Report Larry D. Bowers Department of Laboratory Medicine and Pathology University of Minnesota Minneapolis, Minn. 55455
Applications of Immobilized Biocatalysts
in Chemical Analysis In 1974, Weetall published a REPORT in ANALYTICAL CHEMISTRY documenting the increasing interest in a relatively new concept in catalysis involving enzymes physically or covalently bound to a solid support (1). This article and a subsequent article (2) discussed the applications of immobilized enzymes over the 60 years since it was first observed that enzymes could be adsorbed onto a surface and retain their activity. The decade of the seventies produced an enormous proliferation of literature on the technology and analytical applications of these materials, including more than 20 monographs and more than 1000 research publications. Immobilized enzymes have now moved beyond the novelty stage and have become a mature area of research. Very few reports of a new immobilization chemistry can stir enthusiasm, and the descriptions of new analysis systems have become less and less frequent. With these advances in mind, it seems appropriate to evaluate the use of immobilized enzymes as routine laboratory tools, a prediction made in 1976 (2), and the reasons for the success or failure of the technique. The advantages of immobilized enzymes are numerous. The most obvious is that the enzyme can be readily 0003-2700/86/0358-513A$01.50/0 © 1986 American Chemical Society
separated from the reaction mixture and reused. Thus the effort and expense of preparing immobilized enzymes are offset by their extended use and greater economy. In some cases, there have been assertions that immobilized enzymes are more stable than their soluble analogues. It would be particularly useful if immobilization permitted the use of enzymes in organic solvents or at temperatures above those that would ordinarily cause a loss of activity. This will be discussed in greater detail below. The cost of these advantages is an increase in complexity of the system. When the enzyme is physically localized on a matrix, the substrate for the reaction must diffuse to the enzyme for reaction to occur. This convolution of diffusion and reaction rate makes quanti-
fication of catalytic activity a function of a number of nonenzymic factors. As a result, two types of activity can be measured. Operational or apparent activity is the measured catalytic rate (/imol/min) under conditions similar to those used in the analytical system. This type of activity can be used to optimize the system for an analysis. Inherent or intrinsic activity is the catalytic rate of the enzyme when the physical limitations of mass transport are removed. In order to characterize the properties of the enzyme, compare recovery of enzyme activity from the immobilization mixture, investigate the effects of immobilization on enzyme behavior, or assess the economic and catalytic efficiency of the enzyme, a measure of the inherent activity is required. Clearly, a complete description of the enzyme system requires measurement, and understanding, of both parameters. Effects of immobilization on enzyme properties Physically confining an enzyme to a specific region of a carrier material has a number of effects on the characteristics of the enzymic catalysis. These effects may arise as a result of a specific interaction between the support and the enzyme such that the mechanism
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986 · 513 A
(b)
(a)
S
i
^oant/^
f
\
\
/
/
BPI ^ S | B
Bulk fluid
\
1 I
Substrate
Diffusion
H
_.
7i : Convection
Substrate
17-37μΐη
\
100 A
\ Mass transfer rate (mol/cm
2
s) = const • ([S] bulk - [S] surfaoe ) - dp
%
ta. Dr m + im ι. ** m®.
d[S]
u1fe
dr
L dr2
r dr J
Km + S '
£fl
Figure 1. External (a) and internal (b) mass transfer processes for an immobilized enzyme contained in a spherical support particle
of catalysis is affected. One example of this type of behavior would be an in teraction of the support with the ac tive site of the enzyme. A second po tential cause of a change in enzyme characteristics might be the result of a difference between the bulk properties of solution and the microenvironment near the enzyme. The introduction of a negatively or positively charged group onto the support material shifts the optimum pH for enzyme catalysis due to partitioning of protons either toward or away from the surface. Thus the pH of the bulk solution is either greater than or less than the pH expe rienced by the enzyme. A similar par titioning of substrate toward or away from the surface can also be induced by introduction of a charge or a hydro phobic group onto the support. The possibility of tailoring the enzyme mi
croenvironment to allow the use of en zymes in an otherwise hostile environ ment has not received much attention in the development of analytical sys tems. Effect of diffusion on kinetics. As mentioned above, the use of heteroge neous catalysts introduces the compli cation of diffusion into any consider ation of catalytic rates. The rate of conversion of substrate to product is influenced by both external and inter nal resistance to mass transfer. Exter nal mass transfer is the process of movement of the substrate from the bulk of solution to the surface of the support matrix. Internal mass trans fer resistance is the result of diffusion of the substrate within the support matrix (Figure 1). These phenomena exist for all forms of immobilized en zymes commonly used in analytical
applications. The form of the equa tions describing the mass transfer dif fers depending on whether the immo bilized enzyme matrix is in the shape of a spherical particle, an open tube, or a membrane. The mathematical re lationships relating the enzyme reac tion rate to the parameters that affect it are summarized in Table I. External mass transfer is partly at tributable to diffusion. If the fluid to be analyzed is moving near the surface of the support because of bulk flow or stirring, convective transport will also contribute to the delivery of substrate to the enzyme. As illustrated in Figure la, in the case of external mass trans fer resistance control, the rate of reac tion is a function of the particle diam eter (dp), the diffusion coefficient of the substrate (Ds), and the superficial flow velocity (μ). Clearly this presents
Table I. Concentration of product present at the sensor under first-order reaction conditions Support type
External mass-transfer-limited reaction
Membrane 3
[ 1 , - o = [Slo
Membrane 6
i=nF[S]0
αΟεν/φ 1 + DsVV0
Internal mass-lransfer-llmlted reaction
[P] x = 0 = [S]0(1 - sechViP)
aD.V
aD.V
1 + aDV'/φ
Packed bed of
spherical particles 0
[P]x . . = [S]J 1 - exp ( I
\
°-6aD'V' XI 0(1 + 0.06adp2))]
[p]
= [s] L
I
_ exp (- ^ψΛ \
f ^
d p 2Q J V
coth I * [ p ]x - ο is the product concentration at the sensor surface; a is the enzyme-loading factor (A2[E]/Ds/Cm); Φ is the permeability of the membrane for substrate; V is the volume of the membrane; L is the membrane thickness. 6 Reformatted from Reference 3 for an amperometric electrode; η is the number of electrons and F is Faraday's constant. c [Ρ]χ = β is the product concentration of the exit of the reactor; Vr is the reactor volume; Q is the volumetric flow rate; dp is the support diameter.
514 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
•
1.60 1.40 1.20
·/*
1.00 ο Ε • | 0.80 a. , 0.60 0.40 0.20 0
2
4
6
8 10 12 14 16 18
Flow rate (mUmin) Figure 2. Rate of generation of product as a function of flow rate for immobi lized /3-glucuronidase using a gradientless recirculating reactor Reprinted with permission from Reference 4
several problems. If the amount of ac tive enzyme that has been immobi lized is to be measured accurately, the effects of external diffusion must be eliminated, as the activity measure ment is based on the rate of reaction. This has been accomplished with microreactor systems in which the rate of conversion of substrate can be mea sured as a substrate solution is rapidly flowing through a thin bed of catalyst. The reaction rate as a function of flow
rate for immobilized ^-glucuronidase is shown in Figure 2. Note that the "activity" is threefold greater in the absence of external mass transfer limi tations (e.g., at high flow rates). The reaction rates observed even at the lowest flow rates used in this study are sixfold greater than those observed for particles suspended in bulk stirred so lution. Substantial differences in the recovery of enzyme activity would be calculated based on these various ex perimental conditions. Even greater difficulty would be encountered if the recovery of different-sized particles were compared under diffusion-con trolled conditions. A second problem is that if diffusion limits the rate of re action, the enzyme is not being used efficiently. Again, this has economic rather than scientific implications. In addition to the effect of external mass transfer limitations on the rate of reaction, the other enzyme charac teristics are affected as well. Slow mass transfer invariably increases the apparent Michaelis constant, KMapp(The Michaelis constant is the concen tration of substrate at which half of the maximal reaction rate is ob served.) Thus, mass transfer limita tions have a significant impact on the evaluation of the characteristics of im mobilized enzymes. However, this in crease in Kyp-PP has the analytical ben
• V ^ • • 1 #^k 1 I I I • • » i i i 1^ι#
efit of increasing the linear dynamic range of the immobilized enzyme sys tem relative to its soluble counterpart. It should be pointed out that the ap parent activity and K M ° P P are the op
erational parameters that should be optimized in an analysis system. Un fortunately, much of the work report ed in the analytical literature fails to take mass transfer limitations into ac count. This makes valid comparison of various immobilized enzyme systems extremely difficult, if not impossible. Internal or intraparticle mass trans fer occurs within the interior of the support matrix, a region not accessible to convection from the bulk solution. As shown in Figure lb, the reaction rate will be a function of the radius of the particle, the substrate concentra tion, the diffusion coefficient of the substrate, and the amount of enzyme present. For the analytically useful case in which the reaction rate is first order with respect to substrate, the re action rate is given by Rc = Ds[S]0/dp X [yfad/ coth«Jad/)/2)
where Rc is the reaction rate, [S]o is the concentration of substrate in the bulk of solution, and a is the enzymeloading factor (k2[E]/DsK!^PP). The loading factor is a function of the in-
^ P ^ i ^ l i P ^ i B » • ,ίΡββ»,'ΐΡ
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- 1]
RESEARCH
herent dissociation constant of the product from the enzyme, the concentration of enzyme in the matrix, the diffusion coefficient of the substrate, and the apparent Michaelis constant. The derivation of the equation for internal mass transfer limitations in spherical particles assumes that the enzyme is uniformly distributed throughout the particle. Recent evidence indicates that this may not always be the case. It should be noted that the internal mass transfer limitations on the reaction rate are most deleterious at low substrate concentrations. The above equation would seem to indicate that with increasing internal mass transfer limitations the enzyme that has been immobilized is used less and less effectively; that is, to achieve a twofold increase in reaction rate one must increase the enzyme concentration at least fourfold. An effectiveness factor can be derived for the various circumstances limiting enzyme catalysis in each of the forms in which enzymes are used analytically. This is basically the rate observed in the presence of the ratelimiting factor relative to the rate of reaction in the absence of the limitation. The enzyme-loading factor, a, is of fundamental importance in both the response of the analytical system to substrate and the effective use of
the enzyme. An increase in the loading factor results in an increase in the analytical response but may result in a decrease in the efficiency of enzyme use. An increase in the loading factor might also result in a desirable increase in the amount of time the enzyme preparation can be used. It is not uncommon to set the experimental conditions to maximize the loading factor and accept any consequences. Clearly there are commercial implications of this trade-off that will have a significant impact on the design of immobilized-enzyme instrumentation. Effect on stability. One of the advantages cited when immobilized enzymes are discussed is an increase in stability. However, we have observed, as have others, that this so-called advantage is ambiguous. There are a number of reasons for these equivocal findings. First, there are several types of stability that can be measured. Storage stability is simply the ability of the enzyme to retain its activity under a specified set of storage conditions. Thermal stability is a measure of the ability of the enzyme-matrix system to withstand elevations in temperature, frequently in excess of those that would denature the native protein. In principle, an enzyme system capable of withstanding autoclaving and other types of sterilization could
be developed. Finally, operational stability reflects the ability of the enzyme system to function in the analysis system. It is a function not only of the enzyme, but also the carrier durability, the inhibitor concentrations in the analysis stream, the pH, and other physical characteristics of the analyte solution. Unfortunately, the storage stability is most frequently reported, and comparison of the various types of stability is difficult. A second consideration is the manner in which the stability of the enzyme is measured. As noted above, diffusion can have a significant effect on the observed enzyme kinetics. It can also have a substantial influence on the stability measurements. When the enzyme loading is high and the system is diffusion controlled, the rate of reaction is independent of the enzyme concentration. Thus, a significant amount of enzyme could be inactivated before the diffusional limitations were removed and a decrease in "activity" was measured. Although this is an operational increase in stability, no inherent stabilization of the enzyme has occurred. In economic terms, there may have been no cost savings. Inexplicably, systematic study of the experimental factors leading to increased, controlled stabilization has
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Support function 4~~^OH ά ^ ^ΟΗ
+
CNB
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λ
40mg/g — *Dioxane
?, /=N
^•"•••-^O-C-N
4^~^-ΟΗ+CF3CH2SOjCI
-ΝΗ2
-Ov ;C = NH + N H 2 - R
— Dry acetone Ο Ο ρΗ7.0 +HC-(CH2)3-CH 2.5% Glutaraldehyde
Protein-support conjugate PH9-10
pH 8.5-10
I + H 2 N-R
y - ^ ~ ^ o SO2-CH2CF3 + H 2 N - R •
5 pH 7.5-9.5
4 - ~ - - N = C - ( C H 2 > 3 - C H + H 2 NR •
L·^ »H Λ
H
H - N = C ( C H 2 ) 3 C=NR
Ο Α ?, f~] * " - ~ - C - O H +HO-N 3 \_]
0.20/0 0.2% ΝN-hydroxysuccinimide 0.4% Ν,Ν dicyclohexylcarbodiimide dioxane
i^~~^C-0-N
+ H 2NR
pH5.9 »-
Figure 3. Immobilization reactions commonly used in covalently coupling an enzyme to an activated support matrix with various chemical functionalities been lacking—possibly because en zyme inactivation itself is a complex and poorly understood phenomenon. Although the inactivation process may be different for each individual en zyme, the generally accepted mecha nism of loss of activity involves the unfolding of the three-dimensional structure of the protein, which disas sembles the active site of the enzyme. Processes that would prevent this un folding would be expected to stabilize the enzyme activity, and in fact the re sults support this hypothesis. Much work remains to be done in this area. Investigations into the possibility of stabilizing enzymes by immobilization for use in organic solvents or in organ ic-aqueous solvent mixtures have, in general, met with limited success. Our own studies have indicated that under conditions in which mass transfer lim itations are removed, the immobilized enzyme is not significantly more sta ble than the soluble enzyme (4) .The inactivation problem is exacerbated by certain solvents such as acetonitrile and is less troublesome in hydrogenbonding solvents such as methanol. Martinek and co-workers have taken a different approach, using reversed mi celles of enzyme solution to perform reactions in water-immiscible solvents (5). Interestingly, the use of either miscible (4) or immiscible (5) solvents can increase the observed rate of reac tion because of improved solubiliza tion of either substrate or product. Another interesting point is that the enzyme inactivation process seems to affect only some of the enzyme mole cules because once the initial inactiva tion is observed, the enzyme seems to function in the mixed-solvent environ ment without further loss of activity. Clark and Bailey recently reported on the detection by electron paramagnet ic resonance of two subpopulations of α-chymotrypsin that became inactive at different rates (6). This may ex plain our observations of a differential 520 A ·
loss of activity. As was mentioned ear lier, this area is relatively unexplored. Methods of preparation The chemical and physical methods of immobilization have not changed much over the years. There are basi cally four methods used in immobili zation: chemical cross-linking of the enzyme to itself and to other protein molecules, entrapment of the enzyme within the pores of a support, adsorp tion onto a rigid matrix, and covalent coupling to a matrix. An additional technique, called encapsulation, has not been used in analytical applica tions because of the diffusional limita tions of substrate entering a "globule" of enzyme. No universally useful im mobilization technique has emerged; hence the approach taken to immobi lization is frequently hit or miss. The chemistry of immobilization has been well reviewed in the monographs list ed in the suggested reading list at the end of this REPORT. The decision about which immobili zation technique to use depends on the expected use of the preparation. Covalent attachment of the enzyme to the matrix is generally the most irreversible of the immobilization techniques. A few of the most com monly used reactions are summarized in Figure 3. It has been our experience that for some enzymes the binding chemistry may have a substantial ef fect on the recovery and stability of activity, so a single immobilization scheme may not always yield optimal results. The choice of the immobilization method depends largely on the final application intended for the enzyme. The capacity of the matrix or tech nique to bind enzyme, the mechanical and chemical stability of the matrix, the expense and the difficulty in acti vation of the support, the availability of the support material, and the flow characteristics are all important con
ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
siderations in developing an immobi lized enzyme analysis system. It must be emphasized that there is no best technique for immobilization. Analytical applications Although hundreds of analytical systems using immobilized enzymes have been described, basically they can be divided into two types. In one approach, the immobilized enzyme is in direct contact with the sensor or transducer. In the second approach, the enzyme resides in a flowing stream into which the analyte is inserted, and detection occurs in the stream itself. The differences in these two ap proaches arise from the limitations imposed by the controlling phenome na in the analytical system. Thus, the two approaches can be viewed as ex tremes of a continuum of system de sign rather than as totally indepen dent approaches. Figure 4 illustrates the physical layout of the various ap proaches. Transducer-bound systems. The transducer-bound system is primarily limited by diffusion of substrate to the immobilized-enzyme membrane and by the loading of enzyme in the mem brane. It can be viewed as a reactor at tached to the sensor in which diffusion is the primary form of mass transfer. This approach has been the most pop ular as reflected by the number of pa pers in the literature. The most prom inent approach in this category has been the enzyme electrode, in which an amperometric or potentiometric electrode is used as the sensor. The transducer-bound approach ac centuates the importance of certain enzyme characteristics while minimiz ing others. For example, recovery of activity is relatively unimportant be cause in a typical membrane less than one unit of enzyme activity will be used. This assumes a membrane thick ness of 100-300 μηα and a concentra tion of enzyme sufficient to deliver
(b)
Semipermeable membranes
Enzyme on surface of solid bead
Flowing analyte stream Tubing wall
Enzyme in annulus
(d) Air segmented flowing analyte
Flowing analyte solution
stream
Tubing wall
Figure 4. Typical electrode and reactor configurations used in immobilized-enzyme-based analyses (a) Transducer-bound enzyme system, (b) STRING reactor, (c) packed-bed reactor, (d) open-tubular heterogeneous enzyme reactor
99% of the substrate to the sensor surface as product. Hence, it should be possible to use even an expensive enzyme in this type of system. The response time of the device is also a strong function of the thickness of the membrane. If one considers a substrate with a diffusion coefficient of 10~5 cm 2 /s, the steady-state response time of the electrode will range from 14 to 216 s for the aforementioned membrane thicknesses. The limit of detection of the probe will be a function of the limit of detection of the sensor and the relative rates of diffusion and enzyme reaction. Thus, there are a variety of trade-offs that must be explored. For example, the enzyme rate can be increased relative to the rate of diffusion by increasing the enzyme concentration in the membrane, by increasing the membrane thickness, and by decreasing the permeability of the membrane to substrate. The second approach has an effect on the response time as well as on the magnitude of the response. The last approach will affect the limit of detection as well as the magnitude of the response. The behavior of singlesubstrate and pseudo-single-substrate immobilized-enzyme membranes in contact with sensors has been described and should assist in the design of transducer-bound immobilized-enzyme systems. Recent research in the area of immobilized-enzyme transducers has involved approaches directed at specific problems in the analysis system. For example, a functional implantable infusion pump has been developed that could be used to release drugs or hormones such as insulin on physiological demand if a suitable sensor could be developed. Clearly, the constraints on development of an enzyme electrode
that can be implanted in people are unique. The sensor must be extraordinarily reliable, must be totally dependent on the physiological milieu for supply of all of the substrates required for analysis, and must be biocompatible. Leypoldt and Gough (7, 8) have been extending the theory of enzyme electrodes to the two-substrate case as would be encountered for a glucose oxidase electrode (glucose, O2). By evaluating the model with regard to the various trade-offs, they concluded that limiting the permeability of the membrane for glucose would yield the best results for an electrode that is limited by physiological amounts of oxygen. More recently, Gough and coworkers (9) have described an electrode for glucose in which oxygen can diffuse into the electrode either axially or radially, whereas the glucose can enter the electrode axially only. This may prevent the oxygen depletion observed in previous electrode designs and has important implications for an implantable electrode. Castner and Windgard (10) have been evaluating treatment of the platinum electrode itself to standardize the response to glucose relative to the product of the glucose oxidase reaction. These important advances should lead to the development of an effective treatment for diabetes mellitus if tight control of blood glucose is found to ameliorate the secondary pathology of this disease. The recent use of microorganisms, both native and genetically engineered, for the commercial production of a variety of substances from ethanol to drugs requires frequent analysis of nutrients in the bioreactor containing the microorganisms. This application, of course, has different constraints than those described above. If the sen-
522 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
sor is to be placed inside the bioreactor, there is a need to sterilize it so that the contents of the reactor are not contaminated by another organism. If the microbe used is anaerobic, a source of oxygen would be required if the enzyme used were an oxidase. Cleland and Enfors (11) recently described an externally buffered electrode that uses a continuously flowing stream of fluid within the electrode body. This approach allows O2, H + , or a buffer solution to be added on the inside of the enzyme membrane, thus allowing its use under a variety of conditions. The authors report that the electrode can be sterilized and used in anaerobic sites. Another application of immobilizedenzyme membranes has been the development of a sensor for monitoring air for organophosphates and carbamates. Invented by workers at the Midwest Research Institute (12), the sensor is a pad containing immobilized acetylcholinesterase. When pesticides are present, the enzyme, which is similar to the enzyme causing toxic symptoms in humans, is irreversibly inhibited. The activity of the enzyme on the pad can be monitored with a small instrument and the degree of exposure quantified. Similar approaches are being applied to the detection and detoxification of neurotoxins. In addition to the investigation of specific conditions in particular applications, a unique approach has been taken in the development of microorganism and tissue electrodes. Rechnitz and his co-workers (13,14) have entrapped various bacteria and plant and animal tissue at the surface of an electrode. These electrodes work amazingly well and have unique specificity. Perhaps most interesting is the lack of effort required to prepare the
electrode. Schubert et al. reported on an electrode system in which a 0.1-mm slice of potato was fixed on the surface of an immobilized glucose oxidase electrode to measure phosphate and fluoride by inhibition of the alkaline phosphatase in the potato slice. The response time of 25 s was amazing in light of the discussion above (15). The ability to measure various chemicals that affect the rate of the enzyme reaction, such as inhibitors and activators, presents a wide variety of opportunities for extending the role of sensor analyses. Siegopaul and Rechnitz have reported on an electrode for measurement of thiamine phosphate (16). The analyte activates apo-pyruvate decarboxylase, which in the absence of this cofactor is inactive. This activation results in production of a potentiometrically active product. Because one molecule of the cofactor results in the production of many molecules of the detected species, a significant amplification can be obtained. Electrodes are not the only transducers that have been used in conjunction with enzyme membranes. Fiber-optic probes have been used to conduct the light generated in a chemiluminescent enzyme reaction to a photomultiplier tube. Enzyme membranes have also been affixed to thermistors to monitor the heat gener-
ated by the enzyme reaction, albeit with mixed success. Perhaps one of the most novel systems reported involves the use of the change in enzyme conformation upon binding of the substrate to cause a chemomechanical change sensed by a force transducer! These specific examples are only a few of the numerous inventive analyses reported. Immobilized-enzyme reactors. In contrast to transducer-bound enzyme systems, immobilized-enzyme reactors (IMERs) generally consist of a relatively large amount of enzyme immobilized on a support in the form of a bed or annulus. The advantage of using a large amount of enzyme is that all of the substrate contained in a sample can be converted to product in the relatively short time that the sample is in the reactor. Under first-order conditions, the fractional conversion, X, is given by the relation X = 1 - exp -
(aDaVr/Q)
where a is the enzyme-loading factor, Vr is the volume of the reactor, and Q is the volumetric flow rate. The enzyme-loading factor is very important because increasing it will allow an increase in flow rate or a decrease in the reactor volume. The disadvantage of requiring a large amount of enzyme is
CIRCLE 67 ON READER SERVICE CARD 524 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
the premium it places on the development of a stable preparation of enzyme. This is because a significant amount of time and money is invested in the reactor. A second disadvantage of the IMER is that the capability of complete conversion is offset by the dispersion of the product band, which occurs in the reactor and connecting tubing before the detector. Thus, evaluation of an IMER will depend on both the enzyme activity and the dispersion inherent in the reactor system. In general, three types of reactors have been used—open-tubular gassegmented, packed-bed, and STRING reactors. The dispersion in open-tubular flow systems has been discussed in detail by Snyder and co-workers (17). Basically, the axial dispersion is limited by the gas segmentation, and what dispersion does occur is the result of a thin film of liquid adhering to the wall. It should be pointed out that extrareactor broadening during the removal of the gas segments from the stream was a major contribution to the relatively poor characteristics of the first generation of autoanalyzers. Incorporation of an electronic debubbler has resulted in significant improvement in the dispersion characteristics. The major limitation of gassegmented reactors is the fact that for
a 1-mm-i.d. tube, the surface area available for immobilization is on the order of 30 cm2 per meter of length. This compares with hundreds of square meters per gram of inorganic support material and, as mentioned above, the amount of enzyme immobi lized is related to the available surface area. To circumvent this problem, a number of methods to produce organic or inorganic annuli have been report ed. Unfortunately, the presence of the annulus thickens the stagnant layer at the wall of the tube, requiring greater diffusion of the product to re-enter the fluid stream and resulting in greater dispersion of the product. Nevertheless, a large number of suc cessful demonstrations of open-tubu lar reactors have been reported. Dispersion in packed-bed IMERs is analogous to the dispersion seen in chromatographic columns. Ap proaches to minimizing dispersion in clude improved packing structure, de creased support particle diameter, and optimization of the linear fluid veloci ty. At present, typical particle diame ters in IMERs are 37-74 μηι, and packing techniques are archaic. In our experience, there are other factors that limit the utility of decreasing the particle size and achieving lower dis persion, although the theoretical basis and techniques are well established by
modern high-performance liquid chro matography (HPLC). Most of the work done on the enzyme kinetics in IMERs has assumed a continuous in troduction of substrate, when in actu ality a sample for analysis is intro duced as a pulse. Adachi and his co workers (18) have studied the elution profile for an impulse injection for both a single enzyme and consecutive first-order reactions. The most impor tant characteristic of the reactor in minimizing dispersion was the ratio of the time required to diffuse into the support to the time required to be eluted from the column. When this parameter approaches one, very broad and asymmetric peaks were observed. Fortunately, for the conditions nor mally used in analytical reactors (100-μηι particles, 0.5 X 5-cm reactor), the peaks should be symmetrical and relatively narrow. In essence, we have decoupled the dispersion and reaction rate processes. We have also shown that if the reaction is not totally first order, "kinetic" broadening can occur. Recently a third type of reactor has been described in which solid beads are packed single file in a tube whose inside diameter is one to five times the diameter of the beads. Reijn, Poppe, and van der Linden have studied dis persion in the single-bead STRING reactor (SBSR) (19). The authors con
clude that there is a residence timereaction rate component and a flow rate-reactor length-bead diameter component to the dispersion. The re actor can be optimized using either one of the two components, and the authors conclude that they are inde pendent of each other. Using nonreactive compounds, they conclude that the SBSR has similar dispersion, re agent consumption, and residence time as a more conventional packedbed reactor. Gnanasekaran and Mottola have reported on an immobilized penicillinase SBSR, but no compari son to a packed-bed system was in cluded (20). Because the issue of com parable enzyme loading cannot be eas ily addressed, it is unclear what the role of the SBSR in immobilized-enzyme technology will be. The possibility that the enzyme it self can cause peak broadening has re cently been described. Because the substrate must bind to a specific site on the enzyme to facilitate the reac tion and the product(s) must then be released, it is not inconceivable that the enzyme could influence the peak shape. Slow release of product, for ex ample, would be analogous to a kinetically slow dissociation from a binding site in adsorption chromatography and would result in asymmetric, broad peaks. This phenomenon has been ob-
Ifj union GiKEn
Stopped Flow Rapid Scan Spectrophotometer • Minimum dead time — 500 Msec • 16 spectra measured every 1 msec sequentially • S i m p l e and robust mixing system without syringe • Fluorescence, T-jump and flash accessories. CIRCLE 2 ON READER SERVICE CARD 526 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
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to:
ATAGO B U S S A N C O . , L T D . 7-23, 5-chome Shimbashi, M i n a t o - k u , T o k y o 105 T e l e x : 2 8 4 2 1 P h o n e : (03) 4 3 2 - 8 7 4 1
Table II. Applications of immobilized enzyme reactors Enzyme stystem
Detection system
Comments/results
Creatinine iminohydrolase/ glutamate dehydrogenase 7a-hydroxysteroid dehydrogenase/luciferase/NAD:FMN
UV-VIS photometry
Nylon open-tubular reactor; serum and urine samples Nylon open-tubular reactor; serum samples
Analyte Creatinine Bile acids
oxidoreductase Glucose oxidase; ascorbate oxidase
D-glucose; ascorbate; sucrose Cholesterol Glucose; choline Penicillin Galactose
Aldehyde oxidoreductase Glucose oxidase; choline oxidase Penicillinase Galactose oxidase Microperoxidase
H202
served with the enzymes ^-glucuroni dase, urease, and penicillinase. In the first case, the reaction catalyzed is the hydrolysis of the glucuronide moiety from, for example, a steroid moiety, resulting in a polar and a nonpolar product. In aqueous solution, the ste roid has limited solubility and remains bound to the enzyme, resulting in ex traordinarily broad "peaks." Addition of methanol dramatically improves the solubility of the reaction product and results in a narrowing of the peak and an increase in the rate of reac-
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Calorimetry
Packed-bed reactor
UV-VIS photometry Amperometry
Nylon open-tubular reactor Flow injection system
Potentiometry Amperometry Chemiluminescence
SBSR/flow injection system Packed-bed reactor Packed-bed reactor; 10~ 9 M detection limit
tion.The frequency of occurrence of this phenomenon is unclear, but it could pose a major limitation in achieving rapid analysis with immobi lized enzymes in reactors. Nevertheless, a great many applica tions of immobilized-enzyme reactors have been reported. One of the advan tages of immobilized-enzyme reactors is that they can be used in conjunction with any type of detection system compatible with a flowing liquid stream. As can be seen from Table II, this feature is exploited. Without
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ι 12
ι 14
ι 16
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18
20
22
24
26
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Time (min)
Figure 5. Schematic diagram of an immobilized enzyme postcolumn reactor detec tion system for HPLC The chromatogram illustrates the specific detection of a number of bile acids at a 0.2-μΜ concentration. The peaks, in order of elution, are tauroursodeoxycholic, taurocholic, taurochenodeoxycholic, glycoursodeoxycholic, taurodeoxycholic, glycocholic, taurolithocholic, ursodeoxycholic, glycochenodeoxycholic, cholic, and glycodeoxycholic acids
528 A · ANALYTICAL CHEMISTRY, VOL. 58, NO. 4, APRIL 1986
doubt, the most frequently reported systems are those involving glucose oxidase and urease. This is unfortu nate, because these enzymes are inex pensive in soluble form and thus are unlikely candidates for acceptance in large-volume assays performed in clin ical laboratories. On the other hand, sampling devices that will allow the use of IMERs in the analysis of bioreactor contents such as ethanol and glucose are being developed, which should revitalize the field. One interesting use of immobilized enzymes has been as specific derivatizing reactions for HPLC. The reac tions have used both the pre- and postcolumn approach to derivatization. Figure 5 shows a block diagram of a postcolumn reactor system for quantitation of bile acids in serum. Also shown is a chromatogram ob tained with this system for a 0.2 X 10~6 M bile acid mixture. The classspecific oxidation catalyzed by 3«-hydroxysteroid dehydrogenase allows quantification of the bile acids while not sensing other components that might coelute; the HPLC separation allows quantification of individual bile acids. This tandem coupling of a reversed-phase chromatographic system with an immobilized-enzyme system requires careful co-optimization of both parts of the system. For example, enzyme loading, particle size, and sup port characteristics must be optimized in the reactor to achieve maximum conversion and minimum dispersion. In the aforementioned system, the separation required a pH of 3.5 where as the enzyme reaction was optimal at pH 11. In addition, one must consider the stability of the enzyme upon expo sure to the organic modifier and the solubility of the buffer in the organic modifier. Despite these complications, a number of IMER-based detection systems have been reported, including those for bile acids, cholesterol, cho line and acetylcholine, xanthines, and
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u r e a - b a s e d fertilizers. I m m o b i l i z e d e n z y m e r e a c t o r s h a v e also b e e n u s e d t o q u a n t i f y e n z y m e s e l u t e d from vari ous c o l u m n s . Immobilized e n z y m e s a r e n o t always used on-line, however, a n d a r e c e n t r e p o r t t h a t describes use of t h e e n z y m e t o c o n v e r t benz[a]pyrenes t o a fluorescent p r o d u c t for analysis is a n i n t e r e s t i n g twist. In a d d i t i o n t o t h e i r use in analysis, immobilized e n z y m e s can be used t o m a k e u n s t a b l e r e a g e n t s on-line a n d t o p r e p a r e otherwise difficult t o s y n t h e size m e t a b o l i t e s of d r u g s or o t h e r com pounds.
Conclusion Immobilized e n z y m e s h a v e b e e n used in a v a r i e t y of analytical s y s t e m s with varying degrees of success. T h e r e a r e a n u m b e r of a r e a s , such as t h e sta bility e n h a n c e m e n t p r o v i d e d b y im mobilization, t h a t n e e d m o r e research. M a n y of t h e f u n d a m e n t a l issues re g a r d i n g t h e response of immobilized e n z y m e s y s t e m s h a v e b e e n resolved, a l t h o u g h t h e application of t h e s e prin ciples t o specific, i m p o r t a n t p r o b l e m s h a s y e t t o be d e m o n s t r a t e d . Exciting progress is being m a d e in t h e a r e a s of e n z y m e a n d tissue electrode s y s t e m s . E n z y m e reactors a r e being successful ly used b o t h in analysis a n d in in vivo t h e r a p y for m e t a b o l i c disorders. I m mobilized-enzyme technology h a s m a tured. Commercially it is difficult t o assess t h e success of i m m o b i l i z e d - e n z y m e technology. In 1976, t h e r e were six companies with immobilized-enzyme p r o d u c t s on t h e m a r k e t . Only t h r e e of t h e s e c o m p a n i e s still p r o d u c e i m m o b i lized-enzyme p r o d u c t s , a n d even t h e s e indicate t h a t t h e m a r k e t for t h e s e m a terials h a s been d i s a p p o i n t i n g . W i t h t h e growth of biotechnology, t h e r e is once again a n increasing i n t e r e s t in immobilized e n z y m e s a n d a new surge of activity in t h e field. As m e n t i o n e d , t h e fact t h a t 40% of t h e p u b l i c a t i o n s in t h e i m m o b i l i z e d - e n z y m e l i t e r a t u r e cite t h e use of t h e e n z y m e s glucose ox idase or u r e a s e h a s n o t e n h a n c e d t h e a c c e p t a n c e of immobilized enzymes. I m m o b i l i z e d - e n z y m e technology will only achieve t h e success envisioned in 1976 w h e n it provides economical a n d u n i q u e solutions t o t h e a n a l y t i c a l p r o b l e m s of t h e 1980s.
References (1) Weetall, H. H. Anal. Chem. 1974,46, 602-15 A. (2) Bowers, L. D.; Carr, P. W. Anal. Chem. 1976,48, 544-52 A. (3) Mell, L. D.; Maloy, J. T. Anal. Chem. 1975,47, 299-307. (4) Bowers, L. D.; Johnson, P. R. Biochim. Biophys. Acta 1981, 667, 100-105. (5) Martinek, K.; Mozhaev, V. V.; Berezin, I. V. In Enzyme Engineering—Future Directions; Wingard, L. B.; Berezin, I. V.; Klyosov, Α. Α., Eds.; Plenum Press: New York, 1980; pp. 1-54. (6) Clark, D. S.; Bailey, J. E. Biotechnol.
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Bioeng. 1984,26,1090-97. (7) Gough, D. Α.; Leypoldt, J. K. Anal. Chem. 1979,57,439-44. (8) Leypoldt, J. K.; Gough, D. A. Anal. Chem. 1984,56, 2896-2904. (9) Gough, D. Α.; Luciano, J. Y.; Tse, P.H.S. Anal. Chem. 1985,57, 2351-57. (10) Castner, J. F.; Wingard, L. B. Anal. Chem. 1984,56, 2891-96. (11) Cleland; N.; Enfors, S.-O. Anal. Chem. 1984,56,1880-84. (12) Goodson, L. H.; Jacobs, W. B. In Bio medical Applications of Immobilized Enzymes and Proteins; Chang, T.M.S., Ed.; Plenum Press: New York, 1977; pp. 55-69. (13) Rechnitz, G. Α.; Reichel, T. L.; Kobos, R. K.; Gebauer, C. R. Anal. Chim. Acta 1977,94, 357-65. (14) Rechnitz, G. A. Anal. Chim. Acta 1981, 737,91-96. (15) Schubert, F.; Renneberg, R.; Scheller, F. W.; Kirstein, L. Anal. Chem. 1984,56, 1677-82. (16) Siegopaul, P.; Rechnitz, G. A. Anal. Chem. 1983,55, 1929-33. (17) Snyder, L. R. Anal. Chim. Acta 1980, 774,3-18. (18) Adachi, S.; Hashimoto, K.; Matsuno, R.; Nakanishi, K.; Kamikubo, T. Bio technol. Bioeng. 1980,22, 779-97. (19) Reijn, J. M.; van der Linden, W. E.; Poppe, H. Anal. Chem. 1984,56, 943-48. (20) Gnanasekaran, R.; Mottola, H. Anal. Chem. 1985,57, 1005-1009.
Suggested reading Carr, P. W.; Bowers, L. D. Immobilized Enzymes in Analytical and Clinical Chemistry: Fundamentals and Applica tions; Wiley-Interscience: New York, 1980. Guilbault, G. G. Analytical Uses of Immo bilized Enzymes; Marcel Dekker: New York, 1984. Methods in Enzymology; Mosbach, K., Ed.; Academic Press: New York, 1978; Vol. 44. Weetall, H. H. Immobilized Enzymes, Antigens, Antibodies, and Peptides; Marcel Dekker: New York, 1975. Zaborsky, O. R. Immobilized Enzymes; CRC Press: Cleveland, 1973.
Larry D. Bowers is associate professor of Laboratory Medicine and Patholo gy at the University of Minnesota in Minneapolis and a member of the University-Industry Cooperative Re search Center for Biocatalytic Process Technologies. He received his B.A. degree from Franklin and Marshall College in 1972 and his Ph.D. in ana lytical chemistry from the University of Georgia in 1975. His research inter ests include immobilized enzymes and chromatography in bioanalytical and clinical chemistry.
