Flow injection analysis and in-line biosensors for bioprocess control: a comparison

Journal of Biotechnology, 25 (1992)75-80

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© 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1656/92/$05.00

BIOTEC 00777

Flow injection analysis and in-line biosensors for bioprocess control: a comparison H. Liidi, M.B. G a r n , S.D. H a e m m e r l i , A. M a n z a n d H . M . W i d m e r CIBA-GEIGYAG, Analytical Research, CH-4002Basel, Switzerland

(Received 5 January 1991; revisionaccepted 29 April 1991)

Summary Miniaturization will unify the different approaches chosen for the application of biosensors in bioprocess control. The most versatile system, which in our opinion is flow injection analysis will be the method of choice for the introduction of biosensors in bioprocess control. A lot of experience will be gained for the future development of miniaturized total chemical analysis systems. Flow injection analysis; Bioprocess control; Biosensors

Introduction The complex chemical reactions occurring during growth of microorganisms or ceils used in biotechnological processes are scarcely controlled. Progress is expected from the emerging fields of biosensors and process metrology to get better yields and a constant product quality. The real time analysis of nutrients, metabolites and product(s) is primarily envisaged. With respect to this task, an on-line technique (flow injection analysis) and the in-line use of biosensors are compared. Future developments of both approaches will be outlined with special regard to miniaturization. Correspondence to (and present address): H. Liidi, Ciba Corning Diagnostics Corp., Research & Devel-

opment, 63 North Street, Medfield, MA 02052, USA.

76 The Comparison

Biochemical sensors are often considered as a future t o o l f o r bioprocess control. Most biotechnologists then think of sensors similar to the commonly used pH- or pO2-electrodes. The calibrated sensor is introduced into the bioreactor, sterilized and then measures e.g. glucose or ethanol. The sensor output is amplified using an already available control unit equipped with an additional board. A recent example of this approach is the glucose sensor proposed by Brooks et al. (1987/88), was tried to be commercialized by BIOTRACE. On the other hand, flow injection analysis has proven to be a useful method for on-line process monitoring and control. Different applications have been installed in production plants (see e.g. Gisin and Thommen, 1986). Using flow injection analysis implies to install an analytical instrument containing a sampling device, valves, pumps, detector, reagent bottles etc. as an additional system next to the bioreactor. Furthermore one is obliged to care about a new analytical method, a new instrument and its maintenance. The situation might be compared to the installation of a mass spectrometer for off-gas analysis. The two approaches are further illustrated in Figs. 1 and 2. First experimental data comparing these methods during yeast cultivation have recently been published by Filippini et al. (1991). Though the combination of flow injection analysis and biosensors has been proposed by several authors (see e.g. Brand et al., 1990; Dremel et al., 1989; Lfidi et al., 1990), the two approaches described above represent a different 'analytical philosophy'. The electrode-like system is mainly based on engineering and mechanical tools in combination with a biological recognition system, whereas in flow injection analysis, in addition, a profound knowledge of wet chemical reactions and gradient techniques ( G a r n e t al., 1988) is required. The two main drawbacks of biosensors if used for in-line bioprocess control are the difficulties to sterilize the sensor and the presence of large amounts of interfering and inhibiting substances in a cultivation media. In flow injection analysis, these problems are solved by the introduction of a by-pass containing the sterile barrier, by sample preparation modules, such as variable dilution with a stirred mixing chamber and by using gradient techniques (Fig. 2). In the electrodelike system, the sensor is placed behind a sterilizable membrane just after sterilization and sample preparation is achieved by dialysing the sample through this membrane and in most cases by permanently flushing the sensor with a liquid stream pumped through the 'electrode housing' containing the sensor (Fig. 1). A more detailed comparison is given in Table 1. Depending on the problem and preference for one or the other approach, the statements given in the table are judged as advantages or disadvantages. But it is obvious that the following features, common to both systems, have to be considered to get reliable results over a reasonable period of time under sterile conditions: - sample preparation is required due to interfering and inhibiting substances present in cultivation media;

77 ............

m

b .............

S

Fig. 1. In-line glucose electrode. The figure shows an in-line electrode according to Brooks et al. (1987/1988). S, selector valve; D, detector (enzymatic membrane fixed at electrode); I, data treatment and interpretation. - the sensor must be introduced behind a sterile barrier; and - re-calibrations are necessary for long time reliability and precision/accuracy.

The Trend: Miniaturization

The improvement of analytical performance due to miniaturization of 'total chemical analysis systems' has recently been demonstrated by Manz et al. (1990). In addition the advantages of miniaturized flow injection analysis systems has been proven by Ruzicka and Hansen (1988). With respect to miniaturization the common features of both approaches described above play a very crucial role. Their advantages might now be combined, i.e. the versatility of flow injection analysis and the ease of handling and small size of in-line biosensors. Miniaturization therefore must include sample preparation and liquid handling. A modular system giving the flexibility of flow injection analysis and probably containing

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c__0_/

D

Fig. 2. On-line glucose analyzer using flow injection analysis. On-line glucose analysis using a by-pass and a flow injection system according to G a r n e t al. (1989). F, filter unit; S, standards; MC, stirred mixing chamber (variable dilution); GOD, enzyme reactor containing immobilized glucose oxidase; D, detector; I, data treatment and interpretation. TABLE 1 Comparison of 'electrode-like' sensors and flow injection approach Electrode-like system

Flow injection analysis

Handling

Easy; in situ measurement

Requires know-how in FIA technique; by-pass must be installed

Required skills

Low

High skills required tO start with; 'new' analytical technique

Maintenance

Easy

Easy with high-tech equipment

Sterility

Sterile membrane

Sterile membrane

Flexibility

Low

High; modular construction of sample preparation, derivatization and detection possible

Reliability

Not proven; performance of sterilizable membrane cannot be controlled

Proven by on-line applications

Information content of signal

Small; but constant signal

High; peak shape determinations allows multiple diagnosis

Dynamic range

Depending on several parameters such as flushing rate

5-6 orders of magnitude due to gradient techniques

Re-calibration

Possible, but does not include control of sterilizable membrane (permeation properties)

Easy, does not include control of sterile membrane, but this is not necessary if a sufficient flow rate is guaranteed

Multi-component analysis

Possible, but difficult

Easy due to gradient techniques

79 REAGENT 1 REAGENT2

STANDARD CARRIER 2

DETECTOR MIXINGCOIL MIXINGCOIL PUMP (REAGENS 2) PUMP (REAGENS 1) VALVE PUMP (CARRIER2) PASSIVE MIXER VALVE PUMP (CARRIER I) PUMP (GAS) GAS BUBBLEREMOVER PUMP (STANDARD) PUMP (SAMPLE)

SAMPLE Fig. 3. Conceptional on-line glucose analyzer. Several structured plates are stacked to realize an on-line glucose analyzer. Bold line: sample flow for injection, for more details see Manz (1990). biochemical sensors, but allowing, due to the small size, the introduction i n / a t the bioreactor will be the ultimate goal. It is concluded that miniaturization will finally lead to a 'unified' analytical system.

A Novel Concept A future approach, using a different geometry, than proposed by Ruzicka and Hansen (1988), may look as follows (Fig. 3): Up to several dozens of structured plates containing holes and channels are stacked together to give a defined flow path. Each, in some cases several, of the structured plates contain a defined function such as a pump, a valve, a mixing chamber, a reaction coil or a detector (sensor). The sample is introduced from one side, whereas all the other carrier

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liquids, mobile phases, reagents, optical fibers and electric wires are connected from the opposite side. The size of the entire system could be in the order of 5×5×30 cm (0.75 1) down to 3 x 3 × 2 0 mm (180 ~1). Figure 3 shows a conceptional picture of an on-line glucose analyzer based on a bulk enzymatic reaction with subsequent optical detection of hydrogen peroxide. The flow injection analysis system involved is described by Garn et al. (1989) and a detailed description of the concept including first experimental data will be published elsewhere (Fettinger et al., 1991). As the figure suggests, such a structure would nicely fit into a standard electrode housing.

References Brand, U., Reinhardt, B., Ri.ither, F., Scheper, T. and Schiigerl, K. (1990) Bio-field-effect transistors as detectors in flow-injection analysis. Anal. Chim. Acta 238, 201-210. Brooks, S.L., Ashby, R.L., Turner, A.P.F., Calder, M.R. and Clarke, D.J. (1987/88) Development of an on-line glucose sensor for fermentation monitoring. Biosensors 3, 45-56. Dremel, B.A.A., Schaffar, B.P.H. and Schmid, R.D. (1989) Determination of glucose in wine and fruit juice based on a fibre-optic glucose biosensor and flow-injection analysis. Anal. Chim. Acta 225, 293-301. Fettinger, J.C., Manz, A., Liidi, H. and Widmer, H.M. (1991) Stacked modules for micro flow systems in chemical analysis: concept and studies using an enlarged model. Sensors and Actuators, submitted. Filippini C., Sonnleitner, B., Fiechter, A., Bradley, J. and Schmid R. (1991) On-line determination of glucose in biotechnological processes: comparison between FIA and in situ enzyme electrode. J. Biotechnol. 18, 153-160. Garn, M.B., Gross, H., King, P., Schmidt, W. and Thommen, C. (1988) Extensive flow-injection dilution for in-line sample pretreatment. Anal. Chim. Acta 207, 225-231. Garn, M., Gisin, M., Thommen, C. and Cevey, P. (1989) A flow injection analysis system for fermentation monitoring and control. Biotechnol. Bioeng. 34, 423-428. Gisin, M. and Thommen C. (1986) Industrial process control by flow injection analysis. Anal. Chim. Acta 190, 165-176. Liidi, H., Garn, M.B., Bataillard, P. and Widmer, H.M. (1990) Flow injection analysis and biosensors: applications for biotechnology and environmental control. J. Biotechnol. 14, 71-79. Manz, A. (1990) Vorrichtung zur Aufbereitung oder Vorbereitung von fliissigen Proben fiir eine chemische Analyse. Patent Application CH 67-18419, Switzerland. Manz, A., Graber, N. and Widmer, H.M. (1990) Miniaturized total chemical analysis systems: a novel concept for chemical sensing. Sensors and Actuators B1,244-248. Ruzicka, J. and Hansen, E.H. (1988) Flow Injection Analysis, 2nd Ed. Wiley, New York.