Monica Campas and Jean-Louis Marty
This chapter describes two enzyme immobilization methods based on the biomolecule encapsulation into polymer matrices: the sol-gel technology and the entrapment into the polymer poly(vinyl alcohol) with styrylpyridinium groups (PVA-SbQ). The sol-gel technology is based on the formation of silica matrices of metal or semi-metal oxides through the aqueous processing of hydrolytically labile precursors. The encapsulation into PVA-SbQ involves the photo-cross-linking of the styrylpyridinium groups in order to create the polymer matrix. These networks are chemically stable and do not restrict the enzyme activity. Both bioencapsulation strategies provide simple, easy, and low-cost methods for enzyme immobilization. They are versatile, as matrixes can be tailor-designed and used to entrap a large number of biomolecules. They present numerous applications, including the development of biooptical devices, biosensors and biocatalysts.
Key Words: Entrapment; sol-gel technology; tetramethoxysilane; TMOS; methyltrimethoxysilane; MTMOS; poly(vinyl alcohol) bearing styrylpyridinium groups; PVA-SbQ; photo-cross-linking; polymer matrix; screen-printed graphite electrode.
Biosensors, bioarrays, lab-on-a-chips, biocatalysts, and industrial bioprocesses are some examples of technologies that have as a common key point the immobilization of biological materials such as enzymes, engineered proteins, catalytic antibodies, nucleic acids, and whole cells. All of these biotechnologies require a successful bio-immobilization in terms of resistance to leaking or desorption, long-term stability, stability under adverse experimental conditions, retention of the biomolecule functionality, or activity, accessibility to analytes, fast response times, and sometimes high immobilization density and oriented immobilization.
Among the different methods for enzyme immobilization, encapsulation is of particular interest because of the simplicity of preparation and the biomolecule freedom. The fabrication process is straightforward and reproducible and does not require sophisticated equipment. The encapsulation process is based on the entrapment of the biomolecule in a polymer matrix and there is no covalent association between the network and the biomolecule. This entrapment restricts rotation and unfolding movements but allows substrate recognition and binding as well as catalysis (1-4). Other advantages are the permeability of the matrices, which allows the transport of low-molecular-weight (MW) compounds without leaking of the entrapped biomolecules; the tuneable material porosity, which allows the accommodation of biomolecules of different size; the possibility of chemically modifying the matrix, introducing, for example, electrochemical moieties; the optical properties, which open the possibility to measure absorbance of fluorescence signals; the resistance to chemical, thermal, and biological degradation; and the negligible swelling effects.
In this chapter, two different encapsulation methods are described: the sol-gel technology and the bioencapsulation in a matrix formed by photo-cross-linkable poly(vinyl alcohol)-bearing styrylpyridinium groups (PVA-SbQ). The sol-gel process (see Fig. 1) is based on the ability to form metal-oxide, silica, and organosiloxane matrices of defined porosity by the reaction of organic precursors at room temperature (5). In a first reaction, one or two metal alkoxide precursors (usually, tetramethoxysilane [TMOS; Si(OCH3)4] and/or methyltrimethoxysilane [MTMOS; H3Si(OCH3)3] are hydrolyzed in the presence of water at acid pH, resulting in the formation of silanol (Si-OH) groups. In the second step, the condensation reaction between silanol moieties at basic pH results in the formation of siloxane (Si-O-Si) polymers, creating a matrix in which the biomolecules are entrapped. In other words, the hydrolysis of precursors results in the sol formation and subsequent gelation occurs by their condensation and polycondensation. Finally, the sol-gel is dried, forming a xerogel. In the PVA-SbQ encapsulation (see Fig. 2), the photo-cross-linking of the styrylpyridinium groups of the PVA-SbQ creates a network in which the biomolecule is entrapped
The encapsulation techniques are not restrictive in terms of the type of enzyme that can be immobilized. Sol-gel encapsulation started with the entrapment of glucose oxidase as a model enzyme, using different sol-gel-derived materials (6-9). The versatility of this enzyme has led to the use of different strategies for its detection based on electrochemical and optical measurements. Horseradish peroxidase has also been immobilized using sol-gel processes, leading to biosensors for different analytes, such as hydrogen peroxide (10) and cyanide (11), with high sensitivity, high stability, and high reproducibility. With the purpose to determine phenols, tyrosinase has also been entrapped by sol-gel techniques. This enzyme retained 73% of its original activity after intermittent use for 3 wk when stored in a dry state at 4°C (12).
The PVA-SbQ encapsulation technique has also provided the entrapment of different enzymes. Aldehyde dehydrogenase was immobilized by PVA-SbQ entrapment on the surface of disposable screen-printed graphite electrodes with the purpose of developing a biosensor for the detection of metam-sodium, a slightly
Fig. 1. The sol-gel entrapment of enzymes. (From ref. 26.)
Fig. 1. The sol-gel entrapment of enzymes. (From ref. 26.)
Fig. 2. The PVA-SbQ entrapment of enzymes. (From ref. 13.)
toxic soil fumigant, and its toxic metabolite methyl isothiocyanate (MITC) (13). The enzyme activity was inhibited by the presence of these dithiocarbamate fungicides, although the biosensor showed a relatively low sensitivity (the limit of detection for MITC being 100 ppb), probably as a result of the high amount of immobilized enzyme. This mono-enzymatic biosensor was stable during 23 successive injections, indicating that the retention of the enzyme was successful. Bi-enzymatic biosensors incorporating aldehyde dehydrogenase and diaphorase were also developed for the detection of other dithiocarbamante fungicides (14). The lowest limits of detection were 0.05 ppm and 0.25 ppm for maneb and zineb, respectively. In this case, a 60% desorption of the diaphorase enzyme was observed when using a PVA-SbQ polymer with a low degree of polymerization (1700), which reduced the operational stability by 60% after 10 assays. This operational stability was improved by the incorporation of a cellophane membrane on top of the electrode surface (15). In this case, the biosensor was able to detect as low as 1.48 ppb of maneb. The same bi-enzymatic strategy was used for the detection of acetalde-hyde in wines, beers, and ciders, achieving limits of detection comparables to those obtained with the standard spectrophotometry method (16). Through the co-entrapment of aldehyde dehydrogenase, nicotinamide-adenine dinucleotide (NADH) oxidase and high-MW NADs (NAD-dextran and NAD-polyethylene glycol [PEG]), a reagentless acetaldehyde sensor was developed (17). The high-MW NADs were used to avoid the leaking that would be observed if the low-MW NAD+ cofactor had been used in the encapsulation. The sensor reported limits of detection of acetaldehyde in alcoholic beverages comparable to those obtained by spec-trophotometry. Moreover, the sensor did not show any decrease in the operational stability after 80 successive measurements. D-Lactate dehydrogenase was another of the dehydrogenases encapsulated using PVA-SbQ. The developed biosensor was used for the detection of D-lactate in French and Romanian wines (18). Finally, the entrapment of acetyl cholinesterase allowed the detection of paraoxon (19.1 nM) and chlorpyrifos ethyl oxon (1.24 nM) in the presence of 5% acetonitrile (19).
It is not just different enzymes that can be immobilized using the encapsulation techniques but other biomolecules as well. Antibodies, regulatory proteins, membrane-bound proteins, and whole cells have been entrapped using this generic and versatile technology (20-24). However, each biomolecule requires specific component ratios, and optimization of the experimental parameters always has to be performed.
Despite all the advantages and the promising results achieved, it is necessary to keep in mind that bioencapsulation is still in its early phases. More biocompatible precursors and protocols are needed, shrinkage and pore-collapse effects have to be reduced, and the porosity and mechanical stability of sol-gels has to be improved if better catalytic efficiencies and response profiles are to be achieved (25). In the PVA-SbQ encapsulation, the leaking of the biomolecule from the polymeric network has to be completely eliminated and the reproducibility has to be guaranteed (14,26). In any case, despite the nonoptimized biocompatibility, porosity, mechanical robustness, and long-term stability of the entrapped enzymes, the encapsulation techniques are attractive and reliable tools for bio-immobilization in many practical applications such as bio-optical devices, biotransducers, biosensors, biocatalysts, bioaffinity chromatography, bioelectronics, and bioprocesses.
2.1. Sol-Gel Encapsulation
1. Tetramethoxysilane (TMOS; Sigma, St. Louis, MO). Caution: corrosive.
2. Methyltrimethoxysilane (MTMOS; Sigma). Caution: highly flammable.
3. Milli-Q water.
4. 1 mMHCl. Caution: corrosive.
5. Polyethylene glycol (PEG) with average mol wt: 600 (PEG600; Sigma).
6. Enzyme to be immobilized.
7. Automatic pipets (and automatic pipets special for viscous solutions).
8. Eppendorf tubes.
9. Vortex mixer.
10. Screen-printed graphite electrodes (BIOMEM Group, Université de Perpignan, France).
11. Refrigerator or cold chamber.
2.2. PVA-SbQ Encapsulation
1. Enzyme to be immobilized.
3. Photo-cross-linkable poly(vinyl alcohol)-bearing styrylpyridinium groups (PVA-SbQ) with degree of polymerization: 1700, degree of saponification: 88, SbQ content: 1.1 mol %, solid content: 11 mol %, pH 7.0 (Toyo Gosei Kogyo Co., Chiba, Japan).
4. Automatic pipets (and automatic pipets special for viscous solutions).
5. Eppendorf tubes.
6 Vortex mixer.
8. Screen-printed graphite electrodes (BIOMEM Group, Université de Perpignan), Maxisorp microtiter plates (Nunc, Roskilde, Denmark) or Ultrabind modified polyethersulfone affinity membranes with 0.45 |im pore size (Pall Gelman Sciences Inc., New York).
10. Refrigerator or cold chamber.
1. Mix the reactant solutions TMOS, MTMOS, H2O, HCl (1 mM) and PEG600 in the convenient ratio (see Note 1) in a 1.5-mL Eppendorf tube using automatic pipets (use an automatic pipet special for viscous solutions for PEG600).
2. Cap the Eppendorf tube and sonicate for 15 min to homogenize the mixture.
3. Store the Eppendorf tube at 4°C overnight to allow hydrolysis of the precursors (see Note 2).
4. Dissolve the enzyme in a basic buffer using Milli-Q water (see Note 3).
5. Mix the sol solution with the enzymatic solution in a 50:50 ratio in a 1.5-mL Eppendorf tube and using automatic pipets to start the condensation (see Notes 4-6).
6. Cap the Eppendorf tube and homogenize the mixture using a vortex mixer (see Note 6).
7. Spread the mixture on the support surface using an automatic pipet (see Notes 6-8).
8. Dry the support surface for at least 36 h at 4°C (see Note 9).
9. Rinse the support surface with water prior utilization (see Note 10). While rinsing, carefully look to see if any desorption of the sol-gel from the surface support occurred.
3.2. PVA-SbQ Encapsulation
1. Dissolve the enzyme in the convenient buffer using Milli-Q water.
2. Mix the enzymatic solution with PVA-SbQ in a 50:50 ratio in a 1.5-mL Eppendorf tube and using automatic pipets (use an automatic pipette special for viscous solutions for PVA-SbQ) (see Notes 11 and 12).
3. Cap the Eppendorf tube and homogenise the mixture using a vortex mixer (see Note 13).
4. Spread the mixture on the support surface using an automatic pipet (see Notes 7 and 14).
5. Expose the support surface to neon light for 3 h at 4°C to allow entrapment of enzymes by polymerization (see Note 15).
6. Dry the support surface for at least 36 h at 4°C (see Note 9).
7. Rinse the support surface with water prior utilization (see Note 10). While rinsing, carefully look to see if any desorption of the PVA-SbQ from the surface support occurred.
Precursor Mixture Compositions in |L (Final Volume: 108 mL).
Precursor Mixture Compositions in |L (Final Volume: 108 mL).
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