Idris Yazgan

Biosensor for selective detection of E. coli in spinach using the strong affinity of derivatized mannose with fimbrial lectin.

Escherichia coli (E. coli) contamination in foods and water resources represents a major threat for human health and the environment. This work exploits the strong affinity of mannose-containing oligosaccharides with the fimbrial lectin of E. coli to design novel biosensors. Modified carbohydrate ligands were synthesized by introducing phenyl residues and aliphatic chains to mannose via reductive amination in order to increase both the affinity and selectivity to E. coli compared to other pathogenic bacteria. The synthesized ligands include p-thiolphenyl aminomannose (PTAM), p-carboxyphenyl aminomannose (PCAM), 1-deoxy-1-aminomannopyranoside (DAMP), glucosamine and low molecular weight chitosan bonded to mercapto undecanoic acid. The structures of the ligands were confirmed using (1)H NMR and 1H, (13)C, COZY NMR, and ESI/MS. The ligands were immobilized onto gold electrodes and SPR surfaces using-mercaptoundecanoic acid with glycine as deactivating agent. Two detection mechanisms were tested: (i) metal-enhanced electrochemical detection (MED) and (ii) label-free surface plasmon resonance (SPR) detection. The introduction of phenyl residues and aliphatic side groups to the mannose-containing oligosaccharides produced extremely high affinity for E. coli with detection limit of 1 cfu/mL. The relative selectivity of these ligands for E. coli, Citrobacter freundii, Staphylococcus epidermidis were 100%, 2.6% and 8.6% respectively. The biosensors were validated using spinach leaves at 3.0 cfu/mL. The work provides a generic biosensor for other pathogenic bacteria by enabling multivalent binding, immediate recognition for pathogens as well as inhibition of bacterial growth.

Greener synthesis and characterization, antimicrobial and cytotoxicity studies of gold nanoparticles of novel shapes and sizes

We hereby present a novel approach for the synthesis of gold nanoparticles (AuNPs) using water soluble, naturally-derived flavonoids. Quercetin pentaphosphate (QPP), quercetin sulfonic acid (QSA) and apigenin triphosphate (ATRP) were utilized as reducing agents and stabilizers for the gold nanoparticle synthesis. Synthesis was achieved at room temperature using water as a solvent and it requires no capping agents. The AuNPs were characterized using UV-vis, X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive absorption spectroscopy (EDS), high resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED). The resulting AuNPs were spherical, triangular, cubicle, hexagonal and rectangular in shape. The average particle sizes of 4.85 nm, 9.56 nm and 13.54 nm were obtained for the nanoparticles derived from QPP, ATRP and QSA respectively. The surface plasmon resonance peak of the AuNPs derived from QSA, ATRP and QPP was observed at 541 nm, 544 nm and 547 nm respectively. The AuNPs exhibited excellent antibacterial activities of 99.9%, 100% and 99.9% growth inhibition for Escherichia coli ATCC® 25922™, Staphylococcus epidermidis ATCC® 12228™ and Citrobacter freundii ATCC® 8090 at 104 cfu inoculations. The AuNPs were observed to retain stability after 150 days compared to those reported using conventional approaches of 30 days. This work also provides insights into the mechanism of flavonoid-based nanoparticle synthesis while eliminating the use of hazardous and toxic organic solvents and adopting the use of water as a solvent.

Synthesis and antibacterial characterization of sustainable nanosilver using naturally-derived macromolecules.

Greener nanosynthesis utilizes fewer amounts of materials, water, and energy; while reducing or replacing the need for organic solvents. A novel approach is presented using naturally-derived flavonoids including Quercetin pentaphosphate (QPP), Quercetin sulfonic acid (QSA) and Apigenin Triphosphate (ATRP). These water soluble, phosphorylated flavonoids were utilized both as reducing agent and stabilizer. The synthesis was achieved at room temperature using water as a solvent and it requires no capping agents. The efficiency of the resulting silver nanoparticle synthesis was compared with naturally-occurring flavonoid such as Quercetin (QCR). Results show that QCR reduced Ag(+) faster followed by QPP, QSA and ATRP respectively. This is the first evidence of direct utilization of QCR for synthesis of silver nanoparticles (AgNPs) in water. The percentage conversion of Ag(+) to Ag(0) was determined to be 96% after 35min. The synthesized nanoparticles were characterized using Transmission electron microscopy (TEM), Energy dispersive absorption spectroscopy (EDS), UV-vis spectroscopy, High resolution TEM (HR-TEM) with selected area electron diffraction (SAED). The particle sizes ranged from 2 to 80nm with an average size of 22nm and in the case of ATRP, the nanoparticle shapes varied from spherical to hexagonal with dispersed particle size ranging from 2 to 30nm. Crystallinity was confirmed by XRD and the SAED of (111), (200), and the fringes observed in HRTEM images. Results were in agreement with the UV resonance peaks of 369-440nm. The particles also exhibit excellent antibacterial activity against Staphylococcus epidermidis, Escherichia coli and Citrobacter freundii in water.

Use of Pyranose Oxidase Enzyme in Inhibitor Biosensing

Abstract An inhibition based biosensing system was developed for the determination of glutathione (GSH) and ethanol (EtOH) as pyranose oxidase (PyOx) inhibitors. The PyOx was immobilized in carbon paste electrode and the amperometric detection of hydrogen peroxide through the enzymatic reaction was monitored at + 0.9 V versus Ag/AgCl. In addition to the optimization studies, analytical characteristics and the effect of various compounds on the biosensor response were researched. Finally, the proposed system was applied to analyze GSH and EtOH in real matrices.

Modification of chitosan-bead support materials with l-lysine and l-asparagine for α-amylase immobilization

Maltose syrups have got wide-range utilizations in a variety of applications from bakery to drug-development. α-Amylases are among the most widely utilized industrial enzymes due to their high specificity in production of maltose syrup from starch. However, enzymes are not stable in ex vivo conditions towards alteration in pH, temperature, and such other parameters as high salt concentrations and impurities, where immobilization is required to advance the stability of the enzyme with which approach the requirement of isolation of the enzyme from media is eliminated as well. In this study, Termamyl® α-amylase was immobilized on the none-modified chitosan beads (NMCB), l-lysine-modified chitosan beads (LMCB), and l-asparagine-modified chitosan beads (AMCB) to assess effects of the support material on optimum conditions and kinetic parameters of the α-amylase activity in production of maltose from starch. Immobilization on NMCB, LMCB, and AMCB puts a strong influence on optimum pH, optimum temperature, stability, and kinetic parameters of α-amylase. Modification of chitosan beads with l-lysine and l-asparagine dramatically altered the overall immobilization yield, and enzyme’s response to pH and temperature variations and the kinetic parameters. AMCB provided the best immobilization yield (49%), while LMCB only improved the yield by 2% from 22 to 24%.

Selective Sensing and Imaging of Penicillium italicum Spores and Hyphae Using Carbohydrate–Lectin Interactions

The blue-green mold Penicillium italicum is among the most problematic post-harvest plant infections limiting the integrity of citrus and many other crops during storage and transportation, but there is no sensor for its on-site or field detection. We hereby, for the first time, report the development of novel biomolecular sensor for assessing the presence of P. italicum spores and hyphae using carbohydrate-lectin recognitions. Two approaches were developed: (i) lateral tests using standalone poly(amic) acid (PAA) membranes and glass surfaces and (ii) quantitative tests on 96-well polystyrene plates and paper electrodes. In both cases, the surfaces were functionalized with novel derivatized sugar based ligands while staining was performed with gold nanoparticles. Both approaches provided strong signals for 104 spores/mL of P. italicum isolated from experimentally infected lemons as the lowest-reliable concentration. The 96-well plate-based gave the most sensitive detection with a 4 × 102 spores/mL limit of detection, a linear dynamic range between 2.9 × 103 and 6.02 × 104 spores/mL ( R2 = 0.9939) and standard deviation of less than 5% for five replicate measurements. The selectivity of the ligands was tested against Trichaptum biforme, Glomerulla cingulata ( Colletotrichum gloeosporioides), and Aspergillus nidulans fungi species. The highest selectivity was obtained using the sugar-based gold-nanoparticles toward both the spores and the hyphae of P. italicum. The advanced specificity was provided by the utilized sugar ligands employed in the synthesis of gold nanoparticles and was independent from size and shapes of the AuNPs. Accuracy of the sensor response showed dramatic dependence on the sample preparation. In the case of 5-10 min centrifugation at 600 rpm, the spores can be isolated free from hyphae and conidiophore, for which spiked recovery was up to 95% (std ±4). In contrast, for gravity-based precipitation of hyphae, the spiked recovery was 88% (std 11).

High performance paper-based microbial fuel cells using nanostructured polymers

In this work, we report paper-based microbial fuel cells (MFCs) that produce high power and current densities from one drop of bacteria-containing liquid. The devices feature (i) a simple and versatile fabrication technique by using paper as a substrate and (ii) an exceptional performance by incorporating novel nanostructured polymers, PAA-Poly (amic) acid) and PPDD-Poly(pyromellitic dianhydride-p-phenylene diamine), into the paper substrate. Four 3-D MFC configurations were designed by using different numbers of 2-D sheets of paper layers. Each device integrated four functional compartments (i.e. anode, reservoir, proton exchange membrane, and air-cathode) into one, two, three or four paper layers, respectively. The nanostructured polymers were engineered as a proton exchange membrane to enhance ion traveling efficiency or an oxygen mitigating layer to prevent diverting electrons away from the anode. Among the four MFC devices with different numbers of layers, two-layer paper-based MFC generated the highest current density of 47|UA/cm2 and power density of 4|UW/cm2, both of which are substantially greater than achieved by previous paper-based MFCs and even comparable to that of conventional micro-sale counterparts.