Sol-gel Encapsulation Research Articles

Ultra-thin porous silica coated silver–platinum alloy nano-particle as a new catalyst precursor

Templated sol–gel encapsulation of surfactant-stabilised micelles containing metal precursor(s) with ultra-thin porous silica coating allows solvent extraction of organic based stabiliser from the composites in colloidal state hence a new method of preparing supported alloy catalysts using the inorganic silica-stabilised nano-sized, homogenously mixed, silver–platinum (Ag–Pt) colloidal particles is reported.

Ferricytochrome c encapsulated in silica hydrogels: correlation between active site dynamics and solvent structure

Ferricytochrome c encapsulated in silica hydrogels has been prepared by the sol–gel technique following, with some modifications, the procedure originally developed by Zink et al. A suitable preparation of hydrogels enables to have both ‘wet’ and ‘dry’ samples. Wet samples have a high water content: as the temperature is lowered below ∼260 K water freezes and the samples crack. On the contrary, dry samples have a low water content (hydration h∼0.35): in these conditions water does not freeze even at cryogenic temperatures and the samples remain transparent and non-cracking. The dynamics of ferricytochrome c and its dependence on the surrounding medium have been studied by optical absorption spectroscopy in the temperature range 10–300 K. At each temperature, spectra were collected both in the Soret region and in the near infrared at ∼1.45 μm (the water overtone band); this enables to probe the local dynamics of the protein active site as well as the ‘structure’ of water molecules present in the sample. The data show that sol–gel encapsulation ‘per se’ does not alter the protein active site dynamics, but rather introduces an increased local heterogeneity. At difference, we find a correlation between active site dynamics and water structure: in the wet hydrogel, freezing of water quenches the ensemble of soft modes linearly coupled to the Soret transition; while, in the dry hydrogel, water does not freeze, and an active site dynamic behavior—similar to the non-freezing water/glycerol solution—is observed.

Ferricytochrome c encapsulated in silica hydrogels: correlation between active site dynamics and solvent structure

Ferricytochrome c encapsulated in silica hydrogels has been prepared by the sol–gel technique following, with some modifications, the procedure originally developed by Ellerby et al. (Science 255 1113 (1992)). A suitable preparation of hydrogels enables having both ‘wet’ and ‘dry’ samples. Wet samples have a high water content: as the temperature is lowered below ∼260 K, water freezes and the samples crack. On the contrary, dry samples have a low water content (hydration h∼0.35): in these conditions water does not freeze even at cryogenic temperatures and the samples remain transparent and non-cracking. The dynamics of ferricytochrome c and its dependence on the surrounding medium have been studied by optical absorption spectroscopy in the temperature range 10–300 K. At each temperature, spectra were collected both in the Soret region and in the near infrared at ∼1.45 μm (the water overtone band); this enables probing the local dynamics of the protein active site as well as the ‘structure’ of water molecules present in the sample. The data show that sol–gel encapsulation ‘per se’ does not alter the protein active site dynamics, but rather introduces an increased local heterogeneity. We find a correlation between active site dynamics and water structure: in the wet hydrogel, freezing of water quenches the ensemble of soft modes linearly coupled to the Soret transition; while, in the dry hydrogel, water does not freeze and an active site dynamic behavior—similar to the non-freezing water/glycerol solution—is observed.

Spectroscopically and Kinetically Distinct Conformational Populations of Sol-Gel-encapsulated Carbonmonoxy Myoglobin: A COMPARISON WITH HEMOGLOBIN

We have used sol-gel encapsulation protocols to trap kinetically and spectroscopically distinct conformational populations of native horse carbonmonoxy myoglobin. The method allows for direct comparison of functional and spectroscopic properties of equilibrium and non-equilibrium populations under the same temperature and viscosity conditions. The results implicate tertiary structure changes that include the proximal heme environment in the mechanism for population-specific differences in the observed rebinding kinetics. Differences in the resonance Raman frequency of nu(Fe-His), the iron-proximal histidine stretching mode, are attributed to differences in the positioning of the F helix. For myoglobin, the degree of separation between the F helix and the heme is assigned as the conformational coordinate that modulates both this frequency and the innermost barrier controlling CO rebinding. A comparison with the behavior of encapsulated derivatives of human adult hemoglobin indicates that these CO binding-induced conformational changes are qualitatively similar to the tertiary changes that occur within both the R and T quaternary states. Protein-specific differences in the time scale for the proposed F helix relaxation are attributed to variations in the intra-helical hydrogen bonding patterns that help stabilize the position of the F helix.

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.

Molecular confinement influences protein structure and enhances thermal protein stability.

The sol-gel method of encapsulating proteins in a silica matrix was investigated as a potential experimental system for testing the effects of molecular confinement on the structure and stability of proteins. We demonstrate that silica entrapment (1) is fully compatible with structure analysis by circular dichroism, (2) allows conformational studies in contact with solvents that would otherwise promote aggregation in solution, and (3) generally enhances thermal protein stability. Lysozyme, alpha-lactalbumin, and metmyoglobin retained native-like solution structures following sol-gel encapsulation, but apomyoglobin was found to be largely unfolded within the silica matrix under control buffer conditions. The secondary structure of encapsulated apomyoglobin was unaltered by changes in pH and ionic strength of KCl. Intriguingly, the addition of other neutral salts resulted in an increase in the alpha-helical content of encapsulated apomyoglobin in accordance with the Hofmeister ion series. We hypothesize that protein conformation is influenced directly by the properties of confined water in the pores of the silica. Further work is needed to differentiate the steric effects of the silica matrix from the solvent effects of confined water on protein structure and to determine the extent to which this experimental system mimics the effects of crowding and confinement on the function of macromolecules in vivo.

Sol–gel encapsulation of bacteria: a comparison between alkoxide and aqueous routes

The viability of bacteria in the presence of sol–gel reagents has been studied in order to define the best experimental conditions for the sol–gel encapsulation of E. coli. The β-galactosidase activity of these bacteria, trapped in sol–gel silica matrices, was then analyzed. Two routes, using alkoxide and aqueous precursors, have been used and compared. It appears that the aqueous route is less damaging than the alkoxide one. Moreover the aqueous silica matrix appears to slow down the lysis of cell membranes when bacteria are aged without nutrient.

Sol-Gel Optical Sensors for Glutamate

AbstractThe amino acid glutamate is the major excitatory neurotransmitter used in the nervous system for interneuronal communication. It is used throughout the brain by various neuronal pathways including those involved in learning and memory, locomotion, and sensory perception. Because glutamate is released from neurons on a millisecond time scale into sub-micrometer spaces, the development of a glutamate biosensor with high temporal and spatial resolution is of great interest for the study of neurological function and disease. Here, we demonstrate the feasibility of an optical glutamate sensor based on the sol-gel encapsulation of the enzyme glutamate dehydrogenase (GDH). GDH catalyses the oxidative deamination of glutamate and the reduction of NAD+ to NADH. NADH fluorescence is the basis of the sensor detection. Thermodynamic and kinetic studies show that GDH remains active in the sol-gel matrix and that the reaction rate is correlated to the glutamate concentration.

Sol-Gel Entrapment of Escherichia coli

Whole cell bacteria have been entrapped within sol-gel silica matrices in order to perform bio-catalytic experiments. Escherichia coli have been chosen as a model for sol-gel encapsulation. Transmission electron microscopy shows that bacteria are randomly dispersed within the silica matrix and that their cellular organization is preserved. The β-galactosidase activity of entrapped E. coli was studied using p-NPG as a substrate. The formation of p-nitrophenol was followed by optical absorption. These experiments show that E. coli still exhibit noticeable enzymatic activity after encapsulation in wet gels. They follow the well known Michaelis-Menten kinetic law but their activity decreases in dried xerogels.

UV Resonance Raman Spectra of Ligand Binding Intermediates of Sol-Gel Encapsulated Hemoglobin

We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an alpha14beta15 Trp W3 band difference at 1559 cm(-1). The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm(-1)) UVRR difference bands suggest conformational shifts for the penultimate Tyralpha140 on the F helix, the "switch" region Tyralpha42, and the "hinge" region Trpbeta37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.

Interactions Between sol-gel Encapsulated Myoglobin and Carbon Dioxide in Supercritical CO2

The development of an isolated reaction vessel suitable for use with supercritical CO2 is an important step towards conducting valid spectroscopic experiments because contamination from the high-pressure vessel is avoided. This is crucial when biomolecules are the subject of investigations due to the ease with which contaminants perturb their reactivity. The combination of sol-gel encapsulation of biologically active molecules with supercritical fluid spectroscopy provides an experimental method for studying CO2 interactions previously considered inaccessible. The molecules retain their biological activity even under conditions required to maintain die CO2 in the supercritical state.

Environmental Effect of Sol-Gel Encapsulation on Photochromic andThermocliromic Anils

Photochromic and thermochromicanus have been encapsulated in an organic-inorganichybrid network, produced by theSol-Gel method. It was found that the photochromic anils have been transformed to thermochromic, something observed for the first time in rigid glasses. Spectroscopic investigations indicate a common infrastructure responsible for the coloured xerogel.

Silica sol-gel encapsulation of pancreatic islets.

We developed a biocomposite material containing silica ceramic. The sol-gel technology in which ceramic materials are polymerized from liquid solutions at room temperature and physiologic pH can be used to produce ceramics that have a determined pore size and that contain living organisms or cells. Capsules were stable to extreme acid and base conditions as well as to trypsin in vitro for 6 months. We used insulin-secreting murine islet cells as the first mammalian material for encapsulation. Two approaches to generating successful encapsulation of islets were used: drop-tower sphere generation and emulsion. Sphere diameters of less than 1 mm were associated with positive insulin secretory capacity as documented by a static batch incubation technique. Average pore sizes were 161 A for drop-tower spheres and 105 A for emulsion spheres. Capsules allowed the passage of insulin and cytokines but not the passage of antibody. Implantation of encapsulated islets did not result in fibrosis of the capsule in vivo, and retrieval of capsules after 1 month in vivo documented continued insulin secretory capacity. Further in vivo experiments documented increased survival of transplant recipients despite failure to achieve normoglycemia in all but a few cases. Silica sol-gel encapsulation provides a potentially useful alternative for encapsulation of cells for transplantation or drug delivery, and further work is warranted to develop this potentially useful approach for the treatment of diabetes mellitus.

A sol–gel based fiber optic sensor for local blood pH measurements

This paper describes a fiber optic pH sensor based upon sol–gel encapsulation of a self-referencing dye, seminaphthorhodamine-1 carboxylate (SNARF-1C). The simple sol–gel fabrication procedure is ideal for encapsulation and immobilization of dye molecules onto the end of an optical fiber. A miniature bench-top fluorimeter system was developed for use with the optical fiber to obtain pH measurements. Linear and reproducible responses were obtained in human blood in the pH range 6.8–8.0, which encompasses the clinically-relevant range. The sensor was also shown to have low coating leachability. This sensor will be used as part of an array of sensors being developed to monitor stroke patients.

Biogels of cytochrome c : Cytochrome c peroxidase complex studied by electron paramagnetic resonance spectroscopy

Cytochrome c: cytochrome c peroxidase (Cc: CcP in 1: 1 ratio) complex was successfully encapsulated in sol-gel derived glass. The electron paramagnetic resonance (e.p.r.) and optical absorption techniques were used to characterize the coordination number, spin state, charge-transfer activity and structural orientation of Cc: CcP complex and its constituents. The sol-gel encapsulation of metalloproteins allows, for the first time, the detection of e.p.r. signals of biological systems at room temperature. CcP exhibits an e.p.r. spectrum representing the high spin and purely axial symmetry with parameters at g⊥ ≅ 6 and g∥ ≅ 2 and an electronic absorption spectrum with a descent in spectral intensity of shoulder band at 380 nm and a blue-shifted charge-transfer band at 620 nm. Cc shows an e.p.r. spectrum characterizing a mixture of high spin (g⊥ ≅ 6 and g∥ ≅ 2) and low spin (gx=2.7, gy=2.2 and gz=1.8) components. Upon complexation, Cc:CcP pair displays a single and broad e.p.r. spectrum at g∥ ≅ 2 and a light absorption spectrum with a red-shifted Soret band at 423 nm, a blue-shifted charge-transfer band at 620 nm and an intensified charge-transfer band at 507 nm. These results suggest that the sol-gel encapsulated Cc:CcP complex has the following chemical and physical characteristics: (a) a hexa-coordination, (b) a high-spin state, (c) an active charge-transfer (or redox) pair, and (d) the direction of the g∥ paramagnetic center of Cc : CcP complex lies nearly parallel to that of the heme normal. The structural coordinations of the sol-gel encapsulated Cc, CcP and Cc : CcP are examined. Moreover, the possible use of biogels at the sol, gelation, and xerogel stages during gel processing to control the structural rigidity and spatial separation/orientation of the encapsulated heme proteins and to study their possible routes of long-range electron transfer reactions are also discussed.

Encapsulation of the ferritin protein in sol-gel derived silica glasses

A significant recent development in sol-gel science has been the encapsulation of biomolecules such as proteins and enzymes in optically transparent silica glasses. This paper reports on the encapsulation of an iron (Fe) storage protein, ferritin, to develop a magnetic silica glass. Native ferritin, which has a nanometer-sized microcrystalline Fe oxide core, was encapsulated in optically transparent silica glasses using the sol-gel process. Fe could be released from ferritin but could not be reconstituted into apoferritin when the protein was trapped in the pores of the glass. Transmission electron microscopy of ferritin-doped aged silica gels indicated that crystallinity of the Fe oxide core was retained upon sol-gel encapsulation. Magnetic measurements on ferritin-doped silica gels indicated the material to be paramagnetic, but not superparamagnetic.

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.

Sol–gel encapsulation of metalloproteins for the development of optical biosensors for nitrogen monoxide and carbon monoxide

Three heme proteins; horse heart cytochrome c, myoglobin (Mb) and hemoglobin (Hb), have been encapsulated in a sol–gel matrix and investigated for their potential use as biological recognition centres for the biosensing of NO and CO. It has been shown that the encapsulation of these metalloproteins in sol–gels has no effect on the formation of nitrosyl or carbonyl complexes as observed using UV/VIS spectrophotometry. The metalloproteins in sol–gels could be readily reduced to the FeII state, using excess sodium dithionite, and re-oxidized to the FeIII state on the addition of excess potassium ferricyanide. The observed redox capability within sol–gels enabled formation of the nitrosyl complex in both the FeIII and FeII states of Mb and Hb and in the FeIII state of cytochrome c. The formation of these nitrosyl complexes was shown to be reversible. Similarly, the reversible formation of the carbonyl complex with Mb and Hb in the reduced form has been achieved. Titration of a saturated solution of CO with Hb contained in a sol–gel showed a linear increase in intensity of the α(568 nm) and β(540 nm) absorption bands of the metalloprotein until saturation of the iron centres of the Hb occurred. A similar increase in intensity of the α(562 nm) absorption band of cytochrome c was observed when a saturated solution of NO was titrated against this metalloprotein contained in a sol–gel. The results show that sol–gels can be readily used as a host for the encapsulation of cytochrome c, Mb and Hb and that these metalloproteins can be used as the biological recognition centre for the biosensing of NO and CO at the micromolar level.