Target Hybridization

From LPDwiki

Necessary Materials

1. np-Au/planar Au chips immobilized with probe DNA → working electrode

2. Aliquoted target DNA (C = 100 μM, V = 10 μl) for hybridization with probe DNA

3. 20 μM methylene blue (MB)

3. 25 mM Phosphate Buffer (PB)

4. x1 PBS Solution

4. 75 mM MgCl2

6. Platinum wire → counter/auxillary electrode

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

7. Reference Electrode

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

8. Gamry Reference 600 potentiostat


MB Accumulation

1. Place dry DNA probe functionalized electrode [DNA Probe Immobilization] inside the custom-built Teflon electrochemical cell and assemble the cell. Make sure to make connection for the working electrode using platinum wire and make sure to expose unscratched part of np-Au for testing.

2. Wash the el. cell with 150 μl of 25 mM PB.

3. Incubate el. cell with DNA functionalized electrode with 150 μl of 20 μM methylene blue (MB) for 10 minutes.

4. Wash with 25 mM PB after MB (3 x 150 μl) accumulation to remove unbound MB molecules.

5. Fill chamber with 150 μl of electrolyte (usually x1 PBS).

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

• Equilibration time: 30 seconds

• 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)

10. Save the first measurement.

Take successive SWV measurements until peak signal depletes to about 65% of the first SWV peak signal (takes about 10-15 SWVs).

  • The baseline subtracted peak current from SWV 1 is used for calibration. Baseline subtracted peak current is obtained by subtracting the capacitive baseline from the redox peak current.

11. IMMEDIATELY rinse the el. cell with 25 mM PB (3 x 150 μl) to remove left-over MB molecules. Incubate the el. cell in 25 mM PB until target hybridization experiments.

Target Hybridization

1. Incubate electrode with the desired concentration target DNA prepared in 25 mM PB containing 50 mM MgCl2 for 35 minutes at 37°C in the oven/incubator.

  • 300 nM or 500 nM DNA target solutions are usually used for incubation.
  • Some PB can be substituted with complex media, such as BSA or whole blood for complex media target hybridization studies.

2. Remove cell from the oven, let it cool for 5 minutes. Rinse with 25 mM PB (3 x 150 μl) to remove unhybridized target DNA.

3. Incubate with MB as described in MB Accumulation section (MB regeneration) and obtain SWV measurements as described above.

4. Hybridization efficiency is estimated from the baseline subtracted peak current of the first SWV scans. Hybridization efficiency = (Iprobe – Itarget/Iprobe) X 100.

5. Repeat steps above at increasing target concentrations to obtain calibration curve.