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Applications of Immobilized Enzymes
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In a previous report, Howard Weetall reviewed the chemistry and bio chemistry of enzyme immobilization (1). Since his review emphasized the chemical methods for immobilization, comparisons of the properties of im mobilized and free enzymes with re spect to pH profiles, thermal stability, and Michaelis-Menten constants, we will assume familiarity with these top ics. This report is intended to be an overview of the spectrum of analytical applications for immobilized enzymes in biochemical, environmental, and in dustrial analysis. The principles of im mobilized enzyme electrodes and en zyme reactors will also be discussed. The utility of enzymes as analytical reagents has been well documented (2, 3). Previously, the instability, ex pense, scarcity, and general lack of fa miliarity with these biochemicals have discouraged analytical chemists from using them. In the past decade ad vances in the isolation and purifica tion of proteins have increased the availability of many enzymes. Over 1000 enzymes have been isolated and characterized, thereby providing the basis for selective reactions for deter mining substrates ranging from simple inorganic species such as nitrate and phosphate ions to macromolecules (3, 4). This indicates the analytical po tential inherent in enzyme technology. With the advent of a variety of suc cessful immobilization methods, it ap pears that enzymes are destined to be come routine laboratory tools. The more immediately obvious ad vantages of insolubilizing an enzyme are its easy introduction into and sep aration from a reaction mixture and, more importantly, its reuse. This con1 Present address, Department of Clini cal Pathology, University of Oregon Medi cal School, Portland, Ore.
544 A . ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
Report
Larry D. Bowers 1 and Peter W. Carr Department of Chemistry University of Georgia Athens, Ga. 30602
in Analytical Chemistry veys a number of advantages. For example, the same immobilized enzyme can be used to catalyze a reaction in a large number of samples and still be recovered. Analytically, this is important since it enables the analytical chemist to use large amounts of enzyme to achieve rapid equilibrium type assays at low cost. Another feature which often accompanies the use of immobilized enzymes is increased pH and/or temperature stability, thereby permitting the use of the enzyme under adverse conditions (1). These advantages, combined with the wide variety of enzymes available and the inherent selectivity of enzyme processes, make these materials an important addition to the analytical and clinical laboratory. Methods for Preparing Immobilized Enzymes Immobilized enzymes may be classified by the method used to prepare them. Four distinct approaches which have been extensively studied are summarized in Table I along with their analytically important features. The four techniques are: adsorption onto an insoluble carrier (5-7), covalent crosslinking of the enzyme to itself or a second type of protein, entrapment within a gel matrix (8), and covalent attachment to an insoluble carrier such as glass, cellulose, dextran, or ion-exchange resin. The reader is referred to one of the many excellent recent reviews (1, 9, 10) or monographs (11-19).
bilized enzymes are arbitrarily divided into three categories: solid-state fluorimetric assays, "enzyme electrode" type devices wherein an artificial enzyme membrane is fixed directly to the transducer, and the immobilized enzyme reactor approach with subsequent detection by any convenient method such as colorimetry or amperometry. We will attempt to review the more recent advances in the application of immobilized enzymes to analytical chemistry with particular emphasis on clinical and biochemical measurements. Some important analytical characteristics of these systems are given in Table II. Solid Surface Fluorescence. This methodology is essentially an adaptation of solution fluorescence which is carried out on a semisolid surface. The enzymes necessary to catalyze the formation of a fluorescent material along with all other required reagents are lyophilized onto a silicone rubber pad. After reconstitution the analyte solution is placed on the pad and allowed to diffuse into the gel. An interesting example is found in the analysis of serum urea in which urease, glutamate dehydrogenase, and NADH are entrapped in the pad (21). Upon addition of serum, the hydrolysis of urea is coupled to the removal of NADH by the reactions: urease
Urea + H+ + 2H 2 0 i = ± 2NH 4 + -ketoglutarate + N H 4 + glutamate
Analytical Applications Although the analytical uses of immobilized enzymes have been reviewed elsewhere (1, 10, 14, 15), these compilations have been somewhat limited in scope. For the purposes of this report, analyses based on immo-
+ NADH
^=^ dehydrogenase
+ glutamate + H 2 0 (2) Quantitation is accomplished by monitoring the change in fluorescence as a function of time. A very similar system for the determination of serum
glucose uses the hexokinase/glucose6-phosphate dehydrogenase/NADP system (22). Solid-state fluorescence has also been used to measure the activity of creatine kinase (23). These methods have not generated a great deal of interest as yet. By and large, the systems developed have been for the detection of enzymes rather than substrates and thus have not generally involved the use of immobilized enzymes. The advantages of the technique include small sample volumes, ease of use, and increased reagent stability. The disadvantages include the necessity for dissolution of the reagent on the pad, relatively high levels of background fluorescence, and a somewhat involved pad preparation procedure. Transducer-Bound Immobilized Enzymes. Potentiometric. The concept of combining the selectivity of an enzyme-catalyzed reaction with the convenience and sensitivity of electrochemical methods is generally attributed to Clark and Lyons (24). The operation of artificial membrane enzyme probes is based on the diffusion-controlled movement of substrate through a thin layer of catalyst. The substrate reacts to form a product species which is detected at the sensor surface. Thus far, the reactants or products have been quantitated by potentiometry and amperometry. A schematic representation of these devices is shown in Figure 1. Enzyme electrodes have flourished due to the number of important enzymes producing electroactive species (25) and the availability of compatible ion-selective electrodes. Although the basic operating concept is the same for both amperometric and potentiometric enzyme probes, there are substantial differences in behavior due to the electrochemical con-
ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976 · 545 A
fO Table I. Comparison of Techniques for Immobilizing Enzymes Method
Advantages
Adsorption ^ • • • • • • • Μ
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Chemically simple; can give high initial yield; widely applicable Can make membranes
Proteincross-linking Entrapment Chemically simple; high initial yield; can make membranes; widely applicable; can be lyophilized Covalent Very flexible approach; can achieve bonding good flow properties (glass support); no leakage of protein
Rigid control of conditions to prevent desorption Poor flow properties; low reactivity for high MW materials Poor flow properties; low reactivity for high MW materials; will leach protein slowly Low initial yield of enzyme activity
Table II. Comparison of Immobilized Enzyme Analytical Systems Advantages
Type PROBE Potentiometrlc
Amperometric
REACTORS Column
Tubular
Simplicity of operation; easy to make; uses small amounts of enzyme
Slow response; memory effects; relatively few enzymes compatible with operation (NH 3 , C0 2 ); sensitive to inhibitors and activators; restricted linear range; incomplete conversion
Simplicity of operation; easy to make; wider linear range; more enzymes produce or consume Oa or H2O2; uses little enzyme
Slow response, memory effects; electroactive interferences; sensitivity to materials which consume H 2 02; incomplete conversion
Wide variety of enzymes and detectors; complete conver- ' sion; high throughput
Not economic of enzyme; may have high pressure drop; more elaborate system; some solid supports (porous glass) expensive
Low pressure drop; completely compatible with flow analysis; wide variety of enzymes; high throughput
May be very long for some enzymes; may have long start-up time
sumption of the product at the sensor surface of the amperometric devices. Elegant mathematical treatments of mass-transport-coupled enzyme reac tions in membranes have been devel oped (26-29). Blaedel and coworkers {29) have derived equations which de fine the steady-state concentration of both the substrate and product for po tentiometric sensors. Their model in cludes the effect of film diffusion, i.e., external mass transport and the selec tive phase-partitioning of the sub strate between the external solution and the membrane phase (29). When such complicating factors are disre garded, one obtains the following equationsfor the concentration of product (P) at the sensor surface: [^Isfnsur
Nationwide Service
ZEISS
Limitations
=
f/ 5 ] bulk
+ V 7 T - ; [s\ » Km [PI
- IP]
eu
, " A , cosh q-X - 1 Up cosh αΛ • Mi.uik; \S] « Km (4i
where: V
K,rJK The terms V, Χ, Γ)ρ, and /X represent the enzyme concentration per unit volume of gel (mol/cc-s). the thickness of the enzyme membrane (cm), and the diffusion coefficients of the sub strate and product, respectively (cm'/s), in the membrane phase. The quantity η is the appropriate stoichiometry coefficient for conversion of the substrate to product, e.g., η = 2 for urea when NH:1 or I\"H4+ is detect ed. As is the case with all enzymatic rate analyses, the measured parameter (|^]scnsor) becomes independent of the substrate at very high concentration (see Equation 3 and Figure 2). At high levels of enzyme activity ( \'/Km ) or with thick membranes (X), Equation 4 simplifies to: L'p
when oX > 5 (5)
We have dropped the term [P]bulk to emphasize the linear relationship be tween the product concentration at the sensor surface and the bulk sub strate concentration. A sensor such as an ion-selective electrode will be re sponsive to any material which is con verted to the product, but will also be sensitive to the bulk concentration of product. The advantages of the potentiometric membrane probe are its simplicity, reliability, and low cost. The limita tions of the technique are the result of a number of fundamental factors. First, an enzymatically generated species is required for which an ionselective electrode exists. A second problem is the somewhat limited se lectivity of the electrodes which may result in susceptibility to ionic inter ferences. An excellent example of this problem and its solution is evident in the evolution of several generations of urea electrodes. In the first paper on enzyme-ion-selective electrodes, Guilbault and Montalvo used a layer of polyacrylamide gel-entrapped urease over the surface of a glass cation-selec tive electrode to detect ammonium ions (30). This particular electrode ex hibited response to sodium and potas sium ions as well as to the ammonium ion produced by the hydrolysis of urea, thus necessitating an ion-ex change pretreatment of biological fluid before analysis. To overcome this difficulty, Guilbault et al. showed that a more selective ammonium ion-selec tive electrode (nonactin-silicone rub ber membrane) could be used, provid ed that the sample's potassium level was adjusted appropriately (31). For greater specificity, Anfalt et al. found that urease could be covalently bound to the gaseous diffusion membrane of an ammonia-selective electrode and the free ammonia quantitated (32). A similar approach using an entrapped enzyme layer and a CC>2-selective elec trode was attempted (33, 34). Both of these gas diffusion electrodes are slow (5 min at Ι Ο - 4 Μ), and the membrane will clog in biological matrices. The most recent approach to the analysis of urea is the air gap electrode intro duced by Hansen and Ruzicka in which the pH electrode and its elec trolyte film are separated from the analyte solution by a thin air layer (35). The ammonia produced in the reac tion of the urea with immobilized ure ase beads is trapped in a gas-tight sys tem and diffuses into an electrolyte solution. The change in the pH of this electrolyte is then used to quantitate the substrate (36). Although this is not strictly an "enzyme electrode", but rather a microflow system, it does provide the advantage that the elec trode membrane is not in contact with the solution and therefore does not
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CIRCLE 34 ON READER SERVICE CARD ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976 · 547 A
Internal Reference Electrode
p-Ring
Cellophane embrane
Reference Solution
Entrapped Enzyme Solution
Cation Selective Membrane
H2O2
Glucose.
Gluconic r Acid Enzyme Gel Layer
Plastic Membrane.
Ag Anode
Cathode
Figure 1. Schematic diagram of electrochemical enzyme probes. A: Potentiometric sensor; B: amperometric sensor
Valve
Background
Sa ni pie
Glucose Oxidase Column
To Waste
Cell
Luminol Base
• Nitrogen K3Fe(CN)6
Figure 2. Schematic diagram of immobilized enzyme-chemiluminescent analyzer for serum glucose (62) 548 A . ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
become clogged. Recent work by Blaedel and Kissel in which a urease membrane was joined to an anion-exchange membrane indicates that the selectivity problem of the cation N H 4 + selective electrode might be avoided by using the electrostatic exclusion phenomena of an ion-exchange material (94). Their work demonstrates the powerful advantages inherent in the use of membrane technology for simultaneous separation and detection. A third problem which has been alluded to above is the slow response time of the device. By its nature, the sensor requires diffusion of the substrate and product into and out of the enzyme layer. Such diffusion may be relatively slow, particularly over the distances normally encountered in artificial membranes (0.01 cm), A second problem propagated by this same process is sample carry-over or membrane memory effects which reduce the sample throughput even further. Despite these limitations, potentiometric enzyme electrodes are very much in vogue as illustrated by the multitude of such systems given in Table III. Amperometric. Although the amperometric electrode was the first type of enzyme probe reported in the literature (37), it has only recently received substantial attention. The amperometric probe is based on the proportionality between the observed current and the amount of electroactive species generated in the membrane phase. The oxygen or hydrogen peroxide consumed or produced by certain enzyme reactions in a membrane provides a convenient analytical handle for amperometric detectors. Although the two types of electrochemical transducers are operationally similar, their performance may be significantly different. One of the major distinctions between the potentiometric and amperometric probes is the wider linear range of the amperometric probe, i.e., this sensor can under some circumstances provide a linear response at concentrations much greater than the Michaelis constant of the substrate-enzyme system. This phenomenon can be observed in the potentiometric (71) and amperometric (76) glucose probes which are designed around the use of glucose oxidase. The linear range of the amperometric probe not only exceeds that of the potentiometric probe by an order of magnitude, but also exceeds the Km of the enzyme, behavior which is not generally observed with the potentiometric device. This apparent discrepancy has been explained by the fact that the amperometric electrode removes the reaction product from solution, thereby altering its concentration profile in the
Table III. Potentiometric Enzyme Probes Analyte
Enzyme
L-amlno acids L-amino acid oxidase D-amlno acids D-amino acid oxidase Amygdalin β-glucosidase
Transducer
Comments/results +
Na , K interference; precision 2.5%
66
Cation selective
Ion-exchange pretreatment necessary
67
CN _ ion selective
Very slow response (30 min for 10 - 4 M); enzyme paste used Formation of urea by arginase followed with urea enzyme electrode
Urease
Asparagine Glucose
Asparaginase Glucose oxidase
Cation selective Iodide ion selective
Glutamine Penicillin
Glutamfnase Penicillinase
Cation selective pH electrode
L-tyrosine
C0 2 selective
Urea Urea
L-tyrosine decarboxylase Urease Urease
Urea
Urease
Cation selective Nonactin-silicone rubber ammonium selective Carbon dioxide selective
Urea
Urease
Ammonia selective
enzyme layer. Mell and Maloy, using numerical techniques to solve the boundary value problem, have shown that the current may be limited by ei ther the rate of the enzyme reaction or by the rate of diffusion (38). If the cat alytic rate is limiting and the bulk concentration is low, i.e., [S] « Km, ,. nFAXV
Ref
Cation selective
Arginase
.
+
rm
where i is the steady state current, and n, F, and A have their usual elec trochemical significance. It is inter esting to note that this relationship is similar to the potentiometric response predicted by Equation 4, except that there is no dependence on any diffu sion coefficients. Their calculations indicate that the current is limited by diffusion, i.e., in dependent of V, _the enzyme concen tration when a2X2 becomes larger than unity (38). Thus, at high enzyme concentration and low substrate con centration, the steady-state current is limited by diffusion and is given by: ._nFAD,[S)b»at (7) X An important feature here is the strong inverse dependence on the membrane thickness. At sufficiently high substrate concentration, regard less of the amount of enzyme, the cur rent becomes independent of the sub strate concentration. The calculations of Mell and Maloy were the first to yield theoretical estimates of the re sponse time of an enzyme electrode.
Iodine must be present in solution to react with hydrogen peroxide Linearity to 1 0 - 4 M; poor reproducibility; improved performance; 30-s response C0 2 interference; long response time
70 67 71 72 73.74 33
Na + , K + interference; slow response K + interference
30 31
C0 2 levels in serum give unstable readings; slow response Enzyme linked directly to gas diffusion membrane; excellent selectivity
33, 34
Their work indicates that the steady state will be achieved at times less than about 1.5 X2/D; furthermore, their experimental work indicates that substrate diffusion constants are es sentially the same in the membrane as in water. Guilbault and Nagy have developed a novel phosphate electrode using a dual enzyme amperometric electrode (39). Glucose oxidase and alkaline phosphatase were copolymerized with glutaraldehyde and placed over a plat inum disc electrode. With the reac tions: alkaline
H2O + Glucose-6-phosphate
v
v
phosphatase
Glucose + HPO4 2 H 2 0 + Glucose + 0 :
(8)
glucose
Gluconic acid + H 2 0 2 (9) the bulk phosphate concentration was monitored by its inhibitive effect on Reaction 8, thus lowering both the rate and the net consumption of oxy gen, which were monitored electrochemically. The electrode had a dynamic range of less than a decade with a re sponse time of 3-5 min in the steadystate mode. The electrode was also ad versely affected by glucose and a num ber of oxyacids. Although the amperometric elec trode avoids, to some extent, the limit ed dynamic range and slow response (38) of the potentiometric device, this sensor may be quite sensitive to other electroactive species. In serum a num
550 A · ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
68,69
32
ber of reducing agents such as uric acid, glutathione, and cysteine could interfere with the quantitation of per oxide. Despite these potential difficul ties, a number of amperometric de vices have been developed, some of which are listed in Table IV. Immobilized Enzyme Reactors. The last type of analyzer to be dis cussed here, the immobilized enzyme reactor (IMER), is perhaps the most important because it may be inter faced with virtually any detector, is readily compatible with complex flow systems, and, in certain cases, can be employed under conditions where the substrate is totally converted to prod uct, thereby avoiding the complexities of kinetic analysis. There are a num ber of reactor types used in industry, but these have been reviewed else where (40-43) and will not be consid ered here. The most commonly used reactor in analytical applications is the fixed bed or packed bed reactor which is similar in operation and de sign limitations to liquid chromato graphic columns. A second approach gaining favor is the tubular reactor de veloped by Hornby and coworkers in Great Britain. The methods of detec tion used for quantitation cover the map and include electrochemical (both amperometric and potentiomet ric), photometric, chemiluminescent, and thermal detectors. This versatility has strongly influenced the increasing use of immobilized enzyme reactors. Several groups (44-47) have shown (Continued on page 554 A)
Table IV. Amperometric Enzyme Electrodes Anafyle
Transducer
Glucose
Pt electrode with oxygen membrane Pt electrode
Phosphate
Pt disc electrode
Ethanol L-phenylalanine, Lleucine, L-cysteine, L-tyrosine Ureate (uric acid)
Pt disc
[S]0-[S]L = X m l n i | ^ Pjz,
+ v£ do) υο where S0 and Si represent the sub strate concentration (mol/1.) at the en trance and exit of the column, respec tively, L is the column length (cm), U0 is the superficial velocity (cm/s), e is the column voidage, and V is the en zyme activity per unit volume of col umn (mol/s-cc). For analytical pur poses, the greatest sensitivity will be obtained if the substrate is totally converted to product in the IMER. The fraction of substrate converted, F, can be obtained from the relationship:
+ V~
(11)
Under conditions of high substrate concentration, i.e., [S] » Km, the ex tent of the reaction is simply propor tional to the amount of time spent in the reactor: [S}oF=V(1~()L
Glucose oxidase
(12) *-> CI
Unfortunately, the situation is kinetically much more complex than that presented above. Two categories of problems are inherent in the use of immobilized enzyme reactors. The first class, enzymatic problems, in cludes microenvironmental factors, product and substrate inhibition, and thermal inactivation. These have been discussed at length elsewhere (48, 49). A second set of problems is present in the mass transfer processes which re strict the design of all chemical reac tors. Analogous to the situation which
Comments/results
Depletion of 0 2 at Clark oxygen electrode
Glucose oxidase
Response time < 12 s; direct amperometric measure of H 2 0 2 Alkaline phosphatase, Inhibition by oxyacids; glucose Interference glucose oxidase Alcohol oxidase Methanol interference; linear range is 0-10 mg/dl L-amino acid oxidase Uricase
that the kinetic behavior of a fixed bed IMER containing nonporous ma terial may be described in terms of a simple integrated Michaelis-Menten equation:
[S]0F = Km]n(l-F)
Enzyme
37 75,76 39 77 78
Measures 0 2 depletion at electrode surface; linear 79 range 0-4 mg/dl
exists in modern high-performance liquid chromatography, the effects of inter- and intraparticle mass transfer play a significant role in the efficient use of an immobilized enzyme. Interparticle diffusion is involved in the transfer of analyte from the flow stream through the stagnant layer near the particle to the enzyme carrier surface. Intraparticle diffusion is re sponsible for transport of the sub strate to the region containing the en zyme, e.g., the interior of a porous macroreticular ion-exchange bead. Both of these effects act to decrease the observed reaction rate and the ef ficiency of the immobilized enzyme by imposing additional rate determining factors. The procedure for maximizing col umn efficiency is similar to that em ployed in liquid chromatography. Interparticle resistance to mass transfer can be reduced by decreasing the par ticle size or by increasing the linear velocity of the fluid. This second op tion will improve the sample through put. Intraparticle resistance to mass transfer can be quite severe at high flow and reaction rates. Although the magnitude of the problem is some what enzyme dependent, one calcula tion involving glucose oxidase covalently bound to controlled pore glass indicated that particles smaller than 30 μ in diameter were necessary for a diffusion-free condition to exist (50). Pellicular beads are not a viable alter native for use as supports for immobil ized enzymes because of the small sur face area available for bonding the protein. In using very small porous particles, two problems arise: high pressure drops and difficulties in packing the column. It is generally ac cepted that the pressure drop across a column is proportional to the inverse square of the particle diameter. Thus, more expensive high-pressure equip ment is required. In addition, packing
554 A · ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
Ref
methods such as the balanced density slurry technique, which may or may not be enzyme compatible, are neces sary to obtain reproducible columns of small particles. Because of the com plex nature of these kinetic problems, the reader is referred to the rather ex tensive collection of literature on the subject for more detail (12, 50-59). One interesting problem in mass transfer pertains to the use of tubular reactors. Because of the low surface area available for immobilization and large diffusional distances from the center of the tubes, the first tubular reactors were necessarily very long to achieve any reasonable degree of con version. Recently, Horvath and co workers found that maintenance of slug flow by segmentation with nitro gen bubbles causes significant in creases in the rate of mass transfer and thus increased enzyme efficiency (58, 59). The first successful analytical appli cation of an IMER to chemical analy sis was the determination of urea with immobilized urease by Reisel and Katchalski in 1964 (60). As mentioned previously, the number of IMER sys tems is staggering; therefore, we have attempted to list only some represen tative and novel applications in Table V. Senn et al. have developed a rapid assay system for trace nitrate determi nation which appears on page 954 of this issue (61). As shown in Reaction 1 of that paper, immobilized nitrate re ductase is used to induce the reduc tion of nitrate to nitrite with reduced methyl viologen. The nitrite produced is then measured by a classical reac tion to form an azo dye. A limit of de tection of 15 ppb of nitrate was achieved. Another novel application of immo bilized enzymes has been the quanti tation of blood glucose using a chemiluminescent reaction after oxidation
Table V. Uses of IMER in Analytical Chemistry Analyte
L-aspartic acid
Glucose
Enzyme
Aspartate aminotransferase, malate dehydrogenase Glucose oxidase
Method of detection
UV absorbance
Colorimetry Clark 0 2 electrode Chemiluminescence
Glutamateoxaloacetate transaminase Nitrate Organophosphates; carbamate insecticides Penicillin G Pyruvate; phosphoenolpyruvate Phosphate Sulfate Urea
Uric acid
Hexokinase/glucose-6-phosphate dehydrogenase Hexokinase Glucose isomerase Malate dehydrogenase
Calorimetry UV absorbance
Comments/results
Ret
Noncontinuous flow; sample stopped in column to increase 80 conversion
Conversion required only 600 μΙ of immobilized enzyme; 60-s analysis Peroxide coupled to emission of luminol; linear from 10~ e 10~ 4 M Column coupled to LKB flow calorimeter; very slow Nylon tube reactor; response nonlinear over the range of clinical interest
81 82, 83 62, 84 85 86
Flow enthalpimetry Colorimetry UV absorbance
Linear 10 _ 4 -10~ 2 M; equilibrium assay up to 40 samples/h 64 Differential measurement on autoanaiyzer 87 Nylon tube reactor; product of GOT reaction quantitated by 65 immobilized enzyme; use IMER to make reagent
Nitrate reductase Cholinesterase
Colorimetry Electrochemical
Kinetic mode assay; 15 ppb detection limit Enzyme in cellulose or entrapped in starch; inhibition of enzyme increases potential; 1 ppm detection limit
61 88
Penicillinase Lactate dehydrogenase, pyruvate kinase Alkaline phosphatase Arylsulfatase Urease
Colorimetric UV absorbance
Nylon tube reactor Linear range 1 0 _ 4 - 1 0 _ s M shown
65 89
Colorimetry
Competitive inhibition used to quantitate
90
Colorimetry Colorimetry Differential conductance Flow enthalpimetry
Competitive inhibition used to quantitate Enzyme reaction coupled to Berthelot reaction
90 60, 91 92
Rapid equilibrium analysis; 60 samples/h; enzyme preparation very stable
63
Uricase Urate oxidase
of the sugar and production of hydro gen peroxide hy glucose oxidase. The system described by Bostick and Her cules (62) is shown in Figure 2. Com plete conversion of glucose to gluconic acid and hydrogen peroxide in a col umn which contained either Sephadex or CPG-bound enzyme allows quanti tation by the light emitting reaction between luminol-ferricyanide and per oxide. The lower limit of detection is Ι Ο - 8 Μ peroxide, and a linear re sponse of over four decades in concen tration allows considerable latitude in the analysis procedure. Determination of both serum and urine glucose is possible when a preliminary NelsonSomogy precipitation is carried out. The development of a flow enthalpimetric analyzer using an IMER has been proposed (63). The differential measurement of the temperature change across an adiabatic column packed with CPG-bound urease was the basis for the equilibrium analysis of urea in serum. The system, shown in Figure 3, permits the measurement of less than 1 raM urea in 100 μΐ of serum. A similar system has been de
93 Nylon tube reactor
94
veloped for determination of serum glucose based on its hexokinase-catalyzed phosphorylation (64). One interesting area which has not received significant attention in the analytical community is the enzymemediated formation of expensive or unstable reagents. Hornby et al. (65) have used the system illustrated in Figure 4 to determine the activity of serum glutamate-oxaloacetate trans aminase (SCOT). In this assay the product of the SGOT reaction, oxaloacetate, produced during a fixed time is quantitated by its reaction with NADH in the presence of malic dehy drogenase which was covalently bound to the inside of a nylon tube. The dis appearance of NADH per unit time may be related to the amount of SGOT. Since NADH is much more ex pensive than NAD, it was generated "on-line" using nylon tube-immobil ized alcohol dehydrogenase by the re action: C 2 H 5 OH + NAD+
alcohol
^=±
dehydrogenase
C9H5O + NADH
556 A · ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
(13)
Summary and New Directions Although this review has attempted to survey the broad applicability of immobilized enzymes in analytical chemistry, this is only a small part of the entire effort involving these mate rials. Most of the interest to date has been related to the food and beverage processing fields and in the pharma ceutical industry. Although this tech nology is being developed for indus trial processes, the analytical chemist is certainly a beneficiary. New metho dologies for immobilization are now being investigated which will allow higher yields of enzyme activity and the immobilization of presently in tractable enzymes. We have summarized three distinct areas of application of these materials. At present, however, there are only a few commercially available analyzers designed around immobilized en zymes. The glucose analyzers made by Yellow Springs Instrument Co. and Leeds and Northrup Corp. both em ploy amperometric detectors and im mobilized glucose oxidase. A number
Acknowledgment Eluent
T h e a u t h o r s express their apprecia tion to all who helped in preparing this report.
Pump
To Bridge
Figure 3. Schematic diagram of immobilized enzyme-thermal analyzer for serum urea a) Sample injection valve, b) large insulated bath, c) pre-equilibration coil (30 cm of 0.25 mm i.d. stain less steel tubing coiled on a 7/i6-in. diam. aluminum rod), d) eluent equilibration coil (15 cm of 0.76-cm Teflon tubing), e) stirrer (2 in. diam., rotated at 600 rpm), f) adiabatic reaction column (5 mm i.d. by 2.5 cm iong), g) thermistors fitted in a connector Courtesy of the American Association of Clinical Chemistry (63)
:l·,· till,'.:':'. Sri? r n ;
Figure 4. Schematic diagram of contin uous flow analyzer for SGOT activity using immobilized enzyme system Flow system for automated determination of glutamate-oxaloacetate activity. Pump tubing lines 1, 2, 3, 4, 5, and 6 gave flow rates of 0.42, 0.42, 0.60, 0.60, 0.60, and 2.00 ml/min, respectively. Sample, air, 0.15 M aspartate and 0.1 M α-oxaloglutarate in 0.1 M phosphate, pH 7.6, and 0.1 M phosphate, pH 7.4, pumped through lines 1, 2, 3, and 5, respectively. With packed bed of immobil ized alcohol dehydrogenase inserted at position A, solution of 1.0 mM NAD and 0.2 M ethanol in 0.1 M glycine, pH 10.0, pumped through this line. Delay coil, malate dehydrogenase tube, and packed bed all maintained at 25° Courtesy of Plenum Publishers (65)
of other companies are preparing in s t r u m e n t s utilizing these materials— most notably Corning Glass Works a n d Technicon Corp. Other commer cially available immobilized enzyme products are subsystems for currently used devices, e.g., the recently intro duced Catalinks by Miles Laborato ries, Inc. T h r e e types of tubes (glucose oxidase, catalase, a n d urease) are pres ently available for use on a flow ana lyzer and a fourth, containing uricase, will be released shortly. Immobilized enzymes are available in various forms for the analyst's own i n s t r u m e n t a l modifications from m a n y biochemical supply companies. We believe t h a t the combination of t h e delicate selectivity of enzymes and t h e operational simplicity of using an immobilized reagent will bring about more widespread use of these materi als for a u t o m a t e d analysis and become a useful addition to t h e tools of the analytical chemist.
References (1) H. H. Weetall, Anal. Chem., 46, 602A (1974). (2) G. G. Guilbault, ibid., 42, 334R (1970). (3) H. U. Bergmeyer, Ed., "Methods of Enzymatic Analysis", Vols 1-4, Academ ic Press, New York, N.Y., 1975. (4) T. Ë. Berman, "Enzyme Handbook", Springer-Verlag, New York, N.Y., 1969. (5) J. M. Nelson and E. G. Griffin, J. Am. Chem. Soc, 38, 1109 (1916). (6) C. A. Zittle, Adv. Enzymol. Relat. Areas Mol. Biol., 14, 319 (1953). (7) R. A. Messing, J. Am. Chem. Soc, 91, 2370 (1970). (8) G. G. Guilbault and G. J. Lubrano, Anal. Chim. Acta, 64, 439 (1973). (9) G.J.H. Melrose, Reu. Pure Appl. Chem., 21,83 (1971). (10) O. R. Zaborsky, "Immobilized Enzymes", CRC Press, Cleveland, Ohio, 1973. (11) H. H. Weetall, "Immobilized Enzymes—A Compendium of References from the Recent Literature", Corning Glass Works, Corning, N.Y., and New England Research Application Center, University of Connecticut, Storrs, Conn., 1973. (12) R. A. Messing, Ed., "Immobilized Enzymes for Industrial Reactors", Academic Press, New York, N.Y., 1975. (13) R. B. Dunlop, Ed., "Immobilized Biochemicals and Affinity Chromatography", Plenum Press, New York, N.Y., 1974. (14) M. Salmona, C. Saronio, and S. Garattini, "Insolubilized Enzymes", Raven Press, New York, N.Y., 1974. (15) E. K. Pye and L. B. Wingard, Eds., "Enzyme Engineering", Vol II, Plenum, New York, N.Y., 1973. (16) C. R. Lowe and P.D.G. Dean, "Affinity Chromatography", Wiley, New York, N.Y., 1974. (17) G. R. Stark, "Biochemical Aspects of Reactions on Solid Supports", Academic Press, New York, N.Y., 1971. (18) G. E. Means and R. E. Feeney, "Chemical Modification of Proteins", Holden-Day, San Francisco, Calif., 1971. (19) H. H. Weetall, Ed., "Immobilized Enzymes, Antigens, Antibodies and Peptides", Marcel Dekker, New York, N.Y., 1975. (20) G. G. Guilbault, Crii. Reu. Anal. Chem., 1,377 (1970). (21) J. W. Kuan, Κ. Υ. Lau, and G. G. Guilbault, Clin. Chem., 21, 67 (1975). (22) S. W. Kiang, J. W. Kuan, and G. G. Guilbault, ibid., ρ 1799. (23) Η. Κ. Lau and G. G. Guilbault, ibid., 19, 1045 (1973). (24) L. Clark and A. Lyons, Ann. Ν. Υ. Acad. ScL, 102, 29 (1962). (25) L. C. Clark, in "Enzyme Engineer ing", pp 377-94, L. B. Wingard, Jr., Ed., Wiley, New York, N.Y., 1972. (26) R. Goldman, O. Kedem, and E. Katchalski, Biochem. J., 7, 4518 (1968). (27) R. Goldman, O. Kedem, and E. Katchalski, ibid., 10, 165 (1971). (28) W. J. Blaedel and R. C. Boguslaski, Biochem. Biophys. Res. Commun., 47, 248 (1972). (29) W. J. Blaedel, T. R. Kissel, and R. C. Boguslaski, Anal. Chem., 44, 2030 (1972). (30) G. G. Guilbault and J. G. Montalvo, J. Am. Chem. Soc, 92, 2533 (1970). (31) G. G. Guilbault, G. Nagy, and S. K. Kuan, Anal. Chim. Acta, 67, 195 (1973).
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(32) T. Anfalt, A. Graneli, and D. Jagner, Anal Lett., 6, 969 (1973). (33) G. G. Guilbault and F. R. Shu, Anal. Chem., 44, 2161 (1972). (34) N. Nilsson, A. Akerland, and K. Mosbach, Biochim. Biophys. Acta, 320, 529 (1973). (35) Ε. H. Hansen and J. Ruzicka, Anal. Chim. Acta, 72, 353 (1975). (36) G. G. Guilbault and M. Tarp, ibid., 73,355(1974). (37) S. J. Updike and G. P. Hicks, Nature, 214,986(1967). (38) L. D. Mell and J. T. Maloy, Anal. Chem., 47, 299 (1975). (39) G. G. Guilbault and G. Nagy, Anal Chim. Acta, 78, 69-80 (1975). (40) L. B. Wingard, Ed., "Enzyme Engineering 3", Wiley, New York, N.Y., 1972. (41) A. Bar-Eli and E. Katchalski, J. Biol Chem., 238,1690 (1963). (42) S. P. O'Neil, Rev. Pure Appl. Chem., 23,133(1972). (43) H. H. Weetall, Food. Prod. Dev. (April 1973). (44) T. Tosa, T. Mori, N. Fuse, and I. Chibata, Agric. Biol. Chem., 33,1047 (1969). (45) R.J.H. Wilson, G. Kay, and M. D. Lilly, Biochem. J., 108, 845 (1968). (46) M. D. Lilly, W. E. Hornby, and E. M. Crook, ibid., 100,718 (1966). (47) N. B. Havervala and W. H. Pitcher, Jr., in "Enzyme Engineering", Vol II, E. K. Pye and L. B. Wingard, Eds., Plenum, New York, N.Y., 1974. (48) L. Goldstein and E. Katchalski, Z. Anal. Chem., 243,275(1968). (49) L. Goldstein, Y. Levin, and E. Katchalski, Biochem., 3,1913 (1965). (50) B. Atkinson and D. E. Lester, Biotechnol. Bioeng., XVI, 1299 (1974). (51) T. Kobayaski and M. Moo-Young, ibid., XV, 47 (1973).
(52) K. F. O'Driscoll, I. Hinberg, R. Korus, and A. Kapoulas, J. Polym. Sri., Polym. Symp., 46,227 (1974). (53) B. K. Hamilton, C. R. Gardner, and C. K. Colton, AIChE J., 20, 503 (1974). (54) M. Moo-Young and T. Kobayashi, Can. J. Chem. Eng., 50, 162 (1972). (55) S. Gondo, T. Sato, and K. Kusunoki, Chem. Eng. Sci., 28, 1773 (1973). (56) Β. Κ. Hamilton, L. J. Stockmeyer, and C. K. Colton, J. Theor. Biol., 41,547 (1973). (57) B. J. Robito and J. R. Kittrell, Biotechnol. Bioeng., XV, 543 (1973). (58) C. Horvath, A. Sardi, and B. A. Soloman, Physiol Chem. Phys., 4, 125 (1972). (59) C. Horvath, B. A. Soloman, and J. M. Engasser, Ind. Eng. Chem. Fundam., 12, 431 (1973). (60) E. Reisel and E. Katchalski, J. Biol. Chem., 239,1521 (1964). (61) D. R. Senn, P. W. Carr, and L. Ν. Klatt, Anal. Chem., 48, 954 (1976). (62) D. T. Bostick and D. M. Hercules, ibid., 47, 447 (1975). (63) L. D. Bowers, K. M. Sayers, L. M. Canning, Jr., and P. W. Carr, Clin. Chem., in press. (64) L. D. Bowers and P. W. Carr, Clin. Chem., in press. (65) W. E. Hornby, J. Campbell, D. J. Inman, and D. L. Morris, in "Enzyme Engineering", Vol Π, ρ 401, Ε. Κ. Pye and L. B. Wingard, Eds., Plenum, New York, N.Y., 1974. (66) G. G. Guilbault and E. Hrabankova, Anal. Chem., 42, 1779 (1970). (67) G. G. Guilbault and E. Hrabankova, Anal. Chim. Acta, 56, 285 (1971). (68) R. A. Llenado and G. A. Rechnitz, Anal. Chem., 43, 1457 (1971). (69) M. Mascini and A. Liberti, Anal.
Chim. Acta, 68,177 (1974). (70) H. E. Booker and J. L. Haslam, Anal. Chem., 46, 1054 (1974). (71) G. Nagy, L. H. Von Storp, and G. G. Guilbault, Anal. Chim. Acta, 66, 443 (1973). (72) G. G. Guilbault and F. R. Shu, ibid., 56, 333 (1971). (73) G. J. Papariello, A. K. Mukherji, and C. M. Shearer, Anal. Chem., 45, 790 (1973). (74) L. F. Cullen, J. F. Rusling, A. Schleifer, and G. J. Papariello, ibid., 46,1955 (1974). (75) G. G. Guilbault and G. T. Lubrano, Anal. Chim. Acta, 60, 254 (1972). (76) G. G. Guilbault and G. T. Lubrano, ibid., 64, 439 (1973). (77) G. G. Guilbault and G. T. Lubrano, ibid., 69, 189 (1974). (78) G. G. Guilbault and G. T. Lubrano, ibid., ρ 183. (79) M. Nanjo and G. G. Guilbault, Anal. Chem., 46, 1769 (1974). (80) S. Ikeda, Y. Sumi, and S. Fukui, FEES Lett., 47, 295 (1974). (81) G. P. Hicks and S. J. Updike, Anal. Chem., 38, 726 (1966). (82) M. K. Weibel, W. Dritschilo, H. J. Bright, and A. E. Humphrey, Anal. Biochem., 52, 402 (1973). (83) H. J. Kuntz and M. Stastny, Clin. Chem., 20, 1018 (1974). (84) J. P. Auses, S. L. Cook, and J. T. Maloy, Anal. Chem., 47, 244 (1975). (85) A. Johannson, Protides Biol. Fluids, Proc. Colloq., 20, 567 (1973). (86) D. L. Morris, J. Campbell, and W. E. Hornby, Biochem. J., 147, 593 (1975). (87) J. W. Finley and A. C. Olson, Cereal Chem., 52, 500 (1974). (88) Environmental Protection Agency, Report R2-72-010, August 1972.
Chemtrix digital pH-pIon meter $289. Proof that quality need not be high-priced. Up here in Oregon we keep our overhead low and our standards high. For example, our Type 60A pH-pIon Meter uses the best qual ity components available...yet it costs $ 3 0 6 less than a competing unit. You can pay more, but you won't get more performance than Type 60A gives you. We have considerable applications data to support our claims for Type 60A (which is also great for specific-ions). Furthermore, Chem trix guarantees satisfaction. If Type 60A disappoints you in any way, we'll gladly take it back. Specifications Accuracy: 0.01 p H · p H range: 0 - 1 4 · Millivolt range: ± 1 9 9 9 · Temp compensation: 0 - 1 0 0 ° C · Readout: 7-segment LED · Power: 1 1 0 - 2 2 0 VAC
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CHEMTRIX INC. 135 N.W. Adams, Hillsboro, O r e g o n 97123 Telephone (503) 6 4 8 - 0 7 6 2 CIRCLE 38 ON READER SERVICE CARD 558 A · ANALYTICAL CHEMISTRY, VOL. 48, NO. 7, JUNE 1976
(89) T. L. Newirth, M. A. Diegelman, Ε. Κ. Pye, a n d R. G. Fallen, Biotechnol. Bioeng., XV, 1089 (1973). (90) H. H. Weetall and M. A. Jacobson, Proc. IV IFS Ferment. Technol. Today, 361 (1972). (91) R. A. Messing, Biotechnol. Bioeng., X V I , 525 (1974). (92) W. Dritschilo and M. K. Weibel, Biochem. Med:, 11, 242 (1974). (93) H. Filippuson, W. E. Hornby, and A. McDonald, FEBS Lett., 20, 291 (1972). (94) W. J. Blaedel and T. R. Kissel, Anal. Chem., 47, 1602 (1975). Presented in part at the Benedetti-Pichler Award Symposium honoring Petr Zuman at the Second Annual Federation of Analytical Chemistry and Applied Spectroscopy Societies Meeting in India napolis, Ind.
Larry D. Bowers was borii in York, Pa. He received a BS degree (chemis try) at Franklin and Marshall College in 1972 and a PhD (analytical chemis try) at the University of Georgia in 1975. Presently, he is a postdoctoral associate in the Department of Clini cal Pathology at the University of Or egon Health Science Center in Port land. Dr. Bowers is the author or coau thor of 10 scientific papers.
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Peter W. Carr was born in Brooklyn, N.Y. His bachelor's work was carried out under the direction of Louis Meites at the Polytechnic Institute of Brooklyn. In 1969 he received a PhD in analytical chemistry at the Penn sylvania State University working under the direction of Joseph Jordan. A postdoctoral associate of David Click in the Pathology Department of the Stanford University Medical School from 1968 to 1969, he joined the Chemistry Department of the Uni versity of Georgia in September 1969 and is presently an associate profes sor. Dr. Carr has authored or coauthored some 45 papers in electroanalytical, thermoanalytical, bioanalytical, and clinical chemistry.
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