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GG9.09 - Breast Cancer Detection using Charge Sensors Coupled to DNA Monolayer 
Date/Time:
April 10, 2015   4:30pm - 4:45pm
 
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DNA-based electrochemical sensors can be defined as nucleic acid layers with electrochemical transducers. DNA is especially appropriate for biosensing applications because interactions between complementary sequences are specific and robust to provide a simple and accurate biosensor for patient diagnosis. Electrochemical methods are appropriate for DNA diagnostics, giving a direct electronic signal [1]. But, there is still an extensive discussion about how charge transport occurs over DNA distance. MicroRNAs are small sequences that regulate a wide range of cellular processes. There has been a huge interest in studying their expression in human cancers [2]. We proposed to transform MicroRNA into DNA and develop a �MicroDNA� electrochemical biosensor to detect miR-200a sequence (22-mer) related with breast cancer. Au electrodes were cleaned using standard procedures [3] and experiments were performed at room temperature. 1 �M thiol � modified single-stranded probe sequence was dissolved in a pH 7 DNA solution (1 M phosphate buffer, 1 M NaCl, 5 mM MgCl2 and 1 mM EDTA), and immobilized for 18 h. Sequence 1 mM 6 � mercapto - 1 � hexanthiol in MilliQ water was immobilized for 1 h and the hybridization with complementary strand dissolved in pH 7.4 hybridized solution (10 mM phosphate buffer, 1 mM EDTA, 1 M NaCl) for 1h. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were applied to control biosensor development. CV was carried out in a 10 mL electrochemical cell composed by modified working electrode, Ag|AgCl|1 M KCl reference electrode and platinum wire as auxiliary electrode. The measurements were performed in 5 mM potassium ferrocyanide and 10 mM phosphate buffer, cycled from -0.1 to 0.5 mV at 100 mV.s-1. EIS was performed in the same electrochemical cell used for CV. Frequencies varied from 100 kHz down to 0.05 Hz with 10 mV amplitude and single sine. The solutions employed in each step were studied and proved to be crucial to overall device performance. Specially MgCl2 in DNA immobilization solution neutralizes the negative charges of single-stranded probes and promotes a huge surface coverage. The increase of immobilized single-stranded improves the electrochemical impedance spectroscopy hybridization signal. The analytical curve indicates the percent of hybridization as a function of complementary strand concentration. The results of electrochemical techniques will be also compared to quartz crystal microbalance experiments. This work was funded by CNPq, CAPES and FAPESP Brazilian agencies. Reference [1] T. G. Drummond, M. G. Hill and J. K. Barton, Electrochemical DNA sensors. Nature Biotechnology 21(2003) 1192 � 1199. [2] A. Esquela-Kerscher and F. J. Slack, Oncomirs - microRNAs with a role in cancer. Nat. Rev. Cancer 6 (2006) 259�269. [3]L. M. Fiscger, M. Tenje, A. R. Heiskasen, et al. Gold cleanning methods for electrochemical detection applications. Microelectronic Engineering 86 (2009) 1282 � 1285.
 


 
 
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