Reagentless pH-based biosensing using a fluorescently-labelled dextran co-entrapped with a hydrolytic enzyme in sol-gel derived nanocomposite films

We report on the development of reagentless fluorescence-based sensing films utilizing hydrolytic enzymes co-entrapped with polymers that are labelled with pH sensitive fluorophores. Aqueous solutions of a hydrophilic enzyme (urease) or a lipophilic enzyme (lipase) containing fluorescein or carboxy-seminaphtharhodafluor-1 (SNARF-1), either free or conjugated to a dextran polymer backbone, were mixed with hydrolyzed alkoxysilane solutions and cast onto planar surfaces to form thin, biologically active sol-gel derived films (ca. 500 nm thick). The films also contained various additives, such as methyltrimethoxysilane, dimethyldimethoxysilane, polyethylene glycol or polyvinyl alcohol, to optimize the activity of the entrapped enzymes. The photostability, leaching, pK_a and pH response of the entrapped probes were characterized, as was the performance of the entrapped enzymes, and an optimal set of processing conditions was obtained for each different sensing film. In general, the results indicated that SNARF-labelled dextran was the most useful pH sensitive dye owing to insensitivity to leaching and photobleaching. Furthermore, it was observed that the pK_a and pH response of this probe was insensitive to preparation conditions. The performance of the co-entrapped enzymes was highly dependent on the type and level of additive, but in all cases, it was possible to obtain active enzymes with good performance characteristics. Reagentless sensing films for urea and glyceryl tributyrate (GTB) are demonstrated based on the detection of enzyme-mediated pH changes from films coated onto planar substrates.     

Organically modified sols as pseudostationary phases for microchip electrophoresis

We demonstrate that the selectivity of microchip electrophoresis separations is greatly improved by the presence of organically modified silica (Ormosil) sols in the run buffer. A negatively-charged N-(trimethoxysilylpropyl)ethylenediamine triacetic-acid (TETT)-based sol is used for improving the selectivity between nitroaromatic explosives and a methyltrimethoxysilane (MTMOS)-based sol is employed for enhancing the microchip separation of environmental pollutants, aminophenols. These sols are added to the run buffer and act as pseudostationary phases. Their presence in the run buffer changes the apparent mobility of studied solutes, and leads to a higher resolution. The observed mobilities changes reflect the interactions between the Ormosil sols and the solutes. Relevant experimental variables have been characterized and optimized. The diverse chemistry of Ormosil sols should be extremely useful for tailoring the selectivity of a wide range of electrophoresis microchip separations.     

Photostability of a photochromic naphthopyran dye in different sol-gel prepared ormosil coatings

A photochromic naphthopyran derivative was embedded in sol-gel prepared thin ormosil films. The resulting samples show high transparency and exhibit a strong red colouration upon irradiation with UV light. The photostability of the photochromic molecules is strongly related to the nature of the embedding ormosil matrix. The introduction of organic functional groups into the inner pore surface of the matrix allows tailoring the chemical environment where the dye molecules will be allocated, in terms of the effectiveness of the interaction between the photochromic molecules and the Si-OH groups on the surface of the pores, affecting the stability of the molecules upon prolonged exposition to UV light. The photostability of the molecules was increased in matrices functionalized with larger organic groups, or with larger amount of modifying groups. In this way the photodegradation of the photochromic molecules could be reduced by a factor of 5, as compared with the photodegradation of the molecules in unfunctionalized silica matrix.     

Analytical Biosensing of Hydrogen Peroxide on Brilliant Cresyl Blue/Multiwalled Carbon Nanotubes Modified Glassy Carbon Electrode

A cationic quinine‐imide dye brilliant cresyl blue (BCB) and horseradish peroxidase (HRP) were co‐immobilized within ormosil on multiwalled carbon nanotubes modified glassy carbon electrode for the fabrication of highly sensitive and selective hydrogen peroxide biosensor. The presence of epoxy group in ormosil as organic moiety improves the mechanical strength and transparency of the film and amino group provides biocompatible microenvironment for the immobilization of enzyme. The presence of MWCNTs improved the conductivity of the nanocomposite film. The surface characterization of MWCNT modified ormosil nanocomposite film was performed with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cyclic voltammetry and amperometry measurements were used to study and optimize the performance of the resulting peroxide biosensor. The apparent Michaelis–Menten constant was determined to be 1.5 mM. The proposed H₂O₂ biosensor exhibited wide linear range from 3×10^?7 to 1×10^?4 M, and low detection limit 1×10^?7 M (S/N=3) with fast response time <5 s. The probable interferences in bio‐matrix were selected to test the selectivity and no significant response was observed in the biosensor. This biosensor possessed good analytical performance and long term storage stability.     

Thin-film oxygen sensors using a luminescent polynuclear gold(I) complex

Robust thin-film oxygen sensors were fabricated by encapsulating a lipophilic, polynuclear gold(I) complex, bis{m-(bis(diphenylphosphino)octadecylamine-P,P′)}dichlorodigold(I), in oxygen permeable polystyrene and ormosil matrices. Strong phosphorescence, which was quenched by gaseous and dissolved oxygen, was observed from both matrices. The polystyrene encapsulated dye exhibited downward-turning Stern–Volmer plots which were well fitted by a two-site model. The ormosil trapped complex showed linear Stern–Volmer plots for dissolved oxygen quenching but was downward turning for gaseous oxygen. No leaching was observed when the ormosil based sensors were immersed in flowing water over an 8 h period. Both films exhibited fully reversible response and recovery to changing oxygen concentration with rapid response times.     

有机掺杂溶胶-凝胶法制备pH敏感膜及其光谱响应的研究

使用四甲氧基硅烷（TMOS）和二甲基二甲氧基硅烷（DiMe-DMOS)为共先驱体，采用溶胶－凝胶的有机掺杂，制备对pH具有宽程响应的敏感膜。详细考察了包埋溴酚蓝和溴酚绿的敏感膜对pH的响应值、响应时间、泄漏和可逆性等响应性能指标，并进行了溴酚蓝和溴酚绿在水相与膜内吸收光谱的研究。     

A Novel Amperometric Hydrogen Peroxide Biosensor Based on Horseradish Peroxidase Incorporated in Organically Modified Sol-Gel Glass Matrix/Graphite Paste with Multiwalled Carbon Nanotubes

We herein report an electrochemical hydrogen peroxide sensor based on horseradish peroxidase immobilized in organically modified sol-gel glass (ormosil) with mediator ferricyanide along with multiwalled carbon nanotubes (mwcnts). The ormosil material is converted to fine powder followed by incorporation within graphite paste electrode. The electrochemistry of redox materials encapsulated within ormosil has been studied. The requirement of mwcnts is examined. The ormosil prepared with optimum concentration of mwcnts shows better redox electrochemistry as compared to that made without mwcnts. The biosensor has been characterized by cyclic voltammetry and chroanoamperometry. The performance, stability, and reproducibility of a new peroxide biosensor are reported.     

Ion exchange and ion transport properties of sulfonated organically modified silica hydrogels

Sulfonated ormosil hydrogels (~80% water) were prepared using tetramethyl orthosilicate as a silica precursor and 2(4-chlorosulfonylphenyl)ethyltrichlorosilane to provide sulfonate functionality for ion-exchange and ion conductivity. Ruthenium(III) hexamine was used as a redox probe in electrochemical studies performed on porous carbon fibre paper electrodes impregnated with the gel. The gel-modified electrodes extracted Ru(NH₃)₆³⁺ from solutions in 0.1 M CF₃CO₂Na_(aq) with a partition coefficient of ~36, and with ~100% of the sulfonate sites being accessible for ion exchange. The Ru(NH₃)₆^3+/2+ couple exhibited reversible and facile electrochemistry in the gel, with a Ru(NH₃)₆²⁺ diffusion coefficient of 4.9×10^–8 cm² s^–1 determined by chronoamperometry. This is an order of magnitude higher than the mobility of this complex in Nafion. The hydrogel-modified electrodes were stable for days, and could be repeatedly loaded with Ru(NH₃)₆³⁺.Special Issue to celebrate the 70^th birthday of Professor Zbigniew Galus     

TTF‐TCNQ Functionalized Ormosil Based Electrocatalytic Biosensor: A comparative study on Bioelectrocatalysis

A novel electrocatalytic biosensor for glucose is reported that incorporate encapsulation of tetrathifulvalene‐tetracyanoquinodimethane (TTF‐TCNQ) functionalized organically modified sol gel glass (ormosil). The new ormosil is made using palladium‐linked glycidoxypropyltrimethoxysilane precursor, trimethoxysilane, HCl and TTF‐TCNQ powder at 25 °C. The ormosil is converted into fine powder and incorporated within graphite paste electrode along with glucose oxidase. The bioelectrochemistry of GOD and TTF‐TCNQ functionalized ormosil is examined based on cyclic voltammetry and amperometric measurements. A large electrocatalytic current to the order of 8000 μA/cm² is recorded on the addition of 300 mM glucose. Typical responses of new biosensor are reported. The sensitivity of glucose analysis is found relatively much better as compared to earlier reported glucose biosensors. The role of palladium and TTF‐TCNQ introduction within ormosil and its advantages on bioelectrocatalysis are discussed.     

染料掺杂有机改性硅酸盐的制备与光谱特性

1988年Pavlopoulos等人发现1,3,5,7-四甲基吡咯亚甲基BF₂化合物是一个性能优良的激光染料^[1].它的荧光量子效率高,激光效率比香豆素540A高10%,并具有低的三重态吸收和高的光化学稳定性.因此,在染料激光、生物荧光探针、光动力疗法等方面都有潜在的应用背景^[2~4].