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Öğe Electrochemical based detection of microRNA, mir21 in breast cancer cells(Elsevier Advanced Technology, 2012) Kilic, Tugba; Topkaya, Seda Nur; Ariksoysal, Dilsat Ozkan; Ozsoz, Mehmet; Ballar, Petek; Erac, Yasemin; Gozen, OguzIn this work, a novel electrochemical microRNA (miRNA) detection method based on enzyme amplified biosensing of mir21 from cell lysate of total RNA was demonstrated. The proposed enzymatic detection method was detailed and compared with the conventional guanine oxidation based assay in terms of detection limit and specificity. For the detection of mir21, capture probes and/or cell lysates were covalently attached onto the pencil graphite electrode (PGE) by coupling agents of N-(dimethylamino)propyl-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). Having immobilized the capture probe onto the surface of PGE, hybridization was achieved with a biotinylated (from its 3' end) complementary target. Extravidin labeled alkaline phosphatase (Ex-Ap) binds to the biotinylated target due to the interaction between biotin-avidin and the enzyme converts electro-inactive alpha naphtyl phosphate (the substrate) to electro-active alpha naphtol (alpha-NAP, the product). alpha-NAP was oxidized at +0.23 V vs Ag/AgCl and this signal was measured by Differential Pulse Voltammetry (DPV). The signals obtained from alpha-NAP oxidation were compared for the probe and hybrid DNA. The specificity of the designed biosensor was proved by using non-complementary sequences instead of complementary sequences and the detection limit of the assay was calculated to be 6pmol for cell lysates. (C) 2012 Elsevier B.V. All rights reserved.Öğe Electrochemical Detection of a Cancer Biomarker mir-21 in Cell Lysates Using Graphene Modified Sensors(Wiley-V C H Verlag Gmbh, 2015) Kilic, Tugba; Erdem, Arzum; Erac, Yasemin; Seydibeyoglu, M. Ozgur; Okur, Salih; Ozsoz, MehmetIn the present study, the voltammetric and impidimetric detection of microRNA-21, mir-21 from cell lysates was investigated for the first time by using graphene modified disposable pencil graphite electrodes (GME). The surface characterization of GME was performed via electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Upon passive adsorption of inosine substituted antimicroRNA-21, antimir-21 probe, InP, onto the surface of GME and then solid phase hybridization of InP with mir-21, the target, the electrochemical detection was performed by using Differential Pulse Voltammetry (DPV) and EIS techniques. This developed biosensor, GME has presented a 2.77 times lower detection limit of 2.09 mg/mL (3.12 pmol) with respect to unmodified pencil graphite electrode (GE). Moreover it is capable of analyzing mir-21 in the cell lysates of mir-21 positive breast cancer cell line (MCF-7) contrast to mir-21 negative hepatoma cell line (HUH-7). The proposed electrochemical yes-no system does not require any purification and/or amplification step prior to fast detection of mir-21 from real samples.Öğe Label-Free Electrochemical Detection of MicroRNA-122 in Real Samples by Graphene Modified Disposable Electrodes(Electrochemical Soc Inc, 2016) Kilic, Tugba; Kaplan, Merve; Demiroglu, Sibel; Erdem, Arzum; Ozsoz, MehmetIn the present work, an electrochemical nucleic acid biosensor has been designed for the purpose of detection of miR-122 in real samples, i.e cell lysates. The fabrication of the biosensor has been done first by immobilization of complemantary anti-miR-122 to the surfaces of the graphene (GRP) modified pencil graphite (PGEs) electrodes then solid phase hybridization with either synthetic miR-122 or miR-122 included in total RNA isolated from HUH-7 cell line. The characterization of GRP modification onto the PGE surface has been proved via Raman spectroscopy analysis, Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV). The intrinsic guanine oxidation signal measured via DPV and the charge transfer resistance, R-ct values recorded via electrochemical circle fit option of EIS have been used for hybridization detection. The proposed biosensor with the limit of detection (LOD) 1 pmol is applicable for analysis of certain miRNAs as well as miR-122 from total RNAs isolated from cell lysates. (C) 2016 The Electrochemical Society. All rights reserved.Öğe microRNA biosensors: Opportunities and challenges among conventional and commercially available techniques(Elsevier Advanced Technology, 2018) Kilic, Tugba; Erdem, Arzum; Ozsoz, Mehmet; Carrara, SandroAs being the most extensively studied, non-coding, evolutionary conserved, post-transcriptional gene regulators of genome, microRNAs (miRNAs) have taken great attention among various disciplines due to their important roles in biological processes and link with cancer. Due to their diagnostic value, there have been many conventional methods used in detection of miRNAs including northern blotting, quantitative real time PCR (qRT-PCR) and microarray technology besides novel techniques based on various nanotechnology approaches and molecular biology tools including miRNA biosensors. The aim of this review is to explain the importance of miRNAs in biomedical field with an emphasis on early cancer diagnosis by overviewing both research based and commercially available miRNA detection methods in the last decade considering their strengths and weakness with an emphasis on miRNA biosensors.Öğe A new insight into electrochemical microRNA detection: A molecular caliper, p19 protein(Elsevier Advanced Technology, 2013) Kilic, Tugba; Topkaya, Seda Nur; Ozsoz, MehrnetmicroRNA (miRNA) has drawn a great attention in biomedical research due to its functions on biological processes. Detection of miRNAs is a big challenge since the amount present in real samples is very low and the length of them is short. In this study, for the first time an electrochemical biosensor for detection of mir21 using the oxidation signal of protein 19 (p19) as a molecular caliper was designed. The proposed method enables detection of mir21 in direct, rapid, sensitive, inexpensive and label-free way. Binding specificity of the p19 to 20-23 base pair length double stranded RNA (dsRNA) and direct/water-mediated intermolecular contacts between the fusion protein and miRNA allows detection of miRNA-antimiRNA hybrid structure. The detection of mir21 was achieved in picomole sensitivity through the changes of intrinsic p19 oxidation signals observed at +0.80 V with Differential Pulse Voltammetry (DPV) and the specifity of the designed sensor was proved by control studies. (C) 2013 Elsevier B.V. All rights reserved.Öğe A novel method for sensitive microRNA detection: Electropolymerization based doping(Elsevier Advanced Technology, 2017) Kaplan, Merve; Kilic, Tugba; Guler, Gunnur; Mandli, Jihane; Amine, Aziz; Ozsoz, MehmetIn the proposed study, for the first time, sensitive electrochemical detection of a breast cancer biomarker microRNA (miRNA), mir-21 was achieved via electropolymerized polypyrrole (PPy) modified pencil graphite electrodes (PPy/PGE). The detection of hybridization of electrochemically doped probe miRNA, antimir-21, with its complementary target, mir-21 was monitored by either electrochemical impedance spectroscopy (EIS) via comparison of charge transfer resistance (Rat) values before and after hybridization or by electrochemical reduction signal of an hybridization indicator, Meldola's blue (MDB). The study covers all the optimization steps for hybridization procedure and electropolymerization of pyrrole as well as detection from real samples of breast cancer cell line, MCF-7. The designed sensor shows a high selectivity and a low detection limit of 0.17 nM thanks to electrical conductivity and porous structure of PPy.Öğe OPTIMIZATION OF THERMOSTABLE ALPHA-AMYLASE PRODUCTION FROM GEOBACILLUS SP. D413(Slovak Univ Agriculture Nitra, 2016) Caliskan Ozdemir, Sennur; Coleri Cihan, Arzu; Kilic, Tugba; Cokmus, CumhurThe qualitative and quantitative a-amylase production capacities of six thermophilic bacilli were screened. Geobacillus sp. D413 was selected for enzyme optimization, as it displayed higher alpha-amylase activity. The maximum enzyme activities of D413 and G. stearothermophilus ATCC 12980(T) were observed at the time of 72 h. While the optimal pH of medium for bacterial growth and enzyme production of D413 (pH 7.0) differed from ATCC 12980(T) (pH 8.0), the optimal temperature for enzyme production was 55 degrees C for both. The effects of various carbon and nitrogen sources were determined by changing their concentrations. The highest bacterial growth and enzyme production were sustained by the starch and maltose containing medium. Both bacterial growth and enzyme production were inhibited by NH4Cl. D413 and ATCC 12980T amylases showed optimal activity at 65 degrees C, pH 9.0 and at 65 degrees C, pH 7.5, respectively. They remained active over temperature and pH ranges of 45-75 degrees C and 4.0-10.5. Their activities retained 65% and 54% when incubated at 75 degrees C for 10 min and 98-86.5% and 95-84.5% at pH 4.0-10.5 for 15 h at 37 degrees C. In conclusion, the alpha-amylase production conditions of D413 have been optimized which can be useful in biotechnological processes such as hydrolysis of starch to glucose.Öğe Proteomic-based biomarker discovery for development of next generation diagnostics(Springer, 2017) Khalilpour, Akbar; Kilic, Tugba; Khalilpour, Saba; Alvarez, Mario Moises; Yazdi, Iman K.In the post-genome age, proteomics is receiving significant attention because they provide an invaluable source of biological structures and functions at the protein level. The search for disease-specific biomarkers for diagnostic and/or therapeutic applications is one of the areas that proteomics is having a significant impact. Thus, the identification of a "good" biomarker enables a more accurate early diagnosis and prognosis of disease. Rapid advancements in mass spectrometry (MS) instrumentation, liquid chromatography MS (LCMS), protein microarray technology, and other protein profiling methodologies have a substantial expansion of our toolbox to identify disease-specific protein and peptide biomarkers. This review covers a selection of widely used proteomic technologies for biomarker discovery. In addition, we describe the most commonly used approaches for diagnosis based on proteomic biomarkers and further discuss trends and critical challenges during development of cost-effective rapid diagnostic tests and microfluidic diagnostic systems based on proteomic biomarkers.Öğe Surface Characterization Techniques(Wiley-V C H Verlag Gmbh, 2017) Erdogan, Gokhan; Guler, Gunnur; Kilic, Tugba; Kilic, Duygu O.; Erdogan, Beyhan; Tosun, Zahide; Kivrak, Hilal D.; Turkan, Ugur; Ozcan, Fatih; Gursoy, Mehmet; Karaman, Mustafa; Gursoy, M; Karaman, M
