Nanostructure for detecting cell-free DNA using conductive polymer and the use thereof
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example 1
Experimental Materials and Methods to Synthesize Nanowire Structure
[0165]1-1. Manufacture of Ppy-Coated Au NWs (Ppy / Au NWs)
[0166]An approximately 150-nm-thick Au layer was deposited onto one surface of the AAO template (Whatman; pore diameter, 100 nm) by employing the conventional thermal evaporation technique. Au nanowires were electrochemically grown within the pores of the Au-backed AAO membrane by using a gold-plating solution (Orotemps 24 RTU Rack) and applying cyclic voltammetry in the potential range of −1.1-0 V at a scan rate of 100 mV / s.
[0167]All electrochemical experiments were conducted using a potentiostat / galvanostat (BioLogic SP-150) in 3-electrode cells, in which Ag / AgCl, platinum wire, and the designed platform were employed as reference, counter, and working electrodes, respectively. After fixing on the indium tin oxide (ITO) surface by using a conductive carbon paste, the generated membrane was dissolved in an aqueous NaOH solution (2 M) for 4 h to remove the AAO t...
example 2
Evaluation of Attachment / Isolation Ability to cfDNA Using Nanowire Structure
[0188]Using the feature that a surface of polypyrrole is effectively modified by electrical stimulation, concentration and isolation of cfDNA were attempted by spiking a DNA ladder in plasma. In addition, it was confirmed that cfDNA was attached / isolated in a much higher amount in a nanowire structure than a flat surface.
[0189]In particular, three steps are required to accomplish concentration and isolation of DNA. First, over-oxidation was carried out by applying a high voltage to a polypyrrole nanowire structure, and thus a positively charged surface was induced. Subsequently, DNA capture was attempted at 1.0 V, and then a voltage of 1.3 V was applied after washing three times, thereby releasing concentrated DNA from a surface of the nanowire structure and obtaining the concentrated DNA. As a result, it was confirmed that DNA capture efficiency increased with increasing over-oxidation voltage (see FIG. 4A)...
example 3
Evaluation of cfDNA in Blood of Breast Cancer Patient and Lung Cancer Patient Using Nanowire Structure
[0193]Using blood from a breast cancer patient, cfDNA attached to a surface of the conductive nanostructure was observed using a fluorescent dye, SYBR Green / PICO Green, with a confocal microscope.
[0194]As illustrated in FIG. 7, it can be confirmed that cfDNA is completely attached to a surface of a wire when a nanowire having a length of 1.0 μm is used, and, after the application of electrical stimulation, the attached cfDNA is released and thus fluorescence is not observed.
[0195]In addition, the present inventors compared the concentrations of cfDNAs isolated by the conductive nanostructure of the present invention and Qiagen kit using blood samples from a breast cancer patient and a lung cancer patient.
[0196]As a result, as illustrated in FIGS. 6A and 6B, the amounts of cfDNAs from blood samples of cancer patients are up to ten fold or more with respect to the amount of cfDNA capt...
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