Emine Guler

Polypeptide Functional Surface for the Aptamer Immobilization: Electrochemical Cocaine Biosensing

Electroanalytical technologies as a beneficial subject of modern analytical chemistry can play an important role for abused drug analysis which is crucial for both legal and social respects. This article reports a novel aptamer-based biosensing procedure for cocaine analysis by combining the advantages of aptamers as selective recognition elements with the well-known advantages of biosensor systems such as the possibility of miniaturization and automation, easy fabrication and modification, low cost, and sensitivity. In order to construct the aptasensor platform, first, polythiophene bearing polyalanine homopeptide side chains (PT-Pala) was electrochemically coated onto the surface of an electrode and then cocaine aptamer was attached to the polymer via covalent conjugation chemistry. The stepwise modification of the surface was confirmed by electrochemical characterization. The designed biosensing system was applied for the detection of cocaine and its metabolite, benzoylecgonine (BE), which exhibited a linear correlation in the range from 2.5 up to 10 nM and 0.5 up to 50 μM for cocaine and BE, respectively. In order to expand its practical application, the proposed method was successfully tested for the analysis of synthetic biological fluids.

Electrochemical deposition of polypeptides: bio-based covering materials for surface design

A simple and efficient approach for the electrochemical deposition of polypeptides as bio-based covering materials for surface design is described. The method involves N-carboxyanhydride (NCA) ring-opening polymerization from its precursor to form a thiophene-functionalized polypeptide macromonomer (T-Pala), followed by electropolymerization. The obtained conducting polymer, namely polythiophene-g-polyalanine (PT-Pala), was characterized and utilized as a matrix for biomolecule attachment. The biosensing applicability of PT-Pala was also investigated by using glucose oxidase (GOx) as a model enzyme to detect glucose. The designed biosensor showed a very good linearity for 0.01–1.0 mM glucose. Finally, the antimicrobial activities of newly synthesized T-Pala and PT-Pala were also evaluated by using the disc diffusion method.

A novel functional conducting polymer as an immobilization platform

Here, we present the fabrication of conducting polymer based enzymatic and microbial biosensors. To obtain immobilization platforms for both pyranose oxidase (PyOx) and Gluconobacter oxydans, the graphite electrode surface was modified with the polymer of 4-amino-N-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)benzamide (HKCN) which has free amino groups on the surface for further bioconjugation reactions with the biomolecules. Initially, the electrode surface was covered with HKCN via electropolymerization. Then, either PyOx or G. oxydans cell was stabilized using glutaraldehyde as a cross-linker. After optimization of biosensors, analytical characterization and surface imaging studies were investigated. The change of current depends on glucose concentration between 0.05-1.0mM and 0.25-2.5mM with HKCN/PyOx and HKCN/G. oxydans biosensors in batch systems. Also, the calibration graphs were obtained for glucose in FIA mode, and in this case, linear ranges were found to be 0.01-1.0mM and 0.1-7.5mM for HKCN/PyOx and HKCN/G. oxydans, respectively.

Bio-active nanoemulsions enriched with gold nanoparticle, marigold extracts and lipoic acid: In vitro investigations.

A novel and efficient approach for the preparation of enriched herbal formulations was described and their potential applications including wound healing and antioxidant activity (cell based and cell free) were investigated via in vitro cell culture studies. Nigella sativa oil was enriched with Calendula officinalis extract and lipoic acid capped gold nanoparticles (AuNP-LA) using nanoemulsion systems. The combination of these bio-active compounds was used to design oil in water (O/W) and water in oil (W/O) emulsions. The resulted emulsions were characterized by particle size measurements. The phenolic content of each nanoemulsion was examined by using both colorimetric assay and chromatographic analyses. Two different methods containing cell free chemical assay (1-diphenyl-2-picrylhydrazyl method) and cell based antioxidant activity test were used to evaluate the antioxidant capacities. In order to investigate the bio-activities of the herbal formulations, in vitro cell culture experiments, including cytotoxicity, scratch assay, antioxidant activity and cell proliferation were carried out using Vero cell line as a model cell line. Furthermore, to monitor localization of the nanoemulsions after application of the cell culture, the cell images were monitored via fluorescence microscope after FITC labeling. All data confirmed that the enriched N. sativa formulations exhibited better antioxidant and wound healing activity than N. sativa emulsion without any enrichment. In conclusion, the incorporation of AuNP-LA and C. officinalis extract into the N. sativa emulsions significantly increased the bio-activities. The present work may support further studies about using the other bio-active agents for the enrichment of herbal preparations to strengthen their activities.

Synthesis of an amine-functionalized naphthalene-containing conducting polymer as a matrix for biomolecule immobilization

N-functionalized dithienopyrroles (DTP-NH2) were synthesized and electropolymerized onto a graphite electrode as a novel conducting polymer matrix for biomolecule immobilization. 1H-NMR and 13C-NMR were utilized to investigate the characteristics of the monomer. After that, glucose oxidase (GOx) was immobilized onto the amino-functionalised matrix by means of glutaraldehyde. The surface morphologies of both DTP-NH2 and DTP-NH2–GOx were visualised by using SEM and fluorescence microscopy. The chronoamperometric signals of the electrochemical DTP-NH2–GOx biosensors were measured by monitoring the O2 consumption during an enzymatic reaction in the presence of glucose at −0.7 V. After the optimization of the pH and scan number of the polymer deposition in batch mode, the DTP-NH2–GOx biosensor was also tested in Flow Injection Analysis (FIA) mode. The DTP-NH2–GOx biosensors had a very good linearity between 0.05 and 1.0 mM, and between 0.1 and 2.5 mM for glucose in batch and FIA modes, respectively. Finally, it was applied for glucose analysis in real samples where commercial glucose kits were used as the reference method to verify the data obtained with the proposed biosensor.

Gold nanoparticle loaded phytosomal systems: synthesis, characterization and in vitro investigations

Most medicinal and pharmaceutical herbal extracts are poorly soluble in aqueous moieties and have reduced adsorption by living cells. Liposomal encapsulation of those so called phytosomes could be a solution to overcome this problem. Meanwhile, much research shows that metallic nanoparticles such as gold nanoparticles (AuNPs) exhibit biological activity such as wound healing and antioxidant properties on living cells. Here, we constructed a novel liposomal formulation by encapsulating both Calendula officinalis extract and AuNPs. After the preparation of vesicles using the traditional thin film hydration method within extrusion, the resulting AuNP–phytosomes were characterized by dynamic light scattering size measurements, zeta potential and atomic force microscopy. These vesicles are less than 100 nm in size and have a high encapsulation efficiency for chlorogenic acid and quercetin as the model major molecules of Calendula extract. Furthermore, AuNP–phytosomes exhibited antioxidant and wound healing activity significantly according to the free forms of each encapsulated material and the plain liposome as well as the phytosome form. Moreover, the cellular interactions of the vesicles were monitored using the nano-vesicles prepared by Texas-Red labelled lipids under fluorescence microscopy.

Ruthenium (II) complexes of thiosemicarbazone: synthesis, biosensor applications and evaluation as antimicrobial agents.

A conformationally rigid half-sandwich organoruthenium (II) complex [(η(6)-p-cymene)RuClTSC(N-S)]Cl, (1) and carbonyl complex [Ru(CO)Cl(PPh3)2TSC(N-S)] (2) have been synthesized from the reaction of [{(η(6)-p-cymene)RuCl}2(μ-Cl)2] and [Ru(H)(Cl)(CO)(PPh3)3] with thiophene-2-carboxaldehyde thiosemicarbazon (TSC) respectively and both novel ruthenium (II) complexes have been characterized by elemental analysis, FT-IR and NMR spectroscopy. The peripheral TSC in the complexes acts as an electrochemical coupling unit providing the ability to carry out electrochemical deposition (ED) and to form an electro-deposited film on a graphite electrode surface. The biosensing applicability of complexes 1 and 2 was investigated by using glucose oxidase (GOx) as a model enzyme. Electrochemical measurements at -0.9V versus Ag/AgCl electrode by following the ED Ru(II) reduction/oxidation due to from the enzyme activity, in the presence of glucose substrate. The designed biosensor showed a very good linearity for 0.01-0.5mM glucose. The in vitro antimicrobial activities of complexes 1 and 2 were also investigated against nine bacterial strains and one fungus by the disc diffusion test method. No activity was observed against the Gram-negative strains and fungus, whereas complex 1 showed moderate antibacterial activities against Gram-positive bacterial strains.

Complex Structured Fluorescent Polythiophene Graft Copolymer as a Versatile Tool for Imaging, Targeted Delivery of Paclitaxel, and Radiotherapy.

Advances in polymer chemistry resulted in substantial interest to utilize their diverse intrinsic advantages for biomedical research. Especially, studies on drug delivery for tumors have increased to a great extent. In this study, a novel fluorescent graft copolymer has been modified by a drug and targeting moiety and the resulting structure has been characterized by alterations in fluorescent intensity. The polythiophene based hybrid graft copolymer was synthesized by successive organic reactions and combination of in situ N-carboxy anhydride (NCA) ring opening and Suzuki coupling polymerization processes. Initially, targeted delivery of the graft copolymer was investigated by introducing a tumor specific ligand, anti-HER2/neu antibody, on the structure. The functionalized polymer was able to differentially indicate HER2-expressing A549 human lung carcinoma cells, whereas no signal was obtained for Vero, monkey kidney epithelial cells, and HeLa, human cervix adenocarcinoma cells. After integrating paclitaxel into the structure, cell viability, cell cycle progression, and radiosensitivity studies demonstrate HER2/neu targeting polymers were most effective to inhibit cell proliferation. Importantly, the graft copolymer used had no cytotoxic effects to cells, as evidenced by cell viability and cell cycle analysis. This work clearly confirms that a specially designed and fabricated graft copolymer with a highly complex structure is a promising theranostic agent capable of targeting tumor cells for diagnostic and therapeutic purposes.

An aptamer folding-based sensory platform decorated with nanoparticles for simple cocaine testing

The consumption of illicit drugs such as cannabis, cocaine, and amphetamines is still a major health and social problem, creating an abuse in adults especially. Novel techniques which estimate the drug of abuse are needed for the detection of newly revealed psychoactive drugs. Herein, we have constructed a combinatorial platform by using quantum dots (QDs) and gold nanoparticles (AuNPs) as well as a functional aptamer which selectively recognizes cocaine and its metabolite benzoylecgonine (BE). We have called it an aptamer folding-based sensory device (AFSD). For the fabrication of AFSD, QDs were initially immobilized onto the poly-L-lysine coated μ-well surfaces. Then, the AuNP-aptamer conjugates were bound to the QDs. The addition of cocaine or BE caused a change in the aptamer structure which induced the close interaction of AuNPs with the QDs. Hence, quenching of the fluorescence of QDs was observed depending on the analyte amount. The linearity of cocaine and BE was 1.0-10 nM and 1.0-25 μM, respectively. Moreover, the limits of detection for cocaine and BE were calculated as 0.138 nM and 1.66 μM. The selectivity was tested by using different interfering substances (methamphetamine, bovine serum albumin, codeine, and 3-acetamidophenol). To investigate the use of AFSD in artificial urine matrix, cocaine/BE spiked samples were applied. Also, confirmatory analyses by using high performance liquid chromatography were performed. It is shown that AFSD has a good potential for testing the cocaine abuse and can be easily adapted for detection of various addictive drugs by changing the aptamer according to desired analytes. Copyright

Double fluorescence assay via a β-cyclodextrin containing conjugated polymer as a biomimetic material for cocaine sensing

A double fluorescence μ-well assay that exploits a novel conjugated polymer containing cyclodextrin (CD) as the key component is reported. For the construction of the cocaine bioassay, poly(p-phenylene) with CD units in the main-chain and poly(ethylene glycol) side chains (PPP-CD-g-PEG) was first prepared by Suzuki coupling polymerization and coated on each well as a biomimetic material. Although the polyphenylene backbone is responsible for the fluorescence properties without an additional fluorophore, PEG and CD provide water solubility and selective complexation with cocaine, respectively. A cocaine antibody was used as a secondary recognition compound after labelling with quantum dots (QDs). Most notably, we show that the two-color fluorescence nature of the assay facilitates double measurement from the same sample and the described strategy can be adapted to various sensing systems.