Development of a sucrose enzymatic biosensor

Biotechnology Techniques, Vol 12, No 4, April 1998, pp. 305–307

Development of a sucrose enzymatic biosensor A. M. Salgado., R. O. M. Folly, B. Valdman and F. Valero Departamento de Engenharia Quimica - Escola de Quimica, Universidade Federal do Rio de JaneiroRio de Janeiro C. T. - BI E sl 211 Brazil Departament d’Enginyeria Quimica, Universitat Autonoma de Barcelona - 08193 - Bellaterra Spain An enzymatic biosensor for sucrose determination was developed for on-line and continuous monitoring of sucrose concentration. The sensor was adapted to two different measurement schemes, one continuous and another injection sampling lines. The sensor adapted with the injection sampling line presented a linear measurement range of 5–20 g sucrose/1, good reproducibility, and a high versatility permitting the substitution of the immobilized enzymes when their activity decreased.

Introduction An enzymatic biosensor for sucrose determination has been developed and used in association with a continuous line and a FIA (Flow Injection Analysis) system because of its reliability, high sampling frequency and flexibility (Ruzicka and Hansen, 1988). The biosensor consists of a measuring chamber (Folly et al., 1996) packed with invertase and mutarotase immobilized on glass beads, allowing its connection to a pH electrode that measures the pH variation ocurring during sucrose measurement. The potentiometric biosensor adapted to the continuous system and FIA system was used to monitor the concentration of sucrose solutions, and calibration curve and the static and dynamic characteristics of the sensor were determined.

Injection sampling line The biosensor was connected to the FIA system by tubings of PTFE (0.8 mm i.d) and silicon rubber ( 2.0mm, 4.0mm and 6.0mm i.d) joined by PVC fittings. Distilled water used as the carrier solution was conducted by a multichannel peristaltic pump. The sample was injected in the flow using a 4-way rotating injection valve. The pH change was measured with the same electrode-transmitter system used in the continuous line. The analysed solutions were held at 45°C before entering the reactor. The water flow rate through the reactor was kept at 3.5 and 4.5 ml/ min. Sample solutions Pure sucrose solutions of 6.1 pH units and 0.2 to 50 g sucrose/1 were used as sample solutions.

Materials and methods Potentiometric sensor This comprised a micro-reactor of 1.3 ml measuring chamber with a packing of immobilized, cross-linked invertase and mutarotase allowing the continuous flow of the solution to be analysed. A pH electrode was mounted at the top of the chamber.

Results The complete scheme for the sucrose biosensor is presented in Figure 1. The Figure 1-(A) presents the continuous sampling line scheme used in the experiments for the determination of the dynamic profile and the Figure 1-(B) presents the scheme of the biosensor adapted to a FIA sampling line system used in the determination of the calibration curve of the developed biosensor.

Continuous sampling line The continuous sampling line of silicon rubber (4.0 and 6.0 mm i.d) permitted a sample to be taken by a continuous pump from the enzymatic reactor. The pH change was measured with a pH electrode attached to a SmarpHT-101 transmitter and registered with a recorder. The analysed solutions were held at 45°C before the sample entered the reactor. The flow rate of the sample through the reactor was kept at 3.0 ml/min.

Increasing concentrations of the feed solution were evaluated using this continuous sampling line and the pH values obtained in the outlet of the reactor were measured (Figure 2). Each sucrose solution was pumped through the reactor for 20 min and the variation in pH value obtained was measured every 3 min as difference of potential (d.d.p) of the electrode. The response time observed in the output to attain 98% of its final value was 5 minutes for each disturbance.

© 1998 Chapman & Hall

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Figure 1 Sampling line schemes used for sucrose measurements. (A)- Continuous flow system; (B) FIA system. P 5 pump, C 5 coil, B 5 sucrose biosensor, M 5 pH transmitter, R 5 recorder, v 5 two-way valve, S 5 sucrose solutions , W 5 warming bath, V 5 fourway rotary injection valve, T 5 carrier solution Figure 3 fm11Calibration curves of the sucrose biosensor with the FIA sampling line and a carrier flow of 3.5 ml/min (d) and 4.5 ml/min (n).

Figure 2 Dynamic performance of the sucrose biosensor with the continuous sampling line for sucrose solutions of 0.2 to 3.0 g sucrose/l concentration range.

The sensitivity or gain of the sensor estimated as the final difference of potential attained for each concentration change was of 25 mV/l.g. This gain was obtained when the continuous sampled flow through the biosensor was of 3.0 ml/min. The calibration curve was obtained by injecting sucrose solution samples of 30 ml (Qs) in the range of 5–50 g sucrose/1 by means of the rotary injection valve (V) into the continuous water carrier line (Qc) for both 3.5 and 4.5 ml/min flows. The injections were introduced in cycles of ascendent and descendent concentration ranges and pH values obtained for each injection were recorded as the difference of potentials of the electrode. The water carrier solution was of pH 5.4.

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The calibration curves obtained for the biosensor with the FIA system are seen in Figure 3 for carrier flows of 3.5 and 4.5 ml/min. The observed characteristic was the extension of the linear working range up to 20 g sucrose/1 with the increased carrier flow. The detection limit was 5.0 g sucrose/1 (calculated as 3 times the standard deviation of the background noise of the system) with a correlation coefficient in the linear range of 0.9. The sensitivity or gain of the sucrose biosensor with the FIA system was estimated as 5.62 mV/l.g, for a relative standard deviation of 12% and a good reproducibility was observed in all experiments performed. Conclusions The developed biosensor coupled to a continuous sampling line showed a good reproducibility, a linear gain of 25 mV.l/g for a concentration range of 0.2–3.0 g sucrose/1, and a response time of 5 min. The same biosensor coupled to the FIA system is capable to measure sucrose concentrations within a range of 5–20 g sucrose/1 presenting good linearity and reproducibility and a gain of 5.2 mV.l/g. The developed biosensor is very easy to install and adapt to a fermentor and has an economic operation because it does not use enzymatic or other reagent carrier lines, such as those used in other sucrose FIA analysing systems. References Folly, R.O.M., Valdman, B., Valero, F.and Sol´a, C.(1996). Biotech. Tech.,10, 867–870.

Development of a sucrose enzymatic biosensor Sharmat, A.and Rogers, K.(1994). Meas. Sci. Technology, vol. 5, pp 461–472.

Ruzicka, J.and Hansen, E.H.(1988). Flow Injection Analysis, 2nd ed., Wiley, New York.

Received: 14 November Revisions requested: 12 December 1997/21 January Revisions received: 20 January 1998/2 March Accepted: 3 March

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