[42] The applications of immobilized enzymes in automated analysis

[42]

AUTOMATED ANALYSIS

633

A calibration curve was constructed from a series of standards and was linear from 0.1 to 14 mg/dl. The method is simple and has good precision and accuracy. Comparison of the results obtained by this method with the standard enzymic spectrophotometric method (disappearance of uric acid at 293 nm) gave a coefficient of variation of 0.983. Cholesterol Assay 5s A method has been developed based on the following sequential enzymic reactions: cholesterol Cholesterol esters + H~O ~t~r hydrolaa: fatty acid Jr cholesterol Cholesterol -{- 02

H20~

cholesterol

(37)

, cholest-4-en-3-one -t- H~O2

(38)

peroxidase + homovanillic acid ' fluorescent dimer

(39)

oxidase

The initial rate of fluorescent dimer formation is monitored and is proportional to the concentration of total cholesterol. A linear relationship was found in the range from 0 to 400 mg/dl. Results had good precision and accuracy. Acknowledgment The author gratefully acknowledges the financial support of the National Institutes of Health, Grants GM-17268, GM-18646, and NIAMDD-72-2216, for the research described herein. u N. F. Huang, J. W. Kuan, and G. G. Guilbault, Clin. Chem., in press.

[42] T h e A p p l i c a t i o n s o f I m m o b i l i z e d . E n z y m e s in A u t o m a t e d A n a l y s i s By WILLIAM E. HORNBY and GEORGE A. NoY Enzyme-based analysis embraces those techniques in which enzymes are used as analytical reagents for the specific determination of their respective substrates. In this type of analysis the substrate (analyte) is chemically modified selectively by the enzyme and is determined by measuring the emergence of a specific reaction product. Over recent years enzymes have been accepted widely as important analytical tools, and the techniques of enzyme-based analysis have been applied successfully

634

APPLICATION OF IMMOBILIZED ENZYMES

[42]

to a variety of problems. Enzyme-based analysis has made its greatest impact in the area of clinical chemistry, where large numbers of important metabolites and end products are routinely assayed as aids to diagnosis using enzyme-analytical reagents. Because there is an increasing demand for enzyme-based analysis, particularly in the area of clinical chemistry, significant developments have been made over recent years toward the automation of these techniques. In this paper the use of immobilized enzymes as analytical reagents in automated analysis is discussed. Although the usefulness of enzymes as analytical tools is widely accepted, there still remain some problems associated with their routine application as analytical reagents, and it is because of these problems that immobilized enzymes have begun to be considered as substitutes for enzymes in analysis. There are three features of enzyme-based analysis wherein improvements in existing methodologies are desirable. The first of these features relates to the economics of the analysis. Generally enzymes of analytical grade purity tend to be very expensive because such materials have to be extensively purified in order to remove contaminating enzyme activities that might interfere in and impair the specificity of the analysis. Thus enzyme-analytical reagents are more costly than conventional chemical-analytical reagents, and correspondingly the analyses in which they are involved are more expensive. The high cost of enzyme-based analyses is compounded when it is recalled that current trends in analysis are moving toward automated techniques whereby large numbers of samples can be processed routinely. Thus, in the performance of such automated methods, large quantities of expensive enzyme reagents are consumed concomitantly. The second feature of enzyme-based analysis wherein enhanced performance is possible is associated with the stability of enzyme-analytical reagents. An important characteristic of any analytical method is its reliability and, consequently, the reproducibility of the results that it generates. In order to achieve reliability it is important to use stable analytical reagents. Unfortunately, some purified enzymes tend to have a limited stability in solution, and thus their reliability as analytical reagents under operational conditions will parallel this lability. The final feature to be considered interrelates with both features discussed previously and is a reflection of the need to simplify enzyme-based analyses. Many enzyme-based analytical techniques require the provision of an assortment of auxiliary reagents, which may function either as second substrates and coenzymes in the enzyme reaction itself or may serve to stabilize the enzyme under the operational conditions of the

[42]

AUTOMATED ANALYSIS

635

assay. Furthermore, because of the intrinsic lability of some enzymeanalytical reagents it might not be possible always to construct robust analytical systems. Clearly, the complexity of some of these analyses together with the lability of the reagents can involve extra preparation time for the analysis. This would entail necessarily increased operator time and further increase the cost of the analysis. Thus in summary it can be argued that the more economical use of more stable enzymes in simplified analytical systems is desirable. It is toward this goal that the application of immobilized enzymes in analysis is directed. There are several characteristics of immobilized enzymes in general that both warrant the consideration of their application as enzyme substitutes in analysis and at the same time suggest that the above objectives are realizable. For example, the physical form of immobilized enzymes makes possible an improvement in the economics of enzyme utilization in analysis. Conventionally, soluble or free enzymes are used in analysis on a "one-off" basis; in general, a fixed amount of enzyme is dispensed for each assay and is not recovered after the assay for further use. In this way the full catalytic potential of the enzyme is not being realized. On the other hand, an immobilized enzyme can be deployed easily in such a way that a fixed amount of the derivative can be used for the performance of many assays, thereby effectively reducing the amount of enzyme required for each assay. In practice this can be achieved in one of two ways: The immobilized enzyme can be used in a batch mode and recovered after each assay by processes such as filtration and centrifugation for application in further assays. Alternatively, the derivative can be used in a continuous-flow mode, such as a packed bed or an open tube, and sequential samples of the analyte can be perfused continuously through it. In this way a fixed amount of immobilized enzyme can be used for repetitive execution of a large number of analyses. A second characteristic of immobilized enzymes that has influenced their consideration as analytical reagents relates to the stability of many of these artifacts. The immobilization of an enzyme, particularly by those methods in which the enzyme is either cross-linked or covalently bound to a polymeric matrix, often confers upon it an enhanced stability. Thus in many cases immobilization offers a convenient means whereby enzymes can be stabilized. Since the intrinsic lability of some enzymes represents a drawback to their routine application as analytical reagents, then their use in the same context, but in an immobilized form, is very attractive. The combination of the above two characteristics of immobilized enzymes constitute a third feature of these materials that promotes their

636

APPLICATION OF IMMOBILIZED ENZYMES

[42]

use in analysis. It has already been pointed out that immobilized enzymes can be used in analysis more economically than their watersoluble counterparts. This emanates from the ability to use these materials either repetitively or continuously, owing to their physical form and enhanced stability. In practical terms this represents what might be called a "convenience feature"; i.e., because the same derivative can be used over prolonged periods it obviates the need in the analytical laboratory to prepare enzyme reagents. At the same time this also abates some of the concern ensuing from the lability of some soluble enzyme analytical reagents. Some of the applications of immobilized enzymes in analysis have been reviewed already 1,2 (see also chapters [41] and [43]-[45] of this volume). It transpires that two different strategies have been followed when using these artifacts in automated enzyme-based analytical procedures. By the first of these strategies the auxiliary analytical hardware is constructed de novo around the immobilized enzyme derivative, thereby generating some novel analytical systems. All the analytical systems that fall into this category utilize immobilized enzyme derivative in a continuous-flow mode, i.e., the analyte is perfused through an immobilized structure and determined by measuring either the emergence of a reaction product or the disappearance of a substrate in the effluent stream. The first use of immobilized enzymes in automated analysis by this approach was reported by Hicks and Updike2 They described systems for the automated determination of glucose and lactate that utilized small packed beds of polyacrylamide-entrapped glucose oxidase and lactate dehydrogenase, respectively. In each case the formation of specific reaction products in the effluent from the packed beds was measured colorimetrically. Subsequently, the same authors described a "reagentless" assay for glucose in which the utilization of oxygen was used to measure the glucose in the sample4; this was achieved by continuously monitoring the concentration of dissolved oxygen in the column effluent with a polarographic oxygen electrode. This concept of a reagentless enzyme-based assay has been pursued by other workers; for example, Wiebel et al3 described a prototype apparatus for the analysis of glucose in biological fluids that used a polarographic oxygen sensor on the effluent side of a packed bed of glucose oxidase covalently bound to 1H. H. Weetall, Anal. Chem. 46, 602A (1974). s G. G. Guilbanlt, in "Enzyme Engineering" (L. B. Wingard, Jr., ed.), Vol. I, p. 361. Wiley (Interscience), New York, 1972. *G. P. Hicks and S. J. Updike, Anal. Chem. 38, 726 (1966). 4S. J. Updike and G. P. Hicks, Science 158, 270 (1967). s M. K. Weibel, W. Dritsehilo, H. J. Bright, and A. E. Humphrey, Anal. Biochem. 52, 402 (1973).

[42]

AUTOMATED ANALYSIS

637

porous glass particles, and Bergmeyer and Hagen6 have described for the determination of glucose a recirculating assay system that used glucose oxidase coupled to an acrylic polymer. A novel apparatus for the detection of an assortment of insecticides has been reported by Goodson and Jacobs. 7 Their system is different from those described above in that the analyte is not a substrate of the enzyme, but an inhibitor, and is determined by measuring the inhibition of the enzyme cholinesterase. In practice, this was achieved by entrapping the enzyme in starch gel on the surface of open-pore polyurethane foam and using it in an electrochemical cell that monitored the hydrolysis of the substrate butyrylthiocholine iodide. The application of immobilized enzymes in analysis by the second strategy involves their consideration on the same basis as soluble enzymes, i.e., as analytical reagents. According to this approach, the immobilized enzyme derivative is tailor-made so as to be easily accommodated into an existing analytical system. Thus the objective of this strategy is to make a straightforward substitution of the free enzyme with an appropriate immobilized preparation derived from it. The use of immobilized enzymes in automated analysis by this approach is considered in the remainder of this paper. Enzyme-based analyses can and have been automated in a variety of ways, and consequently several different types of analytical hardware, operating according to different principles, are commercially available. 8-1° In view of the diverse nature of these automated systems, it is not possible to formulate a general protocol for their operation with immobilized enzymes. Thus it seems likely that each system will work optimally only with a particular type of immobilized enzyme derivative, which will have to be constructed so that it is compatible with the operational features of that machine. The ensuing discussion reviews some of the factors that have to be considered when immobilized enzymes are used in continuousflow analyzers of the type pioneered by Technicon. It can be argued that comparable considerations will have to be undertaken when the use of immobilized enzymes in alternative systems is reviewed, and hence in this respect the following may serve as a model. e H. U. Bcrgmeyer and A. Hagen, Z. Anal. Chem. 261,333 (1972).

TL. H. Goodson and W. B. Jacobs, in "Enzyme Engineering" (K. Pye and L. Wingard, Jr., eds.), Vol. 2, p. 393. Plenum, New York, 1974. 8H. U. Bergmeyer and S. Klose, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), 2nd ed., Vol. I, p. 221. Academic Press, New York, 1974. °N. G. Anderson, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), 2nd ed., Vol. I, p. 213. Academic Press, New York, 1974. 1oH. G. Netheler, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), 2nd ed., Vol. I, p. 205. Academic Press, New York, 1974.

638

APPLICATION OF IMMOBILIZED ENZYMES

[42]

Continuous-flow analyzers of the Technicon type operate by continuously collecting discrete samples of the analyte and pumping them into an air-segmented liquid stream that contains the various reagents required for the analysis. The continuously flowing stream then is pumped through a series of coils, thereby inducing a delay in the system that allows the component reactions of the analysis to go to completion. At this point in the system the liquid stream is pumped through a monitor, which usually is a recording spectrophotometer, where the reaction products are measured. When-enzyme based analyses are carried out in these automated systems, the enzyme is included in the continuously flowing reagent stream, and thus it is continuously consumed whenever the instrument is operating. The air segmentation of the liquid stream represents a critical feature of these instruments. It serves to facilitate mixing of the analyte and the various reagents as well as providing a "scouring" effect on the walls of the transmission tube, thus minimizing adjacent sample interaction. Clearly the inclusion of an immobilized enzyme into this type of system must not impair the air-segmentation; i.e., it must be designed to assume a configuration that permits perfusion with an air-segmented liquid stream. With the above considerations in mind, several types of immobilized enzyme reactors have been used in continuous-flow analyzers. For example, Inman and Hornby 11 used derivatives of glucose oxidase and urease, immobilized by cross-linking the enzymes through glutaraldehyde in the pores of nylon membranes, for the automated determination of glucose and urea, respectively; small packed beds of glucose oxidase, urease, and uricase likewise have been used for the determination of glucose, urea, and urate, respectively11,~2; and several fiber-entrapped enzyme derivatives have been used by Marconi e t al. ~3 in similar systems. The use of the above types of immobilized enzyme structures in continuousflow analyzers has not proved entirely satisfactory from the point of view of their overall operational performance. Although these derivatives were capable of sustaining air segmentation of the liquid stream, by virtue of their physical form they demonstrated acceptable precision only at lower rates of analyte sampling. Arguably, from an overall operational standpoint open-tubular enzyme reactors have proved to be very useful enzyme reagents in continuous-flow analysis. This type of immobilized enzyme structure is prepared by covalently binding the enzyme to the inside surface of open ~1D. J. Inman and W. E. Hornby, Biochem. ]. 129, 255 (1972). H. Filippusson, W. E. Hornby, and A. McDonald, FESB Left. 20, 291 (1972). laW. Marconi, F. Bartoli, S. Gulinelli, and F. Morisi, Process Biochem. 9(4), 22 (1974).

[42]

AUTOMATED ANALYSIS

639

tubes made of such materials as polystyrene 14,15 and nylon, le,~7 These structures can be inserted easily in series with the flow system, and thus the enzyme itself is effectively fixed in situ in the system. The system using the immobilized enzyme differs from the corresponding system using the free enzyme only in that the enzyme is no longer included in the continuously flowing reagent stream. In this system the enzyme reaction takes place and the products are generated when the analyte comes into contact with the enzyme on the wall of the tube in the course of its passage through the latter. In effect the enzyme is recovered after each analysis, and so it can be used on a continuous basis for the repetitive analysis of many samples of the analyte. Experimental The use of a nylon tube-immobilized enzyme in a Technicon AA1 system is illustrated in Fig. 1. This describes the flow system used for the automated determination of blood glucose with nylon tube-glucose dehydrogenase. In principle, repeated samples containing glucose are continuously pumped in turn into an air-segmented stream containing the assay buffer. After passing through a small mixing coil, the stream enters a dialyzer module in which the glucose is dialyzed away from blood proteins and collected in an air-segmented recipient stream containing the same buffer. The coenzyme, NAD ÷, is pumped continuously into the recipient stream as it emerges from the dialyzer, and after passing through a small mixing coil it is pumped into the nylon tube-immobilized glucose dehydrogenase. Finally the N A D H formed in the enzyme tube is measured automatically by passing the liquid stream, after removal of the air:, through a flow cell in either a recording spectrophotometer or spectrofluorometer. The nylon tube-immobilized glucose dehydrogenase was prepared using the methods described by Hornby and Goldstein (this volume [9] ). The enzyme was isolated from Bacillus cereus by the method of Sadoff et al., TM and used in the form of a 2 mg m1-1 of solution in 0.1 M phos1~W. E. I-Iornby, H. Filippusson, and D. J. Inman, in "Automation in Analytical Chemistry," p. 56. Techicon Instruments Co. Ltd., 1974. 1~W. E. Hornby, H. Fillipusson, and A. McDonald, FEBS Left. 9, 8 (1970). ~ W. E. Hornby, J. Campbell, D. J. Inman, and D. L. Morris, in "Enzyme Engineering" (K. Pye and L. Wingard, Jr., eds.), Vol. 2, p. 401. Plenum, New York, 1974. x~W. E. Hornby and D. L. Morris, in "Immobilized Enzymes, Antigens, Antibodies and Peptides" (H. H. Weetall, ed.), p. 141. Dekker, New York, 1974. ,s H. L. Sadoff, J. A. Bach, and J. W. Kools, in "Spores" (L. L. Campbell and H. O. Halvorson, eds.), Vol. III, p. 97. Am. Soc. Microbiology, Ann Arbor, Michigan, 1965.

640

[42]

APPLICATION OF IMMOBILIZED ENZYMES

D

SMC 2

~W

3 , 4 • 5

.{

-

SMC

Illllll %%%%WW~

'

{ t .

.

.

.

6

7 SMC W ET

Spectrophotometer (340 nm) or Spectrofluorometer (;~ex 3 6 0 n m ; ;~ern 465 nm)

FIO. I. Flow system used for the automated determination of glucose with nylon tube-immobilized glucose dehydrogenase. The pump tubing lines, 1, 2, 3, 4, 5, 6, and 7 gave flow rates of 0.16, 2~)0, 0.60, 2.00, 0.60, 0.23, and 2.00 ml/min, respectively. Sample; 0.1 M-phosphate, 0~5 M NaCI, pH 7,5; air; 0.1 M phosphate, 0,5 M NaCI, pH 7~5; air and 1~5 mM NAD + were pumped through the tubing lines 1, 2, 3, 4, 5, and 6, respectively. Samples were assayed at the rate of 60 per hour using a 2:1 (v/v) sample/wash ratio. The pump (P) and dialyzer (D) were standard Technicon AA1 modules. The small mixing coils (SMC), the dialyzer module, and the enzyme tube (ET) were maintained at 30°. The spectrophotometer was a Beckman DBGT spectrophotometer fitted with a 1-cm light path flow cuvette, and the spectrofluorometer was a Perkin-Elmer Model 1000 fluorescence spectrophotometer fitted with a 1.6 mm-diameter flow cuvette. W, waste. phate, 0.5 M NaC1, p H 8.0. The nylon tube (2 m X 0.1 cm) was a l k y lated on its inside surface using triethyloxonium tetrafluoroborate and then derivatized by substituting it with adipic acid hydrazide. The h y d r a zide-substituted nylon tube was prepared for coupling by activating it with dimethyl suberimidate, and the enzyme was coupled to it by reaction for 3 hours at p H 8.0 and 4 °. The final product was washed free of

[42]

AUTOMATED ANALYSIS

641

noncovalently bound protein and stored filled with 0.1 M phosphate, 0.5 M NaC1, pH 7.0, at 4 °. In practice, this derivative was deployed in the form of a helix, which was made by coiling it around a 2 em in diameter plastic former. The coiled derivative was inserted in the flow system shown in Fig. 1 by connecting it through standard transmission tubing (0.16 cm bore). In this way the immobilized enzyme can be easily removed from the circuit between runs for storage at 4 ° . In general there are several practical considerations to be borne in mind when using nylon tube-immobilized enzymes in automated analysis. The most important of these are summarized below. 1. Since the methods used for covalently coupling the enzyme to the activated nylon tube involve reaction of nucleophilic groups on the protein, it is important that the enzyme preparation be free of contaminating nucleophiles. Thus, dialyzed or salt-free lyophilized enzymes are preferable. When the starting enzyme is available only as a crystalline suspension in ammonium sulfate, it is necessary to remove the latter before using it in the coupling reaction. The necessary operations for this can be illustrated in the case of the preparation of lactate dehydrogenase for binding to glutaraldehyde-activated nylon tube. Lactate dehydrogenase from rabbit muscle was obtained from the Boehringer Corporation (London). This material has a specific activity of 550 IU/mg and was in the form of a crystalline suspension (5 mg of protein per milliliter of suspension) in 3.2 M (NH4)2S0,. Of the suspension, 1 ml containing 5 mg of protein was centrifuged in a bench-top centrifuge for 5 min at 4°; the supernatant was then carefully decanted off, and the enzyme was dissolved in 2.0 ml of 0.1 M phosphate, pH 7.5. The lactate dehydrogenase solution so obtained was dialyzed for 60 min at 4 ° against 2 liters of 0.1 M phosphate, pH 7.5, and then used immediately in the coupling reaction. 2. The bore of the nylon tube used for the preparation of the derivatives must be of a size that supports the air-segmented liquid stream. For applications in Technicon AA1 and AA2 systems, a bore of 0.10-0.15 cm is desirable. The length of derivatives used depends on a variety of factors, principal among which are the activity of the immobilized enzyme; the total flow rate through the tube, when inserted in the flow system; and the concentration of analyte in the sample. These factors are discussed in greater detail in a later section of this chapter. In general, however, tube lengths in the range of 1.0-2.0 m are satisfactory. Preferably, the nylon tube should be extruded from Type 6 nylon and free of plasticizers. Nylon tube satisfying these requirements is obtainable from Porrex Limited, Hythe, Kent, U.K. 3. When not in use, the nylon tube-immobilized enzymes should be

642

APPLICATION OF IMMOBILIZED ENZYMES

[42]

stored at 4 ° filled with an appropriate buffer. In most cases storage in the assay buffer is acceptable. When derivatives are stored over long periods, it is important to ensure that they do not dry out. If this occurs, then the immobilized enzyme can lose part of its activity. In order to obviate this risk, extended storage is best achieved by sealing the tubes in plastic bags containing a small piece of cotton wool soaked in water. 4. Although it can be demonstrated that nylon tube-immobilized enzymes display an enhanced stability in relation to the corresponding free enzymes, nevertheless it is still necessary to exercise some care in their usage. Thus, if the run-down sequence on the analyzer requires washing through the system with a strong detergent, the derivatives should be removed before such a step. Furthermore, if reactions in the method, subsequent to the enzyme-catalyzed step, require the involvement of extremes of pH or the participation of compounds that can inactivate the enzyme, these conditions must be set up in the liquid stream after it emerges from the enzyme tube. In practice this is achieved easily by pumping such components into the liquid path at some point "downstream" from the enzyme tube.

Operational Examination of Immobilized Enzymes in Automated Analysis Several criteria should be evaluated when the overall operational performance of an immobilized enzyme in automated analysis is assessed. The principal criteria in this respect are discussed below. A c t i v i t y . The enzymic activity of the derivative in part controls the overall sensitivity of the analysis. Clearly, this must be sufficient to permit detection of the analyte in its expected concentration range in the sample. The time course of an enzyme-catalyzed reaction is described by the integrated form of the Michaelis-Menten equation, i.e., F• -- K ln(1 -- F ) -- ksEct;

(1)

where F represents the fractional conversion of substrate to product; S is the initial substrate concentration ( m M ) ; K is the substrate concentration required to produce half the maximum reaction velocity (raM) ; ks is the specific activity of the enzyme (~moles min -1 mg-l) ; Ec is the concentration of enzyme (mg cm-3); and t is the elapsed reaction time (minutes). For an open tube enzyme reactor the corresponding equation is FS -- K I n (1 -- F) -- ktLQ-1

(2)

[42]

AUTOMATED ANALYSIS

643

where F, S, and K have the same significance as above; kt is the tube specific activity (~moles min -1 cm-1) ; L is the length of the tube (cm) ; and Q is the rate of flow of substrate through the tube (cm -a min-0. The term kt, the tube specific activity, can be determined by using Eq. (2). In practice this is done by perfusing a fixed length of derivative at a flow rate the same as that to which it will be exposed in the analysis with substrate solutions of different concentration. At each substrate concentration the fractional conversion of substrate to product is measured, and a graph is plotted of F S as a function of l n ( 1 - F), when kt is determined from the value of the intercept on the ordinate. For a given length of derivative and flow rate, kt determines the amount of reaction that takes place. Thus a minimum value of kt will be demanded if a given fractional conversion of substrate to product is required in the analysis. This can be explained with reference to the automated determination of urate with nylon tube-immobilized uricase in a Technicon AA1 system. In this system the immobilized enzyme operates essentially under zero-order conditions with respect to the analyte because the concentration of urate after dilution in the reagent stream is substantially greater than the value of K in Eq. (2). In this particular case, Eq. (2) approximates to F S = k t L Q -1

(3)

Thus, if a fractional conversion of urate in excess of 0.9 is required, the following relationship must hold: ktL > 0.9SQ This inequality can be satisfied either by increasing the length of the tube or by increasing its specific activity. However, on other accounts it might not be practically expedient to increase the length of the tube beyond a certain length, in which case a minimum value of kt will be demanded. Stability. One of the most potent arguments in favor of using immobilized enzymes in analysis issues from their enhanced stability compared to the corresponding free enzymes. Among other things, it was shown that this enhanced stability makes it possible to use immobilized enzymes over prolonged periods; this in turn makes it possible to improve the economics of enzyme utilization in the assay. Therefore the operational stability of these derivatives must be carefully evaluated. The stability of immobilized enzymes can be studied in a variety of ways. One of the most common of these methods involves subjecting the enzyme to exaggerated denaturing conditions wherein the loss of enzyme activity

644

APPLICATION OF IMMOBILIZED ENZYMES

[42]

is accelerated. It is doubtful whether such accelerated-stability studies are of any real value in assessing the practical operational stability of an immobilized enzyme. In the particular case of the use of immobilized enzymes in analysis, the preferred way to test their operational stability is to subject them to the operational conditions that they will encounter in the analytical laboratory and to measure intermittently their residual activity. The procedure used for assessing the operational stability of nylon tube-immobilized enzymes in automated analysis can be illustrated with reference to the determination of glucose with nylon tube-glucose dehydrogenase in a Technicon AA1 system. The derivative is inserted into the system as shown in Fig. 1, and pooled bovine plasma is sampled repeatedly at the rate of 60 samples per hour. At intervals of between 5 and 10 hr, in which time 300-600 samples are processed, the tube specific activity, kt, is measured as described above by sampling aqueous glucose standard solutions. In this way the durability of the derivative is assessed under realistic operational conditions that truly represent those it will encounter in the routine analytical laboratory. Acceptable operational stabilities of nylon tube enzymes should permit the performance of at least 10,000 separate analyses with a single derivative. It is most unlikely that several thousand determinations of a single analyte would be made in a single uninterrupted operation. What is more likely is that a few hundred analyses at the most will be processed on a daily basis. In this case, in order to capitalize on the full operational stability, the immobilized enzyme will have to be usable over a period of several weeks. Hence, operational stability alone is inadequate in defining the stability of the derivative, and therefore, in addition to assessing the operational stability, the shelf life or storage stability should be tested also. This is done conveniently by storing derivatives filled with assay buffer at 4 ° and measuring their kt values as described above at intervals over a period of several weeks. Sample Rate. When a series of samples are processed in a continuousflow system, there is an interaction between them that arises from the trailing edge of one sample interfering with the leading edge of the following sample. This phenomenon sometimes is called carry-over, and it governs the maximum sample rate. Clearly, if the carry-over is bad, then the amount of analyte in one sample will influence the value obtained for the amount of analyte in the following sample. One way of minimizing carry-over is to lower the sample rate. However, this might not be possible always--for instance, when a minimum sample rate is needed to get through a given work load in a set time. On Technicon AA1 systems, this rate corresponds to 60 samples per hour.

[42]

AUTOMATED ANALYSIS

645

Another way of reducing carry-over in the case of open-tube enzyme reactors is to decrease their length. Again this might not be possible always, since the tube length governs the reaction capacity of the derivative, which in turn governs the overall sensitivity of the assay. Thus tube length can be decreased insofar as it does not compromise the overall sensitivity. With nylon tube-immobilized enzymes, carry-over can ensue if either the substrate or product of the enzyme binds to the wall of the tube. One of the commonest causes of such binding arises from ionic interactions in the case of charged substrates and products interacting with residual charges on the surface of the nylon. Effects of this type can be minimized by increasing the ionic strength of the liquid stream or by changing the chemistry used for attaching the enzyme to the nylon so that the residual charge density on the surface of the tube is reduced. In this respect the use of bis-acid hydrazide spacers has been most useful when enzymes are coupled to Q-alkylated nylon tube (this volume

[91 ). :It has been seen that carry-over is affected by a variety of factors, such as sample rate, tube length, ionic strength, and the method used for immobilizing the enzyme. Thus, all these features can be changed within limits in order to generate a system with acceptable carry-over. Clearly, in attempting to optimize the system with respect to carry-over, it is very useful to be able to make a quantitative assessment of the extent of this effect. The method described by Broughton et al. TM affords a simple means of doing this. Three aqueous standards containing a high concentration of analyte are sampled, then a further three samples conraining a low concentration of the analyte. The value of the analyte in each of the samples is recorded, and a carry-over coefficient is expressed according to the expression, Carry-over coefficient =

value of 1st low sample -- value of 3rd low sample value of 3rd high sample - value of 3rd low sample

Improved carry-over is manifested in lower values of this carry-over coefficient, and thus those systems and effects that yield minimum values for this constant can be selected for further ewaluation. Accuracy. Accuracy is a measure of how the values of samples estimated by the method compare with their true value. Assessment of accuracy is achieved by assaying at least 50 samples of the analyte, both by the method being evaluated and by an established method, and the results thus obtained are used to determine the correlation coefficient. ,gp. M. G. Broughton, M. A. Buttolph, A. H. Gowenlock, D. W. Neill, and R. G. Skentelbergy, J. Clin. Pathol. 22, 278 (1969).

646

APPLICATION OF IMMOBILIZED ENZYMES

[42]

For example, in testing the accuracy of the system shown in Fig. 1 for the automated determination of glucose, the following procedure was adopted: Over seventy samples of blood were collected in fluoride-oxalate bottles and centrifuged; the plasma was collected. The concentration of glucose in each of the samples was then determined both using the flowsystem shown in Fig. 1 and using standard glucose oxidase-peroxidase method. Precision. The precision of an analytical method is a measure of the ability of the method to reproduce the same result repeatedly for one sample. The precision of an assay method can be tested in the following way: Three batches of pooled plasma are each assayed at least 25 times in a random order. From the results so obtained, the standard deviation and coefficient of variation are calculated from the replicate values obtained for each of the plasma pools. The preceding discussion has reviewed and described the most iraportant criteria that have to be complied with in establishing the validity of an analytical method. Only when such trials have been carefully undertaken is it possible to project the usefulness of the system as a viable analytical method. The table summarizes the results obtained OPERATIONAL CHARACTERISTICS OF NYLON TUBE-IMMOBILIZED GLUCOSE DEHYDROGENASEa

Characteristic

Value

Tube specific activity Analyte range assayable Operational stability Storage stability Accuracy

4.5 ~moles of glucose oxidized per minute per meter 1-25 mM glucose Over 5000 assays with a 1 m length of derivative Not more than 10 % loss in activity after 4 months at 4° Correlation coefficient = 0.978; compared with AA2 glucose oxidase-peroxidase method Coefficient of variation = 5.1%; 25 glucose samples; 10.5 mM

Precision

The nylon tube-immobilized glucose dehydrogenase was prepared by coupling the enzyme through glutaraldehyde to adipie acid hydrazide-substituted nylon tube (this volume [9]). The derivative was used in the flow system shown in Fig. 1 for the automated determination of glucose. when the Technicon AA1 system using nylon tube-immobilized glucose dehydrogenase shown in Fig. 1 was tested as an automated method for the determination of glucose. The table represents the "assay credentials" of this particular immobilized enzyme and establishes the usefulness of this structure as an analytical tool.