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.     

Mixed Pyridine-phenol Boron Complex Encapsulated in Polymer/Silica Hybrid Sol-gel Matrix

A novel pyridine-phenol boron complex[（dppy）BF] was encapsulated into polymer/silica composite matrix by sol-gel process. UV-Vis absorption spectra show that this process can control the aggregation structure of complex （dppy）BF. The results of photoluminescence of （dppy）BF in sol-gel composite film indicate that both fluorescence intensity and photostability are markedly increased using this method compared with other methods, which increases the practical significance of such composite film.     

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.     

Preparation, structure, and imaging of luminescent SiO2 nanoparticles by covalently grafting surfactant-encapsulated europium-substituted polyoxometalates

A novel route to the preparation of luminescent silica nanoparticles and coloration for living cells was demonstrated in this article. A europium-substituted polyoxometalate was encapsulated by a hydroxyl-group-terminated double-chain quaternary ammonium cation through an ion replacement process, yielding an organic-inorganic complex with core-shell structure bearing hydroxyl groups located at the periphery. The introduction of -OH groups not only increased the solubility of the complex in polar solvents but also caused it to embed into the inner matrix of silica nanoparticles covalently and be well-dispersed through an in situ sol-gel reaction with tetraethyl orthosilicate. Elemental analysis and spectral characterization confirmed the formation of prepared complexes with the anticipated chemical composition. Scanning and transmission electron microscopy images illustrated the size change of luminescent nanoparticles with smooth surfaces and well-dispersed polyoxometalate complexes inside of the silica matrix. X-ray photonic spectra and ζ-potential measurements revealed the chemical association between the silica matrix and the complex. Luminescent spectral characterization indicated the well-retained photophysical property of Eu-substituted polyoxometalate in silica nanoparticles. The surface amino-modified silica nanoparticles were applied to cell coloration, and the dyed Hela cells were observed through laser confocal fluorescence microscopy.     

界面活化的溶胶凝胶包埋Candida rugosa脂肪酶催化合成维生素E琥珀酸酯

采用界面活化的溶胶凝胶包埋Candida rugosa脂肪酶(CRL)催化合成了维生素E琥珀酸酯.考察了影响溶胶凝胶包埋固定化CRL的因素,获得的最佳固定化条件为:丙基三甲氧基硅烷/正硅酸四乙酯摩尔比为1/1,水与硅烷前体摩尔比为15,酶的添加量为0.5mg/ml,PEG400的添加量为12μl/ml溶胶. 溶胶凝胶包埋的CRL在50℃,18h后其活性仍然保持了70.58%,是游离酶的2.6倍,且稳定性得到了明显的改善.基于CRL的界面特性,采用五种表面活性剂对其进行界面活化.结果表明,采用橄榄油活化的溶胶凝胶包埋的CRL合成维生素E琥珀酸酯的酯化活力最高,相比原酶和未界面活化的溶胶凝胶包埋酶分别提高了6.7和1.43倍.     

Multiple fluorescence spectra and stable encapsulation of 7-hydroxy-4-methylcoumarin and rhodamine 6g in silicate glasses by the sol-gel method

7-Hydroxy-4-methylcoumarin(4-methylumbelliferone, HMC) and Rhodamine 6G(R6G) were encapsulated into silicate polymeric glass prepared by the sol-gel method under acidic, basic, and neutral conditions from tetraethyl orthosilicate. The fluorescence spectra of these molecules encapsulated into the xerogel state depend on the used catalysts. Three types of fluorescence emissions having peak wavelengths of ca. 390 nm, 470 nm, and 550 run, respectively, were observed simultaneously in the xerogel state composed of HMC and R6G which were prepared by acid catalysts. The encapsulated HMC remains stable for more than one year in the prepared xerogel. The results open the way to the development of simultaneous three-band laser emissions. The observation of the fluorescence spectrum of HMC is useful for a molecular level photophysical probe elucidating the structural changes oftetraethyl orthosilicate during sol to gel to xerogel transitions.     

Multiple fluorescence spectra of fluorescein molecules encapsulated in the silica xerogel prepared by the sol-gel reaction

The fluorescence and fluorescence-excitation spectra of fluorescein molecules during the sol-gel-xerogel transitions of tetraethyl orthosilicate (TEOS) solutions have been observed as functions of doped concentration, in the order of 10^-2 and 10^-5 mol dm^-3, and of the sol-gel-xerogel transition time. It was shown that with a lower initial concentration only the cation was encapsulated in the pores of the silica xerogel state during the sol-gel reaction processes. On the other hand, with a high concentration four species, i.e., cation, monoanion, dianion, and dimer, were encapsulated in the silica xerogel during the sol-gel reaction processes. These results have the potential to open the way to the development of simultaneous multiple-band solid-state laser emitting materials.     

Fluorescence study of the fluidity and cooperativity of the phase transitions of zwitterionic and anionic liposomes confined in sol-gel glasses

The current work makes use of different fluorescent reporter molecules and fluorescent spectroscopic techniques to characterize the thermotropic, physical, and dynamical properties of large unilamellar liposomes formed from either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol] (DMPG) encapsulated in sol-gel matrixes. In particular, cooperativity of the phase transition is analyzed from steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), the interfacial properties are studied by measuring the spectral shift of Laurdan, and the structural organization (heterogeneity) of the lipid bilayer is determined from the fluorescence lifetime of trans-parinaric acid (t-PnA). In addition, information regarding order and dynamical properties in the bulk hydrophobic core is obtained from time-resolved fluorescence anisotropy of t-PnA and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)-phenylpropionic acid (PA-DPH). The spectroscopic study reveals that upon encapsulation, the basic thermodynamic properties as well as the fluidity of the lipid bilayer practically remain intact for DMPG liposomes but not for DMPC liposomes, whose lipid bilayer exhibits large gel-fluid heterogeneity. On the basis of these experimental results, electrostatic interactions between phospholipid polar heads and the porous surface of the host matrix seem to play a capital role for the preservation of the structural integrity of encapsulated bilayer.     

Structure and dynamics of lysozyme encapsulated in a silica sol-gel matrix

Proteins entrapped in sol-gel matrices have been extensively studied during the last 15 years, showing that most of them can be encapsulated with retention of their native structure and functionality and with enhanced stability. However, relatively little is known about the structural and dynamical details of the biomolecule-matrix interactions. To achieve this goal, the model protein hen egg white lysozyme (HEWL) has been entrapped in sol-gel matrices prepared from tetraethyl orthosilicate through an alcohol-free sol-gel route, and the photophysical properties of its fluorescent tryptophans have been determined using both steady-state and time-resolved fluorescence techniques. By combining fluorescence spectra, quenching experiments, lifetimes, and time-resolved fluorescence anisotropy measurements, we have obtained information on the structure, dynamics, and solvation properties of the entrapped protein. Our results show that the environment of HEWL within the silica pore as well as its internal dynamics is similar to that in aqueous solution, except that the protein showed no or, depending on conditions, very much slower global motion but retained its internal angularly restricted (hindered) segmental rotation upon entrapment. The experiments carried out at different experimental conditions indicate that, below the isoelectric point of the protein, a strong electrostatic interaction is established between the protein molecule and the negatively charged sol-gel walls, which is ultimately responsible for the total arrest of the overall rotation of the protein, but without significant effect upon its segmental rotational relaxation. The electrostatic nature of the interaction is clearly established since either reducing the positive charge of the protein (by increasing the pH toward its isoelectric point) or increasing the ionic strength of the solution (shielding against the attractive interaction) leads to a situation in which the protein freely rotates within the matrix pore, albeit an order of magnitude more slowly than that in free solution under similar macroscopic solution conditions, and still retains its segmental rotational properties.     

溶胶-凝胶法制备的二氧化硅中肌红蛋白的苯胺羟化酶活性

用溶胶-凝胶制备了多孔透明的二氧化硅包埋肌红蛋白，在还原辅酶Ⅰ二钠盐(NADH)和黄素单核苷酸(FMN)的存在下，用可见光谱法测定了肌红蛋白苯胺羟基化反应的K_m和k_cat。表明包埋在二氧化硅中的肌红蛋白保持了苯胺羟化酶活性。     

Heme reactivity is uncoupled from quaternary structure in gel-encapsulated hemoglobin: a resonance Raman spectroscopic study

Encapsulation of hemoglobin (Hb) in silica gel preserves structure and function but greatly slows protein motion, thereby providing access to intermediates along the allosteric pathway that are inaccessible in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet laser excitation provides probes of heme reactivity and of key tertiary and quaternary contacts. These probes were monitored in gels after deoxygenation of oxyHb and after CO binding to deoxyHb, which initiate conformational change in the R-T and T-R directions, respectively. The spectra establish that quaternary structure change in the gel takes a week or more but that the evolution of heme reactivity, as monitored by the Fe-histidine stretching vibration, ν(FeHis), is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving ν(FeHis) frequencies span the full range of values between those previously associated with the high- and low-affinity end states, R and T. This result supports the tertiary two-state (TTS) model, in which the Hb subunits can adopt high- and low-affinity tertiary structures, r and t, within each quaternary state. The spectra also reveal different tertiary pathways, involving the breaking and reformation of E and F interhelical contacts in the R-T direction but not the T-R direction. In the latter, tertiary motions are restricted by the T quaternary contacts.     

Encapsulation of Bromothymol Blue into a Polysiloxane Network Matrix in Presence of Surfactants

Bromothymol blue (BTB) pH indicator was encapsulated into polysiloxane network using the sol-gel process. Transparent monolithic disks of entrapped BTB were obtained in the presence of cetyl trimethyl ammonium bromide (CTAB) or dodecyl dimethyl amino oxide (Genaminox LA, GLA) surfactant. Ultraviolet visible spectra showed that the encapsulated BTB retains its structure in terms of its response to pH. The kinetic studies reveal that there was a faster response to pH in the case of GLA surfactant than in the case of CTAB. It is found that the co-entrapment of cationic surfactant CTAB or zwitterionic surfactant GLA with TEOS has shifted the equilibrium of the BTB to the ionized form due to the electrostatic interaction between the surfactant and the BTB anion and therefore the base form becomes more pronounced (e.g., shift from yellow to red) than that of sol-gel matrix without surfactant.     

荧光光谱跟踪结冷胶水溶液的溶液-凝胶转变

将异硫氰酸荧光黄(FITC)标记在结冷胶分子链,用荧光光谱跟踪了结冷胶水溶液凝胶化过程中FITC荧光强度和各向异性比的变化.结果表明在结冷胶的凝胶化转变中,FITC的荧光相对强度和各向异性随温度降低而增大,在某一温度荧光相对强度和各向异性比对温度的曲线出现了明显的转折点,这个转折点的温度低于流变温度扫描曲线中G′=G″的温度.利用荧光的方法确定物理交联体系的关于重均聚合度和凝胶分数的相关临界指数γ和β.γ和β不符合Flory-Stockmayer和逾渗模型的预测.     

A new mesoporous micelle-templated silica route for enzyme encapsulation

A new mesoporous micelle-templated silica (MTS) route for enzyme encapsulation is presented. The pore structure is given by a new association oflecithin (double chain surfactant) and dodecylamine as cosurfactant. To enhance and to well protect the enzyme activity, lactose was loaded in the synthesis. The mixed-micelles give after the addition of tetraethyl orthosilicate a well-ordered mesoporous material with a spongelike rigid structure stable after calcination at 550 degrees C. The size of the pores lies between 30 and 40 A, matching well with the size of the lipases. The activity of this heterogeneous catalyst was tested in the hydrolysis of the ethylthiodecanoate. These new biocatalysts were very active, more than hydrophobic sol-gel materials and commercially available sol-gel encapsulated lipase. This new MTS synthesis route allows one to encapsulate in one-step various enzymes, even those that are very fragile.     

Allosteric Regulation of Enzymatic Reactions in a Transparent Inorganic Sol-Gel Material

Glutamate dehydrogenase is encapsulated in a transparent porous silicate matrix by using sol-gel techniques. The inorganic polymer is formed around the enzyme (MW > 300,000 D). The enzyme is active in the material, catalyzes the reaction of L-glutamate to 2-oxoglutarate and follows Michaelis-Menten kinetics. The allosteric regulators ADP and GTP inhibit or activate the reaction; at pH 6, GTP acts as a strong activator and ADP acts as an inhibitor. This system involves a complex series of interactions; the co-enzyme NAD⁺ is required for catalysis, large-scale conformational changes accompany the binding of the substrate and coenzyme to the enzyme, the activators/inhibitors must bind to the enzyme to regulate the reactions, and the substrates and products must diffuse through the matrix to and from the binding site. The influence of the unique matrix on the complex enzymatic system is discussed.     

Peralkylated-beta-cyclodextrin used as gas chromatographic stationary phase prepared by sol-gel technology for capillary column

For the first time, three peralkylated-beta-cyclodextrins (beta-CD), permethylated-beta-CD, perethylated-beta-CD and perpentylated-beta-CD, were coated onto the fused-silica capillary by sol-gel method with simplicity and rapidity. Multiple steps in conventional column preparation technology were avoided. Also, these new columns demonstrated many inherent advantages, the main being the outstanding thermal stability (up to 300 degrees C), high number of theoretical plates, excellent column-to-column and run-to-run reproducibility, and pronounced selectivity for positional isomers and enantiomers. Using n-tridecane as a test reagent (k = 4.18), an efficiency value of 3520 theoretical plates/m was obtained on a sol-gel perpentylated-beta-CD capillary column (15 m x 0.25 mm I.D.). On the basis of the results, we proposed that the peralkylated-beta-CD, which has no terminal hydroxyl group, is encapsulated in the sol-gel network and the whole matrix is chemically bonded to the surface of the fused-silica tubing.     

Manipulating formation and drug-release behavior of new sol-gel silica matrix by hydroxypropyl guar gum

To develop biocompatible sol-gel silica matrix for the encapsulation of biomolecules or drugs, a novel water-soluble silica precursor, tetrakis(2-hydroxyethyl)orthosilicates (THEOS), was used in combination with a water-soluble polysaccharide derivative, hydroxypropyl guar gum (HPGG). We found that the introduction of HPGG could trigger and accelerate the sol-gel transition of THEOS in water and induce rapid formation of homogeneous gel matrix without the addition of any organic solvents or catalysts. Moreover, added HPGG macromolecules had a great influence on the network structure and particle dimension in the silica gel matrix, as confirmed by scanning electron microscope (SEM) observation. From the time sweep rheological measurements, it was found that a higher HPGG amount could lead to shorter gelation time for the sol-gel transition. From the strain and frequency sweep rheological experiments, it was found that the resultant silica matrix containing a higher amount of HPGG exhibited a narrower linear viscoelastic region, a higher dynamic muduli, and greater complex viscosity. In particular, the gel strength of the silica matrix could be modulated by the amount of HPGG. By investigating the controlled release of vitamin B12 from the sol-gel silica matrixes, a strong dependence of the release profile on the amount of introduced HPGG was observed. In this case, a higher HPGG amount resulted in lower release rate.     

Electrochemical and spectroscopic characterization of surface sol-gel processes

(3-Mercaptopropyl)trimethoxysilane (MTS) forms a unique film on a platinum substrate by self-assembly and sol-gel cross-linking. The gelating and drying states of the self-assembled MTS sol-gel films were probed by use of electrochemical and spectroscopic methods. The thiol moiety was the only active group within the sol-gel network. Gold nanoparticles were employed to detect the availability of the thiol group and their interaction further indicated the physicochemical states of the sol-gel inner structure. It was found that the thiol groups in the open porous MTS aerogel matrix were accessible to the gold nanoparticles while thiol groups in the compact MTS xerogel network were not accessible to the gold nanoparticles. The characteristics of the sol-gel matrix change with time because of its own irreversible gelating and drying process. The present work provides direct evidence of gold nanoparticle binding with thiol groups within the sol-gel structures and explains the different permeability of "aerogel" and "xerogel" films of MTS on the basis of electrochemical and spectroscopic results. Two endogenous species, hydrogen peroxide and ascorbic acid, were used to test the permeability of the self-assembled sol-gel film in different states. The MTS xerogel film on the platinum electrode was extremely selective against ascorbic acid while maintaining high sensitivity to hydrogen peroxide in contrast to the relatively high permeability of ascorbic acid in the MTS aerogel film. This study showed the potential of the MTS sol-gel film as a nanoporous material in biosensor development.     

Ligand-induced tertiary relaxations during the T-to-R quaternary transition in hemoglobin

When human hemoglobin is encapsulated in nanoporous silica gels, tertiary and quaternary structural relaxations are dramatically slowed down, allowing the characterization of elusive reaction intermediates. In this work, the conformational and functional changes triggered by CO binding to human deoxyhemoglobin gels were determined in the absence and presence of allosteric effectors. CO rebinding kinetics to human deoxyhemoglobin gels after nanosecond laser photolysis were monitored as a function of time after CO saturation. A maximum entropy analysis of the CO rebinding kinetics shows that the T conformation slowly evolves toward R, with an associated redistribution of tertiary species. The tertiary species are characterized by different CO rebinding rates which are essentially unaffected by the protein quaternary state.     

溶胶-凝胶生物包囊化物的制备与特性

溶胶-凝胶法以其高效、简便、通用性强等特点而越来越广泛地用于生物分子如蛋白质、酶、DNA、RNA、抗体以及细胞等的固定化,所得生物活性材料在生物催化、生物传感、生物医学诊断等领域具有良好的应用前景.本文主要介绍溶胶-凝胶法包埋生物分子的基本过程,包埋后生物分子在凝胶基质中的构象变化和运动状态,被分析物或底物与生物分子活性位点的接触情况以及生物分子的活性和稳定性等.最后,对溶胶-凝胶生物包囊化的发展趋势进行了简单分析。