bulk concentration, and the stirred system quickly achieves equilibrium. Without stirring, the response/recovery is erratic until equilibrium is achieved, which can take minutes to hours. HPAEC was performed on an advanced gradient chromatography system (Dionex Corp., Sunnyvale, CA). Separations were done with a CarboPac-PA1 anion-exchange analytical column (Dionex), preceded by a CarboPac-PA1 guard column at a flow rate of 1.00 mL/min. A second injection valve was placed in line before the microdialysis injection system for manual injection of standards. Pulsed amperometric detection was performed with a model PED unit (Dionex) with a thin-layer electrochemical cell. The PAD wave form was applied to a Au disk working electrode (∼1.54 mm2) with a stainless steel auxiliary electrode and a Ag/ AgCl reference electrode. A detection potential of +100 mV was applied for 440 ms, during which the current was integrated over the last 200 ms. The oxidation and reduction potentials were +800 (180 ms) and -400 mV (350 ms), respectively. All injection volumes were 10 µL unless otherwise noted. Procedure. Before use, each membrane was rinsed with deionized water and hydrated for ∼12 h prior to use. It was then placed carefully in the microdialysis cell, and 4 mL of acetate buffer was placed in the sample cup. The block was perfused with water,
and injections of dialysate were made to ensure no contamination in the cell. An aliquot of concentrated glucose solution was then added to the acetate buffer so that the resulting solution, correcting for dilution, was the appropriate concentration for the enzyme reaction. The block was perfused and injections of dialysate were made until the peak heights were constant and within the linear dynamic range. The peak height was quantitated against external standards, and the percent recovery determined. Performance of a membrane was described as percent recovery of a particular analyte, where
% recovery )
concentration of dialysate × 100 concentration of sample
The minimum time required to fill the sample loop is dependent upon the sample loop size and the perfusion rate. For a 10µL sample loop and a perfusion rate of 10 µL/min, 2 min is required to flush the sample loop with two loop volumes. With 1 min allowed to inject the sample and return the valve to the “load” position, injections can be made as rapidly as every 3 min. To overlap injections without overlapping peaks, injections were made every 5 min. After establishing the percent recovery of the membrane, enzyme was added and injections were made every 5 min beginning at time zero. All resulting peak heights were quantitated against external standards and corrected for recovery. The corresponding concentration-time curve was fitted to a first-order exponential decay curve (i.e., y ) ae-bx) for all the available data, and the first 10% of this best-fit curve was used to determine the initial velocity of the reaction. RESULTS AND DISCUSSION The analytes of interest are carbohydrates, and for membrane characterization, a series of maltooligosaccharides, or glucopolymers, were used. These maltooligosaccharides included glucose (g1), maltose (g2), maltotriose (g3), maltotetraose (g4), maltopentaose (g5), maltohexaose (g6), and maltoheptaose (g7). The molecular weights of these compounds range from 180.2 to 1153.0 for glucose to maltoheptaose. Elution of these compounds is accomplished using anion-exchange chromatography with a polymeric column and sodium hydroxide/sodium acetate eluent. The sodium hydroxide is necessary for the weakly acidic carbohydrates to be anionic for separation and for pulsed amperometric detection. Since acetate is PAD-inactive, it is used as a “pusher” anion to reduce the retention of larger oligosaccharides without any adverse effects on the baseline. In general, isocratic elution is preferred over gradient elution for several reasons. By dispensing with the time required for equilibration of the column after the gradient run, the time points of a reaction can be made closer together and better kinetic information can be achieved. In addition, baseline drift associated with the use of gradients in electrochemical detection is not present, and repeatability of injection under isocratic conditions is also improved; relative standard deviations (RSDs) range from 1.6 to 12% for gradient elution and 0.88 to 3.61% for isocratic elution. All studies were performed well above the limit of quantitation and within the linear range for all analytes. For the glucose oxidase assay, glucose is again eluted isocratically, using 150 mM sodium hydroxide. Under these condi-
Figure 2. Chromatogram of glucose and gluconic acid at 100 µM. Conditions: column, CarboPac PA1 and PA1 guard; mobile phase, 150 mM NaOH.
tions, glucose elutes in 4.27 min (k′ ) 2.01). Gluconolactone, the product of this reaction, elutes in 17.58 min (k′ ) 11.38); see Figure 2. By making injections every 5 min, the overlapping injections do not interfere with one another. The gluconolactone peak is not quantitated due to the low sensitivity of the compound. Additionally, gluconolactone hydrolyses under alkaline conditions to gluconic acid, which may yield two unresolved peaks (see Figure 2), which is deleterious to quantitation. Membrane Characterization. Initial studies focused on the characterization of the membranes to understand their performance with regard to recovery of potential carbohydrate-based substrates and/or products. The most commonly used membranes are made of regenerated cellulose and cellulose ester. Each type is available over a wide range of MWCOs. Using a mixture of glucopolymers from g1 to g7, the percent recovery of these analytes was determined for each membrane type at the MWCO indicated (Figure 3A). As noted by the slope of the individual plots (see Figure 3B), the cellulose ester membranes show greater sensitivity to the molecular weight of the analyte than the regenerated cellulose membranes. The sensitivity of the cellulose ester membranes falls off with increasing molecular size of the analyte. The percent recoveries for the regenerated cellulose membranes are relatively constant over the range of 6000-14 000 MWCO. This observation indicates that the MWCOs of the regenerated cellulose membranes have little meaning under these conditions. Both membrane types show a decreasing percent recovery with molecular size. Another advantage of cellulose ester membranes is their structural rigidity, which may facilitate cell ruggedness. Cellulose ester membranes were used for all studies unless otherwise noted. For a given membrane, it has been shown that percent recovery decreases with increases in the perfusion rate.1 Figure 4 shows the microdialysis of five maltooligosaccharides for a 1000 MWCO cellulose ester membrane, and as expected, recovery is lowest for the highest perfusion rate. The error bars at each of the points denote its standard deviation. Pulsed amperometric detection yields limits of detection (LODs) as low as 200 pM for Analytical Chemistry, Vol. 70, No. 4, February 15, 1998
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Figure 4. Percent recovery vs perfusion rate using a 1000 MWCO cellulose ester membrane. Solutions are the same as in Figure 3.
Figure 3. (A) Effect of MWCO on percent recovery on different glucopolymers. Conditions: solutions (A) g1, (B) g2, (C) g3, (D) g4, (E) g5, (F) g6, and (G) g7. All solutions were 200 µM except g6, which was 3 µM. (B) Resulting slope of curves in (A) with increase in glucopolymer number.
Figure 5. Time plot of glucose oxidase reaction showing decrease of glucose concentration (9), increase of gluconolactone concentration (b), and hydrogen peroxide peak height (O). Conditions as in Figure 2, 1 mM glucose, and enzyme added at t ) 0 (12 µg of GOx/mL of solution).
glucose, with a linear range up to 2 mM. The high sensitivity (i.e., low LODs) of PAD facilitates the use of the lower recoveries at higher perfusion rates. Higher perfusion rates aid in the use of rapid on-line injections, as the sample loop can be flushed out and refilled more quickly during the course of the chromatographic run. In addition, the wide linear dynamic range of PAD allows the use of higher analyte/substrate concentrations in the microdialysis system. For in vivo applications, the perfusion medium is matched as closely as possible to the surrounding matrix in order to minimize concentration gradients other than that of the analyte. In sampling from the enzyme reaction, the matrix is typically a buffer with ionic strength ranging from 10 to 250 mM. To determine what
effect matching the perfusion fluid to the matrix has on the sampling, the ionic strength of the perfusion fluid was changed over a limited range while sampling from a water solution. Changing the ionic strength of the perfusion fluid from 0 to 200 mM NaNO3 has little effect on the percent recovery of glucose, lactose, and deoxyribose. This result becomes advantageous in a situation where the buffer requirements of the enzyme reaction would cause incompatibility problems with the chromatographic system. In such a case, the perfusion fluid could be altered to avoid this, while recovery of analyte is maintained. Next, water was used as the perfusion fluid and sampling was from a buffered solution of varying ionic strength. Again, there was little change
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Figure 6. (A) Continuous chromatogram of glucose (a), hydrogen peroxide (b), and gluconolactone (c) peaks from glucose oxidase reaction (0.5 mM glucose, enzyme added at t ) 0, and 12 µg of GOx/mL of solution); (B) glucose concentration vs time. Note: injections in (A) are marked by subscript for clarity. Conditions: see Figure 2. Plots in (B) are for 2 mM glucose and 1.2 (b) and 12 (9) µg of GOx/mL of solution.
in recovery over a range of buffer ionic strength from 0 to 200 mM NaNO3. Percent recovery was found to differ between like membranes with a range of 15-27% for regenerated cellulose and 30-50% for cellulose ester. The wide range of recoveries for like membranes is a result of product variations (e.g., membrane thickness, tortuosity, and pore distribution). Hence, it is imperative to calibrate the microdialysis system before use. The use of an internal standard is readily applicable to in vitro microdialysis, and its use is encouraged. The internal standard needs to be PADactive and have a low k′ value, which does not interfere with the overlapped injections. In addition, the internal standard must not be a potential substrate or inhibitor for the enzyme of interest. An internal standard for the glucose oxidase reaction was not found to meet all these requirements. Glucose Oxidase Reaction. The glucose oxidase unit activity is defined for pH 5.1 at 35 °C.22 The pH range for glucose oxidase is listed as 4-723 with 5.1 being optimum.22 For our system, the reaction was run at 24 °C, in 50 mM acetate buffer, pH 5.1. The ratio of substrate to enzyme concentration was always >1000-fold to ensure proper enzyme kinetics (i.e., maintains steady-state assumptions pertaining to enzyme saturation). Prior to sampling glucose from the glucose oxidase reaction, the recovery of glucose was determined. After an initial equilibration period, percent recovery remains constant with concentration, and this value can then be used to calculate the concentration of glucose in the cell. The cell shows little or no loss in glucose concentration when sampled over the course of 3 h. Quantitation of glucose peaks was by an external calibration curve. For each reaction, the sampling procedure began by sampling glucose from a glucose solution in the cell, prior to enzyme addition. When the glucose concentration in the dialysate is constant, as indicated by constant peak height, enzyme is added to the cell and injections of the dialysate continue. As the reaction progresses, the glucose peak decreases and peak for H2O2 and gluconolactone appears. (22) Sigma product sheet, Product No. G-7016, Sigma Chemical Co., St. Louis, MO. (23) Bright, H.; Appleby, M. J. Biol. Chem., 1969, 244, 3625.
Figure 5 shows the plot for an extended reaction for the decay of glucose by GOx. Prior to enzyme addition at time zero, the glucose concentration is constant. After addition of enzyme, the substrate concentration decreases as expected. Note, the appearance of gluconolactone and H2O2 mirrors the loss of glucose. Figure 6A shows a chromatogram of repeated injections of microdialysate. Enzyme is added at time zero at the same time as the first injection is made. After enzyme addition, the peak height for glucose decreases, indicating hydrolysis of glucose. Additionally, peaks for hydrogen peroxide and gluconolactone appear in the chromatogram. For clarity of presentation, this chromatogram was obtained at higher enzyme concentration than desired for quantitative work. Glucose standards were run prior to and after the reaction for quantitating the peaks from the reaction. Membrane characterization prior to the reaction yielded a value for percent recovery of glucose for the membrane. This value was used to calculate the concentration of glucose in the microdialysis cell. The calculated concentration at each time point was plotted against time. Figure 6B shows typical plots both at (b) ideal and (9) that used in Figure 6A enzyme concentrations. These data represent the kinetics of the glucose oxidase reaction. The Michaelis constant, or Km, can be determined by determining the kinetics of the enzyme reaction over a range of substrate concentrations. To describe the kinetics without concern over inhibitory effects of the products of the reaction, the initial velocity is used. The initial velocity of the enzyme is the velocity of the reaction during the first 10% of the reaction. Figure 6B shows two plots of glucose concentration versus time representing the reaction at two different enzyme concentrations. A larger enzyme concentration yields a faster decrease in glucose concentration. To obtain a greater number of data points defining the initial decay curve, the lower concentration of GOx was used in obtaining initial velocity values. The tangent to the beginning of this curve (b) is 7.34 µM/min. This rate was determined similarly for a range of glucose concentrations and plotted using the Lineweaver-Burk plot. In this plot, the inverse of the initial velocity is plotted versus the inverse of the substrate concentration, yielding a straight line. The slope is Km/Vmax, the X-intercept is -1/Km, and the Y-intercept is 1/Vmax. Figure 7 is the LineAnalytical Chemistry, Vol. 70, No. 4, February 15, 1998
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Figure 7. Lineweaver-Burk plot. Data derived from enzyme reactions with 1.2 µg of GOx/mL of solution, pH 5.1, 24 °C.
weaver-Burk plot for glucose oxidase. The values obtained in our laboratory are 10 ( 30 mM and 40 ( 150 µM/min for Km and Vmax, respectively. These values agree favorably with those found in the literature, which range from 26 to 110 mM for Km.24-26 The published values were not statistically evaluated but appeared to have similar or greater standard deviations. It should also be noted that the literature values were obtained at higher temperatures (e.g., 27-37 °C) as well as at a higher pH value (i.e., pH 5.6). Enzyme Reactions. Monitoring enzyme kinetics is feasible not only for well-characterized enzymes under ideal conditions but also for enzymes under nonideal conditions (e.g., pH, temperature), with alternative substrates or for new enzymes. More importantly, this approach is uniquely suited to monitoring reactions with substrates and/or products that are not chromophores, fluorophores, or dc-active electrophores. The enzyme lactase is found in consumer products for persons who are lactoseintolerant. The enzyme catalyzes the hydrolysis of lactose to glucose and galactose. The commercial product is available in tablet or drop form. In the latter, several drops are added to a container of milk and allowed to work in the refrigerator for several hours. Figure 8 is a chromatogram of two consecutive injections of a lactose solution. The first injection is dialysate prior to addition of lactase. A large lactose peak is present. The second injection is just past enzyme addition (Ein). Already the lactose peak is smaller, and two peaks have appeared for glucose and galactose. In addition to the peaks from sugars directly related to the reaction, there is an internal standard peak, deoxyglucose. (24) Gibson, Q. H.; Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1964, 239, 3927-3934. (25) Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1965, 240, 2209-2215. (26) Nakamura, S.; Hayashi, S.; Koga, K. Biochim. Biophys. Acta 1976, 445, 294-308.
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Figure 8. Chromatogram of two injections sampled from lactase reaction. Conditions: column, CarboPac PA1; gradient elution, 0-8 min, 20 mM NaOH, 8-20 min, 20 mM NaOH to 20 mM NaOH/54 mM NaOAc. Dairy Ease, 400 µL, added at Ein.
The internal standard is included, when appropriate, to monitor the recovery from the microdialysis cell. CONCLUSIONS The microdialysis cell described here is an alternative to commercially available probes designed for in vivo use. The microdialysis cell allows control over membrane composition, temperature, hydrodynamics, and active dialysis area. It is more rugged than the commercially available probes and can be made from materials appropriate to the application. Microdialysis coupled to HPAEC-PAD has been used to monitor glucose during the glucose oxidase reaction. By following the substrate of the reaction instead of the product, false kinetics due to interfering substrates is avoided. ACKNOWLEDGMENT The authors gratefully acknowledge Bioanalytical Systems personnel for technical discussions on microdialysis. This work was partially supported by a Graduate Research Assistantship (UMBC-DRIF Funds) and a Maryland Industrial Partnership grant/Med Immune, Inc. Received for review October 6, 1997. Accepted December 4, 1997. AC971106O
