Anal. Chem. 1994,66, 2726-2731
Enzymatic Reactor/Room-Temperature Phosphorescence Sensor System for Cholesterol Determination in Organic Solvents Maria JesGs Valencla-Gonzblez and Marta Elena Diaz-Garcia’ Department of Physical and Analytical Chemistry, University of Oviedo, c/ Julih Claveria 8, Oviedo, E-33006 Spain The development and evaluation of a novel cholesterol oxidase reactorhoom-temperature phosphorimetric (RTP) sensor system, operating in a continuous organic flowing stream, for cholesterol determination in food samples is presented. The organic carrier is a mixture of hexane/5% (v/v) chloroform. Cholesterol oxidase is physically retained onto controlled pore glass beads to form the bioactivematerial. Oxygenconsumption during the enzymatic reaction is followed via the changes in the RTP of an oxygen-sensitivemetal chelate retained on an anion-exchange resin. Effects of initial aqueous pH of the enzyme, organic solvent nature, and temperature are examined. Two spatial configurations for the enzyme reactor and the RTP sensor are evaluated. At 35 OC the proposed system is M cholesterol) with a hear response very sensitive (DL 5 X up to 4 X 1O-j M. The relative standard deviation (n = 5) at the 2 mM cholesterol level was 2.5%, and the response time is ca. 2 min. Application of the method to total cholesterol determination in food samples (eggs and butters) and comparison with an enzymatic visible/UV procedure yielded consistent results. Recently, much attention has been given to organic-phase enzymatic From an analytical perspective, this growing interest is due to numerous potential advantages in employing enzymes in organic media. These include the possibility for analysis of poorly water soluble substances, increased thermal stability for some enzyme^,^'^ decreased microbial contamination, and recovery of enzymes by simple filtration (enzymes are insoluble in organic solvents). Also, undesirable side reactions in organic media, as well as substrate and product inhibition, may be reduced. Various methods have been devised to “present” enzymes to organic solvents, for example, direct suspension of enzyme particles in organic media,8 use of covalent modifications to render enzyme molecules soluble in organic solvent^,^ and solubilization in water-in-oil (w/o) microemulsions or adsorption onto solid supports.l0-I2 (1) Saini, S.;Hall, G. F.; Downs, M. E. A.; Turner, A. P. F. Anal. Chim. Acra 1991, 249, 1. (2) Wang, J.; Dempsey, E.; Eremenko, A.; Smyth, M. R. Anal. Chim. Acra 1993, 279, 203. (3) Wang, J. Talanra 1993, 40, 1905. (4) Danielsson, E.; Flygare, L. Sew. Actuarors B 1990, I , 523. (5) Danielsson, E.; Flygare, L.; Velev, T. Anal. Lett. 1989, 22, 1417. (6) Zaks, A.; Klibanov, A. M. Science 1984, 224, 1249. (7) Ayala,G.;Tuena deG6mez-Puyon. M.; G6mez-Puyon, A,;Darszon, A. FEES Leu. 1986, 203, 41. (8) Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141. (9) Takahashi, K.; Yoshimoto, T.; Ajima, A.; Tamaura, Y.; Inada, Y. Enzyme 1984, 32, 235.
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The knowledge that enzymes are catalytically active in some organic solvents has led to the important development of organic-phase enzyme electrodes.’ In fact, several organicphase biosensors have been developed for monitoring phenols in olive oil,l3 cholesterol in butter and margarine,14 and hydrogen peroxide or organic peroxides15J6and for measuring catechols in several alcohols.17 The analytical advantages gained from the use of noncovalently immobilized enzyme reactors operating in nonaqueous media in a flow injection approach have been recently documented.18 In spite of the analytical potential of enzymatic catalysis in organic solvents, there is a lack of information on the use of these biocatalytic systems in the development of optical biosensors and/or enzymatic reactor/optical sensor devices operating in organic solvents. Here, we report a novel flow injection method for the determination of cholesterol in food samples using an immobilized bienzymatic reactor/room-temperature phosphorimetric (RTP) sensor system operating in organic media. The bioactive material consists of cholesterol oxidase (ChOD) and horseradish peroxidase (HRP), coimmobilized on the surface of controlled-pore glass beads. In the recognition process cholesterol reacts with ChOD as follows: cholesterol + 0,
-
ChOD
4-cholesten-3-one + H,O,
(I)
Oxygen consumption was followed via the changes in the RTP of an oxygen-sensitive metal chelate immobilized on an anion-exchange resin packed into a conventional flow-through cell. The resulting signal, which is proportional to oxygen concentration changes, was used for cholesterol quantification in real samples through a previously prepared calibration graph. The hydrogen peroxide generated concomitantly is consumed during the oxidation of a HRP substrate (p-methylphenol) according to (IO) Menger, F. M.; Yamada, K. J . Am. Chem. Soc. 1979, 101, 6731. (1 1) Cambou, B.; Klibanov, A. M. J. Am. Chem. Soc. 1984, 106, 2687. (12) Creagh, A. L.;Prausnitz, J. M.; Blanch, H. W. Enzyme Microb. Techno/. 1993, 15, 383. (13) Wang, J.; Reviejo, A. J.; Mannino, S.Anal. Lett. 1992, 25, 1399. (14) Hall, G. F.; Turner, A. P. F. Anal. Lett. 1991, 24, 1375. (15) Schubert, F.; Saini, S.;Turner, A. P. F. Anal. Chim. Acra 1992, 245, 133. (16) Wang, J.; Freiha, B.; Nascr, N.; Romero, E.; Wollenberger, U.; Ozsoz, M.; Evans, 0. Anal. Chim. Acra 1991, 251, 81. (17) Wang, J.; Lin, Y.; Eremenko, A.; Ghindilis, A,; Kurochkin, I. Anal. Leu. 1993, 26, 197. (18) Braco, L.; DarQ, J. A.; de la Guardia, M. Anal. Chem. 1992, 64. 129.
0003-2700/94/036&2726$04.50/0
@ 1994 American Chemlcal Society
H202+ p-methylphenol
-
HRP
H,O + polymer
(11)
in order to prevent inactivation of ChOD19 and to favor the ChOD reaction in the forward direction. Two spatial configurations were considered for the bienzyme reactor. The first one was a two-step approach in which the biocatalytic beads, inserted into a minicolumn, were physically separated from the RTP sensing phase (packed into the flow cell). The other was an one-step approach in which the biocatalytic beads and the RTP sensing beads were closely packed into the small space of a flow-through cell. These two enzyme reactor/RTP sensor devices are illustrated schematically in Figure 1. The optimization performance of the ChOD-HRP reactor/RTP sensor in organic media involved careful consideration of some important factors such as the nature of the organic solvent, pH, and temperature. The practical use of the proposed assay method was evaluated by analyzing some food samples and comparing the results with those of a popular cholesterol oxidase based colorimetric method.
EXPERIMENTAL SECTION Chemical and Solutions. Cholesterol oxidase (ChOD) from Pseudomonas species (EC 1.1.3.6.)with a specific activity of 2540 units/g of solid was purchased from ICN. Cholesterol estearase (ChE) from Pseudomonusfluorescens(EC 3.1.1.13.) with a specific activity of 1300units/g of solid and peroxidase from horseradish (HRP) (EC 1.11.1.7.)with 96 000 purpurogallin units/g of solid were from Sigma. Sodium acetate and disodium hydrogen phosphate were purchased from Merck. Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (Bis-Tris) was from Sigma. Hexane, isooctane, toluene, and chloroform were obtained from Fluka. All other reagents used were of analytical grade. A visible/UV enzymatic test for cholesterol determination in foodstuffs was obtained from Boehringer Mannheim. Cholesterol standards at different concentrations were prepared by dissolving cholesterol from Lanolin (Fluka) in the carrier solution. The organic carrier consisted of a hexane/ 5% (v/v) chloroform mixture containing 10 mM p-methylphenol (Merck) and saturated with the aqueous buffer BisTris (0.1 M, pH 7). Preparationof the RTP Oxygen Transducer. An aluminum chelate solution was prepared by mixing 0.3 mL of 1000 pg mL-l A1 stock solution and 35 mL of 3 X lt3M ferron (7iodoquinolin-8-ol-5-sulfonicacid) in a 1 00-mL flask. The final solution was diluted to the mark with 0.1 M sodium acetate/acetic acid (pH 5.5) buffer solution. This chelate solution did not deteriorate (stable room-temperature phosphorescence signals) for at least 3 months. The Al-ferron chelate was loaded onto a basic anionexchange resin (Dowex 1 X2-200)“on-line” in the flow system by pumping the chelate solution through the flow cell containing the resin.20 Details of this RTP sensing phase production are under patent.21 The performance of the oxygen transducer in different organic solvents was evaluated by (19) Lee, K. M.; Biellmann, J. F. Bioorg. Chem. 1986,14, 262. (20) Liu, Y. M.; Pereiro-Garcia, R.; Diaz-Garcla, M. E.; Sanz-Medel, A. Anal. Chim. A m 1991, 255, 245.
CARRIER
** VALVE
PERISTAI PUI.,, PERISTALTIC PUMP
SENSOR
7 - m
THERMOSTATED ENZYMATIC REACTOR ’
CARRIER
PERISTALTIC PUMP
FLOW CELL
INJECTION VALVE
ENNMEREACTOR
+
RTP-OXYGEN SENSOR
Flgure 1. (A) Flow system device for the two-step enzyme reactor/ RTP sensor system. (B) Flow system device for the one-step approach.
equilibrating the optical sensing phase with the corresponding water-saturated organic solvent. It was found that after passing the organic carrier solution over the sensing phase for 5-6 h, variations in the RTP intensities were less than 45%. Enzyme Immobilization. The procedure for noncovalent immobilization of either ChOD or ChOD-HRP on controlledpore glass (CPG) beads followed that reported previously by Kazandjan and Klibanov:22100p L of an enzyme solution (25 units for ChOD and 250 units for HRP) in 0.1 M aqueous phosphate buffer (pH 7) was added to 100 mg of CPG (106 pm or finer, Sigma) and spread onto a glass slide. The preparation was left to dry for 15 min under gentle air drift. When not in use, the enzyme preparation was stored at 4 OC. Enzyme/RTP Sensor Assemblies. Using a single-line flow injection system, as Figure 1 illustrates, two arrangements were examined for the siting of the immobilized enzyme and the oxygen transducer. A Pharmacia Model V-7 rotary valve was used for sample introduction. PTFE tubing (0.5 mm i.d.) and fittings were used for connecting the flow-through cell, the rotary valve, the enzyme minireactor, and the carrier solution reservoir. A Gilson Minipuls-2 perisaltic pump equipped with organic solvent resistant Viton tubing was used to generate the flow stream. The immobilized enzyme was located into a minicolumn inserted into the flow stream or, alternatively, inside the flow cell (together with the oxygen-sensing phase). In the first case, the enzyme-CPG beads were packed into a Tygon minicolumn (5 mm i.d. X 3 cm) capped with nylon end fittings. The minireactor (thermostated at 35 “C) was incorporated into the flow system as depicted in Figure 1A. The oxygen(21) Sanz-Medel,A.; Liu, Y. M.; Pereiro Garcfa, R.; Dfaz Garcfa, M. E. Span. Pat. Applic. P92/0264 1, 1992.
Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
2727
I
I
"
A
0
C
D
E
F
G
H
i
Figure 2. Influence of the nature of the organic solvent on the oxygen sensor performance: (A) aqueous medium, (B) Isooctane, (C) toluene, (D) hexane, (E) benzene, (F) dioxane, (0)dlchloromethane, and (H) chloroform). Ir* = I / I M M X 100.
active material was packed into a Hellma Model 176.52 flowthrough cell (25 pL of inner volume). At the bottom of the flow cell, a small piece of nylon net was placed to prevent any possible particle displacement by the carrier. In the second approach, enzyme-CPG beads were mixed with the aluminum-ferron chelate-resin beads in a 1:1 ratio. A portion of this bead mixture was placed into the conventional flow cell and located in the sample compartment of the detector (6 units for ChOD and 60 units for H R P were present in the flow cell) (See Figure lb). All room-temperature phosphorescencemeasurements were made at 600 nm (excitation at 390 nm) with a Perkin-Elmer LS5 fluorescence spectrometer which employed a Xenon pulsed (10-s half-width 50 Hz) excitation source and was equipped with a Perkin-Elmer 3600 data station. The delay time used was typically 0.04 ms. The gate time used throughout was 2 ms. Instrument excitation and emission slits were set at 10 and 20 nm, respectively, throughout this study. Steady-state phosphorescent signals were recorded with a Perkin-Elmer 560 recorder. General Procedures for CholesterolDetermination with the Enzyme Minireactor. Calibration. Cholesterol solutionswere injected through the valve (sampling volume 0.25 mL) into the organic carrier solution (hexane/5% (v/v) chloroform saturated with Bis-Tris buffer, pH 7). Each standard solution was injected three times repeatedly. The flow rate of the carrier was set at 0.30 mL min-l and the minireactor thermostated at 35 OC. Typical flow injection RTP signals were measured and their average peak heights plotted against injected cholesterol molar concentration. All measurements were performed in air-saturated organic solutions. Egg Samples. Homogeneizated sample (1 g) was accurately weighed and placed into a round-bottomed flask. ,Sea sand (1 g) and 20 mL of methanolic KOH (1 M) were added, and the resulting mixture was distilled under reflux for 30 min. The sample was left to cool, filtered, and washed with methanol. Cholesterol was extracted from the sample by shaking the methanolic phase with 50 mL of hexane and 30 mL of water.23 The organic phase was then separated and an aliquot of 5 mL was diluted 1 + 1 with the organic carrier solution. Butter Samples. Sample ( 2 g) was weighed and dissolved in the organic carrier solvent, giving a final volume of 10 mL. 2728
Analytical Chemlstty, Vol. 66,No. 17, September 1. 1994
These solutions were incubated 30 min at 37 OC with 10 units of ChE (immobilized on CPG following the same procedure described for ChOD). The glass beads were then removed, and the resulting solutions were used for the analysis. The cholesterol content in all samples was evaluated from calibration graphs obtained using cholesterol standards. The reference method used for the determination of cholesterol in foodstuffs was applied separately but simultaneously using a commercial kit.
RESULTS AND DISCUSSION Selection of the Organic Solvent. Two factors were taken into account in determining which solvent was most appropriate. The first factor was that the solvent selected should be compatible with the optical transducer. The optical sensor used in this work was based on the strong RTP emission at 600 nm exhibited by the Al-ferron chelate when immobilized on a strong anion-exchanger resin. The RTP emission was reversibly quenched by oxygen in aqueous solution.24 The performance of this transducer in different organic solvents was assessed by monitoring the RTP intensity during some oxygenation/deoxygenationcycles. Results demonstrated that the RTP oxygen transducer was completely reversible to oxygen in all the solvents tested, with typical response times for full signal changes of ca. 1 min. Furthermore, the sensor did not exhibit hysteresis and showed high photochemical stability; no release of the aluminum chelate was observed. As can be seen in Figure 2, RTP intensity in deoxygenated aqueous buffered solution was lower than that obtained in organic media, as should be expected, due to the deleterious effects that water molecules exerted on solid-surface RTPeZ5Concerning the organic solvents, higher signals were obtained when pure isooctane, hexane, and/or toluene was used. Detection limits (estimated as the pencentage of 0 2 which produced an analytical signal equal to three times the relative standard deviation of the RTP intensity in argon-saturated organic solvent) were 0.027, 0.040, and 0.025 mg L-l 02, respectively, with a mean reproducibility of 1%. Tabulated data26for 0 2 solubility at 1 atm partial pressure in each of the solvents studied were used. These results confirmed the possibility of using the oxygen RTP transducer for enzymatic optosensing of cholesterol in organic media. The second important factor was the compatibility of the solvent used with the enzymatic reaction. In order to study the performance of the ChOD reactor, cholesterol standards were injected in the flow system. Three hydrophobic solvents were tested, toluene, hexane, and isooctane, as well as their mixtures with chloroform and dioxane. All organic media were air-saturated and buffer-equilibrated. Oxygen consumption was followed using the oxygen RTP sensor. Results demonstrated that pure toluene and the organic mixtures hexane/5% (v/v) chloroform and isooctane/5% (v/v) chloroform were the most suitable for the enzymatic reactor to work because of the insolubility of the ChOD in these solvents, (22) Kazandjian, R. 2.; Klibanov, A. M. J . Am. Chem. SOC.1985, 107, 5448. (23) Tercyak, A. M. J . Nurr. Biochem. 1991, 2, 281. (24) Liu, Y. M.; Pereiro-Garcla, R.; Valencia-GonzAlez, M. J.; Dlaz-Garcla, M. E.; Sanz-Medel, A. Anal. Chem. 1994, 66,836. (25) Hurtubise, R. J . Phosphorimetry. Theory,Instrumentation and Applications; VCH h b l . Inc: New York, 1990. (26) Emmerich, W.; Battino, R. Chem. Reu. 1973, 73, 1.
-1
100
?
:I -;
Cholesterol 10 mM
Hexane/Chloroform
1
80
.-
B
60
.- 40
20
0
5
10
15
20
30
25
35
40
Time (min)
Flgurr 3. Different response profiles of the two-step reactor/RTP sensor system in different organic media. Ail organic solutions were air-saturated. 25
-
r
~
_
_
~
0
10
20
30
\ 40
50
60
70
I
20
25
I
1
30
35
40
,
45
,
1
50
55
60
V°C)
Flgure 5. Influence of the temperature on the bienzymatic reactor performance. The hexane/5% (v/v) chloroform mixture was airequilibrated.
I
ChOD.HRP
ChoD
01 15
80
ASSAY NUMBER
Flgurr 4. Operational stability of the ChOD reactor working at room temperature: [cholesteroi] lo-* M; 25 units of ChOD. Measurements were performedin air-saturated hexanel5% (v/v) chloroform mixture.
which in turn prevented its release from the CPG beads. On theother hand, thesubstrate wasvery soluble in thesesolvents. In Figure 3, the response curves toward cholesterol in three representative organic media are given. The signals were fully reversible with response times in the order of 2 min. The remaining experiments were therefore conducted in an airsaturated organic mixture hexane/5% (v/v) chloroform. Operational Stability of the ChOD Reactor. Results of the tests with 10 mM cholesterol assays are shown in Figure 4. From these results it was evident that the response remained reasonably constant for 18-20 consecutive assays but then decreased to less than 50% of its original value by assay 40. This fact could be explained by taking into account that the hydrogen peroxide generated during the enzymatic reaction was gradually accumulating in the hydration shell around the enzyme, giving rise to the ChOD inacti~ation.'~Fortunately, a solution to the above ChOD reactor limitation was found, consisting of the coimmobilization of ChOD with HRP and the addition of 10 mMp-methylphenol as H R P substrate into the organic carrier. As can be seen in Figure 4, with this new system the response remained reasonably constant for more than 70 consecutive assays. Hydrogen peroxide was consumed according to reaction 11. In order to investigate the long storage stability of the noncovalently immobilized bienzymatic reactor in nonaqueous medium, the reactor was tested over a period of 15 days, during which 15 calibration assays (1 each day) were performed. For
each assay, eight cholesterol injections were performed. After 120 injections in the 15-day period tested, the RTP signal had decreased to about 65% of its original value. Enzymes are in general very sensitive to temperature and ChOD, particularly in soluble form, and are known not to be very stable in aqueous media, even at room temperat~re.~' A study on the effect of temperature on the enzymatic reactor performance was carried out over the range 20-55 OC. The temperature of the organic carrier passing over the RTP sensor was kept at 20 f 2 OC. Figure 5 depicts the dependence of the signal sensitivity on the temperature of the bienzymatic reactor. Although the analytical sensitivitywas higher around 45 OC, for practical purposes temperatures lower than 40 OC are recommended in order to prevent possible baseline instability. The enzymatic reactor was kept thermostated at 35 OC in a water bath for the rest of this study. pH and Flow InjectionCholesterolSensing. In preliminary experiments, we observed that the dependence of enzymatic activity on the pH of the buffer to which ChOD was exposed prior to immobilization and exposition to the organic medium was consistent with previous reports.14J8 The optimum pH was found to be 7.0. On the basis of this study, a buffer solution of 0.1 M phosphate (which provides a final pH value of 7.0) was used to prepare the starting aqueous enzyme solution. The pH dependence of enzyme activity in anhydrous liquid/solid two-phase systems is a frequently observed phenomenon reported for numerous enzymes. This effect, named "pH memory",8 has been ascribed to the optimization of the protonation state of the protein, which is then retained when it is transferred to an anhydrous organic solvent. Furthermore, the buffering capacity of the microaqueous environment can be controlled by the enzyme itself. Indeed, our results demonstrated that when the organic carrier mixture saturated with either water, Bis-Tris buffer pH 5, Bis-Tris buffer pH 6, or Bis-Tris buffer pH 7 was used, signal output was maintained, which indicated that the enzyme activity of the ChOD-HRP reactor was essentially unaltered. The effect of the carrier flow rate (from 0.20 mL min-* up to 0.70 mL min-') on the RTP signal was studied. It was found that RTP response decreased steadily with increasing (27) Masson, M.; Townshcnd, A. Anal. Chim. Acta 1985, 174,
194.
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2729
Table 1. Analytlcal Characterktlcs for the Proposed Enzyme Reactor/RTP Sensor Devlces
one-step approach room temp DL (MI linear range (M) RSD (%) response time (min)
~
-1
/
I
two-step approach room temp 35 "C
3X10-4
1.4X10-4
up to 10-2
more than 10-2
5 x 10-5 up to 4 X 10-3
3 1.5
2 2
2.5 2
flow rate, as would beexpected owing to thedecreasedsubstrate contact with the enzymatic reactor. A compromise flow rate of 0.30 mL min-l was chosen for the remainder of this study as this gave good sensitivity and a relatively short response time. Since the ChOD reactor required oxygen as the cosubstrate to carry out the cholesterol oxidation, the effect of oxygen tension on the RTP sensor output should be considered. It was observed that a constant RTP background level was obtained when air-equilibrated organic carrier passed over the RTP sensor, indicating a constant oxygen supply. When air-saturated samples containing cholesterol were injected in the system, a local depletion of oxygen was produced as result of the cholesterol oxidation by ChOD. This oxygen consumption was detected by a transient increase in the RTP intensity of the oxygen-sensitive RTP sensor. Analytical signals were calculated by subtracting the background close to the value of the RTP total signal measured. Analytical Features. The performance of the one-step approach for the enzyme reactor/RTP sensor was evaluated under the optimum conditions found for the two-step version except for the temperature. Temperatures higher than 20 f 2 OC resulted in a decrease in the phosphorescence signals. The analytical performance characteristics were evaluated for both designs and results are shown in Table 1. As can be seen,the sensitivityof the two-step enzyme reactor/RTP sensor system working at 35 OC was greatly improved whencompared with that obtained at room temperature. For the one-step version, the sensitivity was 2-fold lower than that obtained for the two-step approach working both at room temperature. This fact could be explained by taking into account that in the first case the effective amount of oxygen-sensitive material packed in the flow cell was ca. 50% of that present in the second case. Figure 6 shows the signal relationship for cholesterol concentration from 0.5 to 4 mM (r = 0.999, n = 6 ) for the two-step enzyme reactor/RTP sensor system. Each point is a mean of three injections of a sample. The inset in Figure 6 shows a set of plots of RTP intensity vs time for several different cholesterol concentrations. These analytical figures of merit are clearly superior to that of the unique organicphase enzyme electrode for cholesterol described up to now'.14 A selectivity study of the method was carried out for those compounds of lipidic nature which could accompany cholesterol in food samples. Two fatty acids, palmitic and stearic, were tested and did not significantlyinterfere in a concentration range up to M. Applicationto Food Samples. In order to assess the validity of the bienzymatic reactor/RTP sensor system, food samples (butters and eggs) were analyzed for their cholesterol content 2730
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50 I
Ana&ticalChemistiy, Vol. 66,No. 17, September 1, 1994
.A
0
2
4
6
Cholesterol Concentration (mM)
Flgure 8. Dependenceof the RTP signal of the two-step reactor/RTP sensor system on the cholesterol concentration. Measurementswere carried out at 35 ' C in an alr-saturated hexane/5% (vlv) chloroform mixture. Table 2. Cholerterol Determlnatlon In Food Sampler.
sample
sensor (mg/g)
vis-UV kit (mg/g)
butter 1
2.35 i 0.04 2.42 i 0.16b 2.56 f 0.05 2.11 i O . 0 4 2.29 i 0.05 2.46 f 0.04 11.70 i 0.17 11.06 f 0.20 11.01 i o . l l
2.32 A 0.01 2.32 & 0.01 2.54 f 0.01 2.09 f 0.02 2.26 & 0.01 2.42 f 0.02 11.60 & 0.07 10.98 f 0.03 11.14 & 0.05
butter 2 butter 3 butter 4 butter 5 egg 1 egg 2 egg 3
Each result is the mean of three determinations. Result obtained with the one-step approach enzyme reactor/RTP sensor.
by following the general procedures detailed in the Experimental Section. The results found (using the two-step approach and working at 35 "C)were compared with those obtained by using a visible/UV enzymatic method for determining cholesterol in foodstuffs (Boehringer test). Measurements using the Boehringer test were made according to the manufacturer's instructions. As shown in Table 2, the two methods gave consistent results for the two samples analyzed. This concordance, in addition to the high sample throughput, indicate that the proposed system is adequate to perform the analysis of cholesterol in foods. In Table 2, the results obtained for a butter analysis using the one-step approach and working at room temperature are also shown. The results demonstrated the potential utility of both designs for the determination of cholesterol in this type of samples. CONCLUSIONS It has been demonstrated for the first time that a enzyme reactor/RTP sensor system is useful to perform enzymatic assays in nonaqueous media. While the concept presented here has been limited to the analyses of cholesterol, these observations are likely to stimulate investigations into other classes of analytes possessing a broad solubility range. Several points should be highlighted. From a practical viewpoint, the ability to easily immobilize and retain the activity of enzymes within an organic solvent should find many applications in optical biosensor technology, e.g., via the use of other sensitive optical detection techniques and/or of multienzymatic systems.
On the other hand, the application of new biocatalysts (either through recent isolation or genetic and protei? engineering) in organic media could also open up new areas of research. From a basic standpoint, several fundamental issues remained unresolved. These include the definition of the parameters that govern enzymatic activity in nonaqueous media, the role of water, and the nature of the effects of organic solvents on biocatalysts. Also, a mechanistic and kinetic description of enzymatic catalysis in organic solvents is needed. Biocatalysts (enzymes and/or antibodies) in nonaqueous media promises to be another important application in the basic study of biochemical analysis.
ACKNOWLEDGMENT We thank Profs. V. Gotor and A. Sanz-Medel for helpful suggestions. Financial support from the “Fundacidn para el Formento en Asturias de la Investigacidn Cientlfica, Aplicada y la Tecnologla” (FICYT) and the “Fondo de Investigaciones Sanitarias de la Seguridad Social (Project 93/0469)” is gratefully acknowledged. Received for review December 7, 1993. Accepted M a y 15, 1994.” Abstract published in Aduonce ACS Abstracts, July 1, 1994.
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