Fluorescent sensors for nitroaromatic compounds based on monolayer assembly of polycyclic aromatics

A class of fluorescent films in which pyrene was assembled, in a monolayer manner, on glass slide surfaces via various flexible spacers of different lengths and substructures was used for the detection of nitroaromatic compounds (NACs) in vapor phase. This design strategy offers several advantages for thin film fluorescent sensory materials. These advantages have been demonstrated experimentally by the sensitive response of the films to the presence of trace amounts of NACs in vapor phase. The fluorescence quenching of the films upon exposure to NACs vapors depends on several factors, including the evaporate rate of the NAC detected, the length of the spacers connecting the sensing element and the substrate surface, and the density of the sensing element on the substrate surface. Further experimentation showed that the sensing process is reversible and free of commonly encountered interference. The sensitive response, reversibility of the sensing process, and freedom from commonly encountered interference of the specially designed films to NACs qualify these materials as promising NACs fluorescent sensory materials.     

A dansyl-based fluorescent film: Preparation and sensitive detection of nitroaromatics in aqueous phase

A novel fluorescent film sensor for the detection of nitroaromatics in aqueous phase has been developed via chemical immobilization of dansyl chromophores on an epoxy-terminated self-assembled monolayer on glass slide surfaces. Chemical attachment of the chromophore on the substrate surface endows the present film a satisfying stability and avoids the leaching of the chromophores. Increase in the length of the spacer connecting the sensing element and the substrate results in a dramatic improvement in the performance of the film compared to those with similar structures. Fluorescence quenching experiments demonstrates that in aqueous medium, the emission of the film is more sensitive to nitrobenzene (NB) than to other nitroaromatics, including 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, p-chloronitrobenzene, m-dinitrobenzene, p-dinitrobenzene, and o-chloronitrobenzene, etc. This exceptional result has been rationalized by considering the possible hindrance effect induced by the compact conformation of the long flexible spacer. It is also demonstrated that the NB quenching is static in nature, and the response of the film to NB is fast and reversible.     

Surface and interface control on photochemically initiated immobilization

Surface and interface properties are important in controlling the yield and efficiency of the photochemically initiated immobilization. Using a silane-functionalized perfluorophenyl azide (PFPA-silane) as the photoactive cross-linker, the immobilization of polymers was studied by adjusting the density of the surface azido groups. Dilution of the photolinker resulted in a gradual decrease in the density of surface azido groups as well as the thickness of the immobilized film. When a nonphotoactive silane was added to PFPA-silane, the film thickness decreased more rapidly, suggesting that the additive competed with PFPA-silane and effectively reduced the density of the surface azido groups. The effect of surface topography was studied by adding a nonphotoactive silane with either a shorter (n-propyltrimethoxysilane (PTMS)) or a longer spacer (n-octadecyltrimethoxysilane (ODTMS)). In most cases the long chain ODTMS shielded the surface azido groups, resulting in a more rapid decrease in film thickness as compared to PTMS treated under the same conditions. As the density of the surface azido groups decreased, the immobilized polymer changed from smooth films to patched structures and, eventually, single polymer molecules.     

Surface-grafted poly(acrylic acid) brushes as a precursor layer for biosensing applications: effect of graft density and swellability on the detection efficiency

Carboxyl groups along poly(acrylic acid) (PAA) brushes attached to the surface of a gold-coated substrate served as the precursor moieties for the covalent immobilization of amino-functionalized biotin or bovine serum albumin (BSA) to form a sensing probe for streptavidin (SA) or anti-BSA detection, respectively. Surface-grafted PAA brushes were obtained by acid hydrolysis of poly(tert-butyl acrylate) brushes, formerly prepared by surface-initiated atom transfer radical polymerization of tert-butyl acrylate. As determined by surface plasmon resonance, the PAA brushes immobilized with functionalized biotin or BSA probes not only showed good binding with the designated target analytes but also maintained a high resistance to nonspecific protein adsorption, especially those PAA brushes with a high surface graft density. Although the probe binding capacity can be raised as a function of the graft density of the PAA brushes or the amount of carboxyl groups along the PAA chains, the accessibility of the target analyte to the immobilized probe was limited at the high graft density of the PAA brushes. The effect was far more apparent for the BSA-anti-BSA probe-analyte pair than for the much smaller biotin-SA probe-analyte pair. The impact of the swellability of the PAA brushes, as tailored by the degree of carboxyl group activation, on both the sensing probe immobilization and analyte detection was also addressed. This investigation demonstrated that PAA brushes having a defined graft density have a promising potential as a precursor layer for biosensing applications.     

Functionalization of silica surface using Chan–Lam coupling

The reaction of base-free Chan–Lam coupling was successfully used for functionalization of surface of mesoporous silica gel. Various aromatic, aliphatic, and heterocyclic compounds were immobilized by a copper-catalyzed reaction of corresponding boronic acids with surface amino groups at mild conditions. Obtained functionalized materials were mesoporous although their surface area decreased after immobilization. The reactivity of some surface functional groups was tested in their characteristic reactions.     

Immobilization of hydroquinone through a spacer to polymer grafted on carbon black for a high-surface-area biofuel cell electrode

We immobilized hydroquinone through a spacer to polymer grafted on carbon black and achieved a high-surface-area biofuel cell electrode. Quinone compounds are well-known to transfer electrons in the respiratory chain and have been considered prospective mediators in biofuel cells because of their relatively negative redox potentials. Evaluation of three different spacer arms tethering hydroquinone to linear polymers revealed that only the hydrophilic and flexible di(ethylene oxide) spacer made it possible for immobilized hydroquinone to transfer electrons from glucose oxidase (GOD) to an electrode; direct immobilization and an alkyl spacer did not. The electrode comprising hydroquinone immobilized through di(ethylene oxide) spacer to polymer grafted on carbon black transferred electrons from GOD to the electrode. The potential at which an anodic current began to increase was more negative by about 0.2 V than that for a vinylferrocene-mediated electrode, while the increase in the anodic current density was of the same order.     

Immobilization of light-harvesting chlorophyll a/b complex (LHCIIb) studied by surface plasmon field-enhanced fluorescence spectroscopy

The major light-harvesting chlorophyll a/ b complex (LHCIIb) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that engage in rapid excitation energy transfer. This property makes LHCIIb potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Such applications would require the immobilization of LHCIIb (or similar dye-protein complexes) on a solid surface. In this work, the immobilization of recombinant LHCIIb is tested and optimized on functionalized gold surfaces via a histidine 6 tag (His tag) in the protein moiety. Immobilization efficiency and kinetics are analyzed by using surface plasmon resonance (SPR) and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The latter was also used to assess the integrity of immobilized LHCIIb by recording Chl b-sensitized Chl a emission spectra. Since His tags have been included in a substantial number of recombinant proteins, the immobilization technique developed here for LHCIIb presumably can be extended to a large range of other membrane and water-soluble proteins.     

Immobilization of the enzyme beta-lactamase on biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol)-coated sensor chips: a study on oriented attachment and surface activity by enzyme kinetics and in situ optical sensing

Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors. In this study, the very efficient enzyme beta-lactamase is used as a model protein. The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme. The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent. beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate. The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface. Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site. Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency. In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics. All tested enzyme variants turned out to be active upon immobilization at the polymeric surface. However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site. No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers.     

A new solid-phase nanoparticle FRET assays based on GO/CS/ZnS nanocomposite film and developed in sensing for bromonium ion

Composite thin films consisting of nano-sized ZnS particles dispersed in chitosan/GO films have been prepared by in-situ method. The films obtained were characterized by FTIR and UV–Vis spectroscopy. The ZnS nanoparticles with 90 nm in diameter were dispersed uniformly in the film matrix. Optical absorption peak due to the size of ZnS particles was observed around 350 nm. The fluorescence emission at 430 nm of the GO/CS/ZnS nanocomposite films is very sensitive to the presence of bromonium ion from aqueous solutions. New solid-phase nanoparticles FRET assays are firstly immobilized on the substrate and then interacted with functionalized acceptor molecules in the solution to trigger the FRET effect to detect Br^–.     

Man-tailored cellulose-based carriers for invertase immobilization

Cellulose-based carriers Granocel were specially prepared and optimised for covalent immobilization of enzymes. The effects of carrier characteristics such as pore size, chemistry of anchor groups and their density on invertase immobilization efficiency were evaluated. It was found that the preferential adsorption/binding of the enzyme to a carrier during coupling and its activity after immobilization depended on microenvironmental effects created by hydrophilic surface of the carrier, functional groups and their activators. The best preparations (activity approx. 300 U/mL, high storage stability) were obtained for NH₂-Granocel activated with glutaraldehyde. It is probably due to Granocel modification with pentaethylenehexamine that gave a 19-atom spacer arm. The enzyme concentration in coupling mixture was optimised as well. The kinetic parameters of sucrose hydrolysis for native and immobilized invertase were evaluated. Compared to the native invertase, K _m value of immobilized enzyme was only twice higher with about three times lower substrate inhibition. Reaction runs in a well mixed batch reactors with native and immobilized invertase showed slightly slower reaction rate in the case of the enzyme covalently bound to Granocel. Very good stability of cellulose-based carrier was proved experimentally by 20 successive reaction runs in a batch reactor.