Amperometric Biosensors Based on Recombinant Bacterial Laccase CotA for Hydroquinone Determination

In this study, stable CotA laccase from Bacillus subtilis 168 was adsorbed on electrode modified with a thiol graphene‐gold nanoparticle (thGP‐AuNPs) nanocomposite film. The novel bacterial laccase biosensor was employed for quantitative detection of hydroquinone (HQ) and the electrochemical properties of this laccase biosensor were investigated. The results indicate that the immobilized CotA shows great oxidation activity towards HQ in the presence of oxygen and the biosensor shows linear electrocatalytic activity in the concentration range from 1.6 to 409.6 μM, with a detection limit of 0.3 μM. Further, the CotA modified electrode, when compared to fungal laccase‐modified biosensors, shows better alkaline stability (retaining approximately 80 % and 70 % of response current at pH 8 and 9, respectively) and reusability (retaining ～87 % of response current after 100 days). The development of this new kind of laccase on a biosensor will offer a novel tool for substance detection applications in hostile environments, especially for industrial pollutants.     

The Thermophilic CotA Laccase from Bacillus subtilis: Bioelectrocatalytic Evaluation of O2 Reduction in the Direct and Mediated Electron Transfer Regime

The thermophilic bacterial laccase CotA from Bacillus subtilis adsorbed on graphite electrodes enables monitoring of the temperature dependent direct biocatalytic O₂ reduction. Its entrapment in two different Os‐complex modified redox hydrogels is the basis for mediated bioelectroreduction of O₂. Besides the temperature and pH dependence of the bioelectrocatalytic response chloride and fluoride inhibition studies demonstrate that the Os‐complexes are bound to the T1 copper centre of the enzyme. The interaction between CotA and the Os‐complex modified polymers prevents inhibition by chloride ions.     

Azo‐Based Fluorogenic Probes for Biosensing and Bioimaging: Recent Advances and Upcoming Challenges

T he use of nonfluorescent azo dyes as dark quenchers in activatable optical bioprobes based on the Förster resonance energy transfer (FRET) mechanism and designed to target a wide range of enzymes has been established for over two decades. The key value of the azo moiety (−N=N−) to act as an efficient “ON–OFF” switch of fluorescence once introduced within the core structure of conventional organic‐based fluorophores (mainly fluorescent aniline derivatives) has recently been exploited in the development of alternative reaction‐based small‐molecule probes based on the “profluorescence” concept. These unprecedented “azobenzene‐caged” fluorophores are valuable tools for the detection of a wide range of reactive (bio)analytes. This review highlights the most recent and relevant advances made in the design and biosensing/bioimaging applications of azo‐based fluorogenic probes. Emphasis is also placed on relevant achievements in the synthesis of bioconjugatable/biocompatible azo dyes used as starting building blocks in the rational and rapid construction of these fluorescent chemodosimeters. Finally, a brief glimpse of possible future biomedical applications (theranostics) of these “smart” azobenzene‐based molecular systems is presented.     

Reactive Polymeric Dyes as Textile Auxiliaries

Commercial and synthetic azobenzene derivatives were used for the synthesis of hydrophilic polymeric dyes. Two strategies based respectively on the polymerization of dye methacrylic derivatives with different monomers and on the functionalization of reactive polymers were investigated. Polymers containing rather small amounts of the selected dyes were generally obtained, very likely because of the electron withdrawing effect of azo chromophores. Almost quantitative conversions were recorded in the reaction of commercial dyes with maleic anhydride/methyl vinyl ether alternating copolymers. Some of the prepared polymeric dyes were preliminarily tested as textile finishing agents.     

Biodegradation of Bisphenol A and Decolorization of Synthetic Dyes by Laccase from White-Rot Fungus, Trametes polyzona

Purified laccase from Trametes polyzona WR710-1 was used as biocatalyst for bisphenol A biodegradation and decolorization of synthetic dyes. Degradation of bisphenol A by laccase with or without redox mediator, 1-hydroxybenzotriazole (HBT) was studied. The quantitative analysis by HPLC showed that bisphenol A rapidly oxidized by laccase with HBT. Bisphenol A was completely removed within 3 h and 4-isopropenylphenol was found as the oxidative degradation product from bisphenol A when identified by GC-MS. All synthetic dyes used in this experiment, Bromophenol Blue, Remazol Brilliant Blue R, Methyl Orange, Relative Black 5, Congo Red, and Acridine Orange were decolorized by Trametes laccase and the percentage of decolorization increased when 2 mM HBT was added in the reaction mixture. This is the first report showing that laccase from T. polyzona is an affective enzyme having high potential for environmental detoxification, bisphenol A degradation and synthetic dye decolorization.     

Controlled Sol–Gel Transitions of a Thermoresponsive Polymer in a Photoswitchable Azobenzene Ionic Liquid as a Molecular Trigger

Producing ionic liquids (ILs) that function as molecular trigger for macroscopic change is a challenging issue. Photoisomerization of an azobenzene IL at the molecular level evokes a macroscopic response (light‐controlled mechanical sol–gel transitions) for ABA triblock copolymer solutions. The A endblocks, poly(2‐phenylethyl methacrylate), show a lower critical solution temperature in the IL mixture containing azobenzene, while the B midblock, poly(methyl methacrylate), is compatible with the mixture. In a concentrated polymer solution, different gelation temperatures were observed in it under dark and UV conditions. Light‐controlled sol–gel transitions were achieved by a photoresponsive solubility change of the A endblocks upon photoisomerization of the azobenzene IL. Therefore, an azobenzene IL as a molecular switch can tune the self‐assembly of a thermoresponsive polymer, leading to macroscopic light‐controlled sol–gel transitions.     

Structure control for fine tuning fluorescence emission from side-chain azobenzene polymers

New fluorescent azobenzene dyes and side-chain polymers have been synthesized and characterized and their photophysical properties studied. A series of azobenzene dyes having different fluorophores such as phenol (S1), phenylphenol (S2) and naphthol (S3) incorporated in them were synthesized. S2 had unusually high fluorescence with a quantum yield of phi f = 0.2 recorded in dichloromethane (DCM), whereas S1 and S3 were found to be weakly fluorescent. The azobenzene dyes were converted into methacrylate monomers having short ethyleneoxy spacers and then free radically polymerized. Phenylphenol-based azobenzene polymer (P2) continued to show fluorescence, whereas fluorescence was completely quenched in the case of phenol (P1)- and naphthol (P3)-based polymers. Phenylphenol, though twisted in the ground state is known to have a more planar geometry in the excited state--a factor that enables it to retain its fluorescence behavior even when it is incorporated as part of an azobenzene unit. In contrast, naphthol, which is a better fluorophore compared to phenylphenol, loses much of its emissive behavior upon coupling to the azobenzene unit. The extent of trans to cis photoisomerization in solution was very low (approximately 17%) for P2 after 30 min of continuous irradiation using 365 nm light, in contrast to approximately 40% for P1 under identical conditions. This is attributed to the steric repulsion brought about by the bulky phenylphenol units that restrict rotation. A 2-fold enhancement in fluorescence emission was observed for P2 upon irradiation by UV light at 360 nm, which relaxed to the original intensity in about 7 day's time. The higher emission of the cis azobenzenes is generally attributed to an inhibition of photoinduced electron transfer (PET) mechanism. The emission of P2 showed a concentration dependence which increased initially and then decreased in intensity with the formation of a new red-shifted peak at higher concentration due to aggregation. Irradiation of the fluorescence quenched highly concentrated (1 x 10(-3) M) sample of P2 showed an enhancement in emission from aggregates at 532 nm.     

Direct and Versatile Synthesis of Red‐Shifted Azobenzenes

A straightforward synthesis of azobenzenes with bathochromically‐shifted absorption bands is presented. It employs an ortho‐lithiation of aromatic substrates, followed by a coupling reaction with aryldiazonium salts. The products are obtained with good to excellent yields after simple purification. Moreover, with the presented methodology, a structurally diverse panel of different azobenzenes, including unsymmetric tetra‐ortho‐substituted ones, can be readily obtained, which paves the way for future development of red‐light‐addressable azobenzene derivatives for in vivo application.     

Reactivity of alkoxysilyl compounds: chemical surface modification of nano‐porous alumina membrane using alkoxysilylazobenzenes

In order to clarify the chemical adsorption properties of alkoxysilyl compounds to metal‐oxide surface quantitatively and to obtain the information on the reaction conditions for the efficient surface modification, the chemical surface modifications of nano‐porous alumina membranes (NPAMs) by typical alkoxysilyl compounds of 4‐(triethoxysilyl)azobenzene, 4‐(diethoxyphenylsilyl)azobenzene, 4‐(ethoxydiphenylsilyl)azobenzene and 4‐(methoxydimethylsilyl)azobenzene were examined. The chemical surface modifications were performed by immersing NPAMs into the solutions of the alkoxysilylazobenzenes. Especially for 4‐(triethoxysilyl)azobenzene, the modification was investigated precisely by changing the solvent, temperature, concentration and water content of the solutions to reveal the effects of the reaction conditions on the adsorption property of alkoxysilyl compounds to metal oxides. The NPAMs having chemically modified surface were prepared successfully by the immersing method, and the alkoxysilylazobenzenes were confirmed to be bound on the NPAM surface through Si? O? Al bonds, which were indicated to be formed mainly by the direct condensation reaction between the alkoxysilyl groups of the azobenzenes and the hydroxy groups on the NPAM surface. The amounts of the azobenzenes adsorbed on the NPAM surface were estimated quantitatively by a visible absorption spectroscopy, and the results showed that the solutions with non‐polar solvents, higher temperatures and higher concentrations are suitable for the efficient surface modification. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis of Azoimidazolium Dyes with Nitrous Oxide

A new method for the synthesis of industrially important azoimidazolium dyes is presented. The procedure is based on a reagent which is rarely used in the context of synthetic organic chemistry: nitrous oxide (“laughing gas”). N₂O is first coupled to N‐heterocyclic carbenes. Subsequent reaction with aromatic compounds through an AlCl₃‐induced C? H activation process provides azoimidazolium dyes in good yields.     

A Sulfonic‐Azobenzene‐Grafted Silica Amphiphilic Material: A Versatile Stationary Phase for Mixed‐Mode Chromatography

A novel sulfonic‐azobenzene‐functionalized amphiphilic silica material was synthesized through the preparation of a new sulfonic azobenzene monomer and its grafting on mercaptopropyl‐modified silica by a surface‐initiated radical chain‐transfer reaction. The synthesis was confirmed by infrared spectra, elemental analysis, and thermogravimetric analysis. This new material was successfully applied as a new kind of mixed‐mode stationary phase in liquid chromatography. This allows an exceptionally flexible adjustment of retention and selectivity by tuning the experimental conditions. The distinct separation mechanisms were outlined by selected examples of chromatographic separations in the different modes. In reversed‐phase liquid chromatography, this new stationary phase presented specific chromatographic performance when evaluated using a Tanaka test mixture. Seven dinitro aromatic isomers, four steroids, and seven flavonoids were separated successfully in simple reversed‐phase mode. This stationary phase can also be used in hydrophilic interaction chromatography because of the existing polar functional groups; for this, nucleosides and their bases were used as a test mixture. Interestingly, the same nucleosides and bases can also be separated in per aqueous liquid chromatography using the same stationary phase. Three ginsenosides including Rg1, Re, and Rb1 were successfully separated in hydrophilic mode. There is the potential for more applications to benefit from this useful column.     

Novel Azo‐Dyes‐Modified Isatin Derivatives: Synthesis,UV/VIS Spectroscopic,and Electrochemical Study

A high‐yield and simple synthesis of certain aminomethylisatins bearing dye fragments via the Mannich reaction of isatin with amino‐containing azobenzenes was reported. It was found that the absence of electron‐donating groups in azo‐dye molecule prevents aminomethylation of isatin. The effect of the incorporation of an isatin moiety with an azobenzene dye in one molecule on its absorption and electrochemical behavior was studied using UV spectroscopy and cyclic voltammetry.     

Decolorization of Synthetic Dyes by Crude and Purified Laccases from Coprinus comatus Grown Under Different Cultures: The Role of Major Isoenzyme in Dyes Decolorization

Coprinus comatus laccase isoenzyme induction and its effect on decolorization were investigated. The C/N ratio, together with aromatic compounds and copper, significantly influenced laccase isoenzyme profile and enzyme activity. This fungus produced six laccase isoenzymes in high-nitrogen low-carbon cultures but much less in low-nitrogen high-carbon (LNHC) cultures. The highest laccase level (3.25 IU/ml), equivalent to a 12.6-fold increase compared with unsupplemented controls (0.257 IU/ml), was recorded after 13 days in LNHC cultures supplemented with 2.0 mM 2-toluidine. Decolorization of twelve synthetic dyes belonging to anthraquinone, azo, and triphenylmethane dyes, by crude laccases with different proportion of isoenzymes produced under selected culture conditions, illustrated that the LacA is the key isoenzyme contributed to dyes decolorization especially in the presence of 1-hydroxybenzotriazol, which was further confirmed by dyes decolorization with purified LacA in the same condition. The crude laccase only was able to decolorize over 90 % of Reactive Brilliant Blue K-3R, Reactive Dark Blue KR, and Malachite Green, and higher decolorization for broader spectrum of synthetic dyes was obtained in presence of redox mediator, suggesting that C. comatus had high potential to decolorize various synthetic dyes as well as the recalcitrant azo dyes.     

A Facile Synthesis of Pechmann Dyes

A facile synthesis of Pechmann dyes has been accomplished by the reaction of substituted N‐phenacyl‐4‐dimethylaminopyridinium halides with dimethyl maleate in the presence of DBU. Based on a related 4‐DMAP elimination product and an isolated monolactone intermediate a reaction mechanism has been proposed. The scope of this synthetic method is determined by the availability of α‐haloaroyl or heteroaroyl derivatives. DBU=1,8‐diazabicycloundec‐7‐ene, DMAP=4‐dimethylaminopyridine.     

Radical CH Alkylation of BODIPY Dyes Using Potassium Trifluoroborates or Boronic Acids

A one‐step synthetic procedure for the radical C?H alkylation of BODIPY dyes has been developed. This new reaction generates alkyl radicals through the oxidation of boronic acids or potassium trifluoroborates and allows the synthesis of mono‐, di‐, tri‐, and tetraalkylated fluorophores in a good to excellent yield for a broad range of organoboron compounds. Using this protocol, multiple bulky alkyl groups can be introduced onto the BODIPY core thus creating solid‐state emissive BODIPY dyes.     

Mechanistic study of trans⇆cis isomerization of the substituted azobenzene moiety bound on a liquid‐crystalline polymer

A mechanistic study of the trans?cis isomerization of the azobenzene moiety in a side‐chain liquid‐crystal polymer system was carried out with six liquid‐crystalline polymethacrylates in which different electron‐withdrawing substituents were attached to the para‐positions of the azobenzene chromophores. Compared to the non‐nitro‐substituted azo polymers, the nitro‐substituted azo polymers exhibited two quite different behaviors: an extraordinarily high reaction rate of the thermal cis–trans isomerization and an unexpected composition of cis–trans isomers obtained from the photochemical trans–cis isomerization process. A potential energy profile for the isomerization process was established on basis of the structures of the proposed transition states and was employed to elucidate the reaction mechanism. The results confirmed that the nitro‐substituted azo polymer system proceeded via a rotation mechanism in either direction of the trans?cis isomerization reaction, whereas the non‐nitro‐substituted species were more likely to follow an inversion mechanism. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 2296–2307, 2001     

Synthesis of β-cyclodextrin derivatives functionalized with azobenzene

Two approaches for the synthesis of β-cyclodextrin and bis(β-cyclodextrin) bearing azobenzene on the primary face are reported. First, the nucleophilic substitution of mono-6-tosyl-β-cyclodextrin by azobenzene anion derivatives was reinvestigated and found to produce mono-3,6-anhydro-β-cyclodextrin as a side product. A slight modification of the reported reaction conditions including the use of Cs₂CO₃ led to a substantial improvement of the yields. In addition, a convenient method based on the application of click chemistry led to 1,2,3-triazole-linked azobenzene-cyclodextrin derivatives in good yields.     

Unprecedented Nucleophilic Attack of Piperidine on the Electron Acceptor during the Synthesis of Push‐Pull Dyes by a Knoevenagel Reaction

An unprecedented nucleophilic addition of piperidine on an electron acceptor, namely, 2‐(3‐oxo‐2,3‐dihydro‐1H‐cyclopenta[b]naphthalen‐1‐ylidene)malononitrile is reported. This unexpected behavior was observed during the synthesis of push‐pull dyes using the classical Knoevenagel reaction. To overcome this drawback, use of diisopropylethylamine (DIPEA) enabled to produce the expected dyes PP1 and PP2 . The optical and electrochemical properties of the different dyes were examined. Theoretical calculations were also carried out to support the experimental results. To evidence the higher electron‐withdrawing ability of this electron acceptor, a comparison was established with two dyes ( PP3 and PP4 ) comprising its shorter analogue.     

Photo-induced structural changes of azobenzene Langmuir-Blodgett films

Structural changes of the Langmuir-Blodgett (LB) films of azobenzene accompanied by photoisomerization are described. First, photoisomerization is explained in terms of 'free volume'. In the polyion complex monolayers of amphiphiles having two azobenzene units at the air-water interface, the area per molecule depends on the polycation species. The fraction of cis-azobenzene in the LB films at the photostationary state under the illumination with UV light increased with increasing area per molecule, which is consistent with the concept of free volume. Second, a counter example of the concept of free volume is presented. Three-dimensional cone-shaped structures developed with trans-to-cis photoisomerization in the polyion complex LB film of a water-soluble amphiphilic azobenzene. These structures appeared and disappeared reversibly by alternate illumination with UV and visible light. The results indicate that the two-dimensional LB film structure exerts significant modification by photoisomerization. This is against the concept of free volume because this concept does not consider the possibility that the two-dimensional LB film structures may change into three-dimensional ones. Finally, photo-induced J-aggregate formation of non-photochromic and photochromic dyes is described. Two cyanine dyes were each mixed with an amphiphilic azobenzene in the LB films. These cyanine dyes are known to form J-aggregates in single-component LB films. In the mixed LB films, the J-aggregate formation was suppressed to some extent. The alternate illumination of the films with UV and visible light caused the photoisomerization of azobenzene in the mixed LB films, which triggered the J-aggregate formation of the cyanine dyes. The J-aggregate formation was accompanied by the development of three-dimensional cone-shaped structures from the film surface. When an amphiphilic merocyanine was mixed with the azobenzene in the LB films, J-aggregate formation was also induced by the alternate illumination with UV and visible light. This J-aggregate formation was also accompanied by a large morphological change: circular domains changed into fractal-like ones. The J-aggregate formation of the dyes and the concomitant morphological change were irreversible. In these cases, the photoisomerization of azobenzene served as a trigger to induce self-organization of the dye molecules.     

Photoswitchable architectural polymer: Toward azo‐based polyamidoamine side‐chain dendritic polyester

A versatile approach to the synthesis of novel polyamidoamine (PAMAM) side‐chain dendritic polyester (SCDPE) possessing azobenzene motifs in the polymeric core is described and displayed reversible cis–trans (E/Z) isomerization upon exposure to UV light. A polymerization reaction was conducted in solution using ester‐terminated PAMAM dendritic diol ( 1a , G 3.5) and azobenzene dicarboxylic acid chloride in the presence of triethylamine. PAMAM dendritic diol 1a as well as SCDPE ( 1 ) were thoroughly characterized by means of IR and NMR (¹H and ¹³C) spectroscopies. The intrinsic viscosity of 1 at 36 °C in CHCl₃ was found to be 0.38 dl/g. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 4182–4188, 2001