The sol-gel encapsulation of enzymes

In the past decade, the encapsulation of enzymes inside inorganic sol-gel matrices has become a generic method to prepare efficient biocatalysts which are easy to recycle. In this review, the sol-gel processes useful for enzyme encapsulation, (mostly sol-gel silica) are outlined. Then, the most recent developments in the applications of such biocatalysts are presented, in particular regarding biosensors and chemical synthesis. Finally, a special attention is addressed to the types of interactions which are considered to prevail between the enzyme or the substrates and products, and the matrix, in these materials.     

Aqueous sol-gel encapsulation of genetically engineered Moraxella spp. cells for the detection of organophosphates

An all-aqueous sol-gel method for encapsulation of bacterial cells in porous silicate matrices towards the development of a biosensor is described. The sol-gel encapsulation of cells is achieved at room temperature and neutral pH. Furthermore, use of sodium silicate as precursor avoids generation of alcohol that can be detrimental to cells in contrast to the traditional alkoxide sol-gel encapsulation process. Moraxella spp. cells engineered to express recombinant organophosphorus hydrolase (OPH) on the cell surface were encapsulated and OPH enzymatic activity was measured for paraoxon hydrolysis. Kinetic parameters (Km and Vmax) as well as pH behavior of surface-expressed OPH were determined to evaluate the effect of encapsulation. Cells encapsulated by the sodium silicate method displayed higher activity retention compared to those by the traditional alkoxide process. Time-course studies over a 2-month period indicate that immobilization through the sodium silicate process led to a reduction in activity of approximately 5% as compared to approximately 30% activity reduction in case of free cells in buffer indicating that immobilization leads to stabilization, a key parameter in biosensor development.     

AFM nanometer surface morphological study of in situ electropolymerized neutral red redox mediator oxysilane sol-gel encapsulated glucose oxidase electrochemical biosensors

Four different silica sol-gel films: methyltrimethoxysilane (MTMOS), tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTOS) and 3-glycidoxypropyl-trimethoxysilane (GOPMOS) assembled onto highly oriented pyrolytic graphite (HOPG) were characterized using atomic force microscopy (AFM), due to their use in the development of glucose biosensors. The chemical structure of the oxysilane precursor and the composition of the sol-gel mixture both influenced the roughness, the size and the distribution of pores in the sol-gel films, which is relevant for enzyme encapsulation. The GOPMOS sol-gel film fulfils all the morphological characteristics required for good encapsulation of the enzyme, due to a smooth topography with very dense and uniform distribution of only small, 50nm diameter, pores at the surface. APTOS and MTMOS sol-gel films developed small pores together with large ones of 300-400nm that allow the leakage of enzymes, while the TEOS film formed a rough and incomplete network on the electrode, less suitable for enzyme immobilisation. GOPMOS sol-gel film with encapsulated glucose oxidase and poly(neutral red) redox mediator, prepared by in situ electropolymerization, were also morphologically characterized by AFM. The AFM results explain the variation of the stability in time, sensitivity and limit of detection obtained with different oxysilane sol-gel encapsulated glucose oxidase biosensors with redox mediator.     

Biopolymer nanoparticle production for controlled release of biopharmaceuticals

Abstract

For drug applications, nanoparticles, used as drug carriers, offer the advantage of controlled release, therapeutic impact and targeted delivery. In drug delivery applications, biodegradable polymers can be extracted from natural sources or prepared synthetically by polymerization. Natural polymers typically have varying compositions and physiochemical properties. As a result, methods which utilize natural polymers to encapsulate drugs are more varied and polymer dependent. The following polymers are discussed in this review article: alginate, chitosan, gelatin, albumin, gliadin, pullulan, and dextran. Specialized encapsulation nanotechnologies will be discussed such as ionotropic gelation, complexation, the reverse microemulsion technique, cross-linking methods, emulsion-dependent methods, desolvation methods and self-assembly methods. For each biopolymer an overview of the structure is presented with the corresponding encapsulation techniques. Understanding the structure of the biopolymer is important as to not only understand the rational for current encapsulation techniques but to continue to develop new encapsulation techniques in pursuit of the ideal drug carrier for application in therapeutic treatments.     

A reagent less fluorescent sol-gel biosensor for uric acid detection in biological fluids

The simultaneous encapsulation of a coupled uricase-peroxidase system and amplex red in a sol-gel matrix allows one to obtain a reagent-less and ready-to-use fluorescent biosensor for the accurate detection of uric acid in highly diluted biological fluids. The detection limit of the prepared biosensor was found to be 20 nM and was linear up to 1 microM. The high sensitivity found for the biosensor permitted a reliable determination of uric acid concentrations in the presence of interfering species (e.g., ascorbic acid) just by sample dilution (up to 50000 for urine and 10000 for serum and blood). The sol-gel encapsulation preserved the hierarchy of the enzyme activity as demonstrated by the performance of the fluorescent biosensor.     

Spectroscopic and electrochemical characterization of cytochrome <Emphasis Type="Italic">c</Emphasis> encapsulated in a bio sol–gel matrix

Sol-gel technique represents a remarkably versatile method for protein encapsulation. To enhance sol-gel biocompatibility, systems envisaging the presence of calcium and phosphates in the sol-gel composition were recently prepared and investigated. Unfortunately, the low pH at which solutions were prepared (pH < 2.5) dramatically limited their application to proteins, because the acidic environment induces protein denaturation. In this paper we apply a new protocol based on the introduction of calcium nitrate to the inorganic phase, with formation of a binary bioactive system. In this case protein encapsulation results versatile and secure, being achieved at a pH close to neutrality (pH 6.0); also, the presence of calcium is expected to enhance system biocompatibility. To determine the properties of the salt-doped sol-gel and the influence exerted on entrapped biosystems, the structural and functional properties of embedded cytochrome c have been investigated. Data obtained indicate that the salt-doped sol-gel induces no significant change in the structure and the redox properties of the embedded protein; also, the matrix increases protein stability. Interestingly, the presence of calcium nitrate appears determinant for refolding of the acid-denatured protein. This is of interest in the perspective of future applications in biosensoristic area.     

Sol-gel trapping of functional intermediates of hemoglobin: geminate and bimolecular recombination studies

The encapsulation of proteins in porous sol-gels is a promising technique for generating, trapping, and probing functionally significant nonequilibrium protein species. An essential step needed in the pursuit of that goal is establishing the degree to which the sol-gel limits conformational change upon adding or removing substrates. In the present study, geminate recombination and solvent phase bimolecular recombination of CO to human adult hemoglobin (HbA) are used as sensitive probes of the degree of conformational constraint within the sol-gel. Two forms of CO saturated encapsulated HbA are generated. In one case, designated [COHbA], the equilibrium form of COHbA is directly encapsulated. In the second case, designated as [deoxyHbA] + CO, the equilibrium form of deoxyHbA is encapsulated and only after the sample has aged is CO introduced to the HbA through the porous sol-gel matrix. Three different preparative protocols are used to generate the sol-gels for each of the two forms of encapsulated COHbA. The kinetic traces obtained from these encapsulated samples allow for an easy evaluation of the extent to which the sol-gel is locking in the initial tertiary/quaternary structure. The results show that the sol-gel encapsulated samples can be used with pulsed laser sources and that one of the tested encapsulation protocols is far superior with respect to conformational locking. This protocol is used to trap and probe nonequilibrium forms such as the liganded T state of HbA, a species whose properties are needed to fully explore allostery in HbA.     

Molecular imprinting in sol-gel matrix

Molecular imprinting is a newly developed methodology which provides molecular assemblies of desired structures and properties and is being increasingly used for several applications such as in separation processes, microreactors, immunoassays and antibody mimics, catalysis, artificial enzymes, biosensor recognition elements and bio- and chemo-sensors. The ambient processing conditions and versatility of the sol-gel process makes sol-gel glassy matrix suitable for molecular imprinting. The progress of sol-gel based molecular imprinted polymers (MIPs) for various applications can be seen from the growing number of publications. The main focus of the review is molecular imprinting in sol-gel matrix and applications of molecular imprinted sol-gel derived materials for the development of sensors. Combining sol-gel process with molecular imprinting enables to procure the sensors with greater sensitivity and selectivity necessary for sensing applications. The merits, problems, challenges and factors affecting molecular imprinting in sol-gel matrix have been discussed. Considerable attention has been drawn on recent developments like use of organically modified silane precursors (ORMOSILS) for the synthesis of hybrid molecular imprinted polymers (HMIPs) and applying surface sol-gel process for molecular imprinting. The development of molecular imprinted sol-gel nanotubes for biochemical separation and bio-imprinting is a new advancement and is under progress. Templated xerogels and molecularly imprinted sol-gel films provide a good platform for various sensor applications.     

Bioencapsulation within synthetic polymers (Part 2): non-sol-gel protein-polymer biocomposites

Since the introduction of sol-gel bioencapsulation and the demonstration that biological function can be incorporated into, and preserved within, polymer matrices, a number of alternative polymers have been used to immobilize proteins. Various enzymes have been trapped in such diverse polymers as epoxy-amine resins, polyvinyl plastics, polyurethane foams and silicone elastomers. Together with sol-gel encapsulates, these biocomposites represent a powerful approach for immobilizing biological materials for applications as biosensors and biocatalysts, and hold promise as bioactive, fouling-resistant polymers for environmental, food and medical uses. Although still at the developmental stage, these biocomposites promise to revolutionize the whole arena of high-performance bioimmobilization.     

Distal heme pocket conformers of carbonmonoxy derivatives of Ascaris hemoglobin: evidence of conformational trapping in porous sol-gel matrices

We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy. The heme-bound CO is used as a spectroscopic antenna to probe the original distribution of conformers in the dioxygen derivative of Ascaris Hb, by utilizing sol-gel encapsulation. The first step is to encapsulate the dioxygen derivative in the porous sol-gel and let the gel age, thus trapping the equilibrium conformational distribution of Ascaris dioxygen Hb. In the second step, the dioxygen ligand is replaced by CO. The sol-gel environment impedes any large scale movements, drastically slowing down the conformational relaxation triggered by the ligation change, essentially "locking in" the initial quaternary and even tertiary structure of the protein. Studying the Fe-CO frequencies of the latter sample allows evaluation of the distribution of the distal heme pocket conformers that was originally associated with the dioxygen derivative. Extending the study to the Ascaris mutants allows for examination of the effect of specific residues in the distal pocket on the conformational distribution. The choice of mutants was largely based on the anticipated variation in hydrogen bonding patterns. The results show that the sol-gel encapsulation can slow or prevent re-equilibration within the distal heme pocket of Ascaris Hb and that the distribution of distal heme pocket conformers for the CO derivative of Ascaris Hb in the sol-gel is highly dependent on the history of the sample. Additionally, we report a detailed study of the CO complex of the mutants in solution for assignment of the various heme pocket conformers, and we present a comparison of the sol-gel data with solution data. The results support a picture in which the dioxygen derivative biases the population strongly toward a tightly packed configuration that favors the network of strong hydrogen bonding interactions, and suggest that Ascaris Hb is uniquely designed for dioxygen capture.     

Use of fluorescence to monitor the incorporation of horseradish peroxidase into a sol-gel derived medium

The solvatochromic dye nile red has been employed to monitor the incorporation of an enzyme (horseradish peroxidase) into a sol-gel derived medium. The fluorescence spectrum of the dye, when incorporated into the enzyme, was analysed as the sum of Gaussian component spectra and relative changes between these component spectra were monitored upon encapsulation of the dye-enzyme system within the host matrix. Activity of the confined enzyme was verified and the effect of temperature was also investigated, through the examination of nile red fluorescence in the sol-gel derived matrix, where a stabilising effect was noted.     

Enzymatic synthesis of lignin-siloxane hybrid functional polymers

This study combines the properties of siloxanes and lignin polymers to produce hybrid functional polymers that can be used as adhesives, coating materials, and/or multifunctionalized thin-coating films. Lignin-silica hybrid copolymers were synthesized by using a sol-gel process. Laccases from Trametes hirsuta were used to oxidize lignosulphonates to enhance their reactivity towards siloxanes and then were incorporated into siloxane precursors undergoing a sol-gel process. In vitro copolymerization studies using pure lignin monomers with aminosilanes or ethoxytrimethylsilane and analysis by 2?Si NMR spectroscopy revealed hybrid products. Except for kraft lignin, an increase in lignin concentration positively affected the tensile strength in all samples. Similarly, the viscosity generally increased in all samples with increasing lignin concentration and also affected the curing time.     

Sol-gel encapsulation of lactate dehydrogenase for optical sensing of L-lactate

Sol-gel encapsulation of lactate dehydrogenase and its cofactor can be employed as a disposable sensor for L-lactate. The sensor utilized the changes in absorbance or fluorescence from the reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. Although, problems such as diminished enzymatic activity and/or leaching of enzyme from the sol-gel matrix occurred, the sol-gel process is sufficiently mild to permit retention of enzymatic activity. The apparent activity of LDH in the sensor is at least 10% of that of the dissolved enzyme. The sensor has a linear dynamic range over the normal physiological L-lactate level and has a long-term storage stability of at least 3 weeks.     

Cell-free synthesis of functional proteins using transcription/translation machinery entrapped in silica sol-gel matrix

Herewith we report the encapsulation of functional protein synthesis machinery in a silica sol-gel matrix. When the sol-gel reaction using alkoxysilane monomers was carried out in the presence of Escherichia coli cell extract, macromolecular protein synthesis machinery in the cell extract was successfully immobilized within a silica gel matrix, catalyzing the translation of co-immobilized DNA when supplied with small-molecular-weight substrates for protein synthesis. The efficiency of protein synthesis was affected by the pore size of the gel structure, which was controlled through the use of appropriate additives during the sol-gel reactions. To the best of our knowledge, this is the first report describing the reproduction of the entire set of complicated biological process within an inorganic gel matrix, and we expect that the developed technology will find many applications in numerous fields such as high-throughput gene expression and the development of multifunctional biosensors.     

Solid-phase assays for small molecule screening using sol-gel entrapped proteins.

With compound libraries exceeding one million compounds, the ability to quickly and effectively screen these compounds against relevant pharmaceutical targets has become crucial. Solid-phase assays present several advantages over solution-based methods. For example, a higher degree of miniaturization can be achieved, functional- and affinity-based studies are possible, and a variety of detection methods can be used. Unfortunately, most protein immobilization methods are either too harsh or require recombinant proteins and thus are not amenable to delicate proteins such as kinases and membrane-bound receptors. Sol-gel encapsulation of proteins in an inorganic silica matrix has emerged as a novel solid-phase assay platform. In this minireview, we discuss the development of sol-gel derived protein microarrays and sol-gel based monolithic bioaffinity columns for the high-throughput screening of small molecule libraries and mixtures.     

Silica sol-gel encapsulation of cyanobacteria: lessons for academic and applied research

Cyanobacteria inhabit nearly every ecosystem on earth, play a vital role in nutrient cycling, and are useful as model organisms for fundamental research in photosynthesis and carbon and nitrogen fixation. In addition, they are important for several established biotechnologies for producing food additives, nutritional and pharmaceutical compounds, and pigments, as well as emerging biotechnologies for biofuels and other products. Encapsulation of living cyanobacteria into a porous silica gel matrix is a recent approach that may dramatically improve the efficiency of certain production processes by retaining the biomass within the reactor and modifying cellular metabolism in helpful ways. Although encapsulation has been explored empirically in the last two decades for a variety of cell types, many challenges remain to achieving optimal encapsulation of cyanobacteria in silica gel. Recent evidence with Synechocystis sp. PCC 6803, for example, suggests that several unknown or uncharacterized proteins are dramatically upregulated as a result of encapsulation. Also, additives commonly used to ease stresses of encapsulating living cells, such as glycerol, have detrimental impacts on photosynthesis in cyanobacteria. This mini-review is intended to address the current status of research on silica sol-gel encapsulation of cyanobacteria and research areas that may further the development of this approach for biotechnology applications.     

Toxicity analysis of encapsulation solutions and polymers in the cultivation of insect cells

Summary Microencapsulation of insect cells, hosts for baculovirus expression systems, requires that the encapsulation reagents and membrane-forming materials be non-toxic to the cells. Various encapsulation polymers (poly-l-lysine, chitosan and alginate) and solutions (KCl, CaCl₂, CHES and sodium citrate) were tested for their toxicity toSpodoptera frugiperda cells. The effects of varying polymer molecular weight and concentration on cell viability were also investigated.     

Binding characteristics of bovine serum albumin encapsulated in sol-gel glasses: an alternative for protein interaction studies

Silica glasses doped with 500-700 microg of bovine serum albumin were prepared by the sol-gel method; two pH conditions (pH 5 and 7) were assayed for protein encapsulation. Both biomaterials showed a highly porous structure, with pore sizes in the range 5-28 nm. Columns packed with the ground biogels were on-line coupled to a C18 HPLC column for evaluation of the entrapped protein binding properties using propranolol. Binding capacities (at saturation) were approximately 3.7 and 7.1 microg of propranolol (drug-protein molar ratios 1.4 and 2.7) for the biogels prepared at pH 5 and 7, respectively. The significant difference indicates increased albumin denaturation upon encapsulation at pH 5. A frontal analysis study was then performed in cartridges packed with biogel prepared at pH 7 to evaluate the protein interaction with naproxen at low concentrations (相似文献   

Electronic nose screening of ethanol release during sol-gel encapsulation. A novel non-invasive method to test silica polymerisation

Porous silica matrices prepared by sol-gel process yield biocompatible materials adequate for encapsulation of biomolecules or drugs. The procedure is simple and fast, but when alkoxyde precursors like tetraethoxysilane (TEOS) are used the polymerisation reaction leads to the formation of alcohol as a by-product, which can produce undesirable effects on the activity of entrapped enzymes or modify a drug release kinetic. Therefore, it is critical to determine that no remnant ethanol is left prior using or storing the obtained biomaterial. In this regard, the technique used in the alcohol determination should be non-invasive and non-destructive to preserve the encapsulation device intact and ready to use. In this work we have successfully used a portable electronic nose (e-nose) for the screening of silica polymerisation process during theophylline encapsulation. TEOS reaction was "smelt" since precursor pre-hydrolysis until the end of ethanol release, sensed directly at the headspace of matrices slabs. Measurements showed that ethanol was negligible since 10th day in polymeric slabs of 10 mm width and 2 cm diameter. This first use of e-nose following a polymerisation reaction opens a wide number of putative applications in pharmaceutical and biochemical fields.     

Thermally induced gelable polymer networks for living cell encapsulation

We report the encapsulation of MIN6 cells, a pancreatic beta-cell line, using thermally induced gelable materials. This strategy uses aqueous solvent and mild temperatures during encapsulation, thereby minimizing adverse effects on cell function and viability. Using a 2:1 mixture of PNIPAAm-PEG-PNIPAAm tri-block copolymer and PNIPAAm homopolymer that exhibit reversible sol-to-gel transition at approximately 30 degrees C, gels were formed that exhibit mechanical integrity, and are stable in H(2)O, PBS and complete DMEM with negligible mass loss at 37 degrees C for 60 days. MTT assays showed undetectable cytotoxicity of the polymers towards MIN6 cells. A simple microencapsulation process was developed using vertical co-extrusion and a 37 degrees C capsule collection bath containing a paraffin layer above DMEM. Spherical capsules with diameters ranging from 500 to 900 microm were formed. SEM images of freeze-dried capsules with PBS as the core solution showed homogenous gel capsule membranes. Confocal microscopy revealed that the encapsulated cells tended to form small aggregates over 5 days, and staining for live and dead cells showed high viability post-encapsulation. A static glucose challenge with day-5 cultured microencapsulated cells exhibited glucose-dependent insulin secretion comparable to controls of free MIN6 cells grown in monolayers. These results demonstrate the potential use of these thermo-responsive polymers as cell encapsulation membranes.