Anal. Chem. 1992, 6 4 , 129-133
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Enzymatic Flow Injection Analysis in Nonaqueous Media Lorenzo Braco
Departamento de Bioquimica y Biologia Molecular, Universitat de ValBncia, E-46100 Burjassot, ValZncia, Spain Jose A. Dards and Miguel de la Guardia* Departamento de Qulmica Analttica, Uniuersitat de ValBncia, E-46100 Burjassot, Valdncia, Spain
A novel enzymatic flow injection approach is proposed, involving the use of noncovalentlyimmobilized enzyme reactors operating in nonaqueous media. The feasibility of this approach was tested and successfully demonstrated using as a model system a coimmobiiized cholesterol oxidase-peroxidase reactor for the determination of cholesterol in toluene. M cholesterol, with The response was linear up to 1.8 X a detection IMt of 10" M. The throughput was 60 samplesh. The reactor was stable for more than 4 months with a hatf-life of 81 days. The strategy developed combines the remarkable benefits offered by enzymology in organic solvents and the advantages inherent In flow injection methods. Among them, the biocatalyst can be immobilized in a rapld, complete and irreversible manner by slmple adsorption to the solld support; poorly water soluble compounds can be readily dissolved In the Wle phase and dlrectly analyzed; and most Importantly, the enrymatlc reactor exhibits an enhanced stabiltty In the water-restricted environment.
INTRODUCTION Flow-injection analysis (FIA) has widely incorporated in the last years the use of enzymes as analytical tools for the determination of numerous compounds of clinical, industrial and biotechnological interest (1-4).This trend has been based on the fact that these biocatalysts add valuable, unique properties such as selectivity and rapidity to the advantages already inherent in flow injection systems. In most cases, the procedure of choice has been the covalent immobilization of the enzyme on a solid support packed in a flow reactor, with a carrier (reaction medium) generally composed of an aqueous buffer (5). Less often, the biocatalyst has been alternatively confined physically in the reactor in a soluble form (1). However, although it must be recognized that the approaches used so far in enzyme-based FIA have introduced substantial improvements relative to nonenzymic procedures, some problems still remain linked to the use of aqueous media as carriers, particularly concerning the low yield and tediousness of covalent immobilization and the deleterious effect that water has been proved to have on enzyme inactivation (6). Moreover, an aqueous environment, traditionally considered as "ideal" for the biocatalyst, can indeed hinder numerous analytical determinations due to the poor water solubility of many organic substrates (7). In this regard, attempts to increase the water solubility of the analyte by either adding a surfactant to the carrier solution or using aquo-organic mixtures are likely to cause undesirable detrimental effects on the enzyme whose stability is sometimes drastically reduced. In this concern, the recent, remarkable findings in enzymology, in nonaqueous media (8-lo),a developing area of enzyme technology of increasing interest, offer a priori a
* To whom correspondence should be addressed.
unique opportunity to alleviate the aforementioned drawbacks. In fact, the fundamentals of enzymology in organic solvents have been recently applied to the development of biosensors (11-13)including enzyme electrodes and enzyme thermistor devices (14).Hence, it seems exciting to verify whether the potential advantages of using enzymes in organic as opposed to aqueous media can also be successfully implemented in flow injection systems. In principle, a strategy based on the use of very low water containing organic solvents as mobile phases instead of the conventional aqueous buffers can benefit from a number of practical advantages (7,15,16), such as (i) molecules poorly soluble in water can be readily dissolved in the carrier, and therefore, directly and easily analyzed in the flow system, (ii) microbial contamination is prevented and side reactions are reduced, (iii) since most proteins are insoluble in organic solvents, the enzymes can be rapidly, conveniently, and irreversibly immobilized by simple adsorption from water onto a solid support and, therefore, not be further leached by the solvent (this procedure eliminates the laborious, time-consuming steps involved in covalent attachment), and (iv) most importantly, since it has been demonstrated that proteins in very low water, monophasic organic media possess a rethe enmarkably increased conformational rigidity (17,18), zymes immobilized in the reactor and operating in nonaqueous media are expected to exhibit an enhanced (thermo)stability (relative to the case when they are dissolved in an aqueous buffer), which in practical terms should result in a long reactor life. In addition, most inactivation processes caused by water, especially if the temperature is raised, will be minimized or suppressed. In the present paper, we present for the first time a novel strategy in enzyme-based FIA, the use of noncovalently immobilized enzyme reactors operating in nonaqueous media. The reactors are rapidly and easily prepared by simple adsorption of the biocatalyst onto controlled pore glass (CPG) beads. As a model system to test this experimental approach, we have selected the analysis of cholesterol in toluene, based on a recently proposed batch procedure involving the coupling of the reactions of two enzymes (19), cholesterol oxidase (COD) and horseradish peroxidase (HFtP). Briefly, cholesterol is first oxidized by molecular oxygen in a COD-catalyzed reaction, releasing stoichiometrically equivalent hydrogen peroxide, which is in turn converted to water by HRP in oxidizing p-anisidine (chromogenic indicator substrate), yielding a colored product which is monitored by means of a flow-cell UV-vis detector. The choice of this enzymatic system, in addition to the intrinsic interest of the analysis of cholesterol in food and clinical samples, arises from the fact that COD has been used in the past in aqueous FIA reactors (20-22) as well as for the development of biosensors (23, 24), which permits a comparison of the results and a more consistent evaluation of the methodology developed. EXPERIMENTAL SECTION Materials. Streptomyces spiralis cholesterol oxidase (E.C. 1.1.3.6) (23 international units (IU)/mg) and horseradish (Ar-
0003-2700/92/0364-0129$03.00/00 1992 American Chemical Sociely
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0.4
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Figure 1. Schematic diagrams of (A) the flow injection system and (B) the system used for batch determinations. Abbreviations: C, carrier; P, pump: V, injection valve: R, enzyme reactor; F, spectrophotometric flow-cell; W, waste: TC, thermostated reaction chamber. Inset: scheme of the enzyme reactor: (a)Teflon fitting screws: (b)
Teflon rigid tubing; (c) nylon filter. moracia rusticum) peroxidase (E.C. 1.11.1.7) (220 purpurogallin units (PU)/mg), as well as controlled pore glass beads (120-200 mesh), cholesterol, palmitic acid, and tripalmitin, were purchased from Sigma (St. Louis, MO). p-Anisidine, bencidine, and spectroscopic grade toluene were obtained from Merck (Darmstadt, FRG). Egg yolk phosphatidylcholine, also from Merck, was purified according to Singleton et al. (25)and showed a single spot by thin-layer chromatography (TLC) analysis. Hydrogen peroxide (33% w/v solution in water) was purchased from Panreac (Barcelona, Spain); the actual H202content was determined by volumetric titration with KMn04. Water was distilled twice and purified through a Millipore Milli-Q fitration apparatus. AU other reagents were of analytical grade quality. Immobilization Procedure. In order to obtain enzymatic preparations of either HRP or COD-HRP noncovalently immobilized on CPG, adsorption of the biocatalyst onto the beads was carried out basically as previously reported for a batch system by Kazandjian et al. (19). A number of microliters of an enzyme solution in 0.1 M aqueous phosphate buffer, pH 7.0 (optimized solution) was added t o an equal number of milligrams of glass powder spread on a watch glass, and the preparation was left to dry for 15 min under a gentle air drift. Instrumentation. The FIA system (Figure 1A) consisted of a Gilson Minipuls 2 perstaltic pump equipped with organic solvent resistant Viton tubing (1.5 mm i.d.), a Rheodyne Teflon rotary valve, and a Teflon tubular reactor with adjacent 100-pm nylon filters at each end (26j, where the immobilized enzyme was packed. Teflon tubing of 0.8 mm i.d. was used to connect the injector, the reactor, and the detector. Spectrophotometric detection was carried out with a diode array Hewlett Packard 8452 A spectrophotometer, equipped with a 1-cm path length, 30-pL, quartz flow cell. The carrier was water-saturated toluene containing M p-anisidine. Samples consisted of solutions of cholesterol or H202 at different concentrations in M p-anisidine water-saturated toluene. It must be noted that no appreciable differences in the results were obtained when phosphate buffer (pH 7.0) saturated toluene replaced water-saturated toluene in either the mobile phase or the sample solvent. It was also verified that different p-anisidine concentrations in both the mobile phase and the
Flgure 2. F I A recording and (inset)analytical calibration curve corresponding to the determination of HzOz in water-saturated toluene using a noncovalentiy immobilized HRP reactor (528 PU). Flow injection conditions: flow rate, 4 mL/min; injection volume, 0.5 mL.
sample in the range from to M did not affect the analytical signal. The batch system (Figure 1B) consisted of a thermostated reaction chamber containing the immobilized enzymatic preparation and typically 5 mL of reaction mixture. Gentle stirring was used to prevent external diffusional limitations. Samples consisted of cholesterol solutions in M p-anisidine watersaturated toluene. The progress of the reaction was followed by circulating by means of a peristaltic pump the filtered reaction mixture (which was recycled) through the spectrophotometer flow-cell and monitoring the time-dependent variation of absorbance at 458 nm. Any other details of experimental conditions are given in the corresponding legends to the figures.
RESULTS AND DISCUSSION Noncovalently Immobilized Nonaqueous HRP Reactor. Before preparing the bienzymic reactor, the behavior of HRP in the flow system was tested for the determination of H202in water-saturated toluene as carrier. The choice of this solvent is based on previous data obtained for HRP in batch experiments in nonaqueous media and on the widely accepted rule that hydrophobic, water-immiscible solvents generally favor enzyme activity (8). Figure 2 shows, as an example, a typical FIA recording obtained after injection of H 2 0 2 standards in the range from 2.0 X to 1.8 X lo-* M. The wavelength a t which absorbance was monitored corresponds to the maximum in the spectrum of p-anisidine oxidation products in toluene, as determined on-line with diode array detection and in previous batch experiments (not shown). A good linearity, corresponding to an equation A = 1970[H202]-0.011 ( r = 0.998) and a limit of detection of 9 X lo-: M were obtained in the experimental conditions assayed. In all cases, a quantitative conversion of H202was achieved in the flow system as deduced from the comparison of the above results with those obtained after injection of samples incubated in toluene in the presence of the enzyme (in a batch system) for a time long enough to ensure a complete reaction. Satisfactory calibration curves were also obtained when other water-immiscible solvents were used such as ethyl acetate or isobutyl methyl ketone (not shown). Thus, it seems that the performance of the HRP reactor in organic solvents is comparable to that in flow injection systems in aqueous media using covalently immobilized biocatalyst (21). Noncovalently Immobilized Nonaqueous COD-HRP Reactor. We next tested the performance of the bienzymic reactor, prepared by coadsorption of COD and HRP on CPG beads from an optimized solution (see Experimental Section), for the determination of cholesterol in the water-saturated toluene flow injection system. Figure 3 depicts, as an example, a typical FIA recording obtained after injection of cholesterol M. Again, standards in the range from 2.0 X 10-' to 1.8 X a good linearity is observed corresponding to an equation A
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
Time (min) Flgure 3. FIA recording and (inset)analytical calibratlon curve corresponding to the determination of cholesterol in water-saturated toluene using a noncovalently coimmobilized COD (23 IU) HRP (231 PU) reactor. Flow injection conditions: flow rate, 3 mL/mln; injection volume, 0.5 mL.
0 1 2 3 4 5
0 1 2 3 4 5
Flow-rate (mL/min)
Flow-rate (mL/min)
Flgure 4. Comparison of the flow-rate dependence of (A) the peak height and (B) the peak width in the nonaqueous F I A determination of (0)H,02 using an HRP reactor and (0)cholesterol using a COD-HRP reactor. Mobile phase: water-saturated toluene. The injection volume was 0.5 mL in all cases. H,O, and cholesterol concentrations were 10-4 M. 0.10
I
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= 86l[cholesterol] 0.005 (r = 0.9994). The limit of detection in this case was lo4 M. Note that this calibration does not correspond to a complete conversion of the sterol (in fact, circa 40% was estimated), which is not surprising taking into account the extremely long time (>7 h) reported for equilibrium to be reached in the batch system (19). In this regard, it is worth emphasizing that the kinetic determination carried out with the flow reactor provides a substantial improvement relative to the batch system as far as the assay time is concerned. On the other hand, at cholesterol concentrations M linearity started to be progressively higher than 2 X lost probably because the reaction rate was not any longer proportional to substrate concentration. Interestingly, when alternative experiments were carried out by using either two single reactors in series, each one containing one of the enzymes, or a single reactor in which the enzymes had been immobilized on different sets of beads and then mixed, no analytical signal was obtained. This result gives evidence of the need for COD and HRP coadsorption and is likely due to the known inhibitory effect of Hz02on the oxidase (27). Since the enzymes are insoluble in toluene and in light of the diffusional limitations of the product inside the support relative to aqueous solution, not only the presence in the reactor of HRP but also its physical proximity to COD is required to eliminate the nascent peroxide and allow for the oxidase to continue its catalytic turnover. In light of the nonquantitative conversion of cholesterol in the COD-HRP reactor, a number of experimental parameters which could affect the conversion degree (and therefore the analytical signal) were further investigated. FIA Parameters. The effect of variables such as injected sample volume and mobile-phase flow rate on the analytical signal was next investigated. In general, an increasing injection volume in the range from 100 to 500 gL provided a signal of increasing area which tended to level off at higher values. As to the influence of the carrier flow rate through the COD-HRP reactor on the cholesterol FIA profiles, Figure 4 shows that both peak height (A) and peak width (B) decrease as the flow rate increases. For comparison, the figure also includes the flow-rate dependent variation of the output signal corresponding to the determination of H202with the same reactor. The decrease in the cholesterol signal with increasing flow rate indicates, as mentioned above, that the reaction of sterol oxidation does not reach completion and that the extent of reaction clearly increases as the residence time of the injected pulse inside the reactor increases. Note, by contrast, the very slight decrease in peak height as a function of flow rate obtained in the determination of H202(see Figure 4A). Interferences in Cholesterol Determination. The interference of different compounds of lipidic nature which can
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Temperature ("C)
Flgure 5. Temperature dependence of the peak height in the nonaqueous determinationof cholesterol using the COD-HRP reactor. Each experimental point was the mean of three independent injections of a lo4 M cholesterol sample. Mobile phase: water-saturated toluene.
accompany cholesterol in food and biological samples was also tested. Three compounds were selected, representative of different lipid classes (fatty acids, triglicerides, and phospholipids), and assayed in toluene, codissolved with the analyte. Whereas palmitic acid and tripalmitin did not significantly interfere in a concentration range up to and 6 X M, respectively, phosphatidylcholine at moderate concentrations (>0.5 g/L) caused a quite severe interference in the cholesterol determination (relative error of about 20%). This finding is likely due to an effect of leaching of the enzyme from the support mediated by phospholipid-based reverse micelles which have been reported to quite readily form in these water-immiscible solvents (28). For this reason, it should be noted a t this point that the analytical methodology proposed should be limited to the determination of analytes (cholesterol in the present case) in phospholipid-free or lowphospholipid-content samples or alternatively used without limitations following a previous selective extraction step. Stability of the Reactor. Enzymes are in general very sensitive to temperature, and particularly, cholesterol oxidase in a soluble form is known not to be very stable in aqueous medium even at room temperature (21). A study of the effect of temperature on the noncovalently immobilized COD-HRP reactor performance was carried out over the range from 10 to 50 "C. Figure 5 depicts the temperature dependence of the peak height in the FIA determination of cholesterol in toluene. An almost linear increase in peak height was observed with increasing temperature from 25 to about 45 "C (mainly due to an increase in the reaction rate and in the mass-transfer efficiency of the substrates in the interior of the packed phase). These results are quite similar to previous data obtained in an aqueous flow system for a covalently immobilized cholesterol reactor (21). Although the analytical sensitivity was higher around 45 O C , for practical pwpwes temperatures lower than 30 "C are recommended in order to prevent some baseline instability.
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Figure 6 . Stability of the COD-HRP reactor to the presence of a strong base (5 M NaOH, A) or a strong acid (5 M HCI, B) in the aqueous solution saturating the mobile phase (toluene). Conditions: injection volume, 0.5 mL; flow rate, (A) 2.7 and (B) 2.5 mL/min; cholesterol and (B) 1.0 X M. concentration, (A) 1.25 X The pH is known to be a critical parameter on enzyme activity and sometimes on enzyme stability in aqueous media both in FIA and in batch systems. In order to investigate the effect on noncovalently immobilized enzyme activity of the pH of the aqueous solution saturating the organic phase containing the analyte, we assayed the COD-HRP reactor in extreme conditions using both as a carrier and as sample solvent toluene saturated with either 5 M aqueous NaOH or 5 M aqueous HC1. As Figure 6A shows, after 90 min of continuous reactor operation (about 250 mL of 5 M NaOH-saturated toluene passed through the reactor) the signal output was maintained, which indicated that enzyme activity was essentially unaltered. A similar behavior was also found for the 5 M HCl mobile phase (see Figure 6B). These results are in general agreement with the known increased stability of proteins in low-water media against presumably "adversen conditions (the protein is kinetically trapped in the organic solvent in a high-rigidity conformation) (8,18) and particularly with the previous observation that addition of a relatively high concentration (up to 0.1 M) of strong acids to lipase in hexane did not appreciably inactivate the enzyme (29). From a practical standpoint, our results demonstrate that extreme pH values in analyte-containing aqueous samples to be extracted with an organic solvent will not affect reactor performance. In order to investigate the presumably long storage stability of the noncovalently immobilized bienzymic reactor in nonaqueous medium, a reactor was prepared containing 23 IU of COD and 726 PU of HRP, which was intermittently tested in water-saturated toluene over a period of 4 months, during which 15 calibration assays were performed. When not in use, it was stored in the same mobile phase at 4 "C. For each assay, a set of standard cholesterol solutions was injected. As a measure of the reactor stability, the slope of the calibration curve was selected because for this particular reactor it was related to the COD activity. Note that if the reaction were complete, a partial loss of enzyme activity due to inactivation could not be immediately detected as a decrease in the calibration slope. Figure 7 shows the reactor stability as a function of the storage time. As an example, also are included the FIA responses corresponding to the initial and final calibration assays. An exponential decay of the slope with elapsed time can be observed. When the data were linearized using an integrated exponential equation a very good fit was obtained. Thus, enzyme inactivation during storage seems to follow a first-order process, the calculated half-life of the reactor being 81 days. After an overall 225 reactions in the 120-day period of the experiment, a sensitivity loss of about
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100
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Figure 7. Storage stability of the noncovalentb immobilized COD-HRP reactor, as determined from the slope of the cholesterol calibration curves. The calibration plots were obtained from triplicate injections of five cholesterol standard solutions in water-saturated toluene. The assay temperature was in all cases 28 'C. Insets: FIA recordings corresponding to the freshly prepared reactor and to the same reactor after 120 days of intermittent use, respectively.
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Time (min) Figure 8. Kinetics of cholesterol conversion measured in water-saturated toluene by batch reaction using (a)a freshly prepared noncovalently immobilized COD-HRP batch reactor and (b) the same reactor after 11 reaction-washing cycles. Condftions: total reaction volume, 5 mL; cholesterol concentration, M. 60% was obtained. These results show an enhanced storage stability of the noncovalently immobilized bienzymic reactor when operated in nonaqueous medium that is significantly greater than that recently reported for a noncovalently immobilized peroxidase electrode used in organic solvents (13) and similar to that reported for a COD flow injection reactor, operated with covalent immobilization by cross-linking to the support and in aqueous medium (21). Comparison of FIA and Batch Behavior of a Nonaqueous COD-HRP Reactor. In order to assess the practical advantages of using a flow injection enzymatic approach in nonaqueous medium as compared to a batch system, the repeated batch determination of cholesterol in toluene with coimmobilized COD-HRP was investigated. For this purpose, 23 IU of COD and 231 PU of HRP were coadsorbed on 215 mg of CPG beads. The enzymatic preparation was suspended in the reaction chamber (see Figure 1B) in each case in 5 mL of a reaction mixture containing M p-anisidine and a M) in different cholesterol concentration (from 10-5to water-saturated toluene. A reaction time of 5 min was necessary to obtain absorbance readings of the same order as those found in flow injection conditions. This indicates that the use of enzymes in nonaqueous FIA provides a higher analytical sensitivity and a drastic reduction in analysis time, probably due to a much higher local concentration of the biocatalyst in the flow reactor. More interestingly, when 11reactions were consecutively carried out using the same enzyme preparation in the batch system, with a bead-washing step between assays, a gradual loss of activity (up to circa 40% in this experiment) was observed after repeated use of the biocatalyst. Figure 8 illustrates, as an example, the kinetics of cholesterol conversion
ANALYTICAL CHEMISTRY, VOL. 64, NO. 2, JANUARY 15, 1992
obtained with the fresh enzyme suspension and with the same preparation after eleven reactions. By contrast, using a freshly prepared flow reactor, more than 50 reactions were carried out during one working session without any sensitivity loss. These results as well as the comparison of Figures 7 and 8 quite convincingly demonstrate the improved stability for repeated utilization of the proposed enzymatic flow reactor. Finally, the FIA methodology proposed revealed to be notably versatile with respect to the other variables such as, e.g., nature of the organic mobile phase or type of chromogenic substrate. Thus, when other water-saturated solvents instead of toluene were used, e.g., ethyl acetate, heptane, isobutyl methyl ketone, or a 1:l (v/v) mixture of ethyl acetate and toluene, satisfactory calibration curves were obtained in all cases, although the analytical sensitivity was lower than that found for toluene, in agreement with previous batch data for the same model system (19). This result is of particular interest because it offers a reasonable possibility of a rational choice of solvent (or solvent mixture), and therefore of water content in the organic phase, according to both the substrate solubility and the method of detection. On the other hand, when bencidine instead of p-anisidine was used as a chromogenic substrate, a similar calibration curve was obtained, A = 520[cholesterol] 0.019 (r=0.99991). In this context, the use of recently reported new chromogenic substrates for HRP (30) or fluorogenic substrates is suggested, which could greatly enhance sensitivity in the nonaqueous FIA determination of cholesterol.
+
CONCLUSIONS In this preliminary work, the feasibility of the use of noncovalently immobilized enzymes to carry out flow injection analytical determinations in nonaqueous media has been successfully demonstrated for the first time, using a model system consisting of coupled cholesterol oxidase and peroxidase. The strategy proposed affords a number of advantages. Some of them derive from the exploitation of enzyme technology in organic media. Biocatalyst immobilization through adsorption onto pore glass (or other suitable supports) can be carried out in a facile, complete, and irreversible manner, in contrast to the more tedious, time-consuming, and often incomplete covalent immobilization procedures. We have shown that noncovalent immobilization is very effective in a nonaqueous flow system and that the enzyme is not desorbed from the support during continuous reactor performance. In addition, poorly water soluble molecules can be directly analyzed in the organic phase. Other advantages are inherent in the FIA methodology: a drastic reduction in both the analysis time and reagent consumption as well as easy automation have been clearly demonstrated in the model system investigated. In particular, the bienzymic flow reactor
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prepared, which represents a notable improvement relative to the equivalent batch system, has proved to be remarkably stable, allowing storage and proper operation for more than 4 months. In summary, the methodology proposed is expected to become a promising approach in enzymatic flow injection analysis. Studies with different enzymes as well as practical applications to the analysis of real samples are currently under progress in our lab.
ACKNOWLEDGMENT We are indebted to Dr. Concepci6n Abad for her critical reading of the manuscript. Registry No. COD, 9028-76-6; HzOz,7722-84-1; peroxidase, 9003-99-0; cholesterol, 57-88-5. REFERENCES Hansen, E. H. Anal. Chim. Acta 1080,257-273. Ruzicka. J.; Flossdorf, J. Anal. Chlm. Acta 10809218, 2 9 1 3 0 1 . EmnOus, J.; Gorton, L. Anal. Chem. 1090,6 2 , 263-266. Asouzu, M. U.; Nonidez, W. K.; Ho, M. H. Anal. Chem. 1000, 62. 708-712. Olsson, 0.; Ogren, L. Anal. Chim. Acta 1083. 745, 71-85. Ahern, T. J.; Klibanov, A. M. Science 1085,228, 1280-1284. Klibanov, A. M. Chemtech 1088. 76,354-359. Zaks, A.; Klibanov, A. M. J . Biol. Chem. 1088, 263, 3194-3201. Deetz, J. S.;Rozzeii, J. D. Trends Biotechnol. 1088, 6 , 15-19. Khmelnitsky, Y. L.; Levashov, A. V.; Klyachko, N. L.; Martinek, K. Enzyme Mlcrob. Technol. l08& 10, 710-724. Hall, G. F.; Best, D. J.; Turner, A. P. F. Anal. Chim. Acta 1988, 273, 113-1 19. Miiabayashi, A.; Reslow. M.; Adlercreutz, P.; Mattiasson, 8. Anal. Chim. Acta 1080,279, 27-36. Schubert. F.; Saini, S.;Turner, A. P. F. Anal. Chim. Acta 1091,245, 133-138. Flygare, L.; Danielsson, B. Ann. N . Y . Acad. Sci. 1989, 542, 485-496. Dordick, J. S . Enzyme Microb. Technol. IO8S, 7 7, 194-21 1. Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng. 1087, 3 0 , 81-87. Zaks, A.; Klibanov, A. M. Science 1084,224, 1249-1251. Braco, L.; Dabulls, K.; Kilbanov, A. M. Proc. Natl. Acad. Sci. U . S . A . W O O , 87, 274-277. Kazandjiin, R. 2.; Dordick, J. S.;Klibanov, A. M. Biotechnol. Bioeng. 1986,2 8 , 417-421. Karube, J.; Hara, K.; Matsuoka, H.; Suzuki, S. Anal. Chim. Acta 1082, 739, 127-132. Massom. M.; Townshend, A. Anal. Chim. Acta 1085, 774, 293-297. Yao, T.; Sato. M.; Kobayashi, Y.; Wasa, T. Anal. Blochem. 1085, 149, 387-39 1. Trettnak, W.; Wolfbeis, 0. S.Anal. Biochem. 1000, 784, 124-127. Tatsuma, T.; Watanabe, T. Anal. Chim. Acta 1001,242, 85-89. Singleton, W. S.; Gray, M. S.; Brown, M. L.; White, J. C. J . Am. Oil Chem. SOC. 1065,4 2 , 53-56. de La Guardia, M. A.; Moraies, A. E.; Dares, J. A. Span. Pat. Appl. 9002666, 1990. Lee. K. M.; Blellmann, J. F. Bioorg. Chem. 1088, 74, 262-273. Walde, P.; Giuiiani, A. M.; Boicelli, C. A.; Luisi, P. L. Chem. Phys. LipMs 1900,53,265-288. Klrchner, G.; Scoilar, M. P.; Klibanov, A. M. J . Am. Chem. SOC. 1085, 707, 70-72. Conyers, S.M.; Kidwell, D. A. Anal. Biochem. 1001, 792, 207-211.
RECEIVED for review July 17,1991. Accepted October 3,1991.
