Skip to main content
. 2020 Apr 28;10(5):45. doi: 10.3390/bios10050045
Protocol A10 by Daems et al. [141]: Immobilization of 24-helix DNA bundle as anchor platform for aptamer immobilization
Reagents

  • PEG 8000 precipitation buffer: 5 mM Tris base, 1 mM Na2EDTA, 505 mM NaCl, 15 w/v% PEG 8000;

  • TEMg buffer: 5 mM Tris base, 1 mM Na2EDTA, 13 mM MgCl2, pH 7.6;

  • Phosphate buffer: 12 mM K2HPO4 and 167 mM K2HPO4, pH 8.3;

  • TNM buffer: 10 mM Tris base, 25 mM MgCl2, 2 M NaCl, pH 8.0;

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

  • 0.01% sodium dodecyl sulfate (SDS).

Method for the one-step assembly and purification (underlined steps are described in Stahl et al. [180])

  1. Prepare the self-assembly reaction mixture, containing 20 nM aptamer, 200 nM of each staple DNA, 5 mM TRIS, 1 mM EDTA, 20 mM MgCl2 and 5 mM NaCl (pH 8);

  2. Incubate the reaction mixture at 65 °C for 15 min, anneal from 60 °C to 40 °C in steps of 1 °C per 2–3 h;

  3. Mix with the PEG 8000 precipitation buffer in a 1:1 (v/v) ratio and adjust to 20 mM MgCl2;

  4. Mix the solution by tube inversion and centrifuge at 16,000× g for 25 min at RT;

  5. Remove the supernatant and dissolve the pellet in TEMg buffer for 30 min at 30 °C;

  6. For the spin column purification, wash the Amicon Ultra 0.5 mL centrifugal filter devices with 500 µL of TEMg (containing 6 mM MgCl2) by centrifugation at 10,000× g for 5 min;

  7. Add 50 µL of the prepared DNA sample to the device together with 450 µL TEMg and centrifuge at 4500 g for 5 min;

  8. Remove the excess of staple strands and recover the nanostructures by reverse spinning of the centrifugal filter device.

Method for immobilization

  1. Activate the thiolated ssDNA linkers with 0.1 M dithiothreitol that is removed with a NAP-5 column afterwards;

  2. Incubate the sensor in 1 µM activated thiolated ssDNA, dissolved in phosphate buffer;

  3. Wash three times in phosphate buffer with 0.01% SDS;

  4. Incubate the sensor with the assembled DNA nanostructure with a concentration of 16 nM (LS) or 83 nM (DE) in TNM buffer at 4 °C overnight;

  5. Wash with TGK;

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

  7. 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.

Protocol A11 by Grabowska et al. [23]: Electrophoretic deposition of rGO/PEI nanocomposite and immobilization of azide-terminated aptamers onto PEI
Reagents

  • Polyethylenimine (PEI);

  • 1-Ethyl-3-(3-imethylaminopropyl)carbodiimide (EDC);

  • tris(3-hydroxypropyltriazolylmethyl)amine (THPTA);

  • N-Hydroxysuccinimide (NHS);

  • Propargylactic acid;

  • Copper(II) sulfate pentahydrate (CuSo4);

  • Sodium ascorbate;

  • 0.1 M phosphate buffer (PBS);

  • Pyrene-PEG, which can be synthesized following the method found in [148].

Method

  1. Create GO/PEI dispersion by stirring graphene oxide powder and PEI, each 1 mg/mL, for 48 h at RT;

  2. Immerse the SPGE in the GO/PEI dispersion and apply a DC voltage of 120 V for 20 s between the SPGE (cathode) and a Pt plate (anode);

  3. Treat the azide-terminated apatamers with a mix of 25 mM EDC, 25 mM NHS, and 20 mM propargylactic acid for 2 h;

  4. Incubate the electrode in a mix of 1 µM aptamer solution, 10 mM CuSO4, 100 mM sodium ascorbate, and 20 mM THPTA for 7 h at RT;

  5. Wash the modified electrode three times with PBS;

  6. Immerse the electrode in 1 mM pyrene-PEG for 2 h at RT.

For the regeneration of the aptasensor, immerse in 0.1 M NaOH (pH 12) for 20 min.