In situ detection of transrenal gene mutations without DNA isolation and amplification using Array Piezoelectric Plate Sensor (PEPS)

Tarih: 17.11.2015
Yer: Boğaziçi Üniversitesi Kandilli Kampüsü AZ-19

~~In situ detection of transrenal gene mutations without DNA isolation and amplification using Array Piezoelectric Plate Sensor (PEPS)
Ceyhun Ekrem KIRIMLI, PhD.
Lakehead University, Canada

About the Seminar:
Low molecular weight (LMW) DNA fragments from distant organs can pass through kidneys and exist in urine as ”transrenal DNA”. Transrenal DNA provides a noninvasive means to access cell-free DNAs from distant organs such as liver, pancreas, colon that are otherwise difficult to obtain. The piezoelectric plate sensor (PEPS) developed is based on 8 μm thick highly piezoelectric lead magnesium niobate-lead titanate (PMN-PT) freestanding layer. For detection, receptor specific to a target analyte is immobilized on the PEPS. Binding of the target analyte to the receptor on the PEPS surface decreases its resonance frequency. In-situ, label-free detection of the target analyte is achieved by monitoring the PEPS resonance frequency shift.
In order to achieve the goal of developing PEPS as a tool for rapid, label-free, multiplexed and highly sensitive biosensor capable of detecting transrenal gene mutations of distant organs in urine, 1762T/1764A double mutation of hepatitis B virus (HBVDM), was chosen as a model. HBVDM a risk factor associated with hepatocellular carcinoma (HCC). Kras point mutation at codon 12, is another model biomarker present in 50% of colorectal carcinomas (CRC). To achieve the goal, a signal processing algorithm was developed to reduce noise in the resonance spectrum to increase the resolution of the detection resonance frequency shift. A flow system was implemented with temperature-controlled compartments to effectively denature the naturally double stranded target DNA so that the target DNA can be detected without the need for DNA isolation, concentration, and amplification. The use of temperature, flow speed and locked nucleic acid (LNA) were exploited to enhance the detection specificity of the mutated DNA against the wild type and an array of PEPS is used to detect multiple target DNAs simultaneously. Results indicated that PEPS could directly detect Kras point mutations in urine with 0.1 aM (60 copies/ml) sensitivity and 1:1000 mutant to wild type specificity without labeling, DNA isolation and amplification.

 About the Speaker
Ceyhun Ekrem KIRIMLI received his PhD. in Biomedical Engineering from the School of Biomedical Engineering, Science and Health Systems of Drexel University. His research involved development of a highly sensitive Piezoelectric Plate Sensor (PEPS, patented), and direct label free detection of bacterial and human DNA without amplication and isolation using PEPS (patents pending). He received a Calhoun Fellowships from the Provost Office of Drexel University covering his tuition and stipend and worked as a full time research/teaching assistant in Drexel University. He was offered a Postdoctoral fellowship from Lakehead University to work with Dr. Aicheng Chen (Fellow of the Royal Society of Chemistry, and International Society of Electrochemistry) on the development of Potentiostat assisted automated Fourier Transform Infrared Spectroscopy system. He received his M.Sc. from the Institute of Biomedical Engineering of Bogazici University. During his master’s he developed a working memory performance assessment test while monitoring the prefrontal cortex hemodynamics by means of Functional Near Infrared Spectroscopy (FNIRS). He worked as a visiting researcher at Yale University with Nobel prize laureate Dr. Sidney Altman on assaying the enzymatic activity of E. Coli RNase P mRNA with point mutations using 32P radioisotope labeled pTyr rRNA as substrate. He received his B.Sc. from the Department of Molecular Biology and Genetics in Bogazici University.