Synthesis and characterization of novel chitosan derivatives (containing dipyridinium quaternary salts) with antimicrobial potential.

Chitosan derivatives are versatile materials, biocompatible and biodegradable, that can be tailor-made to suit specific biomedical applications. In this study, two N-heterocyclic salts (N,N'-diphenacyl-[4,4'-dipyridinium] dibromide (DP) and N,N'-diphenacyl-1,2-bis-(4-pyridinium)ethane dibromide (DPE)) were used for chitosan functionalization to enhance its antimicrobial potential. Physico-chemical characterization of the newly synthesized derivatives (Ch-DP and Ch-DPE) was performed by elemental analysis, spectrometry (UV-Vis, FTIR), electrochemistry (OCP, CV), and electron microscopy (SEM) proving that the highest degree of functionalization was obtained for Ch-DP. The antimicrobial effect of chitosan functionalization was further tested in terms of its interaction with Listeria monocytogenes Scott A, and Staphylococcus aureus ATCC 25923, as Gram-positive bacteria and Escherichia coli ATCC 25922, as Gram-negative bacterium, respectively, showing that the Ch-DP had a good inhibitory activity compared with Ch-DPE.

Highly sensitive detection of glucose via glucose oxidase immobilization onto conducting polymer-coated composite polyacrylonitrile nanofibers.

Current study introduces composite polyacrylonitrile - multiwall carbon nanotubes nanofibers (PAN-MWCNTs NFs) coated with conducting polymers (polypyrrole (PPy) or poly(3,4-ethylenedioxythiophene) (PEDOT)) by chemical vapor deposition for efficient glucose detection. The potential of nanofibrous assemblies and nano-conducting elements in biosensing was explored as pre-processing of NFs with MWCNTs and post-processing with CPs were both employed. These 'core-shell' conducting NFs were further employed as platforms for glucose oxidase immobilization for enzymatic detection of glucose. The performance of the biosensors was closely correlated with the concentration of immobilized enzyme and with the type of conducting polymer. The biosensors showed high sensitivities of 92.94 and 81.72 µA/mM.cm-2 for (PAN-MWCNTs)/ PEDOT and (PAN-MWCNTs)/ PPy accompanied by low limit of detection values of 2.30 and 2.38 µM, respectively. Good operational stability was observed throughout twenty-five consecutive measurements, over 90% activity was maintained for both sensors. This study represents proof of concept for the methodology, showcasing the advantages of nanomaterial synthesis for bio-applications. The work was compared thoroughly with previously reported biosensors showing some of the best results reported to date in terms of analytical characteristics.

Trace-Level Phenolics Detection Based on Composite PAN-MWCNTs Nanofibers.

In view of major concerns regarding toxicity (genotoxic, mutagenic, hepatotoxic) of phenolics, there is an on-going necessity for sensitive and accurate analytical procedures for detection and measurements in environmental field, water, and food quality control. The current study proposes composite polyacrylonitrile nanofibrous assemblies enriched with multi-wall carbon nanotubes (PAN-MWCNTs NFs) as suitable immobilization platforms for cross-linking of tyrosinase in detection of both diphenols and monophenols, which are of much interest in water contamination. The prepared biosensor (Pt/PAN-MWCNTs/Tyr) showed high sensitivity in catechol detection (362.5 mA/M.cm2 ) and moderate sensitivity for the two monophenols (phenol, p-chlorophenol) with 14.79 and 25.46 mA/M.cm2 , respectively. The LOD values of the sensor were in the nanomolar range: 4.42×10-9 , 4.06×10-7 and 1.05×10-7  M for catechol, phenol and p-chlorophenol, respectively, allowing detection of trace amounts of phenolics in spiked water samples.

Next step in 2nd generation glucose biosensors: Ferrocene-loaded electrospun nanofibers.

Glucose determination is one of the most common analyses in clinical chemistry. Employing biosensors for this purpose has become the method of choice for home use for diabetic patients. To limit the impact of dissolved O2 concentration or possible interferences (known hindrances in the classical glucose detection approach), a variety of mediated pathways have been explored. Herein, an ingenious, facile and low-cost approach for immobilization of redox mediator within nanofibrous mats is presented. This '2nd generation' biosensor is able to avoid common issues such as leaching or diffusion barriers whilst providing the necessary close contact between the enzyme and the redox shuttle, for enhancing the detection accuracy and accelerate the response. Polyacrylonitrile nanofibers loaded with carbon nanotubes and ferrocene (PAN/Fc/MWCNT-COOH NFs) have been successfully prepared and applied as biosensing matrices upon cross-linking of glucose oxidase (GOD). The morphology of the NFs was investigated by means of scanning electron microscopy (SEM-EDX) and correlated to the kinetics of mediated electron transfer and to the efficiency in glucose detection, which were evaluated through cyclic voltammetry (CV) and amperometric measurements. The content of Fc was varied from 0.5 to 5.0 wt%, with optimum biosensor performance at 1.0 wt% exhibiting a linear range up to 8.0 × 10-3 M with sensitivity of ~27.1 mAM-1 cm-2 and 4.0 µM LOD. Excellent stability (RSD 2.68%) during 40 consecutive measurements along with insignificant interference and accurate recovery in real sample analysis (~100%) make for a very reliable sensor that can easily render itself to miniaturization and has the potential for a wide range of practical applications.

The influence of sugar-protein complexes on the thermostability of C-reactive protein (CRP).

The purpose of the present study was to evaluate de influence of protein-sugar complexation on the stability and functionality of C-reactive protein, after exposure to constant high temperatures, in order to develop highly stable positive controls for in-vitro diagnostic tests. C-reactive protein is a plasmatic protein used as a biomarker for the diagnosis of a series of health problems such as ulcerative colitis, cardiovascular diseases, metabolic syndrome, due to its essential role in the evolution of chronic inflammation. The sugar-protein interaction was investigated using steady state and time resolved fluorescence. The results revealed that there are more than two classes of tryptophan, with different degree of accessibility for the quencher molecule. Our study also revealed that sugar-protein complexes have superior thermostability, especially after gamma irradiation at 2 kGy, the protein being stable and functional even after 22 days exposure to 40 °C.

Wound healing applications of creams and "smart" hydrogels.

Although superficial wounds are often easy to treat for healthy individuals, there are some more severe types of wounds (burns, ulcers, diabetic wounds, etc.) that are a challenge for clinicians. A good therapeutic result is based on the delivery of a treatment at the right time, for the right patient. Our goal was to sum up useful knowledge regarding wound healing and wound treatments, based on creams and hydrogels with various active ingredients. We concluded that both preparations have application in preventing infections and promoting healing, but their efficacy is clearly conditioned by the type, depth, severity of the wound and patient profile. However, due to their superior versatility and capability of maintaining the integrity and functionality of the active ingredient, as well as it is controlled release at site, hydrogels are more suited for incorporating different active ingredients. New wound healing devices can combine smart hydrogel dressings with physical therapies to deliver a more efficient treatment to patients if the indications are appropriate.

Facile copper-based nanofibrous matrix for glucose sensing: Eenzymatic vs. non-enzymatic.

The current study aims to provide a valid comparison between glucose detection efficiency with an enzymatic and a non-enzymatic sensing platform. A low-cost nano-matrix for glucose sensing was developed by drop-coating copper nanoparticles (Cu NPs) onto a polyacrylonitrile (PAN) electrospun nanofibrous assembly. The PAN NFs/Cu NPs matrix was optimized regarding electrospinning time and Cu NPs content and employed as a non-enzymatic sensor or further modified by cross-linking of glucose oxidase (GOD) for the development of an enzymatic sensor. The non-enzymatic glucose sensor was three times more sensitive (300 mAM-1cm-2) than the enzymatic one (81 mAM-1cm-2) with similar limit of detection values (5.9 and 5.6 µM, respectively). Incorporation of MWCNTs improved both the LOD (3.3 µM) and the operational stability of the non-enzymatic configuration (RSD 7.3%). The interference effect proved insignificant for the enzymatic sensor due to the innate catalytic selectivity whilst the non-enzymatic sensor acquired selectivity due to the nanofibrous PAN matrix and Nafion coating. The non-enzymatic PAN NFs/Cu NPs sensor was chosen for the detection of glucose in real blood serum samples whilst the PAN NFs/Cu NPs/GOD sensor was applied for glucose detection in fruit juices, both proving recovery results close to 100%.

The effect of copolymerization and carbon nanoelements on the performance of poly(2,5-di(thienyl)pyrrole) biosensors.

The development of biosensing interfaces based on copolymerization of benzenamine-2,5-di(thienyl)pyrrole (SNS-An) with 3,4-ethylenedioxythiophene (EDOT) is reported. Both homopolymer P(SNS-An) and copolymer P(SNS-An-co-EDOT) films were prepared and evaluated, in terms of biosensing efficiency, upon incorporation of carbon nanoelements (carbon nanotubes and fullerene) and cross-linking of glucose oxidase. The copolymer revealed superior performance as a biosensing interface as compared to the homopolymer structure or previously reported P(SNS) biosensors. The analytical characteristics and stability studies were performed both at cathodic potential, monitoring O2 consumption, as a result of catalytic reaction of glucose oxidase towards glucose and at anodic potential, following the oxidation of the H2O2 produced during the catalytic reaction. Whilst the measurements on the positive side offered an extended linear range (0.01-5.0 mM), the negative side provided sensitivity up to 104.96 µA/mMcm-1 within a shorter range. Detection limits were as low as 1.9 µM with Km value of 0.49 mM. Lastly, the most performant biosensing platforms, including copolymeric structure and CNTs were employed for analysis in real samples.

Utilization of enzyme extract self-encapsulated within polypyrrole in sensitive detection of catechol.

Utilization of polyphenol oxidases (laccase, tyrosinase) in biosensor technology is an efficient approach towards phenol detection, which is significant in numerous fields such as environmental monitoring, food industry etc. The use of crude extract instead of pure enzyme eliminates the need for costly and laborious processes of enzyme separation and purification. This study employs polyphenol oxidase extract, biosynthesized by white-rot fungi Trametes pubescens (TP) for the development of amperometric biosensors for catechol detection. The catalytic activity of the crude extract was firstly used to induce the bio-synthesis of conducting polymer - polypyrrole (Ppy), resulting in the self-encapsulation of the enzyme extract within the conducting material. The viability and biological integrity of the enzyme extract was preserved after the synthesis and was able to efficiently detect phenolic compounds such as catechol. Comparative evaluations between the biosynthesized Ppy based biosensor (bio-Ppy) and the biosensor based on bio-PPy with additional enzyme extract (bio-Ppy-TP) were performed. Lastly, the performance of these two biosensors was compared with that of a third one, based on chemically synthesized Ppy with enzyme extract (chem-Ppy-TP). All three types of biosensors proved high efficiency for catechol detection at low concentration (1-60 µM) and were employed for real sample detection in fruit wines showing linear correlation with the spectrophotometric results obtained with the Folin-Ciocalteau standard test.

Cell-assisted synthesis of conducting polymer - polypyrrole - for the improvement of electric charge transfer through fungal cell wall.

In this research we report the biological synthesis of electrically conducting polymer - Polypyrrole (Ppy). Cell-assisted enzymatic polymerization/oligomerization of Ppy was achieved using whole cell culture and cell-free crude enzyme extract from two white-rot fungal cultures. The selected fungal strains belong to Trametes spp., known laccase producers, commonly applied in bioremediation and bioelectrochemical fields. The biocatalytic reaction was initiated in situ through the copper-containing enzymes biosynthesized within the cell cultures under submerged aerobe cultivation conditions. The procedure was inspired by successful reports of laccase-catalyzed pyrrole polymerization. The usage of whole culture and/or crude enzyme extract has the advantage of overcoming enzyme purification and minimizing the liability of enzyme inactivation through improved stability of enzymes in their natural environment. Spectral and electrochemical techniques (UV-vis spectroscopy, infrared spectroscopy; cyclic voltammetry (CV)) and pH measurements provided insight into the evolution of pyrrole polymerization/oligomerization and the electrochemical features of the final product. Microscopy techniques (optical microscopy and scanning electron microscopy (SEM)) were primary tools for visualization of the formed Ppy particles. The relevance of our research is twofold: Ppy prepared in crude enzyme extract results in enzyme encapsulated within Ppy and/or Ppy-modified fungal cells can be formed when polymerization occurs in whole cell culture. The route of biocatalysis can be chosen according to the desired bioelectrochemical application. The reported study focuses on the improvement of charge transfer through the fungal cell membrane and/or cell wall by modification of the fungal cells with conducting polymer - polypyrrole.

Modification of Aspergillus niger by conducting polymer, Polypyrrole, and the evaluation of electrochemical properties of modified cells.

The enhancement of bioelectrochemical properties of microorganism by in situ formation of conducting polymer within the cell structures (e.g. cell wall) was performed. The synthesis of polypyrrole (Ppy) within fungi (Aspergillus niger) cells was achieved. Two different Aspergillus niger strains were selected due to their ability to produce glucose oxidase, which initiated the Ppy formation through products of enzymatic reaction. The evolution of Ppy structural features was investigated by absorption spectroscopy, cyclic voltammetry and Fourier transform infrared spectroscopy.

The Arabidopsis onset of leaf death5 mutation of quinolinate synthase affects nicotinamide adenine dinucleotide biosynthesis and causes early ageing.

Leaf senescence in Arabidopsis thaliana is a strict, genetically controlled nutrient recovery program, which typically progresses in an age-dependent manner. Leaves of the Arabidopsis onset of leaf death5 (old5) mutant exhibit early developmental senescence. Here, we show that OLD5 encodes quinolinate synthase (QS), a key enzyme in the de novo synthesis of NAD. The Arabidopsis QS was previously shown to carry a Cys desulfurase domain that stimulates reconstitution of the oxygen-sensitive Fe-S cluster that is required for QS activity. The old5 lesion in this enzyme does not affect QS activity but it decreases its Cys desulfurase activity and thereby the long-term catalytic competence of the enzyme. The old5 mutation causes increased NAD steady state levels that coincide with increased activity of enzymes in the NAD salvage pathway. NAD plays a key role in cellular redox reactions, including those of the tricarboxylic acid cycle. Broad-range metabolite profiling of the old5 mutant revealed that it contains higher levels of tricarboxylic acid cycle intermediates and nitrogen-containing amino acids. The mutant displays a higher respiration rate concomitant with increased expression of oxidative stress markers. We postulate that the alteration in the oxidative state is integrated into the plant developmental program, causing early ageing of the mutant.