Preparation of sandwich-structured graphene/mesoporous silica composites with C8-modified pore wall for highly efficient selective enrichment of endogenous peptides for mass spectrometry analysis

In this study, sandwich-structured graphene/mesoporous silica composites (C8-modified graphene@mSiO(2) ) were synthesized by coating mesoporous silica onto hydrophilic graphene nanosheets through a surfactant-mediated cocondensation sol-gel process. The newly prepared C8-modified graphene@mSiO(2) nanocomposites possess unique properties of extended plate-like morphology, good water dispersibility, highly open pore structure, uniform pore size (2.8 nm), high surface area (632 m(2) /g), and C8-modified-interior pore walls. The unique structure of the C8-modified graphene@mSiO(2) composite nanosheets not only provide extended planes with hydrophilic surface that prevents aggregation in solution, but also offer a huge number of C8-modified mesopores with high surface area that can ensure an efficient adsorption of peptides through hydrophobic-hydrophobic interaction between C8-moified pore walls and target molecules. The obtained C8-modified graphene@mSiO(2) materials were utilized for size selectively and specifically enriching peptides in standard peptide mixtures and endogenous peptides in real biological samples (mouse brain tissue).     

Entrapment of biomolecules in sol-gel matrix for applications in biosensors: problems and future prospects

An emerging area that has attracted increased attention in recent years is the development of biosensors based on sol-gel-derived platforms which must be predicated on an understanding of the short and long-term interactions between the biorecognition elements and evolving sol-gel matrix. This review focuses on the growing field of entrapment of biomolecules such as proteins, enzymes and antibodies in sol-gel matrices prepared from alkoxide precursors. Basic aspects of sol-gel, its advantages and disadvantages, factor affecting the sol-gel-derived thin films, strategies for improving entrapment of biomolecules in sol-gel materials and their organic modifications are discussed. Organically modified silane precursors have the ability to tune physical and chemical properties with desired characteristics of sol-gel preparations by simply changing different precursors and their molar ratio. The usefulness of optical method especially time-resolved fluorescence spectroscopy for the characterization of internal environment of sol-gel as well as dynamics of proteins within the sol-gel is highlighted. Significance and designing of new biocompatible sol-gel precursors with the purpose of making the glassy matrix more compatible with entrapped biomolecules has been described. Considerable attention has been drawn on problems and future prospects of sol-gel matrix for entrapment of biomolecules for applications in biosensors.     

Biosorption of gold by immobilized fungal biomass

The characteristics of polyvinyl alcohol (PVA) and calcium alginate as immobilization matrices were examined and compared for the uptake of gold by a fungal biomass. PVA-immobilized biomass showed superior mechanical strength and chemical stability. In addition, PVA beads were also stable under a wider range of pH (1-13). The lower mass transfer resistance in PVA beads was evident from kinetic studies which showed a significantly shorter period of time for the immobilized PVA beads to achieve 80% gold removal as compared with immobilized alginate beads. Calculated rate constants and maximum rates for the uptake of gold by both immobilized PVA and immobilized alginate biosorbent revealed a much more rapid uptake phenomenon by the former. BET analyses also indicated a larger surface area and larger pore size distribution in PVA beads, further indicating a lower resistance to mass transfer. Gold biosorption in the immobilized PVA bead could be modeled by both the Langmuir and Freundlich adsorption isotherms.     

Sol-gel powders and supported sol-gel polymers for immobilization of lipase in ester synthesis

Candida rugosa lipase was entrapped in hybrid organic–inorganic sol-gel powder prepared by acid-catalyzed polymerization of tetramethoxysilane (TMOS) and alkyltrimethoxysilanes, and used in catalyzing esterification reactions between ethanol and butyric acid in hexane. Optimum preparation conditions were studied, which are gels made from propyltrimethoxysilane (PTMS)/TMOS molar ratio=4:1, hydrolysis time of silane precursor=30 min, water/silane molar ratio=24, enzyme loading=6.25% (w/w) of gel, and 1 mg PVA/mg lipase. The percentage of protein immobilization was 95% and the resulting lipase specific activity was 59 times higher than that of a non-immobilized lyophilized lipase. To prepare magnetic lipase-immobilized sol-gel powder (MLSP) for easier recovery of the biocatalyst, Fe₃O₄ nanoparticles were prepared and co-entrapped with lipase during gel formation. This procedure induced surface morphological change of the sol-gel powder and showed adverse effect on enzyme activity. Hence, although only 9% decrease in protein immobilization efficiency was observed, the corresponding reduction in enzyme activity could be up to 45% when sol-gel powder was doped with 25% (v/v) Fe₃O₄ magnetic nanoparticles solution. Lipase-immobilized sol-gel polymer was also formed within the pores of different porous supports to improve its mechanical stability. Non-woven fabric, with a medium pore size of all the supports tested, was found to be the best support for this purpose. The thermal stability of lipase increased 55-fold upon entrapment in sol-gel materials. The half-lives of all forms of sol-gel-immobilized lipase were 4 months at 40 °C in hexane.     

Spectroscopic characterization of the oxyferrous complex of prostacyclin synthase in solution and in trapped sol-gel matrix

Prostacyclin synthase (PGIS) is a member of the cytochrome P450 family in which the oxyferrous complexes are generally labile in the absence of substrate. At 4 degrees C, the on-rate constants and off-rate constants of oxygen binding to PGIS in solution are 5.9 x 10(5) m(-1).s(-1) and 29 s(-1), respectively. The oxyferrous complex decays to a ferric form at a rate of 12 s(-1). We report, for the first time, a stable oxyferrous complex of PGIS in a transparent sol-gel monolith. The encapsulated ferric PGIS retained the same spectroscopic features as in solution. The binding capabilities of the encapsulated PGIS were demonstrated by spectral changes upon the addition of O-based, N-based and C-based ligands. The peroxidase activity of PGIS in sol-gel was three orders of magnitude slower than that in solution owing to the restricted diffusion of the substrate in sol-gel. The oxyferrous complex in sol-gel was observable for 24 h at room temperature and displayed a much red-shifted Soret peak. Stabilization of the ferrous-carbon monoxide complex in sol-gel was observed as an enrichment of the 450-nm species over the 420-nm species. This result suggests that the sol-gel method may be applied to other P450s to generate a stable intermediate in the di-oxygen activation.     

Mediator-free phenol sensor based on titania sol-gel encapsulation matrix for immobilization of tyrosinase by a vapor deposition method

A novel amperometric phenol sensor was constructed by immobilizing tyrosinase in a titania sol-gel matrix. The tyrosinase entrapped sol-gel film was obtained with a vapor deposition method, which simplified the traditional sol-gel process and avoided the shrinkage and cracking of conventional sol-gel-derived glasses. This matrix provided a microenvironment for retaining the native structure and activity of the entrapped enzyme and a very low mass transport barrier to the enzyme substrates. Phenol could be oxidized by dissolving oxygen in presence of immobilized tyrosinase to form a detectable product, which was determined at -150 mV without any mediator. The phenol sensor exhibited a fast response (less than 5 s) and sensitivity as high as 103 microA/mM, which resulted from the porous structure and high enzyme loading of the sol-gel matrix. The linear range for phenol determination was from 1.2x10(-7) to 2.6x10(-4) M with a detection limit of 1.0x10(-7) M. The apparent Michaelis-Menten constant of the encapsulated tyrosinase was calculated to be (0.29+/-0.02) mM. The stability of the biosensor was also evaluated.     

Flow injection fluorescence immunoassay for gentamicin using sol-gel-derived mesoporous biomaterial

Sol-gel-derived mesoporous biomaterials were used for the first time in the flow-injection fluorescence immunoassay system. Anti-gentamicin antibody was immobilized in a mesoporous sol-gel material using tetramethoxysilane as a precursor and poly(ethylene glycol) as a template. The sol-gel glass was used to develop an immunoaffinity column for the flow-injection immunoassay of gentamicin. Little unspecific adsorption of gentamicin on the sol-gel and no antibody leaching under harsh elution conditions were found. The immunoassay is based on the competition between gentamicin and fluorescein isothiocyanate-labeled gentamicin for a limited number of encapsulated antibody binding sites. NaOH solution of 5 x 10(-3)mol/L is used for the regeneration of encapsulated antibody binding sites after each measurement, which allows the immunoreactor to be used for up to 20 times without any loss of reactivity. Sample preconcentration is not needed and a single assay can be performed within 10 min. The calibration for gentamicin has a working range of 250-5000 ng/mL with a detection limit of 200 ng/mL, which is close to that of the fluorescence immunoassay and fluorescence polarization immunoassay using the same reactants. Comparison of the results from this method with that obtained from HPLC showed an excellent correlation.     

Preparation of crosslinked macroporous PVA foam carrier for immobilization of microorganisms

In this work, a new and economical way to prepare macroporous poly(vinyl alcohol) (PVA) foam was explored by adding calcium carbonate as a pore-forming agent and using epichlorhydrin as a chemical crosslinking agent to improve foam stability. The mixture for foam formation has been optimized to obtain macroporous PVA foam carriers with uniform apertures, narrow distribution of pore sizes, and good elasticity. The crosslinked PVA foam (CPVAF) carrier demonstrated better chemical and thermal stability, as well as larger specific surface area and diffusion coefficients than the traditional PVA (TPVA) carrier. Nitrifying bacteria were used to test the suitability of CPVAF and TPVA carriers for immobilized microorganisms. CPVAF carriers supported higher biomass density and microbial activity than TPVA carriers. At the same biomass density, the higher nitrification rate of CPVAF carriers was attributed to excellent mass transfer of the substrate (and oxygen) between the bulk solution and the immobilized microorganisms.     

Equilibrium and kinetics studies of adsorption of copper (II) on chitosan and chitosan/PVA beads

The adsorption of Cu(II) ions from aqueous solution by chitosan and chitosan/PVA beads was studied in a batch adsorption system. Chitosan solution was blended with poly(vinyl alcohol) (PVA) in order to obtain sorbents that are insoluble in aqueous acidic and basic solution. The adsorption capacities and rates of Cu(II) ions onto chitosan and chitosan/PVA beads were evaluated. The Langmuir, Freundlich and BET adsorption models were applied to describe the isotherms and isotherm constants. Adsorption isothermal data could be well interpreted by the Langmuir model. The kinetic experimental data properly correlated with the second-order kinetic model, which indicates that the chemical sorption is the rate-limiting step. The Cu(II) ions can be removed from the chitosan and chitosan/PVA beads rapidly by treatment with an aqueous EDTA solution. Results also showed that chitosan and chitosan/PVA beads are favourable adsorbers.     

Characterization of sol–gel encapsulated lipase using tetraethoxysilane as precursor

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of the precursor tetraethoxysilane (TEOS) in the presence of polyethylene glycol (PEG) as additive. The properties of silica and their derivatives with regard to mean pore diameter, specific surface area, mean pore size, weight loss upon heating (thermogravimetric analysis, TGA) and ²⁹Si and ¹³C NMR are reported. The pH optimum shifted from 7.8 to 6.7 and optimum temperature jumped from 36 to 60 °C upon enzyme encapsulation. Encapsulated lipase in presence of PEG (EN-PEG) exhibited higher stability in the range of 37–45 °C, but from 50 to 65 °C the EN-PEG was inactivated after seven cycles. Hydrolytic activity during long-term storage at room temperature decreased to 50% after 94 days. High diffusional resistance was observed for large oil concentration reducing hydrolytic effectiveness by 60% in the case of the encapsulated lipase. NMR, pore size and specific surface area data suggested an active participation of the lipase enzyme during gelling of the silica matrix. This lead to reduction of available Si–OH groups, larger pores and smaller surface area. Larger pores increase substrate diffusion that correlates well with higher hydrolytic activity of the TEOS–PEG sol–gel matrix encapsulated enzyme in comparison with other sol–gel supports.     

Screen-printed electrodes modified with glucose oxidase immobilized in hybrid organosilicon sol-gel matrix

A new polymer composition was developed based on hybrid sol-gel material for the immobilization of enzymes on the surface of screen-printed carbon electrodes modified with Prussian blue. The existing and developed methods for the glucose oxidase (GO) immobilization are compared. Highly stable bioelectrodes were shown to be obtained by enzyme immobilization on a hybrid composition consisting of solgel/poly vinyl alcohol (PVA) (up to 60 sequential chains) and agar gel (up to 45 sequential chains). The range of glucose concentrations detected during enzyme immobilization in a hybrid sol-gel/PVA composition or agar gel without dilution of a sample was 1.0–5.9 μM and 3.6–6.3 μM, respectively. An analysis of wine products was conducted. The results obtained using the proposed biosensors were shown to differ insignificantly from those obtained by high-performance liquid chromatography (the correlation coefficient was 0.9998).     

Influence of the alkyl-substituted silane precursor on sol–gel encapsulated lipase activity

Lipase from Candida rugosa was encapsulated within a chemically inert sol–gel support prepared by polycondensation of three precursor types (tetraethoxysilane (TEOS), methyltrimethoxysilane (MTMS) and polydimethylsilane (PDMS)) in the presence and absence of polyethylene glycol (PEG) and polyvinyl alcohol (PVA) as additives. Silica and their derivatives were characterised with regard to mean pore diameter, specific surface area, pore size distribution (BET method), weight loss upon heating thermogramivemetric analysis (TGA), chemical composition Fourier transform infrared spectroscopy (FT-IR), and catalytic activities. Immobilisation yields based on the recovered lipase activity vary from 3.02 to 31.98% and the highest efficiency was attained when lipase was encapsulated using TEOS in the presence of the PEG. Further information was obtained by testing the derivatives in esterification reactions and a different reactivity profile was found. Better performance was obtained with derivatives containing lipase encapsulated within gels prepared with MTMS as precursor in the presence of PEG. This lipase preparation exhibits increased esterification activity (155 μmol g⁻¹ min⁻¹), up to of three times greater than that prepared with TEOS (52 μmol g⁻¹ min⁻¹), and almost twice that prepared with MTMS/PDMS (89 μmol g⁻¹ min⁻¹) as precursors.     

Glucose biosensor prepared by glucose oxidase encapsulated sol-gel and carbon-nanotube-modified basal plane pyrolytic graphite electrode

A new glucose biosensor has been fabricated by immobilizing glucose oxidase into a sol-gel composite at the surface of a basal plane pyrolytic graphite (bppg) electrode modified with multiwall carbon nanotube. First, the bppg electrode is subjected to abrasive immobilization of carbon nanotubes by gently rubbing the electrode surface on a filter paper supporting the carbon nanotubes. Second, the electrode surface is covered with a thin film of a sol-gel composite containing encapsulated glucose oxidase. The carbon nanotubes offer excellent electrocatalytic activity toward reduction and oxidation of hydrogen peroxide liberated in the enzymatic reaction between glucose oxidase and glucose, enabling sensitive determination of glucose. The amperometric detection of glucose is carried out at 0.3 V (vs saturated calomel electrode) in 0.05 M phosphate buffer solution (pH 7.4) with linear response range of 0.2-20 mM glucose, sensitivity of 196 nA/mM, and detection limit of 50 microM (S/N=3). The response time of the electrode is < 5s when it is stored dried at 4 degrees C, the sensor showed almost no change in the analytical performance after operation for 3 weeks. The present carbon nanotube sol-gel biocomposite glucose oxidase sensor showed excellent properties for the sensitive determination of glucose with good reproducibility, remarkable stability, and rapid response and in comparison to bulk modified composite biosensors the amounts of enzyme and carbon nanotube needed for electrode fabrication are dramatically decreased.     

铜对三叶草-土壤酶系统的影响

通过盆栽实验研究了重金属Cu污染对植物(三叶草)-土壤酶(脲酶、蔗糖酶、过氧化氢酶和多酚氧化酶)系统的影响.结果表明,随着Cu浓度增加,脲酶、蔗糖酶、过氧化氢酶和多酚氧化酶活性均逐渐减小,与Cu浓度有高度相关性,蔗糖酶>多酚氧化酶>脲酶>过氧化氢酶.在处理浓度不变情况下,酶活性随时间而变化,且呈现低Cu浓度(<00 mg·kg^-1)时4种酶活性均有所上升,而Cu浓度增高(00～3 000 mg·kg^-1)时各酶活性逐渐下降的趋势.统计分析表明,在每一梯度浓度上,4种酶在Ⅰ、Ⅱ、Ⅲ组内均存在显著差异性(P<0.01),与植物受重金属Cu污染时的生长情况一致.随着Cu浓度增加,土壤pH值逐渐下降,而电导率上升;同一Cu浓度下的pH值和电导率均随时间呈缓慢上升趋势,统计分析显示,二者在Ⅰ、Ⅱ、Ⅲ组内均存在显著差异性(P<0.01).土壤pH值和电导率与4种土壤酶活性有高度相关性,多酚氧化酶>蔗糖酶>过氧化氢酶>脲酶.这4种酶同时可作为检测土壤环境质量的指标.     

Silica nanocasts of wood fibers: a study of cell-wall accessibility and structure

The porosity and the available surface area of a lignocellulosic fiber can influence the accessibility and reactivity in derivatization and modification reactions because the porous cell-wall network determines the upper size limit for molecules that can penetrate and react with the interior of the wall. To obtain information concerning the accessibility of the porous cell wall of wood fibers, surfactant-templated sol-gel mineralization has been examined. Wood and kraft pulp samples of Norway spruce were impregnated with a silica sol-gel and subsequently heated (calcined) and transformed into structured mesoporous silica. Microscopy studies (environmental scanning electron microscopy, transmission electron microsopy, TEM) on the silica casts showed that the three-dimensional architecture of the wood and pulp fiber cell wall was revealed down to the nanometer level. Image analysis of TEM micrographs of silica fragments from the never-dried pulp revealed complete infiltration of the cell-wall voids and microcavities (mean pore width 4.7 +/- 2 nm) by the sol-gel and the presence of cellulose fibrils with a width of 3.6 +/- 1 nm. Cellulose fibrils of the same width as that shown by image analysis were also identified by nitrogen adsorption measurements of the pore size distribution in the replicas.     

Combining submerged electrospray and UV photopolymerization for production of synthetic hydrogel microspheres for cell encapsulation

Microencapsulation within hydrogel microspheres holds much promise for drug and cell delivery applications. Synthetic hydrogels have many advantages over more commonly used natural materials such as alginate, however their use has been limited due to a lack of appropriate methods for manufacturing these microspheres under conditions compatible with sensitive proteins or cells. This study investigated the effect of flow rate and voltage on size and uniformity of the hydrogel microspheres produced via submerged electrospray combined with UV photopolymerization. In addition, the mechanical properties and cell survival within microspheres was studied. A poly(vinyl alcohol) (PVA) macromer solution was sprayed in sunflower oil under flow rates between 1-100 μL/min and voltages 0-10 kV. The modes of spraying observed were similar to those previously reported for electrospraying in air. Spheres produced were smaller for lower flow rates and higher voltages and mean size could be tailored from 50 to 1,500 μm. The microspheres exhibited a smooth, spherical morphology, did not aggregate and the compressive modulus of the spheres (350 kPa) was equivalent to bulk PVA (312 kPa). Finally, L929 fibroblasts were encapsulated within PVA microspheres and showed viability >90% after 24 h. This process shows great promise for the production of synthetic hydrogel microspheres, and specifically supports encapsulation of cells.     

Protein array consisting of sol-gel bioactive platform for detection of E. coli O157:H7

Sol-gel-derived bioactive platform was fabricated for detection of pathogenic microbes, E. coli O157:H7. Design flexibility of sol-gel technique and ease of fabrication can fulfill to create the surfaces with structural and chemical features that are compatible with biomaterials such as antibody, enzymes, etc. In this study, the bioactive platform was prepared based on the silica gels, which were produced by hydrolyzing tetraethylorthosilane (TEOS) in ethanol. The mercaptopropyl triethoxysilane (MPTS) was mixed with the TEOS solution for the surface functionalization of bioactive platform. During TEOS hydrolysis, the modified thin film was prepared by sol-gel dip coating. Antibody against E. coli O157:H7 was immobilized with a configuration of protein array using piezo-type dispensing system. Surface morphology of the prepared bioactive platform was analyzed using atomic force microscopy (AFM). The antibody-antigen interaction was investigated with fluorescence microscopy and sandwich type immunoassay using fluorescein isothiocyanate (FITC)-labeled antibody. The results showed that antibody was sequestered within the sol-gel-derived bio-gel due to physical adsorption. The measurement of E. coli O157:H7 was done using the fabricated antibody surface. The fluorescence intensity was proportional to the concentration of E. coli O157:H7, of which the detection limit was 10(2)CFU/ml.     

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.     

Various properties of urease encapsulated into liposomes

The enzymic activity of plant urease encapsulated into liposomes from egg lecithin was studied. Liposomes contained 3-5% of the initial enzymic preparation. Incorporation of urease into liposomes increases the permeability of the lecithin membrane for urea. The liposome membrane provides protection of the incorporated material from the inhibitory action of heavy metal ions. Kinetics of the reactions catalyzed by the free enzyme and encapsulated one is different. Km for the encapsulated enzyme is 1 X 10(-3) M and for free urease--4 X 10(-4) M, that is related to limited substrate mass transfer rate and as a result of it due to inhomogeneity of the catalysis proceeding in liposomes.     

Simultaneous determination of renal clinical analytes in serum using hydrolase- and oxidase-encapsulated optical array biosensors

An optical array biosensor encapsulated with hydrolase and oxidoreductase using sol-gel immobilization technique has been fabricated for simultaneous analysis and screening of multiple samples to determine the presence of multianalytes which are clinically important in relation to renal failure. Urease and creatinine deiminase were used to detect urea and creatinine, while glucose oxidase and uricase were coimmobilized with horseradish peroxidase to quantify glucose and uric acid. Moreover, the concentrations of analytes in fetal calf serum were measured and quantified using the developed sensing system. The array biosensor showed good specificity for the simultaneous analysis of multiple samples for multianalytes without obvious cross-interference. The analytical ranges of the four analytes were between 0.01 and 10mM with detection limits of 2.5-80 microM. High precision with relative standard deviations of 3.8-9.2% (n=45) was also demonstrated. The reproducibility of array-to-array in 3 consecutive months was 5.4% (n=3). Moreover, the concentrations of analytes in fetal calf serum were 5.9 mM for urea, 0.13 mM for creatinine, 3.3mM for glucose, and 0.15 mM for uric acid, which were in good agreement with results obtained using the traditional spectroscopic methods. These results demonstrate the first use of a sol-gel-derived optical array biosensor for simultaneous analysis of multiple samples for the presence of multiple clinically important renal analytes.