Mikrochim. Acta 120, 231-242 (1995)
Mikrochimica Acta 9 Springer-Verlag 1995 Printed in Austria
Biocatalysis and Biorecognition in Nonaqueous Media. Some Perspectives in Analytical Biochemistry Lorenzo Braco Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias Biol6gicas, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain
Abstract. Biocatalysis and, to a lesser extent, biorecognition in non-aqueous media (including organic solvents as well as supercritical fluids and gases) constitute at present an exciting research area which has already demonstrated its biotechnological potential in numerous, varied applications. Less attention, however, has been paid to its analytical possibilities, even though many advantages have been postulated and a wide range of poorly water-soluble analytes are present in samples (or waste materials) from food and drink, petrochemical, pharmaceutical, military and other industries. The main approaches, developed in recent years to exploit the use of enzymes, antibodies or antibody mimics in water-restricted environments for analytical purposes, as well as possible future directions are briefly discussed. Key words: non-aqueous enzymology, antibodies, flow-injection analysis, biosensors, molecular imprinting, review. Biological macromolecules involved in biocatalysis (enzymes) and in biorecognition (antibodies) have undoubtedly become, in recent years, of routine, widespread use, as valuable analytical tools in the determination of numerous compounds of clinical, nutritional, environmental, industrial and general biotechnological interest. In particular, enzymes have found applications in kits for titrations, immunoassays, reactors for chromatographic or flow-injection analysis (FIA) and biosensors. Apart from their ever-increasing availability, accompanied in most cases by relatively low costs, their success comes from a number of inherent advantages in that they provide, under moderate reaction conditions, substantial rate accelerations with remarkable substrate selectivity (including chemo-, regio- and enantiospecificity). However, an efficient use of enzymes still has to overcome a number of limitations, particularly those concerning biocatalyst operational stability (essential for longterm repeated use or automated procedures) and in many instances the poor solubility of the analytes. A routine, conventional way to alleviate stability limitations has been immobilization of the enzyme, preferably through covalent bonds (with the option of uni- or
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multi-point attachment, the latter usually yielding more stable preparations), although normal methods of immobilizing the biocatalyst may be to a variable extent detrimental to its performance, the most efficient procedures being typically laborious and time-consuming. More recently, protein engineering-based strategies have been reported, directed at designing "robust" biocatalysts able to resist even rather aggressive conditions [1] (e.g. by creating strategically positioned intramolecular bonds or by modifying the polarity or charge density of the protein surface), which open up exciting possibilities for analytical applications in the near future, not to mention the direct use of "naturally" stable enzymes from extremophilic organisms. As regards substrate solubility limitations, the common recourse of adding either a miscible co-solvent or a surfactant to the reaction (carrier) buffer may often be counter-productive, since it is known that such additives usually have deleterious effects on the enzyme. In this context, the promising area of non-aqueous enzymology [2, 31 offers a number of genuine advantages (see below) worth exploring in depth by analytical (bio)chemists. In the following sections, the different approaches addressed so far (as well as some future prospects) to make use of biological macromolecules (enzymes, antibodies, catalytic antibodies) or their mimics in water-restricted environments for analytical purposes will be briefly discussed. The basic principles and rules of the general behaviour of enzymes in non-aqueous media (crucial for optimizing biocatalyst performance), e.g. role of water, solvent selection or 'memory' effects, will not be detailed as they can be found in several reviews [2-4].
Non-Aqueous Enzymology:General Aspects and Advantages At present, the notion that enzymes can retain their activity in a variety of non-conventional (i.e. non-aqueous) media is fully accepted [,1-4]. Moreover, this notion may not be so unconventional if one remembers that many types of enzymes are known to perform their natural function in relatively hydrophobic environments (inserted in or bound to phospholipid bilayers) where water activity is diminished. In a strict sense, within the definition of a non-aqueous environment, low-water organic solvents [1-41, supercritical fluids [51, gases [6], solvent-free liquid substrates or even solvent-free eutectic mixtures of substrates [-7] should be considered. Different approaches have been reported in the last decade for placing a functional enzyme in an organic medium: as a powder suspension [2, 81, incorporated into reverse micelles [,9], "solubilized" by amphiphilic polymer-modification [-101, among others. The approach commonly referred to as enzymology in monophasic organic media, i.e. the direct use of bare enzymes (optionally adsorbed on supports) suspended in organic solvents, where they remain active while retaining a high degree of selectivity, has become a widely accepted choice because of its straightforwardness and inherent advantages [3], especially some novel, interesting properties potentially exhibited by the enzymes under these conditions [2-4]. The nonfamiliarized reader must keep in mind at this point that, although a relatively high percentage of water-miscible solvent added to an enzyme solution is usually harmful (denaturing), in the case of an enzyme suspended in a predominantly anhydrous (preferably non-polar) solvent the protein is 'kinetically trapped' in the active
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conformation it had in the solution that it was lyophilized from; in other words, the enzyme should denature in the organic solvent (on thermodynamic grounds) but it cannot (on kinetic grounds) because of water restriction and, hence, a lack of conformational flexibility needed for unfolding. Although most of the advantages referred to above have already been exploited in numerous biotechnological conversions, some particularly attractive features from a bioanalytical perspective are: (i) the enhanced biocatalyst (thermo)stability (which can extend both the operational temperature range and the enzyme lifetime), (ii) the increased solubility of non-polar substrates (which can extend the analyte range), (iii) the solvent-induced changes in enzyme efficiency or substrate specificity (which can potentially extend detection linearity by changes in the apparent Michaelis-Menten constant), (iv) the elimination of potential microbial contamination and (v) the prevention of detachment of non-covalently-bound (simply adsorbed) enzyme from a support (which simplifies immobilization schemes), derived from the general insolubility of proteins in organic solvents.
Analytical Applications of Enzymes in Non-Aqueous Media In spite of all the above recognized benefits, the number of reports where enzymes have been used for analytical purposes in non-aqueous environments is still quite limited, especially when compared with the profusion of work carried out in aqueous phases, from enzyme reactors (pre- or post-column) in chromatographic determinations or flow systems to varied enzyme sensors, not to mention batch applications. Some possible reasons come to mind to account for the present remarkable imbalance in the use of aqueous and non-aqueous media in enzyme-mediated analytical methods. First of all, although it has been known for more than two decades that enzymes could function in organic solvents, only recently have the fundamentals of non-aqueous enzymology, and therefore the possibility of a rational exploitation of this technology, begun to be understood. Thus an inertia based on the "conventional" use of enzymes in all-water media is to some extent understandable, and usually influences the choice of the operating medium in favor of the aqueous one. Second, the behaviour of many types of enzymes in water-restricted environments still remains to be explored. Future research in this direction will probably expand the options of non-aqueous media for analytical problems not easily tractable with conventional methodologies. Third, many real samples are aqueous or aqueous-based, which sometimes implies either inefficient partitioning of the analyte into the non-aqueous enzyme environment or a previous extraction step into the organic phase. Nevertheless, an organic extraction prior to analysis may be indeed beneficial in some instances, e.g. as a pre-concentration step for a poorly water-soluble analyte, or when the analyte is present in a rather unmanageable, viscous matrix (hydrophobic or not) which imposes serious diffusional limitations. Fourth, the use of organic solvents sometimes implies drawbacks in relation to safety aspects (some are hazardous or explosive), disposal aspects (as toxic waste) or cost. In this regard, the possibility of using non-toxic, biocompatible lipid-based (organic) solvents (see, e.g. [11]) in analytical applications has been hardly explored. In any case, in recent years interest in the field has grown and numerous contributions have been reported describing actual or potential applications, in an
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effort to evaluate the possibilities of transferring the (ever-increasing) fundamental knowledge of non-aqueous biocatalysis to solve analytical problems. Although this review intends by no means to be exhaustive in compiling the work carried out so far, some different types of approach (logically paralleling in most cases the already established methods in aqueous assays) are considered below:
(i) Batch Analyses in Organic Media Some work has been done on batch determinations where the enzyme was directly introduced in the non-aqueous medium and product formation was monitored by color appearance, e.g. in the assay of cholesterol in different organic solvents using cholesterol oxidase [COD] coupled to horseradish peroxidase [HRP] (both adsorbed on porous glass beads) with p-anisidine as chromogenic substrate El2], or in the simple but ingenious use of HRP homogeneously dispersed in chromogen-containing solidifiable paraffin mixtures (of selectable melting point by varying the composition) as a prototype temperature-abuse sensor applicable to foods, pharmaceuticals or other heat-sensitive materials [13].
(ii) Flow-Injection Enzyme Reactors Operating in Organic Media Paradoxically, little attention has been paid to the translation of the profusely used flow enzyme reactors (in both FIA and chromatographic analyses) into nonaqueous conditions of operation. The first systematic evaluation of the possibilities of implementation of enzymatic flow-injection analysis in non-aqueous media has recently been reported [14]; the previously described cholesterol assay in organic solvent [12] was adapted to flow conditions as a model system. In that work, the feasibility of a non-covalently immobilized enzyme reactor [NIER] in non-aqueous FIA determinations was demonstrated and a notable improvement obtained relative to the equivalent batch system. As an example, Figs. 1A and 1B illustrate FIA recordings (a diode array UV-Vis flow-cell was used for detection) corresponding to calibration curves for H 2 0 2 and cholesterol, respectively, obtained with either a monoenzymic (HRP, Fig. 1A) or a bienzymic (co-immobilized HRP-COD, Fig. 1B) reactor operated with p-anisidine-containing water-saturated toluene as carrier. It is interesting to note that, for the cholesterol assay, when alternative experiments were performed using either two single reactors in series (each 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 produced. With the CODHRP reactor, we obtained a linear response up to 2 x 10 - 4 M cholesterol with a detection limit of 1 ~tM and a throughput of 60 samples per hour. In addition, the aforementioned advantages derived from using enzymes in anhydrous media were confirmed, particularly that concerning stability. Figure 1C depicts the storage stability of the non-covalently immobilized COD-HRP reactor, which could be reliably operated for more than 4 months. Taking into account how fast and simple the preparation (by simple adsorption of the enzymes to the support) and operation ofa NIER can be, and the large number of poorly water-soluble analytes present in many types of samples, it might be expected that the scope of applications of enzyme reactors in non-aqueous FIA will
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increase in future. Very recently, a COD-NIER operating in an organic flow system has been successfully used, either coupled on-line to a room-temperature phosphorescence (RTP) sensor or directly built in the flow cell as a flow-through enzymic RTP-sensor, in the determination of total cholesterol in food samples [-15].
(iii) Organic-Phase Enzyme Electrodes Enzyme-based biosensors [16] in organic phases, particularly organic-phase enzyme electrodes, OPEEs (or preferably in the future, microelectrodes), undoubtedly constitute the facet to which most attention has been devoted in the recent past. It is quite evident that OPEEs offer, in principle, opportunities for the direct analysis of many poorly water-soluble analytes in a variety of challenging sample matrices. An excellent discussion of the different technical approaches used to prepare enzyme electrodes for organic phases and an evaluation of the potential applications of non-aqueous biosensors in numerous areas have been reported by Saini et al. [17]. Most of the electrodes described so far have made use of some "model" enzymes in non-aqueous studies (mainly peroxidases or other oxidases), or even whole fungal or plant tissue preparations, for the determination of peroxides or phenolic compounds in a variety of organic media (see, e.g. [-18-23] and also the list of references in
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Table 1 in [24]). As an example of practical applications, OPEEs have been successfully used in the rapid determination of phenol content in olive oil samples (tyrosinase-based sensor) [25], of cholesterol in butter and margarine samples (COD-based sensor) [26], of phenol and peroxide antiseptics in anti-infective formulations (tyrosinase- and peroxidase-based sensors) [27] or of secondary alcohols in untreated gasoline samples (alcohol dehydrogenase-based sensor) [24]. Of special mention in the use of OPEEs is the possibility of using mediators insoluble in organic solvents (which can be simply co-immobilized with the enzyme by direct adsorption) to obtain reagentless sensors [28]. Apart from the obvious efforts to improve technical aspects involving more suitable organic-phase supporting electrolytes and mediators or optimized biocatalyst supports and more sophisticated immobilization strategies, work in the future is likely to be directed at broadening the spectrum of candidate enzymes for non-aqueous biosensors and at complementing the so-far almost exclusive use of electrochemical transduction with other methods (optical, calorimetric, etc.), possibly better suited for some applications. In this respect, the relevant analytical applications and future prospects of organicphase optical biosensors (OPOBs) have been recently reviewed [29], and their use has been proposed in areas such as food technology and testing, clinical diagnostic or water contamination control. In particular, OPEEs offer numerous opportunities in flow analysis. In a recent review [24], Wang has discussed the advantages of electrochemical enzyme-based flow detectors: apart from all the aforementioned benefits of non-aqueous enzymology, they permit a direct, rapid flow analysis of challenging samples without the need for tedious sample pretreatment, which makes them attractive for quality control testings and industrial process control. Several recent applications of OPEEs in flow analysis can be found, mainly based on the use of tyrosinases and peroxidases [25, 27, 30, 31]. On the other hand, there is no absolute need for the analyte to be a substrate of the enzymic reaction: it can be alternatively determined on the basis of its ability to modulate enzyme activity, i.e. as an enzyme effector (activator or inhibitor). This indirect biosensing strategy has been exploited to develop an OPEE for the on-line determination of trace water in organic solvent [32], since small amounts of water are known to enhance enzyme activity in non-aqueous media [-2-4]. This approach, proposed as an attractive alternative to the well-known Karl Fischer titration method, has been also successfully applied to the quantitation of moisture in food samples [33]. The same strategy has been used for the organicphase on-line monitoring of several enzyme inhibitors [34].
(iv) Enzyme-Based Gas-Phase Biosensors Apart from organic solvents, the potential analytical applications of biocatalysis in the gas phase [6] have also raised considerable interest, particularly in the development of biosensor devices [6, 16]. After all, a gas can be considered as a non-aqueous environment where virtually dehydrated enzymes (provided they retain their "essential" water) can display activity towards volatile substrates [35]. As an example, the use of alcohol oxidase, peroxidase and a chromogenic substrate adsorbed on an Avicel support has been proposed for the direct determination of ethanol or formaldehyde in breath or air [36]; the use of a piezoelectric crystal coated with
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formaldehyde dehydrogenase plus co-factors (NAD + and reduced glutathione) has permitted the quantitation of formaldehyde concentrations in vapor phases with excellent sensitivity and selectivity [37]; or an immobilized (entrapped) alcohol oxidase enzyme electrode has been constructed which exhibited high selectivity for ethanol in the presence of other gas-phase chemicals and covered the concentration range encountered in breath after alcohol drinking [38]. Enzyme-based gas-phase biosensors can be applied, e.g. in on-line monitoring of analytes in the effluent of gas-solid enzyme or micro-organism bioreactors or directly in "field" determinations. However, their general usefulness in sensitive and selective direct analyses of gaseous samples potentially containing diverse toxic gases, anaesthetics, hazardous vapors or other threatening agents still remains to be assessed and is likely to concentrate part of the near-future efforts in the field of applied gas-phase biocatalysis. Finally, it seems worth mentioning a recent report on non-aqueous capillary electrophoresis [39] using formamide as solvent (although several other solvents were proposed), where the advantages and potential of this approach are analyzed. Although proteins were not used in that work, the possibility can be envisaged a priori of, for instance: (i) incorporating enzymes in the system to facilitate detection or (ii) incorporating protein coatings to assist separations of, for example, poorly water-soluble enantiomers (similarly to protein coatings used in chromatographic chiral stationary phases), as an alternative to the current chiral selector-based strategies in aqueous media.
Analytical Possibilities of Antibodies and Abzymes in Non-Aqueous Media Of course, enzymes are not the only biomolecules of value in analytical biochemistry. Antibodies [Abs], particularly the monoclonal ones which provide an exquisite level of selective biorecognition, offer a powerful analytical tool which has already been successfully exploited in different approaches in numberless applications in conventional aqueous media. Furthermore, it is reasonable to expect that Ab-based analytical procedures will soon greatly benefit from the current advances in Ab technology, which will offer custom-made, more selective and stable, higher-affinity biomolecules obtained by means of considerably less laborious protocols. Interestingly, Abs have recently also proved to be operative in organic media [40], thus confirming their potential for general non-aqueous analysis. Several advantages have already been postulated for the use of immunosensors in organic environments [17], e.g. sample preconcentration which would expand the detection limits, greater stability and operational temperature range, or the challenging possibility of modulating antigen-Ab interactions to render them reversible without compromising (by denaturation) the functional conformation of the immunoglobulin binding site. However, it must be taken into account that if there is no analyte conversion (just binding) only piezoelectric, optical and some electrochemical transducers can be used. Moreover, problems in the use of Ab-based biosensors often arise with non-specific binding; in this case, apart from appropriate presaturation of the support with protein(s), the use of adequate control experiments is mandatory. On the other hand, some preliminary results on the use of Abs for the direct assay of gas-phase haptens are very encouraging with regard to the application of
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Ab-coated piezoelectric crystals as immunochemical gas-phase sensors, of evident interest for the in situ detection and quantitation of, for instance, environmental pollutants or illegal drugs. As an example, piezoelectric crystals coated with Abs against parathion have been successfully employed in a reversible, relatively fastresponse manner for the selective determination of the pesticide concentration (at ppb levels) directly in the gas phase [41], with an acceptable lifetime for the non-covalently immobilized Ab. It is noteworthy that the frontiers commonly invoked until some years ago between enzymes and antibodies have recently become rather blurred. Thus, opportunities (predominantly based on protein engineering strategies) to rationally generate tailor-made enzymes endowed with superior selectivities are more and more apparent, and Abs exhibiting catalytic activity, i.e. catalytic Abs (catAbs) or "abzymes", are at present a consolidated, promising area of research [42], with great potential in many areas, particularly in synthetic chemistry. Again, organic media have also proved to be practicable for catAbs, as demonstrated in several encouraging reports where different abzymes were shown to be moderately active in reverse micellar or aqueous-organic media [43-45]. Although their analytical potential is at this moment far from having been explored in depth, the inherent value of catAbs (which combine tailor-made selectivity and catalytic activity) raises an optimistic expectation on their possibilities of implementation in the analytical field.
The Potential of Molecular Imprinting Techniques Finally, among the approaches developed to generate synthetic recognition systems (or biorecognition mimics) specific for a given molecule (e.g. crown ethers or cyclodextrins), a brief reference is merited to the emerging powerful methodology of molecular imprinting, which basically consists of creating a selective recognition site (cavity) in a macromolecule or macromolecular network by means of a print molecule (template). Molecular imprinting has been implemented following essentially two different strategies (Fig. 2 and Table 1). (i) Ligand-induced "memory" or (bio)imprinting has been applied to enzymes and in general proteins and other natural and synthetic macromolecules [46-50] and exploits the drastically enhanced conformational rigidity of the macromolecule in an anhydrous organic solvent [2-4]. In the case of enzymes (Fig. 2A), the approach has been applied to proteases and made use of competitive inhibitors (amino acid derivatives) to "tune" the active site conformation: this altered conformation in aqueous solution is presumably preserved (memory) during freeze-drying and in the enzyme powder in anhydrous solvents, and significant activity enhancements in non-aqueous media have been reported using this method [46-48]. In this respect, we have recently introduced the (bio)imprinting of lipolytic enzymes using lipid-water interfaces as templates [51], which permits non-aqueous activations of 2 or more orders of magnitude, depending on the substrate [51-52]. In the case of non-enzymic proteins (or other macromolecules) (Fig. 2A), the strategy consists of generating ligand (template)-binding sites (cavities) in an unrelated macromolecule in aqueous solution, which will be preserved after freeze-drying and washing out (with an anhydrous organic solvent) of the template, provided that
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the macromolecule is maintained in anhydrous environments [49]. In this case, the approach allows quite specific adsorbents to be obtained and has been proposed in, for instance, organic-phase chromatographic separations [49, 50_], although its potential for enzyme mimics and for biosensors remains to be assessed. (ii) Conceptually quite similar, but technically more versatile and probably with more realistic current possibilities of analytical application, is the so-called molecular imprintin9 of polymers (covalent or non-covalent) (Fig. 2B), a straightforward approach mimicking natural biorecognition, which permits polymers to be generated with a pre-determined selectivity towards a given template molecule [53]. In this case, if the non-covalent approach is used, a cocktail of functional monomers agglutinated around the template by non-covalent interactions (ionic, hydrogen bonding, hydrophobic, etc.) is polymerized by conventional procedures to produce (after washing out of the template) a polymeric three-dimensional network containing cavities presumably complementary (both in shape and functionalization) to the print molecule. It is noticeable that, for technical reasons, most of the work carried out so far with imprinted polymers has made use of predominantly non-aqueous media. Molecularly imprinted polymers [MIPs] have potential applications in different areas, as tailor-made separation materials (e.g. in chiral resolutions), catalytically active polymers or enzyme mimics, and as antibody- (or receptor-) binding-site mimics, with general implications in analytical biochemistry and particularly in the construction of substrate- (analyte-) selective sensors. As an example of this potential, Mosbach and colleagues [-54] have elegantly reported on several anti-drug (theophylline, diazepam and others) MIPs which can be successfully used as stable, low-cross-reactivity, sensitive alternatives to antibodies in conventional immunoassays. More recently, this group has described the use of the molecular imprinting technique to generate thin-layer chromatography plates for the rapid chiral separation of several amino acid derivatives [55] or as a selective,
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Table 1. Strategies employing the molecular imprinting approach (A) Molecular (No)imprinting of enzymes, non-enzymic proteins and other macromolecules Features: - based on conformational rigidity of the protein (macromolecule) in anhydrous media - pre-existing macromolecule (backbone) template (ligand) induces changes in macromolecule conformation - very low-water media are needed for operation - simple - good selectivity in the case of enzymes notable non-aqueous rate enhancements can be obtained -
-
Proposed for: - improving enzymic reaction rates in non-aqueous media (or even altering substrate specificity) - bioadsorbents - chromatographic separations
(B) Molecular imprinting of polymers (covalent or noncovalent) Features: - polymerization occurs in the presence of the template (the cross-linked backbone is created in situ around the template) - typically used in organic solvents, but in principle not necessarily - good selectivity excellent stability - more versatile -
Proposed for: - bioadsorbents - chromatographic separations (column, thin layer) - enzyme mimics antibody mimics (e.g. as RIA" alternative) - biosensors -
RIA: radioimmunoassay. solid-extraction sample e n r i c h m e n t step which simplifies further analysis [56]. Interestingly, in the field of biosensors it is evident t h a t M I P s can offer great potential to create c u s t o m - t a i l o r e d binding sites as robust substitutes for the sensor biological c o m p o n e n t in cases where a suitable biomolecule is either unavailable or exhibits very p o o r stability. Some examples of the preliminary w o r k carried o u t to assess the possibilities of M I P s as part of future sensing devices have been given [53]. Hopefully, technological i m p r o v e m e n t of m o l e c u l a r i m p r i n t i n g a n d i m p l e m e n t a t i o n of suitable t r a n s d u c t i o n systems m i g h t p r o m o t e M I P s as reasonable alternatives in the search for future-generation, long-lived, universal biosensors able to operate in a q u e o u s as well as n o n - a q u e o u s environments. To conclude, we expect t h a t future directions in analytical biochemistry research in n o n - a q u e o u s m e d i a will benefit f r o m the currently available diversity of tools potentially operative in these e n v i r o n m e n t s (enzymes, Abs, catAbs, M I P s , etc.) a n d from their c o n s t a n t i m p r o v e m e n t . It is likely t h a t the n u m b e r of applications in areas of interest such as e n v i r o n m e n t a l m o n i t o r i n g a n d food, petrochemical a n d military
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industries (where poorly water-soluble analytes are so common) will increase substantially in the near future as our knowledge of the fundamentals of biocatalysis and biorecognition in water-restricted media expands, although much still remains to be explored in assessing the full potential of this technology for analytical determinations. Acknowledgements. The author is grateful to I. Mingarro for the artwork. This work was supported by Grant PB93-0359 from Direcci6n General de lnvestigaci6n Cientifica y T6cnica (Spain).
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