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. 2020 Apr 28;10(5):45. doi: 10.3390/bios10050045
Protocol A9 by Meirinho et al. [120]: Immobilization of biotinylated aptamer via streptavidin
Reagents

  • 200 mM 3,3′-dithiodipropionic acid (DPA) solution;

  • 200 mM N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC) solution;

  • 100 mM N-hydroxysuccinimide (NHS) solution;

  • 100 mM, pH 8.5 ethanolamine (ETA) solution;

  • Phosphate buffer saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.47 mM KH2PO4), pH 7.4;

  • 4 nM biotinylated aptamer solution in PBS buffer.

Method

  1. Incubate cleaned electrode with DPA solution for 30 min at RT and then rinse with deionized water;

  2. Treat with EDC and NHS solutions (1:1 v/v) for 1h at RT;

  3. Incubate with streptavidin solution overnight at 4 °C;

  4. Treat with ETA for 20 min at RT;

  5. Heat up the aptamer solution to 95 °C for 5 min, cool down to 4 °C for 5 min and at RT for 10 min;

  6. Place 5 µL aptamer solution on the electrode for 40 min at RT and rinse with PBS buffer.