Instrumentation
Klaus Mosbach Ben@ Danielsson Pure and Applied Biochemistry
Chemical Center, University of Lund P.O. Box 740. S-220 07 Lund 7 Sweden
Thermal Bioanalyzers in Flow Streams Enzyme Thermistor Devices In recent years the development of the technique of enzyme immobilization (1)has led to bioanalytical devices in which the "sensing" enzymes are placed in close proximity to the actual measuring component, the transducer. A number of examples of such a combination of enzyme and transducer have now been reported, in particular, the concept of the enzyme electrode (2). Apart from the obvious possibility of repeated use of the immobilized biocatalyst, the advantages gained by such an arrangement include higher sensitivity, quicker response, probable stabilizatiou of the enzyme, and the possibility of applying such analytical devices in continuous flow operations. The same advantages have also been observed in the combination immobilized enzymethermometric probe. In addition, the calorimetric principle of analysis possesses unique universality, since most enzymic reactions are accompanied by considerable heat evolution in the range of &lo0 kJ/mol (Table I). However, the generality of calorimetry could also be a serious limitation, as all enthalpy changes of a system are registered without discrimination, although the inherent specificity of biological processes, such as enzymic reactions, more than adequately compensates for the use of such a nonspecific method of detection. Thus, a number of applications of the analysis of biochemical systems using d o r i metric measurements, both batch and flow-through type, have been reported (3). However, in spite of the great potential of this detection principle, application of calorimetry to biochemical analysis has not gained widespread use, probably due to the rather high 0003-27W/80/0351983A$01.W/0 @ 1980 American Chemical Sociely
instrument costs of commercially available microcalorimeters. In the following report we wish to describe our attempts. and those of other research groups, to develop simple, inexpensive devices utilizing immobilized biocatalysts. In one report a combination immobilized enzymethermal analyzer (small volume calorimeter) was described, in which the enzyme was attached directly to a thin foil placed on the surface of a small Peltier element, after which the s m ple solution was added (4). The sensitivity of such a system was poor, however, and it has not been applied to continuous analysis. It appears that a common intermediate step taken in the development that led eventually to useful thermal hioanalyzers in flow streams involved the proximal arrangement of the enzyme to the ther-
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mal transducer (either by cross-linking the enzyme around the transducer, allowine an enzvme film to be formed. or by e;trapm&t of the enzyme in a ' dialysis bag enclosing the transducer) (5.6).Soch devices, also called thermal enzyme probes (TEP) (7)have usually been used under batch conditions. "hiis also holds true for some recent alternative TEP developments (7a. 7 b ) . Although clearly useful, the low sensitivity obtained with the early devices-due to escape of evolved heat to the surroundings-made these systems somewhat unattractive. A solution to the problem entailed the use of flow-through systems, because this allows evolved heat literally to be transported to or along the thermal probe, thus minimizing heat losses. To this end, the enzyme was immobilized to
I Table 1. Molar Enthalplesof Some Enzyme-CatalyzedReactions Emnu , cata(ase cholesteml oxidase Glucose oxidase Hexokinase 1 Lactatedehydrogenase 1 Ttypin ~
~
urease i Urlcsse ~
BC
1.11.1.6 1.1.3.6 1.1.3.4 2.7.1.1 1.1.1.27 3.4.21.4 3.5.1.5 1.7.3.3
I*lb.b.(.
Hydrogen peroxide Cholesterol Glucose Qlucoae Na-pyrwate
Benzoyl-l-arginineamide Urea urate
-AH (kJlm0l)
R . h m r
100.4 52.9 80 27.6 62.1 27.8
6.6 49.1
a
b C
d b e e b -
N . h D. P.: K W . L. A. ANI. Bbo%m. tm4S 47C78. @) Rh*.N. N.: V-. D..S. Cm. Mm 1977.24, 141CtO. : (C) Rehma 12. : (a) ~
ANALYTiCAL CHEMISTRY. VOL. 53, NO. 1, JANUARY 1981
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Thermistor EnzymePeltier Element
1
Enzyme Tubing
Y
Thermistor
Stationary
(4
II
Stationary
Enzyme Column
T
Sample
Enzyme
c
Thermistor
Flow
or flow (W
(C)
Figure 1. Schematic of: (a)small volume calorimeter, (b) thermal enzyme probes (TEP). and (c)enzyme thermistor or immobilized enzyme flow enthalpimetric analyzer support particles, preferably packed in small columns surrounding the probe, as exemplified by the enzyme thermistor ( 8 , 9 )or by the immobilized enzyme flow-enthalpimetric analyzer (IO).Recent modifications of the original TEP method now also utilize a laminar flow ( 2 1 ) . An obvious additional advantage gained hy such a flow-through arrangement is the possibility for continuous analysis. With these latter systems, end-point determinations are usually carried out; that is, the reactions are allowed to go rapidly to completion using an excess of biocatalyst. Kinetic (rate) measurements can also be made by measuring the ascending slope of the temperature peak, although this is less appealing, as such measurements are heavily influenced by external factors such as pH and temperature, and therefore require frequent calibration. We wish to discuss the instrumentation and application of thermal bioanalyzers in flow streams using the enzyme thermistor device developed by us as a model. It should be added that related devices developed almost simultaneously are very similar in their analytical capacity and basic setup (IO, loa, 22-24). In Figure 1a schematic presentation of the various devices developed is given.
Instrumentation In its simplest form a single thermistor device is used (Figure 2). A small plastic column (0.2-1.0 mL) contains the immobilized enzyme preparation, usually with the enzyme bound covalently to small, porous glass beads. The column is mounted in a Plexiglas housing, surrounded by an airspace for thermal insulation. in a holder de84A
signed for convenient change of columns. A small, glass-encapsulated thermistor (e.g., Veco type 41A28,1.5 X 6 mm, 10 kR a t 25 "C, temperature coefficient - 4.4%, Victory Engineering Corporation, Springfield, N.J.) fixed at the tip of a stainless steel tube (2-mm outer diameter) constitutes the temperature probe, which is placed a t the top of the column. Buffer is continuously pumped through the enzyme thermistor using a peristaltic pump after passing through a heat exchaneer made of a Diece of thin-walled staizess steel tubing (4W50 cm long, 0.8-mm inner diameter). The heat exchanger coil sits in a water-filled plastic cup. The purpose of this arrange-
mentis to minimize temperature fluctuations in the solution passing through the column. These temperature fluctuations are on the order of *O.0lo in the water baths normally used and less than "C around the thermistor. The temperature signal is registered with a potentiometric recorder cou. pled to a commercially available Wheatstone bridge. It delivers a 100 mV signal for akmperature change of 0.02O on the m a d sensitive range. We have also used bridges of our own design with somewhatbetter resolution. In order to increase baseline stability and insensitivity to nonspecific heat production caused by dilution or in-
Sample Buffer
Thermistor Enzyme Column
1
Figure 2. Schematic representation of an enzyme thermistor system (can be used a s a single thermistor device or, if required, in the split-flow arrangement including referencecolumn indicated to the right)
ANALYTICAL CHEMISTRY. VOL. 53. NO. 1. JANUARY 1981
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!any Columns are rapidly destroyed y moisture in the carrier *$,and this estrumion often occurs unrurpeetedly : is common t o find 100ppm of water I Carrier g a m ; and although this is a nall amount of water. it can caure rapi ydrolvois of phases such as polyesterr, FAP, SP-1000. as well as many others. he snlution is simple , . . install a long )be filled with molecular sieve in the irrier p r line prior t o the column as asurance against carrier gar moisture. 1 fact, most of you probably have such evices fitted into your rystfemr. but then was the last time YOU changed the iolecular sieves? It i s like any inruranc Dlicy - Y O U must continue to pay the remiumsorthe insurance laprer! I f yo o not now have a regular maintenance :hedule. begin now b y changing the a p each time you change the tank of w i e r sar.
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Figure 3. Photograph of a complete enzyme thermistor setup from the prototype series. From left to right: recorder, pump, and enzyme thermistor connected to Wheatstone bridge
teractions with the enzyme support, a split-flow enzyme thermistor arrangement can he applied (Figure 2) (1.5). In this case two columns are usedone containing the immahilized enzyme and the other, the reference column, containing support material only. or, if required. inactivated enzyme. Nunspecific effects are assumed tu he equal in the two columns. SI) that the differential temperature signal recurded by the twu thermistors a t the outlets ofthe columns represents the true signal uriginating from the enzymic reaction. Two piimps are used in this arrangement since it is important that the flow through each column he identical. Immersion of the device in a good water hath gives sufficient accuracy for a great numher of analyses. However, to allow use as a convenient routine instrument, the water hath may he replaced with a temperature-cuntrolled metal block. This unit, which includes a new Wheatstone bridge. now produces a t maximum sensitivity a 100-mV change in the recorder signal for a temperature change of IO-:< "C (16),thus permitting measurement of temperature changes down to 10-5 "C. Electrical and other calibrations have shown that as much as 80%of the total heat evolved in our semi-adiahatic device can optimally he registered as a change in temperature. This implies that for a given substrate present a t a concentration of 1 mM and with a molar enthalpy change fur the enzymic reaction of80 kJImol. a peak height corresponding to 10-2 "C iir higher will he obtained. and a temperature resolution of IO-.' "C gives 1% accuracy in the measurement. This unit allows two different colm n s to he used (provided no refer-
CIRCLE 193 ON READER SERVICE CARD
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ANALYTICAL CHEMISTRY. VOL. 53.
NO. 1. JANUARY 1981
ence column is required), thus permitting the simultaneous analysis of two components in a sample. The enzyme thermistor can quickly (i.e., within 15 min) he recharged with a new enzyme column when required. The complete enzyme thermistor setup is shown in Figure :4. Procedure
The enzyme thermistor is initially equilihrated hy pumping buffer through the system at a flow rate of 0.5-2.0 mI,/min, whence samples (USIIally as short pulses) are intruduced into the cuntinuous fluw via a threeway or chromatography injection valve. Typical heat responses to the introduction of urea to immohilized urease are shown in Figure 4. When the pulse length is increased, a plateau or steady state representing thermal equilihrium is uhtained, resulting in a higher amplitude. Generally, sample virlumes in the range of 0.25-1.0 ml. have heen used, giving a sample handling capacity 111 15-330 samples/h, although these figures have not been op. timized. More samples per hour could he analyzed by reducing the sample size (down to IOpI,). It should lie added that, regardless of pulse length. the temperature peak has heen found to he a suitahle measure of reaction heat. Linear correlation hetween the height of the temperature peaks and substrate concentration is uhtained. The integral of the temperature peak is alsu linearly related tu suhstrate concentration and may even give more accurate data than peak height, hut is more difficult to ohtain. In additiun, the slope of the ascending temperature peak is a useful measure o f suhstrate concentration and may he of particular value in automated systems.
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Flgure 4. Recorder tracing obtained in the determination of urea with one of the earlier devices containing urease covalently bound to glass particles. Urea Sam ples were introduced for 1 min or for 12 min at a flow rate of 0.75 mLlmin
Enzyme lmmoblllzalion Techniques Any of the five common immobilization techniques can he used (Figure 5). depending on the support, the hiological species, and the desired enzymetransducer configuration. The technique most widely adopted is covalent attachment, usually to controlled-pore glass heads hecause of their high enzyme-binding capacity and good stability toward pressure. Cross-linked Sepharose is also useful. When crude solutions are applied, as for example in the analysis of lipemic sera in clinical samples, of samples from microbiological fermentations, or of waste water samples (for environmental control), a flow system with a low tendency to clog should be used. In these cases it is advantageous to use nylon tubing as enzyme support. The enzyme can he covalently coupled, using glutaraldehyde, to the inside of
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the nylon tubing, thus allowing passage of particulate matter present in the sample. Entrapment techniques, for instance in the three-dimensional lattice of polyacrylamide, are the procedures of choice for cell immobilization (17). Adsorption has not been applied to any extent because of the risk of leakage. However, if the adsorption step is followed by cross-linking, then this is a convenient way of fixing the biocatalyst directly onto the transducer or probe. Bioaffinity binding, a special form of adsorption, appears to have considerable notential. For instance. after covalenily binding the lectin ’ concanavalin A to a support, these preparations will strongly and biospecifically bind glycoproteins such as acetylcholine esterase (18)or even cells such as red blood cells by interacting with the glycoproteins on the
cell wall (19).An advantage of the latter procedure is the possibility that the biocatalyst, if denatured, can he dissociated from the support and the column recharged with new enzymes or cells (reversible immobilization). Furthermore, in immunological applications, Covalently bound antibodies are allowed to affinity-hind their corresponding antigens (20).Techniques involving aggregation by chemical cross-linking to large particles and subsequent packing in small columns have not yet been attempted. There is room for further improvement of support matrices, especially concerning their mechanical stability in flow-streams. Furthermore, they should exhibit no nonspecific binding properties and allow a high load of biocatalyst to he hound. The thermal analyzers discussed here require an excess of catalytic power in relation to substrate present, partly because of the basic principle of measurement and partly to compensate for possible denaturation taking place. A number of tricks can be applied to amplify heat formation. Although many enzymic reactions have sufficiently high molar enthalpies, permitting determinations with devices such as the enzyme thermistor with a sensitivity down to 10-5 M, some reactions produce very little heat. For example, hydrolysis of an ester by trypsin evolves almost no heat. Nevertheless, this reaction can be followed calorimetrically because the hydrolytic step produces a proton that can protonate a suitable buffer (e.g., Tris) and the protonization heat can then be registered (8).Another method of amplifying the heat signal is to coimmohilize sequentially operating enzymes. This means that when the substrate is converted in the primary enzyme-catalyzed reaction, a second sequentially acting enzyme in close proximity to the first continues the “transformation” of the former substrate molecule. The heat signals from the enzyme reactions are superimposed and
L,
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Flgure 5. Schematic drawings of the five major types of immobilized enzyme preparations: (a)covalentl,
~entrapment, ,
(c)adswption, (d) affinity-binding, and (e)cross-linking ANALYTICAL CHEMISTRY, VOL. 53. NO. 1, JANUARY 1981
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1 .o
0.5
Time (h)
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Figure 6. Change of pump speed (red) to maintain glucose concentration in the effluent(blue) at a preset concentration of 65 mM afler passing whey (lactose) through a column containing immobilized lactase (23 registered as a measure of the first enzyme’s substrate concentration. This superimposed signal makes the analysis more sensitive. Thus, the sensitivity toward glucose was increased about two-fold with a system where glucose oxidase (glucose 0 2 gluconolactone HzOz)was coimmobilized with catalase (HzOz 1f2 0 2 HzO). Furthermore, the coimmobilized sequentially acting multistep enzyme systems offer other advantages, for example, faster substrate conversion, as compared with the situation where the enzymes are immobilized on separate polymer heads, and better efficiency a t low substrate concentrations (21).
+
+
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Applications The usefulness of the enzyme thermistor and similar devices has been tested in the following areas: clinical analysis, process control, fermentation analysis, environmental control, and recently as an instrument allowing monitoring of chromatographic separations. At the outset it can be said that the results obtained so far are indeed highly promising. Table I1 summarizes a number of systems that have been studied with the enzyme thermistor described here. A great variety of different suhstances have been determined. In principle, the chances are excellent of finding a suitable candidate for a particular biochemical substrate to he analyzed among the more than 2100 different enzymes that have been characterized so far and allotted a specific number. Although the sensitivity of the different systems given in the table has not been optimized in many of the cases listed, the concentration of a number of compounds of clinical interest such as glucose or urea already lies well within the detection range. The same measuring device can also be applied with slight modifications to the growing area of immuno9OA
chemical analysis, Le., antigenlantibody determination. For this alternative procedure we have suggested the name thermometric enzyme-linked immunosorbent assay (TELISA) (20). In principle, the column of the enzyme thermistor is filled with an immunosorbent such as antibodies immobilized on Sepharose CL 4B. The antigen to be determined and an enzyme-labeled antigen are introduced into the flow, whence the amount of enzymebound antigen remaining hound to the column is a function of the content of antigen. The less antigen present in the sample, the more enzyme-labeled antigen will he found in the column, and thus more heat will be evolved after the subsequent introduction of the enzyme’s substrate into the flowstream. Excellent sensitivity down to 10-13 m o a has been found with the TELISA technique (Table 11). The determination of soluble enzymes that are of interest in the clinical area and are of interest for enzyme manufacturers as well can also be carried out with a slightly modified version of this unit. In this case the enzyme column is replaced with a piece of Teflon tubing forming a “reaction coil” with a volume of about 1mL in which the soluble enzyme to be analyzed reacts with its substrate. The sample solution containing the enzyme and an appropriate substrate solution (with the substrate in excess) are each passed through a heat exchanger prior to being mixed, and rapidly passed through a new short heat exchanger (to eliminate heat generated on mixing) before entering the reaction coil. The temperature a t the outlet of the reaction coil is continuously measured with a thermistor. Linear correlation has been found between temperature response and enzyme activity for a variety of enzymes (22).
In the area of process control and
ANALYTICAL CHEMISTRY, VOL. 53, NO. 1, JANUARY 1981
fermentation analysis, thermal bioanalyzers in flow streams also appear to have excellent potential. Most analytical processes used in process control so far, except for pH, pO2, and pCOa determinations, are discontinuous. In most cases the goal in the development of analytical processes should be to make them continuous, as this reduces the costs of sample handling and personnel costs, and gives much more information per unit time. In addition, determination directed to a specific component formed or consumed in a process is to be preferred to indirect estimates based on changes in, for example, pH or p0z. In one model study to test the suitability of the unit for continuous analysis, the effluent from an enzyme column containing &galactosidase was monitored (23)(Figure 6). In the reactor, lactose present in the whey applied was hydrolyzed to glucose and galactose, and the glucose level in the effluent was measured using a gluchse oxidase/catalase thermistor. The heat signal registered by the thermistor was used, via a control unit, to regulate the flow of substrate through the enzyme reactor. Thus, it was possible to keep the product composition (e.g., glucose) in the effluent constant, despite clogging occurring in the column. In another study, penicillin present in fermentation broth was analyzed with a unit containing immobilized penicillinase. Furthermore, with the enzyme thermistor unit it is possible not only to continuously monitor penicillin as it is formed in a fermentation as well as substrate being consumed, but also to simultaneously register the overall thermal behavior of such a fermentation process, yielding a thermogram (power-time curve), which provides additional valuable information. Most of these cases are complex systems containing particulate matter and ways have to be found to prevent
ANALYTICAL CHEMISTRY. VOL. 53. NO. 1. JANUARY 1981
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0New Approaches to
assays important to the study of aromatic metabolism in animals and plants. Recent developments, including the use of LCEC for enzyme acitivity assays and the detection of unsaturated fatty acids, as well as unique advantages of post-column reactions and column switching techniques, are discussed.
Fluorescence Measurements Gary D. Christian,Department of Chemistry, University of Washington, Seatt/e. WA 98195
Alfred H. Free, Ames Company Division, Miles Laboratories, Inc., E/khari,IN 46514
Rapid scanning multichannel detectors may be used for fluorescence analysis of complex mixtures, and for fingerprinting of fluorescing 24 Illustrations. molecules in high performance liquid 37 Illustrations. chromatography and thin layer Localized Elemental Analysis by Couple:d Contributions of Clinical Analytical chromatography experiments. Instrumentation Back Scatter Electron Microscopy and X Chemistry to the Quality of Life (GARVAN such as the video fluoreometer consisting of a Irradiation Analysis MEDAL AWARD ADDRESS) polychromatic optical system computer Martin Rubin. Department of Siochemistry, HeUen M. Free,Ames Company Diuision, interfaced to a u.v.-enhanced silicon intensified Georgetown Uniuersity, Washington. D.C.20007 Miles Laboraton'es, Inc., Nkhart, IN 46514 target vidicon detector, is discussed. The distribution and movement of inorganic Urinalysis, kidney dialysis, and neo-natal inborn 50 Illustrations. species in the complex compartmentalization error testing to prevent mental retardation are among the analytical techniques discussed. OMeasurements of Drugs and Metabolites within the cell can be distorted by analfical methods which depend on cellular disruption, Future areas for clinical analytical chemistry in Body Fluids and Tissue Homongenates fractionation, and organelle isolation. The use of may include Sudden Infant Death Syndrome, Using Liquid Chromatography with Robinson back scatter electron microscopy with geriatrics and evaluation of a pending disease Electrochemical Detection simultaneously emitted X-rays collected in a before it occurs. Peter T. Kisslnger, Depariment of Chemistry, wavelength dipersive spectrometer is explored 37 Illustrations. Purdue Uniuersity, West Lalayette, IN 47907 for the examination of pathologic tissues. Liquid chromatography with electrochemical tissues. Trends in Clinical Chemistry in 44 Illustrations. detection (LCEC) is suitable for a number of Relation to Medicine F. WMam Sunderman, Institute for C/inica/Science r"""""""""'""""""""""---------------I Hahnemann Medical College and Hospital, 230 To order entire Clinical Analytical symposium including cassette album and illustrations for $89.00,; North Broad Street, Philadelphia, PA 19102 I check the box below. Or, check the individual tapes of your choice@ $13.50.Send payment along I I . I A brief historical survey of medicine and wth this ad to: American Chemical Society, Department 903,l 155 Sixteenth Street, N.W., I chemistry is followed by a look at the future of I Washington, D.C., 20036. VISA and Mastercharge are accepted. California residents add 6% state I I clinical research. use tax. Prices include postage and handling. I Contributions of clinical analytical chemistry to the improvement of world health are described.
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0 Colorimetric Immunoassays with Flavin Adenine Dinucleotide a s the Label Robert J. Carrlco,Ames Company Diuision,
Miles Laboratories, Inc., Nkhart. IN 46514
A new homogeneous competitive binding assay
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method employing flavin adenine dinucleotide as the label has been developed for haptens and proteins. Method has been demonstrated with measurements of theophylline and I immunoglobulin G in human serums. I
26 Illustrations.
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its entering. and thus clogging, the analytical device. This can he accomplished either with a dialysis memhrane of the type Technicon is using in its Auto Analyzer system, or by employing enzymes hound to the inside of nylon tubing instead of using regular columns. With the former arrangement the important requirement for sterility in fermentation processes can also he met. Thermal analysis can also he applied to both discontinuous and continuous environmental control analysis, in the latter case acting as a kind of toxi-guard. One may also utilize immobilized intact microbial or other cells (e.g., the microbe thermistor) ( I 7). organelles, or multienzyme systems placed in the column in case a more general detection of poisonous material is required. With such a system a large number of potentially toxic compounds would he detected as they affect the overall metabolism. leading to decreased heat generation. Alternatively, if detection of a specific toxic compound is required, then enzymes are preferable. For instance, concentrations of Hg2+ down to 0.2 pph could he determined due to the inhibitory action on immobilized urease (24).
Conclusion We feel that in other areas also, thermal flow stream analysis will be put to use increasingly in the future. One is the application for pure thermochemical analysis (25).For routine biochemical analysis, enzyme thermistors or corresponding probes incorporating biological sensors will prohahly find application. One specific example that comes to mind is the continuous
monitoring of chromatographic separations of both enzymes or low molecular weight compounds. Such a device would he a useful supplement or alter. native to conventional spectrophotometric analysis normally using a UV monitor (26). It is difficult to evaluate the usefulness of the thermal probes described here in relation to other related bioanalytical devices such as enzyme electrodes. Certainly all the various probes will find their specific niche. An advantage of thermal probes is the fact that they utilize the most general detection principle, although the sensitivity is somewhat limited, IO@ M, except when used in the TELISA technique. What makes them highly attractive is the possibility of analyzing turbid particulate or colored sampies. In addition, there is the fact that the need for auxiliary enzymic reactions, often involving expensive coenzymes and required in spectrophotometric analyses, is ohviated, hecause heat formed by the primary enzymic reaction can he registered directly.
lius, S.; S e r e , P. A,; Danielsson, B. In "Enzyme Engineering"; I'ye. E. K.; Wingard. I.. B.. Jr., Eds.; Plenum: New York, 1974;Vol. 2,p 151. ( 7 ) Weaver. J. C.; C w n e y , C. L.: Fulton. S. P.; Sehuler. P.; Tannenhaum. S.R. Hiorhim. Hir,phjs. Acto 1976.4.52. 285-91. ( 7 4 Tran-Minh. C.: Vallin. D. Anal.
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Klaus Mosbach ( r i g h t ) is profrssor and head of the Departmrnt of Pure and Applied Riochemistrj, Chemical Center. 1Jniver.sity of Lund. Sureden. His research interests are in solidphase biochemistry, including m zyme technology. enzymology, affinit y chromatography. and bioanalytical devices.
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