Electrografting Study Protocols

From LPDwiki

Necessary Materials

1. np-Au/planar Au chips (22 mm X 22 mm) → working electrode

2. Aliquoted probe DNA (C = 100 μM, V = 35 μl)

3. 20 μM methylene blue (MB)

4. 0.5 M TCEP (tris(2-carboxyethyl)phosphine)

5. Zeba Spin Desalting Columns

  • Product Number: 89882

6. 25 mM Phosphate Buffer (PB)

7. 75 mM MgCl2

8. 1 mM MCH (6-Mercapto-1-hexanol)

9. Platinum wire → counter/auxillary electrode

  • Product Number: 45093
  • 0.25 mm (0.010 in) dia, 99.9% (metals basis) length: 50 cm

10. Reference Electrode

  • Product Number: ET073-1
  • Refillable miniature Ag/AgCl reference electrode

11. Gamry Reference 600 potentiostat


DNA Probe Thiol Reduction and Quantification

1. Take out probe aliquot from -20 °C and let it thaw and equilibrate at room temperature for 20 minutes.

2. Add 30 μl of the DNA stock solution to 90 μl of 25 mM PB to prepare 25 μM dilution. Vortex gently (5 seconds) and spin down (4 seconds).

3. Keep 20 μl aside for control absorbance measurement of the unreduced probe.

4. Add 4 μl of 0.5 M TCEP to 100 μl of unreduced DNA probe solution (~25 μM). Mix and vortex for 5-10 seconds.

5. Leave reaction mixture at room temperature for 2 hours.

  • Cover with Al foil.

6. Filter the mixture using zeba spin column. Centrifuge at 4000 rpm for 2 minutes.

  • Note, zeba spin columns need to be centrifuged at 4000 rpm for 2 minutes to get rid of desalting solution, before DNA solution is added.
  • Make sure to label formed slanted side before adding DNA solution. Labelled slanted side should face outside in the microcentrifuge during DNA centrifugation.
  • Don't forget to balance the microcentrifuge.

7. Measure absorbance of reduced DNA probe using Biotek UV-Vis.

  • Expected concentration after reduction ~ 30 μM.

DNA Probe Electrografting and Measurements

1. Cut np-Au/planar Au chips (22 mm X 22 mm) into four squares. Use one square per experiment.

2. Prepare piranha solution (1 : 4; hydrogen peroxide: sulphuric acid) and leave it to cool for 10 minutes. Clean the electrodes by immersing for 20 seconds each.

  • Rinse with DI water and store in DI water until DNA probe functionalization.

3. Plasma treat electrodes for 1 minute prior to probe electrografting.

4. Place the electrode inside the custom-built Teflon electrochemical cell and assemble the cell.

5. Fill chamber with 150 μl of 5 μM DNA probe solution (100 μl of 25 mM PB + 400 μl of 75 mM MgCl2 + 100 μl of 30 μM reduced probe DNA).

  • Volume of different components of 5 μM DNA probe immobilization solution was determined using the following specifications:
    • Vfinal = 600 μl
    • C(probe DNA)final = 5 μM
    • C(MgCl2)final = 50 mM
    • Rest is 25 mM PB

6. Assemble Ag/AgCl reference electrode, platinum wire counter electrode and connect the cell to the Gamry Reference 600 potentiostat.

7. Check the open circuit potential (OCP) to make sure there is electrical continuity between the working and reference electrodes.

  • A high OCP > 1 V indicates a short. This may be due to poor working electrode contact or bubbles in the electrolyte or damaged reference electrode.
  • Start trouble shooting first by refilling electrolyte making sure to remove bubbles. If that doesn’t solve the problem, disassemble the cell and check working electrode contact. If this fails, finally test with a different reference electrode.

8. Measure OCP for 60 s to make sure the system is stable. 10 - 20 mV change in OCP in this 60 s is acceptable.

9. Start DNA probe electrografting by running Chronoamperometry (CA) script on potentiostat with the following parameters:

• Pre-step voltage (V): 0.04 vs Eref

• Pre-step delay time (s): 10

• Step 1 Voltage (V): 0.8 vs Eref

• Step 1 time (s): 60 (this is subject to change, depending what electrografting time needs to be tested - saturation happens at ~10 minutes).

10. Rinse with 25 mM PB to remove free floating DNA probes.

11. Incubate the electrode in the electrochemical cell with back-fill agent, 1 mM mercaptohexanol for 20 minutes (this is the optimal time found to work well for different morphologies, but higher MCH incubation times, e.g. 30 minutes work well for unannealed np - Au as well) to passivate the surface that has not been covered by probe DNA.

12. Rinse electrodes thoroughly with 25 mM PB to remove non-specifically bound DNA.

13. Incubate DNA probe functionalized electrodes with 150 μl of 20 μM methylene blue (MB) for 10 minutes.

14. Wash with 25 mM PB after MB accumulation to remove unbound MB molecules.

15. Obtain probe electrochemical signature via square wave voltammetry (SWV) with the following parameters:

• Scan range: 0 to – 500 mV

• Step size: 4 mV (batch mode), 10 mV (microfluidics)

• Amplitude: 40 mV

• Pulse frequency: 18 Hz (unannealed np-Au); 30 Hz (annealed np-Au); 60 Hz (planar Au)

16. Copy SWV file in the Gamry Data folder to the desired location under the desired file name.