Huseyin Akbulut

Synthesis and characterization of conducting polymers containing polypeptide and ferrocene side chains as ethanol biosensors

A novel approach for the fabrication of a biosensor from a conducting polymer bearing polypeptide segments and ferrocene moieties is reported. The approach involves the electrochemical copolymerization of the electroactive polypeptide macromonomer and independently prepared ferrocene imidazole derivative of dithiophene, on the electrode surface. The polypeptide macromonomer was synthesized by the simultaneous formation of N-carboxyanhydride (NCA) and ring opening polymerization of N-Boc-L-lysine (α-amino acid of the corresponding NCA) using an amino functional bis-EDOT derivative (BEDOA-6) as an initiator. Alcohol oxidase was then covalently immobilized onto the copolymer coated electrode using glutaraldehyde as the crosslinking agent. The intermediates and final conducting copolymer before and after enzyme immobilization were fully characterized by FT-IR, 1H-NMR, GPC, cyclic voltammetry, SEM and EIS analyses. The newly designed biosensor which combined the advantages of each component was tested as an ethanol sensing system offering fast response time (9 s), wide linear range (0.17 mM and 4.25 mM) and low detection limit (0.28 mM) with a high sensitivity (12.52 μA mM−1 cm−2). Kinetic parameters KappM and Imax were 2.67 mM and 2.98 μA, respectively. The capability of the biosensor in determining ethanol content in alcoholic beverages was also demonstrated.

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.

Synthesis, characterization and targeted cell imaging applications of poly(p-phenylene)s with amino and poly(ethylene glycol) substituents

A novel approach for bioconjugation associated with a fluorescent conjugated polymer is demonstrated. For this purpose, a conjugated polymer, poly(p-phenylene) (PPP), with lateral substituents, namely primary amino groups and poly(ethylene glycol) (PEG) chains, as a potential building block for polymer bioconjugates was synthesized and characterized. The synthesis was achieved through Suzuki polycondensation reaction in the presence of Pd(PPh3)4 catalyst by using independently prepared PEG and amino functionalized dibromo benzenes in conjunction with benzene diboronic acid. For the evaluation of the bioactive PPP labeled with folic acid (FA) as a potential targeted cell imaging probe, HeLa and A549 cancer cells were used. Cytotoxicity assay showed that the polymer was not toxic to either of the cells. Additionally, the fluorescence images showed that, depending on the level of the FA receptors on the cell surfaces, the fluorescent intensity in HeLa cells was obviously higher than A549 cells when treated with FA conjugated PPP-NH2-g-PEG polymer. The resulting FA/PPP-NH2-g-PEG conjugate was successfully used as a bioconjugate for targeting and specifically imaging FA receptor positive HeLa human cervical cancer cells.

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.

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.

Polypeptide with electroactive endgroups as sensing platform for the abused drug 'methamphetamine' by bioelectrochemical method.

Affinity-type sensors have emerged as outstanding platforms in the detection of diagnostic protein markers, nucleic acids and drugs. Thus, these novel platforms containing antibodies could be integrated into the monitoring systems for abused drugs. Herein, we established a novel detection platform for the analysis of a common illicit drug; methamphetamine (METH). Initially, a fluorescent-labeled polypeptide (EDOT-BTDA-Pala), derived from L-alanine N-carboxyanhydride (L-Ala-NCA) via ring-opening polymerization using 4,7-bis(2,3-dihydrothieno[3,4-b][1,4]dioxin-5-yl)benzo[c][1,2,5]thiadiazole-5,6-diamine (EDOT-NH2-BTDA) as initiator, was employed as a glassy carbon electrode (GCE) covering host, in order to immobilize the METH-selective antibody. Prior to the examination of analytical features, GCE/EDOT-BTDA-Pala/Antibody surface was successfully characterized in the way of electrochemical (cyclic voltammetry and electrochemical impedance spectroscopy) and microscopic techniques (scanning electron microscopy and fluorescence microscopy). As for the analytical characterization, linearity and limit of detection (LOD) were found as 10-100µg/mL with an equation of y=0.0429x-0.2347, (R2=0.996) and 13.07µg/mL, respectively. Moreover, sample application using artificial urine, saliva and serum samples spiked with METH (10, 25, 50µg/mL) were performed and LC-MS/MS system was used for further confirmation. The described platform can be adapted to monitor the other types of abused drugs by using suitably selected biorecognition elements.

Poly(p-phenylene) with Poly(ethylene glycol) Chains and Amino Groups as a Functional Platform for Controlled Drug Release and Radiotherapy

Conventional cancer treatments such as chemotherapy, radiotherapy, or combination of these two result in side effects, which lower the quality of life of the patients. To overcome problems with these methods, altering the drug properties by conjugating them to carrier polymers has emerged. Such polymeric carriers also hold the potential to make tumor cells more sensitive to radiation therapy. Herein, poly(p-phenylene) (PPP) polymer with poly(ethylene glycol) (PEG) chains and primary amino groups (PPP-NH2 -g-PEG) is synthesized and conjugated with anticancer drug Doxorubicin (DOX). pH dependent drug release experiments are performed at pH 5.3 and pH 7.4, respectively. Cell viability studies on human cervix adenocarcinoma cells show that lower doses of DOX inhibit cell proliferation when conjugated with nontoxic doses of PPP-NH2 -g-PEG polymer. Additionally, PPP-NH2 -g-PEG/Cys/DOX bioconjugate significantly increases radiosensitive properties of DOX. It is possible to use lower doses of DOX when conjugated to PPP-NH2 -g-PEG in combination with radiotherapy.

Synthesis and application of a novel poly-L-phenylalanine electroactive macromonomer as matrix for the biosensing of ‘Abused Drug’ model

Polypeptide-functionalized macromonomers are very fascinating candidates for the modification of various surfaces of different sizes as well as geometry, and have attracted considerable attention for biomolecule stabilization, biomedical device fabrication and bioanalytical applications. In the present work, synthesis and characterization of a novel poly-L-phenylalanine-bearing electroactive macromonomer (EDOT–BTDA–PPhe) were carried out. In the following steps, a glassy carbon electrode was covered with this material, and then cocaine aptamer was immobilized to obtain a biofunctional surface for the biosensing of ‘Abused Drug’ model. Aptamers attached to polypeptide side chains on the macromonomer with good orientation are induced for conformational change into three-way junction form as a result of selective binding of cocaine or its metabolite. This aptamer folding-based conformational response provides detectable signals due to formation of a compact interface which restricts electron transfer of redox probe. The stepwise modification of the surface was confirmed by electrochemical techniques. At the final step, the aptasensor was applied for the electrochemical detection of cocaine and its major metabolite, benzoylecgonine (BE), which exhibited a linear correlation between 1.0 and 10 nM and between 0.5 and 10 μM respectively. The proposed methods were successfully employed for the analysis of synthetic biological fluids.

Functional poly(p-phenylene)s as targeting and drug carrier materials

ABSTRACT Polymers have a substantial attention in drug delivery systems owing to the diverse intrinsic advantages. It is important to carry the drug to the target site and release to exert its effects. Herein, poly(p-phenylene)s with amino and poly(ethylene glycol) substituents (PPP-NH2-g-PEG) were used as a carrier for doxorubicin (DOX), an anticancer drug, and haloperidol, a sigma receptor targeting ligand. Both human cervix adenocarcinoma cell line (HeLa) and human keratinocyte cell line (HaCaT) having different Sigma receptor 1 (SigmaR1) expression levels were compared. HeLa was found to express twofold SigmaR1 compared to HaCaT cells. Cell imaging studies showed that, DOX cell uptake was higher in HeLa cells when targeted with haloperidol. GRAPHICAL ABSTRACT