Extending DNA from a Single Cell using Integrated System of Electro-osmosis and AFM

This study developed an integrated system of electro-osmotic flow and atomic force microscope (AFM) for human endothelial cell line (ECV304) lysis and DNA extension. The ECV304 cells were incubated in an M199 medium supplemented with 15 percent fetal bovine serum in a standard incubator at 37℃. The cell seeding substrata was a glass of fabricated SU 8 photo-resist structure. Before cells were cultured on the glass, the substrata surface was coated with collagen (Rat Tail Tendon Type I, dissolved in 5 mM acetic acid). After the cells were cultured on the glass for 3 hours, the experimental procedure began with displacing the cell culture medium to a 100 mM glucose solution and placing the glass on the AFM's stage. A silicone resin sheet with a straight-shaped opening was mounted on the substrate to form the electro-osmotic flow chamber. Two Al electrodes were immersed within the bends made at two ends of the straight-shaped opening. The AFM tip continuously applied force onto the cell membrane (once per second of loading rate) for cell destruction at the set point of 20 nN, and the force curve was measured simultaneously. When the cell destruction was confirmed, the electric field was applied to create electro-osmotic flow for DNA extension. Experimental results showed that continuous applied force on the cell membrane by the AFM tip destroyed the cell. The calculated Young's modulus in the low range between 0 nm and 20 nm cantilever deflection were 1.71±0.52 kPa for the intact cell and 13.04±2.37 kPa for the necrotic cell. The electric field was applied 11 minutes later, and DNA fibers emerged from the necrotic cell, which was stretched as long as 130 μm.

並列關鍵字

Atomic force microscope AFM ； Cell destruction ； Electro-osmosis ； DNA extension

國際替代計量

全文下載

主題瀏覽