Analytical Enzyme Reactors

For reasons of economy, application of enzymes as immobilized, i.e. reusable, preparations in clinical chemistry and process control has become increasingly common. Three basic types of analytical enzyme reactor have been described (Mottola, 1983)  

In packed bed reactors the enzyme-catalyzed reaction is carried out in a column of 100 pl-10 ml volume. The column is filled with tiny particles bearing the immobilized enzyme. The continuously formed reaction product is indicated colorimetrically or electrochemically. Enzyme carrier materials with advantageous flow behavior are porous glass with pores of a defined size, organic polymers, like nylon powder, and inorganic polymers. 

In single bead string reactors the enzyme is bound both to the reactor wall and to a carrier within the reactor. Compared with open tubular reactors this reactor type provides a higher conversion at lower sample mixing. 

The combination of an enzyme reactor with direct electrochemical product indication provides a relatively simple flow-through setup. 

for glucose measurement excellent parameters such as a coefficient of variation (relative standard deviation, CV) of 0.2-0.6%, have been achieved by using a flow injection analysis (FIA) device combined with a GDH reactor and an electrode modified for NADH indication (Fig. 39) (Appelqvist et al., 1985). 

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Such interferences can be compensated by using difference measurements, which usually require a reference transducer. An elegant alternative is the use of enzymatic anti-interference systems containing enzymes in front of the sensor that catalyze the conversion of the disturbing compounds to inert products. Such systems have been developed in conjunction with analytical enzyme reactors as well as enzyme electrodes. 

By incorporating the entire analytical scheme (enzyme reaction and electrochemical detection) into the flow system a great improvement in precision can be realized. Sample manipulation is minimized because only a single injection into the flow system is required versus sampling of aliquots for the off-line method. Precision is also improved because the timing of the enzyme reaction and detection are much better controlled in the flow system. Finally, less of both enzyme and sample are needed with on-line enzyme reactor methods. 

L. Korecka, Z. Bilkova, M. Holeapek, J. Kralovsky, M. Benes, J. Lenfeld, N. Mine, R. Cecal, J.-L. Viovy, and M. Przybylski, Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments. Journal of Chromatography, B Analytical Technologies in the Biomedical and Life Sciences 808, 15-24 (2004). 

L Gorton, G Marko-Varga. In S Lam, G Malikin, eds. Analytical Applications of Immobilized Enzyme Reactors. London Blackie Academic Professional, Chapman Hall, 1994, pp 1-21. 

System C is used when an immobilized enzyme reactor (IMER) is introduced into system B. The analyte(s) separated by HPLC is converted to a suitable species for CL detection with an IMER, and then mixed with the CL reagent. In this system, a buffer solution as a mobile phase and an ion-exchange-type column are preferable for an enzyme reaction. 

Enzymes can be used in several ways in chromatographic applications to improve selectivity or to enhance the detector response. Applications may involve enzymes with either a broad specificity toward a group of related compounds or a high specificity toward a particular compound. In the field of drug residue analysis, most current applications concern enzymatic reactions taking place in separate reactors incorporated in LC systems before or after the analytical column. Reactors with immobilized enzymes have proven to be suitable in such continuous flow systems. 

Yang L, Janie E, Huang T, Gitzen J, Kissinger PT, Vreeke M, Heller A. Applications of wired peroxidase electrodes for peroxide determination in liquid chromatography coupled to oxidase immobilized enzyme reactors. Analytical Chemistry 1995, 34, 1326-1331. 

The recent developments in the determination of acetylcholine and choline, the advantages and the disadvantages of a variety of analytical methods used in the analysis, were reviewed and discussed by Maruyama et al. [7]. Hanin published an overview for the methods used for the analysis and measurement of acetylcholine [8], Shimada et al. presented a review, including the applications of some reactors in the analysis of choline and acetylcholine. The immobilized enzyme reactors were used for detection systems in high performance liquid chromatography [9],... 

Yao et al. reported a flow injection analytical system for the simultaneous determination of acetylcholine and choline that made use of immobilized enzyme reactors and enzyme electrodes [25]. Acetylcholineesterase-choline oxidase and choline oxidase were separately immobilized by reaction with glutaraldehyde onto alkylamino-bonded silica, and incorporated in parallel as the enzyme reactors in a flow injection system. The sample containing acetylcholine and choline in 0.1 M phosphate buffer (pH 8.3) carrier solution was injected into the system. The flow was split to pass through the two reactors, recombined, and mixed with 0.3 mM K4Fe(CN)6 reagent solution before reaching a peroxidase immobilized electrode. Because each channel had a different residence time, two peaks were obtained for choline and total acetylcholine and choline. Response was linear for 5 pM-0.5 mM choline, and for 5 pM 1 mM acetylcholine plus choline. The detection limits were 0.4 pM for choline and 2 pM for acetylcholine. 

Analytical column and post column immobilized enzyme reactor supplied as part of a ACh/Ch assay kit. Sodium phosphate buffer pH 8.5 containing Kathon CG (Rohm and Haas, PA, USA), [1 mL/min]. Electrochemical at + 0.5 V versus Ag/AgCl. Human plasma and peritoneal dialysis effluent. [185]... 

L. Gorton, G. Marko-Varga, E. Dominguez and J. Emneus, In S. Lam and G. Malikin (Eds.), Analytical Applications of Immobilised Enzyme Reactors. Blackie Academic Professional, London, 1994, pp. 1-21. 

An enzyme electrode is basically a dense package of dialyzer, enzyme reactor, and electrode (detector). Enzymes introduce analytical selectivity due to the specificity of the signal-producing interaction of the enzyme with the analyte. They enhance the equihbrium formation of chemical reactions. For example, splitting of H2O2 is accelerated by a factor of 3 x 10 in the presence of catalase. Turnover numbers can be as fast as 6 X 10 s (carbonic anhydrase) where cat/ m approaches the diffusion limited value of 10 s ... 

The manifold into which the upper chamber is inserted does not depend on the initial state of the sample, but only on the characteristics of the pervaporated analytes, the type of detector used and its position along the manifold. Depending on the particular type of detector used, auxiliary channels will have to be included to bring the pervaporated species into contact with appropriate reagents in order to obtain products to which the detector will respond. Integrated detection and pervaporation requires altering the pervaporator but simplifies the overall manifold. As shown below, preconcentration units, solid-phase reactors (mainly enzyme reactors) and various other devices can also be connected in-line in the manifold when required. 

For packed-bed enzyme reactors, these results show that low flow rates ensure quantitative conversion of substrate into detectable products. The variation of K m with flow rate indicates that lower flow rates produce higher K m values, so that the linear region of the saturation kinetic curve extends to higher substrate concentrations at lower flow rates. This effect becomes significant when complete conversion does not occur during the residence time of the analyte. 

Lam, S., Mallikin, G., Eds. Analytical Applications of Immobilized Enzyme Reactors Blackie Academic and Professional Glasgow, U.K., 1994. 

L. Ogren, Enzyme Reactors in Analytical Detection Systems. Theory and Applications. Univ. of Lund, Sweden (1981). (Ph.D. Thesis). 

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