Skip to main content
. 2020 Apr 28;10(5):45. doi: 10.3390/bios10050045
Protocol A3 by Jolly et al. [78]: Spherical AuNPs on 11-amino-1-undecanethiol SAM
Reagents
  • 1 mM 11-amino-1-undecanethiol (AUT) dissolved in pure ethanol;

  • 1 mM mercaptohexanol (MCH);

  • gold nanoparticles (20 nm, stabilized suspension in 0.1 mM PBS, reactant free);

  • aptamer solution: thiolated aptamer and MCH (ratio 1:50) in 10 mM PBS, pH 7.4. Prior to preparation, heat up the aptamers to 95 °C for 10 min and gradually cool down to RT.

Method
  1. Immerse the cleaned electrode in the AUT solution and incubate for 16 h at 4 °C;

  2. Wash with pure ethanol and MilliQ water

  3. Incubate in MCH solution for 1 h at RT;

  4. Incubate in AuNP solution in an inverted position overnight;

  5. Incubate in aptamer solution for 2 h at RT;

  6. Rinse with ultra-pure water.

For the bioassay, proteins were dissolved in 10 mM PBS, pH 7.4.
Protocol A4 by Daems et al. [141]: 3D DNA tetrahedron as anchor for aptamer immobilization
Reagents
  • 1 µM stock solution of each oligonucleotide;

  • TM buffer: 20 mM Tris base, 50 mM MgCl2, pH 8.0;

  • 3 mM Tris-(2-carboxyethyl)-phosphin (TCEP);

  • TGK buffer: 25 mM Tris base, 192 mM glycine, 5 mM K2HPO4, 0.1% Tween 20 and 0.15 w/v% BSA, pH 8.3.

Method
  1. Create the tetrahedron solution by mixing 164 µL of TM buffer and 20 µL of TCEP with 4 µL of each oligonucleotide solution;

  2. Heat to 95 °C for 2 min and cool to 4 °C (ramp speed −0.5 °C/min);

  3. Incubate clean electrode with 200 µL tetrahedron mixture at 4 °C overnight;

  4. Wash three times with TGK buffer;

  5. For the bioassay, immerse the electrode in 120 µL TGK buffer for 5 min to reach a stable baseline.

Immerse in thrombin solution (concentrations ranging 15.5–248 nM were investigated) in TGK buffer for 20 min, followed by a washing step of 3 min in TGK buffer.