Stopped-Flow and Rotating Bioreactors in the

Anal. Chem. 1998, 70, 3679-3684

Continuous-Flow/Stopped-Flow and Rotating Bioreactors in the Determination of Glucose Alicia V. Lapierre, Roberto A. Olsina, and Julio Raba*

Department of Analytical Chemistry, National University of San Luis, Chacabuco y Pedernera, C.P.(5.700), San Luis, Argentina

The high sensitivity that can be attained by enzymatic amplification via substrate cycling has been verified by on-line interfacing of a rotating bioreactor and continuousflow/stopped-flow/continuous-flow processing [Raba, J.; Mottola, H. A. Anal. Biochem. 1994, 220, 297-302]. The determination of glucose levels was possible with a limit of detection of 0.2 fmol‚L-1 in the processing of as many as 30 samples per hour. Determination at such low levels is of interest in several situations encountered in fermentation biotechnology and clinical chemistry, and this determination in culture broths illustrates the capabilities of the proposed approach. The glucose oxidase/ glucose dehydrogenase coupled system was used by immobilizing glucose oxidase (EC 1.1.3.4) on the top of a rotating disk while glucose dehydrogenase (EC 1.1.1.47) was immobilized on the top part of the flow-through cell. Substrate cycling was realized via NADH/NAD+ that, in conjunction with glucose dehydrogenase, regenerates glucose, the substrate in the glucose oxidase-catalyzed reaction. This cycling permits generation of H2O2 (detected at Pt ring electrode concentric to the rotating disk) beyond stoichiometric limitations. This permits a 100fold increase in the sensitivity for glucose determination when compared with the determination involving no substrate cycling. The determination of the presence of relatively low concentrations of glucose is of importance in several areas, such as the food industry, and in the monitoring of diabetic diets. Accurate and rapid determination of low levels of glucose in fermentation biotechnology should permit intracellular determinations, and this should provide essential information to enhance fermentation rates. Also of relevance is the determination of glucose in tear fluid as a noninvasive detection of diabetes mellitus.1 Few approaches have been documented in the area of biosensing by exploiting enzymatic amplification via substrate cycling.2 Product accumulation, accomplished by enzyme-cofactor interaction, results in a very powerful tool to detect very low concentrations of a given substrate.3 Schubert et a1.4 have shown * To whom correspondence should be addressed: (e-mail) [email protected], (fax) +54-652-30224. (1) Jin, Z.; Chen, R.; Colon, L. Anal. Chem. 1997, 69, 1326-1331. (2) (a) Bates, D. L. Trends Biotechnol. 1987, 5, 204-209. (b) Avrameas, S. J. Immunol. Methods 1992, 50, 23-33. (3) Mottola, H. A. Kinetic Aspects of Analytical Chemistry; Wiley: New York, 1988; p 67. S0003-2700(97)00819-6 CCC: $15.00 Published on Web 07/17/1998

© 1998 American Chemical Society

that the enzyme amplification system used in the work described here can determine glucose at concentrations as low as 8 × 10-7 M, with the enzymes co-immobilized on a membrane of gelatin and oxygen consumption monitored amperometrically. In this paper, we show that the limit of detection for this system can be lowered considerably if the enzymatic properties of glucose oxidase and glucose dehydrogenase5 and the same substrate cycling are utilized using a rather recently introduced strategy.6 Such a strategy allows not only for very convenient substrate amplification but also for an effective use of immobilized active centers.7 The advantages of this strategy, which can be extended to other enzymatic systems and also to enzyme immunoassays, are further shown here by comparison with determinations involving glucose oxidase only.8 The approach described in this paper is important for the determination of glucose in extracellular medium when the levels are very low and other methods are not suitable due to relatively low sensitivity. It has to be pointed out that a large number of samples can be processed by means of the proposed method, which shows adequate sensitivity, low cost, versatility, simplicity, and effectiveness. The determination of glucose at very low concentrations in extracellular fluid is of particular interest in bacteriology.9 Conventional monitoring procedures (e.g., colorimetric determination) fail to provide useful information after 7 days of incubation. The strategy proposed here, however, is capable of providing useful information where conventional monitoring procedures fail. Experimental data presented in this paper verify this assertion. MATERIALS AND GENERAL METHODS Reagents and Solutions. The water used for solution preparation was deionized and further purified by distillation in a borosilicate glass still with a quartz immersion heater. All reagents used, except as noted, were of analytical reagent grade. The enzyme glucose oxidase (GOx, EC 1.1.3.4), from Aspergillus niger (type VII), containing 1.25 × 105 IU‚g-1 of solid, D-(+)glucose, and β-nicotinamide adenine dinucleotide (β-NADH) (4) Schubert, F.; Kirstein, D.; Schro¨der, K. L.; Scheller, F. W. Anal. Chim. Acta 1985, 169, 391-396. (5) Raba, J.; Mottola, H. A. Crit. Rev. Anal. Chem. 1995, 25 (1), 1-42. (6) Raba, J.; Mottola, H. A. Anal. Biochem. 1994, 220, 297-302. (7) Richter, P.; Lo´pez Ruiz, B.; Sańchez-Cabezudo, M.; Mottola, H. A. Anal. Chem. 1996, 68, 1701-1705. (8) Matsumoto, K.; Baeza Baeza, J. J.; Mottola, H. A. Anal. Chem. 1993, 65, 636-639. (9) Agar, D. W. In Microbial Growth Rate Measurement Techniques: Comprehensive Biotechnology, 1st ed.; Robinson, C. W., Howel, J. A., Eds.; Pergamon Press: New York, 1985; Chapter 17.

Analytical Chemistry, Vol. 70, No. 17, September 1, 1998 3679

Figure 2. Block diagram of the continuous-flow system and detection arrangement. P, pump (Gilson Minipuls 3 peristaltic pump, Gilson Electronics, Inc., Middleton, WI); C, carrier buffer line; SI, sample injection; W, waste line; R&DC, reactor and detector cell; WE, Pt ring working electrode; RE, reference electrode (Ag/AgCl, 3.0 M NaCl); AE, auxiliary electrode (stainless steel tubing); D, potentiostat/ detection unit (LC-4C, Bioanalytical Systems, West Lafayette, IN); R, recorder (Varian, model 9176, Varian Techtron, Springvale, Australia).

Figure 1. Schematic representation of components in the doublebioreactor flow cell. A, assembled reactor; B, upper cell body; C, lower cell body; D, top view of lower cell body; a, fixed bioreactor film (with immobilized GDH); b, rotating bioreactor (with film of immobilized GOx); c, Pt ring working electrode; d, O-ring; e, electrical connection; f, auxiliary electrode (stainless steel tubing); g, reference electrode (Ag/AgCl, 3.0 M NaCl); h, electrical connection. All measurements are given in millimeters.

disodium salt were purchased from Sigma Chemical Co. (St. Louis, MO). Glucose dehydrogenate (GDH, EC 1.1.1.47), from Bacillum megaterium (200 units‚mg-1), and glutaraldehyde (25% aqueous solution) were purchased from Merck Quı´mica Argentina SAIC (Buenos Aires, Argentina). 3-Aminopropyl-modified controlledpore glass (APCPG), with a mean pore diameter of 1400 Å, a surface area of 24 m2‚g-1, and containing 48.2 µmol‚g-1 of amino groups, was from Electro-Nucleonics (Fairfield, NJ). The Trinder method determination kit (Catalog No. 14001) was from Wiener Laboratories (Rosario, Argentina). Aqueous solutions were prepared using purified water from a Milli-Q system, and the samples were diluted to the desired concentrations using a 10-mL Metrohm E 485 buret (Metrohm AG, Herisau, Switzerland). Procedure for Enzyme Immobilization. Glucose oxidase (10.00 mg of enzyme preparation in 0.50 mL of 0.10 M phosphate buffer, pH 7.00) and GDH (10.00 mg of enzyme preparation in 0.25 mL of 0.10 M phosphate buffer, pH 7.00) were immobilized as described earlier.8 After being washed with purified water and 0.10 M phosphate buffer of pH 7.00, the enzyme preparations were stored between uses in the same buffer at 5 °C. The immobilized GOx and GDH preparations were totally stable for at least 1 month and 2 weeks of daily use, respectively. Flow-Through Reactor/Detector Unit (Double Bioreactor). Figure 1 illustrates the design of the double-enzyme reactor/ detector system. In this case, a modification of the system described in ref 6 was introduced. The body for the reference electrode was located at the top of the closed cell. With this modification, the problem that may be created by air bubbles is minimized. Glucose oxidase is immobilized on the top of the 3680 Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

rotating reactor. The reactor at the upper part of the cell consists of a film of double-sided tape with CPG-immobilized GDH. The distance between the two reactors with immobilized enzymes is about 1 mm. For the unamplified determinations, only GOx, immobilized on the rotating disk reactor, was used. Rotation of the lower reactor was effected with a common laboratory magnetic stirrer (Metrohm E649 from Metrohm AG, Herisau, Switzerland) and controlled with a variable transformer with an output between 0 and 250 V and a maximum amperage of 7.5 A (Waritrans, Buenos Aires, Argentina.). The potential applied to the stationary Pt ring electrode for H2O2 detection was +0.600 V vs an Ag/AgCl, 3.0 M NaCl reference electrode. Flow System. A Gilson Minipuls 3 peristaltic pump (Gilson Electronics, Inc., Middleton, WI) was used for pumping, sample introduction, and stopping of the flow. Figure 2 illustrates schematically the components of the single-line continuous-flow setup. The pump tubing was Tygon (Fisher AccuRated, 1.0 mm i.d., Fisher Scientific Co., Pittsburgh, PA), and the remaining tubing used in the rest of the setup was 1.0-mm-i.d. Teflon (ColeParmer, Chicago, IL). Other Apparatus. All pH measurements were made with an Orion expandable ion analyzer model EA 940 (Orion Research Inc., Cambridge, MA), equipped with a glass combination electrode (Orion Research Inc.). RESULTS AND DISCUSSION The oxidation of glucose to gluconic acid in the presence of NAD+ and of glucose dehydrogenase as catalyst is considered one of the dual-enzyme arrangements of choice for measuring glucose.3 The overall reactions make use of the following: GOx

glucose + O2 98 gluconic acid + H2O2 GDH

gluconic acid + NADH 98glucose + NAD+

(1) (2)

where GOx and GDH represent glucose oxidase and glucose dehydrogenase, respectively. Glucose oxidase helps the production of gluconic acid from the analyte, and GDH helps to regenerate glucose which can be oxidized again by reaction 1. Reaction 2 involves a stereospecific net transfer of a hydride ion

between the substrate and the C-4 of the pyridine ring of the coenzyme and the exchange of a proton with the medium.10 The formation of hydrogen peroxide can be easily detected amperometrically by its oxidation at a platinum electrode. As the result of this catalytic cycling, a chemical amplification is in operation, and product accumulation results in enhanced signals, affording very low limits of detection. The attainable conversion efficiency that controls such a limit of detection is, however, dictated by the type of enzyme reactor used. When packed reactors are used in continuous-flow configurations, either a very high enzyme loading for effective use or a long residence time in the reactor (large reactors) is required, which is detrimental for an effective accumulation of products to obtain enhanced signals at the detecting unit. Moreover, these situations favor an increase in the dispersion of the sample plug, also detrimental for effective exploitation of the amplification process.7 In these types of reactors, the value of the apparent MichaelisMenten constant, K′M, of the immobilized enzyme preparation is normally larger than that for the free enzyme in solution. This is the result of diffusional limitations that produce a decrease in the initial rate of the catalyzed reaction. A recently introduced strategy for amplification by substrate cycling consists of a rotating enzyme reactor and a stationary detection unit.6 This effectively circumvents the limitations of a large K′M. A more complete reagent homogenization is achieved, because the cell works as a mixing chamber by facilitating the arrival of substrate at the active sites and the release of products from the same sites. The net result is high initial rate values (see Table 4). The main advantages of this system are its simplicity and the ease with which it can be utilized for the determination of glucose at very low levels. Moreover, this strategy is easily adapted to continuous-flow processing. The implementation of continuous-flow/stopped-flow programming and the location of two facing independent reactors (Figure 1), each containing one of the immobilized enzymes involved in the sequence illustrated earlier, permits (a) reduction of K′M, (b) instantaneous operation under high initial rate conditions, (c) easy detection of accumulated products, and (d) relatively low enzyme loading conditions. According to the theoretical considerations offered by Kulys et al.,11 the amplification factor G is given, in this case, by the following equation:

G ) K1K2/2(K1 + K2)Dturb

where K1 ) (IR)max/K′M(GOx), K2 ) (IR)max/K′M(GDH), and (IR)max is the maximun initial rate. Dturb is defined as a diffusion coefficient characterizing mass transfer in a turbulent fluid. In the experimental conditions proposed here, laminar flow predominates within sample plugs containing the analyte until reaching the detection point, when the flow is stopped and a turbulent regime controls mass transfer.7 Effect of Reactor Rotation and Continuous-Flow/StoppedFlow Operation. In both cases (amplified and unamplified systems), if the reactors in the cell are devoid of rotation, as shown (10) Gorton, L.; Cso¨regi, E.; Domıńguez, E.; Emmeús, J.; Jo ¨nsson-Pettersson, G.; Marko-Varga, G.; Persson, B. Anal. Chim. Acta 1991, 250, 203-248. (11) Kulys, J. J.; Sorochinski, V. V.; Vidzinzinnaite, R. A. Biosensors 1986, 2, 135-146.

Table 1. Effect of Reactor Rotation Velocity on Initial Rate, Measured under Stopped-Flow Conditionsa unamplified system

amplified system

rotation velocity, rpm

initial rate, µA‚s-1

linear regression standard deviation



170 240 420 840 900

0.20 0.50 1.20 2.23 4.25

(0.05 (0.02 (0.02 (0.03 (0.01

7.10 16.33 32.15 64.10 80.07

(0.05 (0.07 (0.02 (0.01 (0.01

a Each value of initial rate is based on triplicate of six determinations. In both cases, flow rate was 1.00 mL‚min-1, cell volume was 400 µL, sample size was 250 µL, and glucose concentration was 0.10 mM, with 0.10 M phosphate buffer of pH 7.00. The flow was stopped for 60 s during measurement.

Figure 3. Effect of reactor rotation under stopped flow (amplified system). At A, the rotation of the lower reactor was interrupted, and at B, the rotation was reinitiated (900 rpm). i ) Injection, s ) flow stopped, f ) flow continued. Flow rate, 1.00 mL‚min-1; cell volume, 400 µL; glucose concentration, 0.10 mM; 0.10 M phosphate buffer of pH 7.00.

in Table 1, there is practically no response, because diffusional limitations control the supply of H2O2 to the Pt ring electrode. If a rotation of 900 rpm is imposed on the reactor located at the bottom of the cell (with immobilized GOx), the signal is dramatically amplified, as illustrated in the same table. As shown in Figure 3, if the lower reactor is devoid of rotation, the response is lower because diffusional limitations control the enzymecatalyzed reaction. Rotation of both reactors (implemented with a modified upper body cell) was tried earlier.6 However, this interferes with the transport of H2O2 to the point of detection, and the resulting signal is smaller than the one obtained with rotation of only the reactor located at the bottom of the cell. If the reactors are interchanged [i.e., the one with immobilized GOx is located at the top of the cell (fixed) and the one with GDH is located at the bottom and rotated], transport of the analyte to the detection point is also impaired, and the signal is about 30% lower. These observations point to prevalent hydrodynamic conditions with convective streamline transport similar to the one taking place at rotating disk electrodes with the radical modifications explained in a previous paper.7 Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

3681

Table 2. Effect of Reactor Rotation Velocity on Initial Rate, Measured under Continuous- and Stopped-Flow Conditions for (a) the Amplified System and (b) the Unamplified Systema continuous-flow programming

continuous-flow/stopped-flow/ continuous-flow programming

rotation initial linear regression velocity, rate, standard rpm µA‚s-1 deviation



170 240 420 840 900

2.20 6.50 8.18 10.20 14.25

(a) Amplified System (0.02 7.10 (0.01 16.33 (0.01 32.15 (0.03 64.10 (0.01 80.07

(0.05 (0.07 (0.02 (0.01 (0.01

170 240 420 840 900

0.02 0.05 0.11 0.20 0.40

(b) Unamplified System (0.01 0.20 (0.02 0.50 (0.01 1.20 (0.03 2.23 (0.01 4.25

(0.05 (0.02 (0.02 (0.03 (0.01

a Flow rate, 1.00 mL‚min-1; cell volume, 400 µL; sample size, 250 µL; glucose concentration, 0.10 mM; 0.10 M phosphate buffer of pH 7.00. The flow was stopped for 60 s during measurement. Each value of initial rate is based on triplicate of six determinations.

Table 3. Effect of Cell Volume on Initial Rate, Measured under Stopped-Flow Conditionsa unamplified system

amplified system

cell volume, µL

initial rate µA‚s-1


initial rate µA‚s-1


400 425 475 500 525 550 700 750 1000

5.20 4.50 2.20 1.23 0.25 0.12 0.05 0.0052 0.00015

(0.05 (0.02 (0.02 (0.03 (0.01 (0.02 (0.02 (0.0001 (0.00002

87.10 64.33 32.15 24.10 18.07 9.09 5.00 3.00 1.21

(0.05 (0.07 (0.02 (0.01 (0.01 (0.02 (0.05 (0.01 (0.02

a Each value of initial rate is based on triplicate of six determinations. In both cases, the reactor rotation velocity was 900 rpm; flow rate, 1.00 mL‚min-1, glucose concentration, 0.10 mM; 0.10 M phosphate buffer of pH 7.00. The flow was stopped for 60 s during measurement.

Figure 4. Effect of sample size on initial rate, measured under stopped-flow conditions. Each value of initial rate is based on triplicate of six determinations. In both cases, flow rate was 1.00 mL‚min-1, cell volume was 400 µL, glucose concentration was 0.10 mM, and 0.10 M phosphate buffer of pH 7.00 was used. The flow was stopped for 60 s during measurement. Table 4. Values of K′M (Apparent Michaelis-Menten Constant) for the GOx/GDH System (Determination As Discussed in the Text; Temperature 20 ( 1 °C) unamplified system rotation velocity, rpm 170 240 420 840 900

K′M, mMa

K′M, mMa


10.25 9.50 5.87 2.23 1.25

(1.54 (2.23 (1.29 (0.30 (0.10

0.051 0.033 0.015 0.010 0.007

(0.005 (0.007 (0.002 (0.001 (0.001

free enzymes (GOx/GDH) in Solutionb 12.00 (0.02 22.20

Table 2 shows the results obtained under continuous-flow and under continuous-flow/stopped-flow/continuous-flow programming for the amplified system (a) and the unamplified system (b). These responses indicate that the effectiveness of the biocatalytic action of the immobilized enzyme preparations is greater under rotation of the reactor at the bottom of the cell. The interplay of the different kinetic situations realized in these cases has been discussed by Richter et al.7 Effect of Cell Volume and Sample Size. Depending on the volume of the cell in contact with the reactors, the overall process becomes controlled by diffusion (large volumes) or by the chemical kinetics of the enzyme-catalyzed reactions (small volumes). The cell volume was changed from 400 µL to 1 mL by inserting a spacer between the upper and lower halves of the cell. The rate of response, as expected, decreased linearly with an increase in cell volume due to the dilution effect favored by rotation and the fact that the measured current is directly 3682 Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

amplified system


free enzyme (GOx) in Solutionb (0.05

a Each value of K′ is based on triplicate of six different substrate M concentration determinations. b K′M estimated by using the o-dianisidine glucose determination kit (Catalog No. 510-DA, Sigma, St, Louis, MO).

proportional to bulk concentration (Table 3). The smallest cell volume of 400 µL was adopted for further studies. As Figure 4 shows, in both cases the rate of response increases linearly with the sample size up to 250 µL. For convenience, a sample size of 250 µL was used to evaluate other parameters. The Apparent Michaelis-Menten Constant. As noted at the beginning of this section, rotation is expected to decrease the values of the apparent Michaelis-Menten constant, K′M, since the catalytic efficiency is increased, with a corresponding increase in initial reaction rate. Table 4 summarizes the values obtained for K′M in both systems obtained at five different rotation velocities. Flow was stopped for 60 s during measurement. The calculation

of K′M was performed under conditions in which [substrate] . K′M. As a consequence, the following applies, assuming that the Briggs and Haldane scheme12 is in operation:

Table 5. Comparison of Results Obtained with the Proposed Method and with the Method of Trindera Trinder method

(1/S) ) (m/[glucose]) + n where S is the rate of response and K′M ) m/n. The apparent constant is thus obtained from the slope and intercept of the plot of 1/S vs 1/[glucose]. This is a graphical approach similar to the Lineweaver-Burk plot. Effects of pH and Glucose Concentration. In reference4, the pH dependence of the electrode response, studied from pH 6.00 to 8.00, shows a maximum at pH 7.00 in phosphate buffer 0.1 M, containing 1.0 × 10-3 M sodium azide. The results of the experiments performed during this work reflect a different pH profile. The rate of response dramatically increased (almost doubled) from pH 6.50 to 7.00 and continued a moderate increase up to pH 8.00, the highest value tested. This behavior is probably the result of a combination of factors, such as changes in the properties of the immobilized enzymes, the buffer capacity in the vicinity of the enzyme active site, the type of immobilization, and the pH dependence of H2O2 detection.13 A linear relation was observed between the rate of response and the glucose concentration in the range of 1 nM-5 fM at 900 rpm. The equation of the regression line obtained at 900 rpm was

rate of response (nA‚min-1) ) 8.33 + 422.11Cglucose,mM The regression coefficient for plots of rate vs concentration was typically equal to 0.999, and the detection limit (estimated from 3 standard deviations of the blank) was ∼1.00 × 10-16 M. The relative standard deviation for 16 successive injections was 0.83% with a 5.00 nM solution of glucose and a constant NADH concentration of 1.00 × 10-3 M. Effect of NADH Addition. The rate of response increases linearly with NADH concentration and levels off above a concentration of 1.00 × 10-3 M (concentration considered to be optimum for the determination). No further amplification was observed above 1.00 × 10-3 M. Higher concentrations of NADH result in some decrease in signal, probably due to product inhibition in the GDH-catalyzed step due to saturation. The increase in rate in the linear region is expected to be proportional to the activity of immobilized GDH and to the conversion of gluconic acid to glucose; thus, for every glucose molecule, several peroxide molecules are produced. In this way, the current exceeds that found in the absence of NADH. The spontaneous hydrolysis of gluconic acid may explain this saturation behavior at high NADH concentrations, and the signal follows the following relationship between 1 × 10-2 and 2 × 10-3 M:

rate of response (nA‚min-1) ) 15.33 + 488.60CNADH,mM with a regression coefficient of 0.997. (12) Briggs, E. G.; Haldane, J. B. S. Biochem. J. 1925, 19, 338-339. (13) Baeza Baeza, J. J.; Matsumoto, K.; Mottola, H. A. Quim. Anal. 1993, 12, 12-17.

day

glucose, g‚L-1

0 3 5 7 9 10 11

4.43 3.36 2.56 2.24 no reaction no reaction no reaction

linear regression standard deviation (0.02 (0.03 (0.01 (0.01

proposed method glucose, g‚L-1


4.50 3.42 2.53 2.18 2.63 × 10-4 5.50 × 10-7 1.90 × 10-8

0.01 0.01 0.03 0.01 2.1 × 10-6 3.0 × 10-9 1.1 × 10-10

a Trinder, P. Ann. Clin. Biochem. 1969, 6, 24-27. Determination of glucose was done in culture broth of Pseudomonas mendocina. Flow rate, 1.00 mL‚min-1; cell volume, 400 µL; sample size, 250 µL; 0.10 M phosphate buffer of pH 7.00. The flow was stopped for 60 s during measurement. Each value of initial rate is based on triplicate of three determinations.

Determination of Glucose in Culture Broth. Glucose was determined in a culture broth of Pseudomonas mendocina, a bacillus Gram-negative of ample environmental distribution. It shows great avidity for the carbohydrates used (mainly glucose) during its logarithmic growth. The determination of such low concentrations requires a very sensitive method for the evaluation of the changes produced in the bacterial cell. An experiment was designed inoculating the microorganisms in tubes containing 20 mL of the culture broth from a subculture in inclined agar for 18-24 h, followed by incubation at 35 °C during 11 days. After this, aliquots were taken periodically and centrifuged at 13 000 rpm during 1 h in order to separate the bacterial biomass, and the supernatant was analyzed. Table 5 shows the results obtained after applying the Trinder method, compared with results obtained using the approach recommended here. According to the enzymatic method of Trinder, after the seventh day of cultivation there is no detectable glucose. Nevertheless, we demonstrated the existence of the carbohydrate when the method developed here was applied. CONCLUSIONS The usefulness of enzymatic amplification by substrate cycling for the determination of very low concentrations of glucose is demonstrated. In practice, such a determination is of interest in clinical chemistry and fermentation technology. The monitoring of glucose in extracellular fluid is presented to illustrate that the proposed approach (the use of rotating bioreactors and continuous-flow/stopped-flow processing) is capable of providing quantitative information when the conventionally used methods fail. The simple strategy presented here can equally well be applied to immunoenzymatic determinations and provides a fast and cost effective means of providing quantitative information at extremely low concentrations levels. ACKNOWLEDGMENT The authors appreciate financial support from the Universidad Nacional de San Luis and the Consejo Nacional de Investigaciones Cientı´ficas y Tećnicas (CONICET), Lic. Hugo J. Centorbi (Area Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

3683

de Microbiologıá. U.N.S.L.) for providing the culture broth of Pseudomonas mendocina. One of the authors (A.V.L.) acknowledges support in the form of a fellowship from the Universidad Nacional de San Luis, Argentina.

3684 Analytical Chemistry, Vol. 70, No. 17, September 1, 1998

Received for review July 29, 1997. Accepted May 12, 1998. AC9708194

Stopped-Flow and Rotating Bioreactors in the

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