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
Immerse the cleaned electrode in the AUT solution and incubate for 16 h at 4 °C;
Wash with pure ethanol and MilliQ water
Incubate in MCH solution for 1 h at RT;
Incubate in AuNP solution in an inverted position overnight;
Incubate in aptamer solution for 2 h at RT;
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
Create the tetrahedron solution by mixing 164 µL of TM buffer and 20 µL of TCEP with 4 µL of each oligonucleotide solution;
Heat to 95 °C for 2 min and cool to 4 °C (ramp speed −0.5 °C/min);
Incubate clean electrode with 200 µL tetrahedron mixture at 4 °C overnight;
Wash three times with TGK buffer;
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. |