THE DEVELOPMENT OF ENZYMATIC SENSORS FOR THE CONTINUOUS MONITORING OF GLUCOSE AND SUCROSE

THE DEVELOPMENT OF ENZYMATIC SENSORS FOR THE CONTINUOUS MONITORING OF GLUCOSE AND SUCROSE

R. Folly ¹ , A. Salgado ¹ , B. Valdman ¹ and F. Valero ²

¹Escola de Química/UFRJ - Cidade Universitária, C.T. Bloco E sala 211,CEP- 21945-900,

Rio de Janeiro - Brazil - phone:[phone]/fax:[phone]

E-mail [e-mail]

²Unitat d’Enginyeria Química, Universitat Autónoma de Barcelona - Bellaterra 08193 - Spain

E-mail: [e-mail]

(Received: June 11, 1997; Accepted: October 30, 1997)

Abstract - Enzymatic sensors for the continuous measurement of glucose and sucrose, with the use of an enzymatic microreactor and a continuous flow dilution line for the medium, are studied in this work. The effects of the best conditions for medium and sampling flow through the sensor were analysed for a glucose concentration range from 0.05 to 0.2 g/l , which was possible to extend to a range from 5.0 to 120 g/l using a dilution and sampling circuit with the FIA analysis methodology.

Keywords: Development, continuous monitoring, enzymatic sensors.

INTRODUCTION

The development of sensors and analysing systems that allow the quick and precise measurement of glucose and sucrose concentrations at a low cost is of great importance in many applications in the medical area, as well as in industrial fermentations and the food industry. In the industrial area, the great interest is concentrated in the development of analytical instruments which can measure "on line" the substances that are present in bioreactors, thus permitting the monitoring and control of bioprocesses that use glucose and/or sucrose as the substrate. Biosensors based on enzymatic reactions connected with analysing systems have been largely used for this purpose, mainly because of the selectivity for the substrate and low response times that these sensors present as their main characteristics (Sethi, 1994 ; Alva et al., 1991).

The biosensor can be defined as an analytical instrument which uses a biological catalyst in close contact with a suitable transducer that converts the biochemical signal that is produced in a biological reaction into a quantifiable electrical signal (Turner et al., 1987 ; Bowe, 1985). As typical biological components different materials such as enzymes, organelles, membrane components and whole cells are used. As transducers the most frequently utilized are: potentiometric and amperometric electrodes, termistors and optical receptors. The biological component is normally immobilized in close contact with the transducer surface to increase the response, decrease the interferences and allow its reutilization. After the conversion of the biochemical signal into a quantifiable electrical signal by the transducer, it is itself measured by a biochemical detector which can be easily connected to a computer or to an automated digital system (Brooks et al., 1991).

The high level of substrate concentration present in the bioreactors is the main limitation of the use of enzymatic biosensors, as most of the enzymatic reactions occur in low concentrations of substrate. Thus, a dilution to an optimum concentration for the enzymatic reaction and a draining system are needed for the sample withdrawn from the bioreactor. This step of dilution must be well developed, and thoroughly investigated, because it can introduce signal oscillations producing analysis errors that will certainly change the final results (Valero et al., 1990).

This work presents the experimental assays carried out for the development of enzymatic sensors of glucose and sucrose, based on enzymatic reactions that convert glucose by glucose oxidase and sucrose by invertase (Xu et al., 1989).The development of the sampling line is presented and the results of calibration and the influence of operating conditions are analysed. The concentration of glucose analysed in the calibration tests of the glucose biosensor covered the range from 0.05 to 0.2 g/l , which was possible to extend up to a range from 5.0 to 120 g/l of glucose , using a sampling circuit with the FIA methodology analysis.

EXPERIMENTAL SETUP AND RESULTS

The enzymes utilized in this work were glucose oxidase and invertase from Sigma and the glucose and sucrose solutions were prepared with analysis grade reagents. For analysis and protein determination the Lowry Method was used , and for the measurement of the enzyme conversion the colorimetric method, based on the Merck glucose oxidase kit, was utilized. The immobilization method for the enzyme in the Sigma glass-supported aminopropyl was of the covalent junction type utilizing 2.5% glutaraldeid (Valdman et al., 1992). The instruments utilized in the experimental tests were specified in each test presented.

The first development stage was a preliminary study of the best conversion conditions for the enzymes utilized. Preliminary experiments were carried out in order to define optimal values for temperature, pH and range of glucose and sucrose concentrations. The results obtained and analysed for the sucrose conversion by the invertase are shown in Figures 1a, 1b , 1c.

An enzymatic sensor developed to continuously measure glucose concentration in a Fermenter (Folly, 1996) was actually been extended to measure sucrose concentrations based on the excellent results described previously.

The glucose sensor consisted of a fixed-bed microreactor with glucose oxidase immobilized in glass pearls and through which the sampling fluid was pumped with a Milan peristaltic pump. In this microreactor, a pH electrode (Digimed) was installed to measure the pH change occuring in the glucose reaction present in the solution flowing through the microreactor with the immobilized glucose oxidase. The pH of the reaction was measured with a pH industrial transmitter (Smar). Experiments were carried out to define the optimal operational conditions of the enzymatic sensor. The best values for reaction temperature, pH and glucose solution concentration of the fluid flowing through the microreactor were then determined. The results obtained in these experiments are shown in the Figures 2a , 2b , 2c.

Figure 1a: Effects of temperature changes on sucrose conversion.

Figure 1b: Effects of sucrose concentration changes on conversion at 45 ^oC.

Figure 1c: Effect of pH changes on sucrose conversion at 45° C.

Figure 2a: Effects of temperature changes on glucose conversion.

Figure 2b: Effects of glucose concentration changes on conversion at 37° C.

Figure 3 shows the schematic diagram of the continuous sampling line developed and utilized for the dynamics tests and the calibration of the sensor. Figure 4 shows the calibration curve obtained for the best specified range of concentration obtained from 0.05 to 0.2 g/l, and Figure 5 presents the response curve obtained for a step change input in the glucose solution concentration from 0.1 to 0.2 g/l.

To extend the utilization range of the sensor, another sampling line was developed adapted to an FIA system using the Splitter Technique (Valero et al.,1991). The sampling flow was measured by the sensor of 7.25 ml/min, which made possible an increase in the glucose concentration range to 5 to 120 g/l. The sampling line developed with this methodology is shown in Figure 6 and is composed of one Masterflex eight-channel peristaltic pump , PTFE tubes with ID= 0.8 mm , Omnifit four-way injection valves and a pH meter with a Pharmacia recorder. The calibration curve obtained for this sensor adapted to an FIA system is shown in the Figure 7.

Figure2c: Effects of pH changes on glucose conversion at 37° C.

Figure 3: Schematic diagram of the continuous sampling line.

Figure 4: Calibration curve for the glucose sensor.

Figure 5: Response curve for the dynamics test of the glucose sensor.

Figure 6: Schematic diagram of the sampling line of the sensor adapted to an FIA system.

Figure 7: Calibration curve for the sensor adapted to a FIA system.

CONCLUSIONS

Tests showed that changes in medium conditions had a considerable influence on the conversion of sucrose solutions by invertase. The buffer acetate with a pH of 5.0 and a temperature of 50° C were the most favourable conditions for larger conversions (50% sucrose).

The biosensor used for measurement of glucose shows very good results, both with continuous measurements and in glucose injection measurements. For continuous flow of the sampling fluid, the concentration range used was from 0.05 to 0.2 g/l, and this range was extended to 5.0 to 120 g/l with the adaptation of this sensor to FIA methodology.

In both cases, the sensor presents a response time of 10 minutes. A similar methodology can be studied for application of a multienzymatic microreactor (glucose oxidase , invertase and mutarotase enzymes) for the continuous measurement of sucrose.

REFERENCES

Alva, S.; Gupta, S.S.; Phadke, R.S. and Govil, G., Glucose Oxidase Immobilized Electrode for Potentiometric Estimation of Glucose, Biosensors & Bioelectronics, 6, p.663 (1991).

Bowe, C.R., An Introduction to the Concepts and

Technology of Biosensors, Biosensors, 1, p.3 (1985).

Brooks, S.L.; Higgins, I.J.; Newman, J.D. and Turner, A.P.F., Biosensors for Process Control, Enzyme Microb. Technology, 13, p.946 (1991).

Folly, R.O.M., Projeto e Desempenho de Biosensor Enzimático de Glicose, Ph.D.diss., Universidade Federal do Rio de Janeiro, Brazil (1996).

Sethi, R.S.,Transducer Aspects of Biosensors, Biosensors & Bioelectronics, 9, p.243 (1994).

Turner, A.P.F.; Karube, I. and Wilson, G.S., Biosensors: Fundamentals & Applications, Oxford University (1987).

Valdman, B.; Folly, R.O.M.; Leite, S.F. and Rocha-Leão, M.H.M., Desenvolvimento de Biosensores, 7^o Sem. Instrum. SINST/PADCT, FINEP(ed), São Paulo, Brazil (1992).

Valero, F.; Lafuente, J.; Poch, M. and Solá, C., On-Line Fermentation Monitoring Using Flow Injection Analysis, Biotechnology & Bioengineering, 36, p.647 (1990).

Valero, F.; Poch, M.; Solá, C.; Santos Papa, R.A. and Costa Lima, J.L., On-Line Monitoring of Lipases Production in Fermentation Process, Biotechnology Techniques, 5, p.251 (1991).

Xu, Y.; Guilbault, G.G. and Kuan, S.S., Sucrose Enzyme Electrode, Analytical Quemistry, 61, p.782 (1989).

• Brooks, S.L.; Higgins, I.J.; Newman, J.D. and Turner, A.P.F., Biosensors for Process Control, Enzyme Microb. Technology, 13, p.946 (1991).
• Folly, R.O.M., Projeto e Desempenho de Biosensor Enzimático de Glicose, Ph.D.diss., Universidade Federal do Rio de Janeiro, Brazil (1996).
• Turner, A.P.F.; Karube, I. and Wilson, G.S., Biosensors: Fundamentals & Applications, Oxford University (1987).
• Valdman, B.; Folly, R.O.M.; Leite, S.F. and Rocha-Leăo, M.H.M., Desenvolvimento de Biosensores, 7^o Sem. Instrum. SINST/PADCT, FINEP(ed), Săo Paulo, Brazil (1992).
• Valero, F.; Poch, M.; Solá, C.; Santos Papa, R.A. and Costa Lima, J.L., On-Line Monitoring of Lipases Production in Fermentation Process, Biotechnology Techniques, 5, p.251 (1991).
• Xu, Y.; Guilbault, G.G. and Kuan, S.S., Sucrose Enzyme Electrode, Analytical Quemistry, 61, p.782 (1989).

Publication Dates

• Publication in this collection
  06 Oct 1998
• Date of issue
  Dec 1997

History

• Accepted
  30 Oct 1997
• Received
  11 June 1997