Atrazine degradation by Pseudomonas strain ADP entrapped in sol-gel glass

Sol-gel entrapment was evaluated as a method for immobilization of an atrazine degrading Pseudomonas. It was found that the bacterium lost much of its atrazine degrading activity upon immobilization. However, partial activity could have been restored by amendment of nutrients. Bacteria immobilized using a prehydrolysis technique for the preparation of the sol-gel, retained better activity in comparison to bacteria immobilized using a composite calcium alginate/sol-gel procedure. Further study is underway to improve the activity of sol-gel entrapped bacteria.     

Optimization of tetramethoxysilane-derived sol gel entrapment protocol stabilizes highly active chlorophyllase

Entrapment of membrane proteins is a challenging task compared to that involving soluble proteins. Chlorophyllase, a membrane protein, was successfully entrapped in tetramethoxysilane-derived sol-gel. Pre-gel sol typically consists of an aqueous suspension of chlorophyllase, precursors including tetramethoxysilane and/or methytrimethoxysilane, and sodium fluoride as catalyst. To obtain a highly active entrapped enzyme preparation, the effects of various immobilization parameters, including the chemical compositions of pre-gel sol (water/silane ratio, precursor type and proportions, enzyme loading, sodium fluoride concentration), and sol-gel process parameters (aging and drying time and approach) have been investigated. Chlorophyllase demonstrated the highest activity in gel derived from a pre-gel sol with water/silane ratio of 30 and enzyme loading of 0.257 mg_protein/g_gel, and showed moderately lower activity in organically modified sol-gel than that in hydrophilic sol-gel. The effects of water/silane ratio and precursor combinations on the activity of entrapped chlorophyllase were also studied by examining the pore morphology of gel via nitrogen adsorption-desorption. Longer aging time leads to an entrapped chlorophyllase preparation with higher activity. Chlorophyllase preparation demonstrated negligible activity after air-drying for 12 h while lyophilized chlorophyllase preparation demonstrated 8, 4 and 4 times higher activity than air-dried, vacuum-dried and solvent-dried preparations. Chlorophyllase demonstrated 30% higher activity in the improved sol-gel protocol than that from a non-optimized sol-gel protocol developed in a previous study.     

Phycobiliproteins encapsulated in sol-gel glass

Light transducing phycobiliproteins are encapsulated in optically transparent sol-gel matrices. Absorption and fluorescence spectroscopies are used to characterize the effect of the sol-gel encapsulation on the conformation and aggregation states of the three major phycobiliproteins present in phycobilisomes: phycoerythrin, phycocyanin, and allophycocyanin. It is found that the effects of sol-gel entrapment on the spectroscopic properties are significantly different for the three phycobiliproteins. The results indicate that phycoerythrin undergoes only minor change in its native structure when entrapped in sol-gel. However, significant changes in conformation and aggregation state occur when phycocyanin and allophycocyanin are entrapped in sol-gel matrices. A thin film of sol-gel encapsulated phycoerythrin is also coated on an optical fiber surface and strong fluorescence from the evanescent wave excitation is detected. The potential applications of sol-gel encapsulated phycobiliproteins in biosensors are discussed.     

Matrix Effects on Selective Chemical Sensing by Sol-Gel Entrapped Complexing Agents

Changes in the selectivity of molecular recognition systems on sol-gel entrapment are reported. Thermodynamic effects are exemplified by studies of the metal-ion complexing agent Eriochrome Cyanine R (ECR). In aqueous solution this binds strongly and selectively to Al3+, whereas in a TMOS-based sol-gel matrix it is selective for Cu3+. Thermodynamic effects, due to restricted translational freedom of water molecules or different solvent structure and isolation of ligands, can explain these observations. Effects of entrapment on molecular recognition by a large conformationally flexible molecule have been studied using a tris-terminated PAMAM dendrimer. The dendrimer conformation and its complexation with Cu2+ changes on entrapment, and binding of aromatic carboxylic acids such as ibuprofen can be detected by changes in visible absorption and surface plasmon resonance using spun films of the sol-gel composite. These effects show that in addition to providing a porous entrapment matrix of good optical quality, sol-gels may be used to alter the binding characteristics of the entrapped receptors.     

Stabilizing structure-switching signaling RNA aptamers by entrapment in sol-gel derived materials for solid-phase assays

Structure-switching, fluorescence-signaling DNA and RNA aptamers have been reported as highly versatile molecular recognition elements for biosensor development. While structure-switching DNA aptamers have been utilized for solid-phase sensing, equivalent RNA aptamers have yet to be successfully utilized in solid-phase sensors due to their lack of chemical stability and susceptibility to nuclease attack. In this study, we examined entrapment into sol-gel derived organic-inorganic composite materials as a platform for immobilization of structure-switching fluorescence-signaling RNA aptamer reporters, using both the synthetic theophylline- and naturally occurring thiamine pyrophosphate-binding RNA aptamers as test cases. Structure-switching versions of both aptamers were entrapped into a series of sol-gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials, and the target-binding and signaling capabilities of these immobilized aptamers were assessed relative to solution. Both immobilized aptamers demonstrated sensitivity and selectivity similar to that of free aptamers when entrapped in a composite material derived from 40% (v/v) methyltrimethoxysilane/tetramethoxysilane. Importantly, this material also conferred protection from nuclease degradation and imparted long-term chemical stability to the RNA reporter systems. Given the versatility of sol-gel entrapment for development of biosensors, microarrays, bioaffinity columns, and other devices, this entrapment method should provide a useful platform for numerous solid-phase RNA aptamer-based devices.     

Silica/Proteoliposomal Nanocomposite as a Potential Platform for Ion Channel Studies

The nanostructuration of solid matrices with lipid nanoparticles containing membrane proteins is a promising tool for the development of high-throughput screening devices. Here, sol-gel silica-derived nanocomposites loaded with liposome-reconstituted KcsA, a prokaryotic potassium channel, have been synthesized. The conformational and functional stability of these lipid nanoparticles before and after sol-gel immobilization have been characterized by using dynamic light scattering, and steady-state and time-resolved fluorescence spectroscopy methods. The lipid-reconstituted KcsA channel entrapped in the sol-gel matrix retained the conformational and stability changes induced by the presence of blocking or permeant cations in the buffer (associated with the conformation of the selectivity filter) or by a drop in the pH (associated with the opening of the activation gate of the protein). Hence, these results indicate that this novel device has the potential to be used as a screening platform to test new modulating drugs of potassium channels.     

Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports

Enzymes were first immobilized on inorganic supports through silane coupling agents over 25 yr ago. Since that initial report, literally hundreds of laboratories have utilized this methodology for the immobilization of enzymes, antigens, antibodies, receptors, and other high and low mol wt compounds. Today silane coupling is one of the commonly used techniques in the arsenal of the biochemist for the binding of material of all sorts to inorganic surfaces. Inorganic materials come in a variety of shapes, sizes, and characteristics. Today silane coupling is one of the most used coupling methods for the preparation of biosensing devices. Sol-gel entrapped enzymes are also produced by the application of silane technology by the polymerization of the silane to form glass-like materials with entrapped protein. This review will discuss the general preparation and characterization of silane coupled proteins with special emphasis on enzymes and describe in detail the actual methods for the silanization and specific chemical coupling of proteins to the silanized carrier.     

Silicate Xerogels with Dopant-Induced Chirality

A series of silicate xerogels with entrapped chiral amino acids have been obtained via sol-gel technology. The transparent, glassy samples obtained exhibit chirality in the bulk due to the presence of the entrapped asymmetric molecules. Measurements of the optical activity of the doped xerogel samples revealed that the entrapment did not significantly influence the optical activity observed for liquid solutions of the amino acids. Thus, the sol-gel method enables the preparation of amorphous optical materials exhibiting properties of strictly spatially defined molecular systems. Apart from the obvious optical applications, such porous materials with asymmetric centers might find interesting applications in chiral chemical syntheses and separations.     

The effect of polymeric supports and methods of immobilization on the performance of an optical copper(II)-sensitive membrane based on the colourimetric reagent Zincon

A comparative study on the effect of different immobilization methods and matrix materials on the performance of copper(II)-sensitive membrane layers is presented. The indicator dye Zincon was immobilized in hydrophilic and hydrophobic polymers by various methods including: (a) physical entrapment of the Zincon-tetraoctylammonium ion pair in plasticized PVC, hydrogel, polystyrene, ethyl cellulose, poly-HEMA, AQ-polymer and in sol-gel glass; (b) electrostatic immobilization on an anion exchanger cellulose; and (c) covalent immobilization on cellulose via a sulfatoethylsulfonyl reactive group. The response to copper(II) ion was evaluated kinetically via the initial slope of the change in absorbance within 1 min. Layers made of hydrogel and PVC provide the highest sensitivity, while covalent immobilization is the most reproducible one, and sol-gel layers display the best mechanical stability.     

Effect of sol-gel confinement on the structural dynamics of the enzyme bovine Cu,Zn superoxide dismutase

Immobilization of proteins in sol-gel glasses has allowed the development of a new generation of robust and sensitive analytical devices as well as contributes to the investigation of the effect of molecular confinement on the structure of proteins. The immobilized protein usually preserves its structural integrity and functionality, while interactions with the matrix and its surface seem to contribute to alter its dynamics and stability. With the aim of better understanding the nature of such interactions, we have encapsulated the enzyme bovine Cu,Zn superoxide dismutase (BSOD), negatively charged at physiological pH, in a sol-gel matrix and the photophysical properties of its single tyrosine have been determined using both steady-state and time-resolved fluorescence techniques. Fluorescence spectra, quenching experiments, fluorescence lifetimes, and anisotropy measurements indicate that immobilization does not lead to any major conformational change, at least in the region of protein where the tyrosine residue is located. In addition, fluorescence anisotropy decays recorded above and below the isoelectric point of the protein indicate that, at neutral pH, well above its isoelectric point, the entrapped BSOD freely rotates within the matrix pore, but showing a different rotational behavior as compared with that in the bulk aqueous solution. However, below the isoelectric point, the global motion of the protein is totally hindered upon entrapment. Electrostatic interactions with the gel matrix, changes in water viscosity, and protein-to-pore size ratio are discussed as possible factors responsible for this behavior.     

Screening of antagonists based on induced dissociation of a calmodulin-melittin interaction entrapped in a sol-gel derived matrix

Sol-gel derived materials offer a unique advantage for the development of sensing and screening platforms in that they allow for the entrapment of multiple species within a confined space. In this work, we show that it is possible to entrap an intact protein-peptide interaction, consisting of bovine calmodulin (bCaM) and melittin, into a sol-gel derived silicate material. Fluorescence emission data demonstrate that the entrapped complex behaves similarly to the complex in solution, and can undergo reversible dissociation upon introduction of the denaturant guanidine hydrochloride. Screening of antagonists of the bCaM-melittin complex was accomplished based on induced dissociation of the entrapped complex, which was followed by measuring the loss of sensitization of Tb(III) luminescence originating from energy transfer from the Trp of melittin to Tb(III) bound in the loops of bCaM. This study shows that entrapped protein-peptide complexes can be used as targets for drug screening or for fluorescence-based biosensing.     

Entrapment of parathion hydrolase from Pseudomonas spp. in sol-gel glass

The present work reports on the entrapment of parathion hydrolase from Pseudomonas spp. into the sol-gel glass matrix. Enzyme entrapment was studied in the range of 0.01–0.25 U, and compared with the activity of the free, non-immobilized enzyme. The reaction catalyzed by the entrapped enzyme was almost two orders of magnitude slower than with the free enzyme. Addition of surfactants slightly increased the parathion hydrolysis rate, and the addition of ethanol almost doubled it. However, this increase of reaction rate cannot by itself explain the decrease of activity, suggesting that irreversible damage to the enzyme during gelation, rather than diffusion limitation throughout the gel-glass structure, is the main cause for the decrease of activity. Regardless of damage to the enzyme during gelation, the remaining entrapped active fraction displayed stability even after eleven days, during successive cycles of the same entrapped enzyme batch, each for 24 h. The sol-gel entrapped enzyme retained relatively good activity for several months when stored as a dry powder, and over a year when kept in buffer solution, at ambient conditions. The results obtained may give a rise to the use of entrapped parathion hydrolase for simple on-field bio-detection of organophosphates.     

On chip single-cell separation and immobilization using optical tweezers and thermosensitive hydrogel

A novel approach appropriate for rapid separation and immobilization of a single cell by concomitantly utilizing laser manipulation and locally thermosensitive hydrogelation is proposed in this paper. We employed a single laser beam as optical tweezers for separating a target cell and locating it adjacent to a fabricated, transparent micro heater. Simultaneously, the target cell is immobilized or partially entrapped by heating the thermosensitive hydrogel with the micro heater. The state of the thermosensitive hydrogel can be switched from sol to gel and gel to sol by controlling the temperature through heating and cooling by the micro heater. After other unwanted cells are removed by the high-speed cleaning flow in the microchannel, the entrapped cell is successfully isolated. It is possible to collect the immobilized target cell for analysis or culture by switching off the micro heater and releasing the cell from the entrapment. We demonstrated that the proposed approach is feasible for rapid manipulation, immobilization, cleaning, isolation and extraction of a single cell. The experimental results are shown here.     

A Neural Network Model in the Calibration of Glucose Sensor Based on the Immobilization of Glucose Oxidase into Polypyrrole Matrix

The immobilization of enzymes on an electrode surface is of great importance in bioelectrochemistry. The entrapment of enzymes into a polymer matrix is simple and a speedy technique for the production of biosensors. This procedure of enzyme immobilization by electropolymerization has a great significance in fabrication of micro sensors in the preparation of multiplayer devices. In current study, glucose oxidase enzyme that is specific for the glucose determination was entrapped into polypyrrole matrix containing p‐benzoquinone in PIPES buffer and glucose sensitivity of the biosensor was investigated. Then, artificial neural network analysis was done for the nonlinear calibration plot. This implementation can be used for the sensor failure detection, as well. The estimation power of the neural network used in the direct and inverse calibration modelling was examined by statistical methods. It presented the good performance for the estimation power.     

Biomolecule immobilization techniques for bioactive paper fabrication

Research into paper-based sensors or functional materials that can perform analytical functions with active recognition capabilities is rapidly expanding, and significant research effort has been made into the design and fabrication of bioactive paper at the biosensor level to detect potential health hazards. A key step in the fabrication of bioactive paper is the design of the experimental and operational procedures for the immobilization of biomolecules such as antibodies, enzymes, phages, cells, proteins, synthetic polymers and DNA aptamers on a suitably prepared paper membrane. The immobilization methods are concisely categorized into physical absorption, bioactive ink entrapment, bioaffinity attachment and covalent chemical bonding immobilization. Each method has individual immobilization characteristics. Although every biomolecule–paper combination has to be optimized before use, the bioactive ink entrapment method is the most commonly used approach owing to its general applicability and biocompatibility. Currently, there are four common applications of bioactive paper: (1) paper-based bioassay or paper-based analytical devices for sample conditioning; (2) counterfeiting and countertempering in the packaging and construction industries; (3) pathogen detection for food and water quality monitoring; and (4) deactivation of pathogenic bacteria using antimicrobial paper. This article reviews and compares the different biomolecule immobilization techniques and discusses current trends. Current, emerging and future applications of bioactive paper are also discussed.     

Enzyme-modified nanoparticles using biomimetically synthesized silica

The entrapment of enzymes within biomimetic silica nanoparticles offers unique and simple immobilization protocols that merge the stability of proteins confined in solid phases with the high loading and reduced diffusion limitations inherent to nano-sized structures. Herein, we report on the biomimetic silica entrapment of chemically derivatized horseradish peroxidase for amperometric sensing applications. Scanning electron microscopy shows evidence of the formation of enzyme-modified nanospheres using poly(ethylenimine) as a template for silicic acid condensation. When these nanospheres are directly deposited on graphite electrodes, chemically modified anionic peroxidase shows direct electron transfer at 0 mV vs Ag|AgCl. Microgravimetric measurements as well as SEM images demonstrate that negatively charged peroxidase is also entrapped when silica precipitates at gold electrodes are modified with a self-assembled monolayer of poly(ethylenimine). Electrostatic interactions may play a crucial role for efficient enzyme entrapment and silica condensation at the PEI template monolayer. The in-situ biomimetically synthesized peroxidase nanospheres are catalytically active, enabling direct bioelectrocatalysis at 0 mV vs Ag|AgCl with long-term stability.     

A simple procedure for the preparation of fritless columns by entrapping conventional high performance liquid chromatography sorbents

A rapid and direct method for immobilizing conventional high performance liquid chromatography (HPLC) packing material inside fritless capillaries has been developed. Due to the simple composition of the entrapment matrix (tetraethoxysilane, alkyltriethoxysilane, ethanol and water), straightforward manufacturing procedure and modest equipment requirement, the method can readily be transferred to any laboratory and easily automated. The entrapment procedure has minimal influence on the structure and chromatographic properties of the original reverse-phase sorbent. Various immobilization solutions have been tested, and a comparison between columns entrapped with different immobilization mixtures and conventional packed capillaries is presented. High efficiency separations were obtained using tert-butyl-triethoxysilane entrapped columns in both capillary electrochromatography (reduced plate heights of 1.1-1.4 were measured) and microliquid chromatography (reduced plate heights of 2.2-2.6 were observed) formats. Elimination of frits, stabilization of the packed bed and on-the-fly customization of column length render mechanically robust columns that are remarkably stable over time, from which manufacturing imperfections can be removed easily.     

Controlling sol-gel properties enhancing entrapped membrane protein activity through doping additives

Chlorophyllase, a membrane protein, was entrapped in tetramethoxysilane (TMOS)—derived sol-gel and activity examined through a modification of sol-gel properties with doping additives. Polyvinyl alcohol or polyethyleneglycol as uncharged polymeric additives reduced the activity of entrapped chlorophyllase by 60 and 70% respectively, explained by restricted accessibility of chlorophyllase, as revealed by nitrogen desorption experiments. Entrapped chlorophyllase demonstrated 10% activity yield in polyethylenimine-doped gel relative to a polyethylenimine-free control, explained by electrostatic interaction between positively charged polyethylenimine and negatively charged chlorophyllase, considering that polyethylenimine led to a gel with a slightly reduced specific surface area and pore volume, but 20% larger pore size. When chlorophyllase was introduced into sol-gel together with various amounts of glycerol, it demonstrated only slightly lower activity in those gels, even though sol-gel demonstrated decreased specific surface area and pore volume with increasing amount of glycerol. Lipase, as additive, reduced activity of entrapped chlorophyllase, explained by its effect on gel formation, and thus gelation time and gel properties, whereas activity of sol-gel entrapped chlorophyllase associated with monogalactosyl diglyceride (MGDG), was increased by 40%. This study provides an in-depth understanding of the change in sol-gel properties as affected by a wide variety of additives, and the consequent affect on the activity of the membrane protein chlorophyllase.     

A comparative study of immobilization techniques for urease on glass-pH-electrode and its application in urea detection in blood serum

Different techniques have been used (physical adsorption, physically entrapped sandwich and microencapsulation) for the immobilization of urease enzyme in tetramethylorthosilicate (TMOS) derived sol-gel matrix on the sensing surface of glass-pH-electrode. No significant leaching of enzyme occurs from the microencapsulated and physically entrapped enzyme sandwich films. Potentiometric techniques have been used for the estimation of urea concentration in each instance. Various parameters of biosensor performance have been studied which indicates that microencapsulation technique is a better method of enzyme immobilization in sol-gel films derived from TMOS. The advantage of microencapsulated biosensor over others include higher sensitivity (dpH/dp(C) = 2.4), lower detection limit of 52 μg mL⁻¹, larger linear range (0.01-30 mM) of urea determination and reasonably long-term stability of about 25 days with 80% response signal.     

Fabrication of poly(ethylene glycol)‐based hydrogels entrapping enzyme‐immobilized silica nanoparticles

In this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3‐aminopropyltriethoxysilane (APTES), and the resultant SNP‐immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000 Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent K_m of SNP‐immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long‐term stability test demonstrated that SNP‐immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non‐entrapped SNP‐immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six‐fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP‐immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright © 2009 John Wiley & Sons, Ltd.