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Anal. Chem. 1999, 65, 845-847
TECHNICAL NOTES
Organic-Phase Enzyme Electrode for the Determination of Trace Water in Nonaqueous Media Joseph Wang' and A. Julio Reviejot Department of Chemistry, New Mexico State University, Las Cruces, New Mexico 88003
INTRODUCTION The ability of enzymes to function in various nonaqueous media112 has led to the development of organic-phase biosensors.3 Such novel sensing devices have received considerable attention recently because they can greatly expand the scope of biosensors toward many previously inaccessible analytes (Le. substrateswith poor water solubility) and toward many challenging samples matrices and hostile environments (e.g. butter, olive oil, gasoline). In addition, the organic-phase operation can offer improvements in the thermostability of enzymes4 (andhence extended sensor lifetimes), reduced side reactions, and greatly simplified immobilization schemes. These capabilities and improvements have been illustrated recently for monitoring of phenols,sse hydrogen and organic peroxides,'+ or cholesterollOusingvarious enzyme-and tissuebased amperometric electrodes. In this paper we report on a unique application of organicphase biosensor aimed at determining trace water in nonaqueous media. Residual water is known to have an indirect role upon the biocatalytic activity in organic media.2J1 While the exact role of water is not yet fully understood, it has been attributed to the maintenance of the hydration layer and noncovalent interactions essential for the biocatalytic activity.2 The enzyme activity, and hence the response of various organic-phase enzyme electrodes, have been shown to be strongly dependent upon the water content.2.3J Water can thus be viewed as an enzyme effector, and accordingly can be determined in a manner analogous to biosensing measurements of other effectors (such as activators or inhibitors).12 The positive modulation (enhancement) of the enzyme activity by low water levels is thus exploited in the following sections for developing a simple, rapid, and sensitive organicphase biosensor for monitoring trace water in organic media. The new biosensing approach offers an attractive alternative Corresponding author. address: Department of Analytical Chemistry, Faculty of Chemistry, Complutense University, 28040 Madrid, Spain. (1) Klibanov, A. M. CHEMTECH 1986,16,354. (2) Dordick, J. 5.Enzyme Microbiol. Technol. 1989, 11, 194. (3) Saini, &;Hall, G. F.; Downs, M. E.;Turner, A. P. Anal. Chim.Acta + Permanent
1991, 249, 1. (4) Zaks, A.; Klibanov, A. M. Science 1984,224, 1249. (5) Hall,G. F., Best, 0. J.; Turner, A. P. Anal. Chim.Acta 1988,213, .2+n
110.
(6) Wang, J.; Naser, N.; Kwon, H.; Cho, M. Anal. Chim. Acta 1992, 245,133. (7) Schubert, F.; Saini, S.; Turner, A. P. Anal. Chim. Acta 1992,245, 133. ( 8 )Wang, J.; Wu, L. H.;Angnes, L. Anal. Chem. 1991, 63, 2993. (9) Wang, J.; Freiha, B.; Naser, N.; Romero, E. G.; Wollenberger, U.; Ozsoz, M.; Evans, 0. Anal. Chtm. Acta 1991,254, 81. (10) Hall,G . F.; Turner, A. P. Anal. Lett. 1991, 24, 1375. (11) Zaks, A.; Klibanov, A. M. J. BioZ. Chem. 1988,263, 8017. (12) Dolmanova,I.; Shekhovtaova,T.; Kutcheryaeva, V. TaZanta 1987, 34, 201. 0003-2700/93/03659845$04.00/0
to the well-known Karl Fischer titration method,13.14 which requires special equipment and suffers from various interferences and side reactions. EXPERIMENTAL SECTION Apparatus. Amperometric experimentswere performedwith an EG&GPAR Model 264Avoltammetricanalyzer,in connection with a X-Y-t recorder (Model RXY, Bioanalytical Systems (BAS)). Batch experiments were carried out in a 10-mL electrochemicalcell (ModelVC-2, BAS). The working (enzyme) electrode, the Ag/AgC1(3M aqueous NaCl) reference electrode (ModelRE-1,BAS),and platinum wire auxiliaryelectrodejoined the cell through holes in its Teflon cover. A magnetic stirrer and a 8-mm-longstirringbar provided the convectivetransport during amperometric measurements. The flow injection system consisted of a 50-mL syringelcarrier reservoir,held by the syring pump (Model341B, Sage),a Rainin Model 5041sample injection valve (20-pLloop), interconnecting Teflon tubing, and a glassy carbon thin-layerflowdetector (Model TL-5, BAS). The referenceand auxiliary electrodeswere located in a downstream compartment (Model RC-2, BAS). Electrode Preparation. The glassy carbon disk electrode (ModelMF2012, BAS) and the glassy carbon thin-layer detector were modified by covering the surface with a 10-pLaliquot of the mixed enzyme/polymeraqueous solution and drying (for 10min) at 45 "C with a heat gun (kept ca. 50 cm from the surface). The mixed enzyme/polymer solution was prepared by dissolving 2 mg of tyrosinase in 200 pL of the 1.4% Eastman AQ 55D solution. Reagents and Procedure. Tyrosinase (EC 1.14.18.1, 2400 units/mg (Sigma), phenol (Baker), acetonitrile, 2-propanol, 1-butanol, tetraethylammonium p-toluenesulfonate (TEATS) (Aldrich), and ethanol (U.S.Industrial Chemicals) were used without further purification. The poly(estex-sulfonic acid) polymer (Eastman AQ 55D, 28% dispersion) was received from Eastman Chemical Products; prior to mixing with the enzyme it was diluted 20-fold with deionized water. Experimentswere performed (at room temperature) in organic solvents (containing 1mM phenol and 0.1 M TEATS) by holding the tyrosinase electrode at 4.25 V and allowing the transient current to decay. The phenol and the electrolyte were dissolved directly in the organic test solution. The organic carrier solution (in flow injection experiments) also contained 1mM and 0.1 M of the substrate and electrolyte, respectively.
RESULTS AND DISCUSSION The organic-phase water biosensor is based on the stimulation by water of the biocatalytic hydroxylation of phenol to o-quinone by tyrosinase (polyphenol oxidase). Tyrosinase has been selected for this task because of its inherent activity and stability in various nonaqueous media.516 By exposing the tyrosinase electrode to a constant concentration of the phenol substrate, the electrode offers a low-potential amperometric detection of the water content. (13) Fischer, K. Angew. Chem. 1935,48,394. (14) Nordin-Andersson, I.; Cedergren, A. Anal. Chem. 1985,57,2571. Q 1993 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993
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flguro 1. Measurements of water In 1-butanone (a), 2-propanol (b), and ethanol (c). (A)Current-time recordings at the tyrosinase electrode to the substrate additlon (1 mM phenol), followed by successive IncrementsIn thewater content (in 0.1 % v/v steps). Operathg potential, -0.25 V; electrolyte, 0.1 M TEATS. Enzyme loading, 240 unlts. Also shown B, resulting standard additions plots.
Figure lAdisplays current-time recordingsobtainedduring measurements of the water content of 1-butanol (a), 2-propan01 (b), and ethanol (c). The first current “jump” reflects the response to the substrate addition (1mM), and hence indirectly correlates to the residual water level. Subsequent current responses are for successive increments of the water level (in 0.1 % v/v steps). The tyrosinase electrode responds rapidly to these changes in the water content, producing steady-state signals within 20-40 s. Detection limits of 0.010.05% v/v water are indicated from the favorable signal-tonoise characteristics associated with the operation at -0.25 V. (Itshould be pointed out that enzymes remain catalytically active at significantly lower water levels.1-3) The standard additions plots resulted from these experiments are shown in Figure 1B. All plots exhibit an initial linear dependence, with a leveling off at higher water levels. Such different profiles reflect the different water requirements of tyrosinase in the different solvents. It is well-known that the amount of water essential to support the biocatalytic activity of a given enzyme in organic media is solvent dependent (in accordance with the optimal level of hydrationh2 Wide variations have thus been observed for different solvents. The water levels calculated from these standard additions plot correspond to 0.77 (1-butanol), 0.18 (2-propanol), and 0.08 % (ethanol) v/v. Such values are in good agreement with the labeled impurity levels (1 (a) and 0.2% (b) v/v). No response was observed for analogous measurements without the enzyme (not shown). Various experimental parameters affecting the response of the water biosensor have been examined. Figure 2A shows the effect of the substrate concentration upon the calibration plots for water in acetonitrile. The sensitivity increases rapidly with the phenol concentration at first (between 0.25 and 1.0 mM), and then it levels off. Such nonlinear (Michaelis-Menten type) substrate concentrationdependence
0.5
1.o
.5
Figuro 2. Effect of phenol concentration (A) and enzyme surface loading (B) upon the measurement of water In acetonitrile. Phenol concentratlon,0.25 (A (a)),0.50 (A (b)), 1.O (A (c)),and 3.0 (A (d)) mM. Enzyme loading, 120 (B (a)),240 (A, B (b)), and 480 (B (c)) u n b . Successbe 0.1 % v/v Incrementsof the water content, wlth background
subtraction. Other conditions. as In Figure 1.
is expected for biocatalytic reactions. All subsequent work was thus carried out in the presence of 1.0 mM phenol. Note the different shape of the calibration plots in acetonitrile (versus those in alcohols, Figure 1). Figure 2B displays the effect of the enzyme surface loading upon the response for water. The enzyme was immobilized within an Eastman AQ 55D polymeric coating, known for its stability in various organic medial5 and for its ability to entrap enzymes on electrodes.l6 As expected, the response increases upon increasing the enzyme loading between 120 and 480 unite. The enzyme/EastmanAQ immobilization scheme is also very reproducible. Six different coatings used for measuring 0.6% v/v water in acetonitrile yielded a mean current response of 85 nA, a range of 81-94 nA, and a relative standard deviation of 5.7%. An operating potential of -0.25 V yielded the most favorable signal-to-noise characteristics for monitoring the enzymatically-generatedquinone product and was used in all subsequent work. Such low operating potential also minimizes potential interferences from coexisting electroactive species. No water contamination(leak)from the reference electrode was observed over prolonged periods. The fast response of the water biosensor can be exploited for high-speed flow injection assays, as often desired in many practical situations. Figure 3A displays the amperometric response of the tyrosinase thin-layer detector to injections of acetonitrile solutions containing increasing water levels (0.21.0% (v/v), a-e). The enzyme electrode respondsvery rapidly to these dynamic changes in the water content. T h e peak half width (- 15 e) allows an injection rate of 60 samples/h. The flow-injection peak current is proportional to the water content over the entire range (slope, 34.1 nA/% (v/v)); correlation coefficient, 0.999).A detection limit around 0.05% (v/v) can be estimated based on the signal-bnoise characteristics of these data. The water flow detector offers also a (15) Gennett, R.; Purdy,W. C. Anal. Chem. 1990,62,2166. (16) Carr-Brion,K.MoistoreSensorinProceos Control;Elsevier: New
York, 1986. (17)Wang,J.; Leech, D.; Ozaoz, M.; Martinez, S.; Smyth,M. Anal. Chzrn. Acta 1991, 245,139.
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3.8%. The flow injection behavior indicates a rapid onfoff modulation of the enzymatic activity (i.e., a reversible sensor characteristic).
CONCLUSIONS
1
-q
e
Time Ftguro 8. Flow injection measurements of water In acetonitrile. (A) injectionsof acetonitrilesoiutlons containing increasing levels of water In 0.2% (v/v) steps (a-e). (B) Repetittve injections of an acetonitrile edutlon contalnlng 0.6% (v/v) water. The acetonitrile carrier and mmpb edutlons also contained 1 mM phenol and 0.1 M TEATS. Operatlng potential, -0.25; Row rate 1.0 ml/mln.
high degree of reproducibility. Figure 3B show 40repetitive injections of an acetonitrile solution containing 0.6% (v/v) water. The water peak remains stable throughout this prolonged series (and is not affected by the slight drift of the baseline). The mean peak current found is 20.8 nA,with a range of 19.0-22.5 nA, and a relative standard deviation of
The experiments described above confirm the expectation that the amplification imparted by water on the organicphase enzymaticactivity can be exploitedfor monitoringtrace levels of water in nonaqueous media. Unlike numerous chemical and physical methods for water determinations," the present device represents the first biosensing probe for this task. It does not require any special equipment and is not susceptibleto interferencesor side reactionscharacterizing Karl Fischer titrations. Such indirect water biosensing represents a unique application of organic-phase biosensors. While the concept of water sensing has been illustrated in connection with an amperometric tyrosinase electrode,other biosensora (based on different enzymes and transducers) may be useful for the same task. The characteristic procedure parameters must be adjusted to suit the requirements of each case. The advantages accrue from this organic-phase water detection (particularly the sensitivity, selectivity,speed and simplicity)hold great promise for many practical applications. Hence, the field of organic-phase biosensors offers many unique analytical opportunities.
ACKNOWLEDGMENT Stimulating discussions with Professor M. Meyerhoff are gratefully acknowledged. A.J.R. acknowledges a fellowship from the Spanish Ministry of Science and Education.
RECEIVEDfor review November 3, 1992. Accepted December 8, 1992.
