Anal. Chem. 1980, 52, 505-508
505
Thermal Enzyme Probe with Differential Temperature Measurements in a Laminar Flow-Through Cell Scott P. Fulton' and Charles L. Cooney' Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139
James C. W e a v e r * Harvard University-MIT, Division of Health Sciences and Technology and Departments of Physics, and Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 139
However, the concept of a convenient probe is still attractive and the column end-point devices also have some disadvantages. With respect to the latter, we note that the measurement of temperatures on the order of 3-5 H O C with single thermistors has been demonstrated in well-stirred aqueous solutions ( I 7). Further, under the laminar flow condition expected in a column, comparable or better performance is expected. However, differential measurement seems mandatory for low-level temperature measurements in columns (8). This necessity arises because both an enzyme column and a reference column are needed to discriminate effectively between the desired signal and the deleterious effects of ambient temperature fluctuations, nonspecific heats of adsorption, mixing, etc. A split-flow (dual column) system (8) can be used, but it is difficult t o maintain identical flow rates in two packed columns, especially if particulate matter and small gas bubbles are present in the sample. Indeed, time-dependent flow resistance is a general problem expected with the column end-point method, while a more open flow geometry may be relatively immune. A series (separated upstream and downstream) thermistor arrangement can also be used with columns (91, but this configuration causes adverse time delays between the thermistors. T h e existence of time delays and the existence of nonspecific heats of adsorption and incoming stream temperature variations together greatly reduce t h e effectiveness of what would otherwise be a differential measurement. Thus, for low-level measurements, the series configuration is generally unacceptable. With this background in mind, we now describe briefly some experiments with a TEP in an open (cross-section) flow system that is intended t o provide a quiet, laminar flow and thereby to eliminate the noise associated with the turbulent fluctuations that occur in a "well-stirred" system (17, 18). We also address the problem of bubble motion and nucleation on or near the thermistors, since bubbles have vastly different thermal properties than the aqueous solution, sometimes producing significant noise in the form of large, intermittent temperature variations as bubbles come and go. In this system, we have achieved rms noise levels (for differential temperature, A T , measurement) of about 2-3 @"C for both the base line before sample injection, a n d also for t h e steady-state response after a change in concentration of the enzyme's substrate (urea). This AT resolution corresponds M (signal-to-noise to a urea detection limit of about 1 X of two) for the present system. Recently described urease TEPs (used in an "immersion" mode rather than in a quiet, laminar stream) have approxiM ( 2 ) and 5 X mate detection limits of 1 X M (1) urea. The present device's better urea detection limit is ob"C tained despite a fivefold lower sensitiivity (about 4 x M-', compared to about 2 X 10 "C M-') than the recently reported immersion TEPs for which explicit temperature
Experiments were conducted with a flow-through cell that provides quiet, laminar flow past a thermal enzyme probe (TEP). The TEP consists of two thermistors, with enzyme (urease; E.C. 3.5.1.5) immobilized into one. I n contrast to TEPs in which well-stirred flow produces dominant temperature noise which is associated with turbulent fluctuations, the present arrangement allows observation of behavior under quiet, steady-flow condltlons. I n addition, continuous removal of some of the dissolved gas from the entering stream prevents motion or nucleation of otherwise troublesome bubbles near the TEP. Under these conditions, the device resolves rms temperature differences of 2-3 p ° C between the two thermistors, corresponding to a concentration resolution of about M urea.
Recently, interest has been renewed in developing analytical devices in which immobilized enzyme is placed on or near a probe-like thermal sensor ( I , 2). As discussed previously (341, such devices allow us to ask whether a convenient probe combining the specificity of enzyme catalysis with the generality of temperature measurement can be realized. Important aspects of such inquiries include not only consideration of fabrication techniques, but also investigations of temperature measurement, and the thermal enzyme probe (TEP) sensitivity (change in temperature difference per concentration of substrate, e.g., "C M-I). I t is particularly important to determine those sources of noise t h a t , in combination with the sensitivity, limit detection of the substrate concentration. Further, given the impressive results (7-16) obtained by combining immobilized enzymes and thermal measurements with flow-through columns (in which reactions are usually completed), one should consider comparisons of the T E P and t h e enzyme thermistor (column end-point method). For example, effects t h a t alter the sensitivity by modifying the reaction rate are important for a kinetic method. Since immobilized enzyme probe devices are almost always based on kinetics rather than on reaching an end point, effects that alter reaction rates (e.g., pH, temperature) usually change a probe's sensitivity, while column end-point devices are relatively unaffected. In addition, end-point columns are about two orders of magnitude more sensitive than a TEP. Specifically, if t h e total enthalpy change associated with an enzyme-catalyzed reaction is lH N lo4 cal-mol-', the sensitivity is about 10 "C M-' for columns and about 0.3-3 X 10 "C M for a T E P ( 4 ) . Thus, the column end-point device is decidedly superior for many applications. 'Department of Interdisciplinary Science. Department of Nutrition and Food Science. 0003-2700/80/0352-0505$01 O O / O
C_
1980 American Chemical Society
506
ANALYTICAL CHEMISTRY VOL. 52, NO. 3, MARCH 1980 4
DRIVE ( G A S OR VACUUM
DEGASSING
2
CONTROLLER WHEATSTONE BRIDGE
Ly-BATH
ELECTRONIC TEMPERATURE CONTROLLER
316 STAINLESS STEEL
FITTING
Figure 1. Block diagram of the flow-through thermal enzyme probe system
measurements are reported ( 1 ) . EXPERIMENTAL E n z y m e Immobilization. Enzyme was immobilized by cementing lyophilized particles of polyacrylamide gel (to which the enzyme was previously covalently bonded) onto the thermistor surface ( 4 ) . Urease (urea amidohydrolase; E.C. 3.6.1.5) was obtained commercially in lyophilized gel form (Enzygel-500 Urease, Boehringer-Mannheim, GmbH). Contact cement (Kontakt 2000. Uhu-Fismar, GmbH), diluted about 1:4 in methylene chloride, was used to coat the tip of the probe, which was then rolled in the gel powder and dried for 1 h. The enzyme-glue layer was about 100 pm thick. The reference thermistor was prepared in a similar fashion but was not rolled in the enzyme powder. C h e m i c a l s . Tris buffer (0.05 M) with M ethylenediaminetetraacetate (EDTA) was used a t p H 7.2 and 7.4. Samples consisted of urea dissolved in buffer. Reagent grade chemicals were used throughout. Equipment. The present TEP system was designed to allow study of AT measurement problems. Although the abilities to introduce urea concentration changes and to provide conditions consistent with the presence of an enzyme-catalyzed reaction a t one of the thermistors are important, priority was given to improving A T measurements when we designed the apparatus. This emphasis requires identification and reduction of major sources of A T noise. In this apparatus, enzyme is immobilized on the surface of one of a pair of thermistors, and both are placed in an open-flow channel in a small stainless steel cylinder (Figures 1 and 2), comprising a flow-through cell. This cell, together with a length of stainless steel tubing that provides approximate thermal equilibration of the incoming sample stream, is surrounded by a stirred, temperature-controlled (-&lo-' "C) oil bath. The bath temperature is either 30 or 35 "C. Highly accurate values O C ) was desired, to were not sought; instead constancy minimize AT noise. In contrast to a previously described apparatus which employed well-stirred, turbulent flow ( 4 ) ,the present apparatus provided quiet, laminar flow through the cell containing the thermistors. Thermistors are sensitive to flow variations since a thermistor's dissipation constant (power dissipated per temperature rise; W OC-') is a function of flow; such behavior occurs because thermistors require voltage excitation to measure their resistance. The associated electrical power dissipation causes a temperature rise that affects the resistance. Since increased flow rate past a thermistor provides additional cooling, variable flows cause a variable thermistor resistance and an associated equivalent temperature noise. As previously shown (17,181 with turbulent flow, this resistance can be the dominant source of noise for typical thermistors excited with powers in the range lo4 to lo-* W. Therefore, the present system is designed to provide quiet, laminar flow with negligible flow rate variations. Our belief that laminar
Figure 2. Cross-section views of the flow-through thermal enzyme probe cell. Top: transverse section: bottom: vertical section
flow existed is based on the observation that a sharp increase in A T noise was reproducibly observed when V 2 0.73 to 1 mL min-', with the exact threshold varying for different thermistors and their spacings. Early experiments with both this system and another (29) showed that mechanical pumps do not provide adequately smooth flow. Peristaltic pumps cause large periodic apparent A T variations. Even syringe pumps with adjustable Teflon O-rings produce AT noise in the form of erratic signals, presumably caused by frictional start-stop motions. However, smooth flow was obtained by using a 3-L stainless steel vessel filled mostly with buffer and overpressured (500 Torr) a t the top through an inlet from a pressure-regulated nitrogen gas source. The movement or nucleation of bubbles near the thermistors comprises another significant AT noise source. This problem quite possibly may be associated with our solution to the flow variability problem; excess nitrogen can dissolve in the buffer. Downstream at lower pressures, bubble nucleation caused by excess dissolved nitrogen (or any pre-existing dissolved gas) is likely. The vastly different thermal properties of gas and liquid suggest that movement or nucleation of bubbles near a thermistor also causes its dissipation constant to vary with time, again producing AT noise. By using a continuous degassing unit, we eliminated AT noise associated with bubbles. The degasser consisted of membrane interface (Figure 3) designed for a mass spectrometer (191, with a mechanical rough pump (Welch Duo-Seal) substituted for the mass spectrometer vacuum system. Two sampling systems were used to test the response of the device to urea concentrations. In the first, a 5-mL sample loop with a sample injection valve was used (Unimetrics, Inc.; Model =10108). In the second, the sample loop was replaced with a continuously stirred 500-mL flask into which concentrated urea was injected to produce stepwise increases in urea concentration. The temperature difference between the thermistors was measured with a dc Wheatstone bridge. The thermistors (Series P60) were obtained as matched pairs from Thermornetrics, Inc., Edison, N.J. At 30 OC, their resistance was 2 X lo4 Q, with a resistance coefficient of about 4 X OC-'. The excitation voltage (across an individual thermistor) was 2.3 V. Each thermistor protruded about 10 cm from the walls into the stream. Tests, in which the voltage across one of the thermistors was varied, were first conducted without enzyme. By using the second thermistor as a receiver, we determined conditions of thermistor separation (3 x IO-' cm) and flow rate ( 2 6 X lo-' mL/min) that produced negligible thermal cross-talk between the thermistors. If heat generation associated with the enzyme reaction were partially measured by the nonenzyme thermistor, a smaller A T signal would result.
ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
+
,
m
507
1
i
Figure 4. Thermal enzyme probe ATsignal in response to a 7.5 X M urea solution using the sample loop method with a volume flow rate of 0.7 mL min-'
Figure 3. Cross-section of the continuous degasser's solution-vacuum interface. The body of the interface is stainless steel. Epoxy (Emerson-Cuming 2651) or solder is used to attach the inlet and outlet tubes, which are 0.16cm 0.d. stainless steel. Two Viton O-rings provae the liquid and vacuum seals. The membrane exposed to the liquid is 25-pm-thick MEM-213 (General Electric Co.),backed mechanically by 1-pm pore Nuclepore filter (Nuclepore Co.), and then a stainless steel disk with 0.16-cm holes. The vertical gap between the membrane and stainless steel underpiece is h = lo-' cm, except at the outer collection annulus, which has a 0.16 X 0.16 cm cross-section. The area of membrane, rrR2, exposed to liquid is 1.3 cm'. A single membrane has been used for up to six months without incident Finally, the thermistor pairs were specified to have a good common mode rejection ratio with respect to temperature (CMRRT). By definition, the CMRRT of a thermistor pair is the ratio of common temperature change t o the associated apparent A T change. An approximate expression for CMRRT is
CMRRT
N
&/Au
Figure 5. Thermal enzyme probe ATsgnal in response to urea solutions M urea with a volume with a change from 2.5 X to 2 . 8 X flow rate of 0.6 mL min-' in the sampling flask system 1000_
800 -
0
-
600.
a
400.
0 0
+
0
o o 0
0
0
2 00
O 0
00
(1)
0 0
where CY = aR/RaT is the fractional sensitivity of a thermistor, I c u is the difference in the thermistors' fractional sensitivities, and u is the average fractional sensitivity for each thermistor of the pair. Unmatched negative temperature-coefficientthermistors typically have a CMRRT of about 10; thus, a 1 " C change in the solution causes an apparent ST of lo-' "C. The present, specially ordered thermistor pairs have a CMRRT of about lo3, so that fluctuations in solution temperature (about that of the oil bath, "C) should generate A T noise at the lo4 "C level. Although we have not carefully established the origin of the noise that limited the apparatus to a rms A T resolution of 2-3 X lo4 "C, we believe that it is primarily coupling through the CMRRT. The CMRRT defines the coupling of: (1)imperfect solution stream temperature equilibration, (2) bath temperature fluctuations, and (3) in combination with dissipation constants, even that of flow-rate variations (if the flow is not smooth). Since thermistors are not usually sought or specified for high CMRRT, it is not yet clear what CMRRT values can reasonably be obtained in the future.
RESULTS AND DISCUSSION Figure 4 shows the A T signal response of the system when a 5.0-mL substrate loop was used to obtain a response to a 7.5 X M urea concentration change. The response shows first a small negative peak caused by the substrate loop compliance and a n associated, transient change in the flow rate; a second larger peak, presumably caused by the heats of mixing and adsorption; and third a steady-state AT signal after about 3 min. I t was observed t h a t upon switching back to buffer, the time required to return to base line was longer
IO-
10-2
2
I
lo-z
3.
U R E A CONCENTRATION (mole l i t e r - ' )
Figure 6. A response curve with a volume flow rate of 0.7 mL min-', using the sample flask method. The symbol size indicates the approximate size of peak-to-peak error bars. The sensitivity is about 4 X lo-' "C M-' at low concentrations, in agreement with a simple model (ref. 4) for a diffusionally limited device than 3 min and t h a t t h e return time increased with urea concentration. The reason for this asymmetry is not presently known, b u t may be related t o the use of a porous, irregular layer of immobilized enzyme. The small pulsations in the AT signal are produced by periodic flow variations caused by the mechanical pump used in early work; these pulsations are absent when the gas pump/reservoir is used to achieve a quiet flow. Figure 5 shows a signal in a later experiment using the flask injection system and a different thermistor pair. There is a M urea. T h e transient positive spike step increase of 3 X is also caused by the sudden change in flow rate after injection. (The sign of the transient response cailsed by flow-rate changes depends on the matching of the particular thermistors and is not important.) This system has a peak-to-peak AT resolution of about 1.5 X "C and a corresponding estimated 2-3 x lo4 "C rms AT resolution. From the empirically determined sensitivity (Figure 6), this resolution corresponds M urea. t o a signal-to-noise of two a t
508
Anal. Chem. 1980, 52, 508-511
Figure 6 shows a A T vs. urea concentration response curve for the sample loop system. T h e sensitivity, a(An/aC,. is 5 X "C M-'; this is of the order of magnitude expected for a diffusionally limited device ( 4 ) and is about two orders of magnitude less sensitive than devices that carry the reaction t o completion (7-14). In conclusion, this study demonstrates the ability to make relatively high resolution AT measurements with an open-flow channel configuration and under conditions compatible with immobilized enzyme use. This configuration has allowed us to address several significant effects on A T measurements: (a) the common mode rejection ratio to temperature (CMRRT) of a thermistor pair, which partially decouples variations in temperature of the incoming stream from A T measurements; (b) t h e CMRRT coupling between flow variations and A T caused by the thermistors' dissipation constants; (c) the motion or nucleation of bubbles near the thermistors; and (d) the thermal cross-talk that can occur between closely spaced thermistors. These findings suggest that future TEP devices, employed as immersible probes in a well-stirred solution, will need to address several problems. First, since temperature variations with time will occur for turbulent flow, better CMRRT values are needed. Second, since turbulent flow also imposes a spatial, fluctuating temperature gradient, closely spaced thermistors appear to be desirable. Therefore, higher average velocities and smaller excitation powers are required to reduce thermal cross-talk. However, reduced excitation power renders other sources of noise significant (17). Low-duty cycle, pulsed excitation may help, since t h e average power could be significantly reduced. In short, while experiments that directly address these issues in turbulent flow are desirable, the present study suggests that a limit for AT measurements in T E P s t h a t are immersed in a well-stirred flow may not have been reached. Further, this work is also relevant to column devices; given their intrinsically
higher sensitivity of about 10 "C M-I, if comparable AT measurements can be made in more open columns with quiet, laminar flow and degassing where necessary and permissible, then significantly improved detection limits (below lo4 M) should be obtained.
LITERATURE CITED (1) Rich, S.; Ianniello, R. M.; Jespersen, N. D. Anal. Chem. 1979, 57, 204-206. (2) Tran-Minh, C.; Vallin, D. Anal. Chem. 1978, 50, 1874-1878. (3) Cooney, C. L.; Weaver, J. C.; Tannenbaum, S. R.; Faller, D. V.; ShieMs, A . In "Enzyme Engineering", Vol. 2, Pye, E. K.. Wingard, L. B., Jr., Eds.; Plenum: New York, 1974; pp 411-417. (4) Weaver, J. C.; Cooney, C. L.; Fulton, S. P.; Schuler, P.; Tannenbaum, S. 13.Biochim. Biophys. Acta 1976, 452,285-291. (5) Weaver, J. C.; Cooney, C. L.; Tannenbaum, S. R.; Fulton, S. P. In "Biomedical Applications of Immobilized Enzymes and Proteins", Vol. 2. Chang, T. M. S., Ed.; Plenum: New York, 1977: pp 191-205. ( 6 ) Cooney, C. L.; Weaver, J. C.; Fulton, S. P.; Tannenbaum, S. R. In "Enzyme Engineering", Vol. 3, Pye, E. K.. Wingard, L. B., Jr., Eds.; Plenum: New York, 1978; pp 432-436. (7) Mosbach, K.; Danielsson, B.; Borgerud, A,; Scott, M. Biochim. Biophys. Acta 1975, 403, 256-265. (8) Mattiasson, B.; Danielsson, B.; Mosbach, K . Anal. Lett. 1976, 9 , 2 17-234. (9) Mattiasson, 8.; Danielsson, B.; Mosbach, K . Anal. Lett. 1978, 9 , 867-889. (10) Danielsson, E.; Gadd, K.; Mattiasson, B.; Mosbach, K . Anal. Lett. 1978, 9 , 987-1001. (11) Mattiasson, 8. FEBS Lett. 1978, 85, 203-206. (12) . . Bowers. L. D.: Cannina, L. M.; Savers. C. N.; Carr. P. W. Clin. Chem. 1976, 22, 1314-131g (13) Bowers, L. D.; Carr, P. W. Clin. Chem. 1976, 22, 1427-1433. (14) Schmidt, H.-L.: Krisam, G.;Grenner, G. Biochim. Biophys. Acta 1978, 429. . 283-290. (15) Mattiasson, B. FEBS Lett. 1977, 77, 107-1 10. (16) Mattiasson, B.; Borrebaeck, C. FEBS Lett. 1978, 85, 119-123. (17) Bowers, L. D.; Carr, P. W. Thermochim. Acta 1974, 70, 129-142. (18) Fulton, S. P. Master's Thesis, Massachusetts Institute of Technology, Cambridge, Mass., 1977. (19) Weaver, J. C.; Abrams, J. H. Rev Sci. Instrum. 1979, 50, 478-481. -~
~
RECEIVED for review September 19,1979. Accepted November 12, 1979. This work was supported by United States Public Health Service Biomedical Research Support grant no. S07RR-007047-10.
Oxalate Determination by Immobilized Oxalate Oxidase in a Continuous Flow System Renze Bais," Nicholas Potezny, John B. Edwards, Allan M. Rofe, and Robert A. J. Conyers Division of Clinical Chemistry, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000
A rapid, one-step procedure is described for the determination of oxalate. Oxalate oxidase, immobilized in a continuous-flow system, converts oxalate to hydrogen peroxide which is detected by a color reaction with 4-aminophenazone. The assay Is Sensitive to oxalate concentrations as low as 5 1 M and is hlghty reproducible. A range of inorganic ions, alcohols, mons and polycarboxylic acids, sugars, and nltrogenous substances do not interfere with the assay. Although ascorbate and NADH did not affect the enzyme reaction, they did reduce the color formation with 4-aminophenazone. The immobilized enzyme system can, however, be readily adapted to more specific detection systems.
Oxalate is found in a wide range of biological and nonbiological materials ( I ) and is used in a variety of manufacturing ( 2 ) and analytical procedures ( 3 ) . In t h e determination of oxalate, techniques such as volumetric analysis, precipitation, 0003-2700/80/0352-0508$0 1 OO/O
solvent extraction, colorimetry, fluorimetry, chromatography, radioisotope methods, ion selective electrodes, enzymes, and mass spectroscopy have all been used ( I ) . Most of t h e published methodologies have the disadvantage t h a t t h e sample must be pretreated before the actual oxalate analysis can be performed. This increases the complexity of t h e analytical procedure and consequently the sensitivity, specificity, and reproducibility of the determination often suffers. As analytical reagents, enzymes provide a degree of specificity and sensitivity available only in other more expensive chemical and physical methods such as mass spectroscopy. In addition, immobilization of the enzyme on an inert surface permits reutilization of t h e enzyme thus reducing t h e cost when used routinely ( 4 ) . If the immobilized enzyme is coupled with a continuous flow system, handling is minimized and reproducibility is enhanced. Oxalate decarboxylase (E.C. 4.1.1.2) and oxalate oxidase (E.C. 1.2.3.4) are two enzymes used in the determination of oxalate. However, the methods described involve either a S 1980 American Chemical Society
