User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/07/15

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July 15, 2013

DNA and AuNP Attachment Procedure

Adapted from Instantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a pH-Assisted and Surfactant-Free Route with Supporting Information containing the specific procedure used.

Calculations

  • Final concentration of buffer in solution: 10mM, from 268mM sodium citrate
    • (268mM)V=(10mM)(500uL)
    • V=18.7uL 268mM sodium citrate
    • (500-18.66)/2=240.7uL each of AuNP and DNA
  • Final AuNP concentration
    • (500uL)C=(240.7uL)(29.67nM)
    • C=14.28nM
  • Final DNA concentration
    • 75DNA:1AuNP
    • (14.28)×75=1.071uM
  • Original DNA concentration (before combining with AuNP and sodium citrate)
    • (240.7)C=(1.071uM)(500uL)
    • C=2.225uM
  • DNA concentration (before extraction)
    • (250uL)C=(500uL)(2.225uM)
    • C=4.45uM
  • Final concentration of buffer in solution: 10mM, from 100mM sodium citrate
  • (100mM)V=(10mM)(350uL)
  • V=35uL 100mM sodium citrate
  • (350-35)/2=157.5uL each of AuNP and DNA
  • Final AuNP concentration
    • (350uL)C=(157.5uL)(29.67nM)
    • C=13.35nM
  • Final DNA concentration
    • 75DNA:1AuNP
    • (13.35)×75=1.0014uM
  • Original DNA concentration (before combining with AuNP and sodium citrate)
    • (157.5uL)C=(1.0014uM)(350uL)
    • C=2.225uM
  • DNA concentration (before extraction)
    • (157.5uL)C=(350uL)(2.225uM)
    • C=4.95uM

Extraction Procedure

  • Combine 250uL 4.45uM DNA (diluted in PBS) with 250uL 100nM DTT in 4% TEA
  • React for 10 minutes
  • Extract DTT with 4 2mL aliquots of ethyl acetate (DTT will partition into ethyl acetate, DNA will partition into aqueous solution)
  • Combine appropriate volumes of AuNP, DNA, and sodium citrate buffer simultaneously; vortex immediately
  • React for 10 minutes
  • Centrifuge 4 times, extracting the supernatant each time and then redispersing in 500/350uL 50mM HEPES buffer (for the 350/500uL solutions; buffer volume should be equal to solution volume)
    • Note: centrifugation times were inconsistent in today's procedure, because we were hesitant to run too long or at too high speed, which has previously caused the nanoparticles to stick to the sides and bottom of the epitubes. Ideal speed and time was found to be approximately 6 minutes at 10000 rpm
  • Take absorbance of final solution and three supernatants

Absorbance Data

<br.> Corrected using HEPES buffer as a reference solution.<br.> <br.> <br.> Replicated; corrected using HEPES buffer as a reference solution.

Notes

Previously, the extracted DNA and AuNP had been mixed and allowed to react for 5 minutes before adding the sodium citrate buffer. However, we think this may be affecting the aggregation of the AuNPs so in this procedure we added the buffer immediately to the AuNP and DNA solution. Additionally, we referred back to the literature and realized the final concentration of sodium citrate buffer in solution needs to be 10mM when we had previously been doing 20mM. Changing this and adding the buffer immediately allowed for the DNA/AuNP attachment, as evident in the spectra. I had some difficultly extracting the supernatants, as the nanoparticles are easily disturbed after centrifugation. Because of this, hardly any absorbance was observed for the final sample. The procedure will be replicated, and concentrations of DNA and AuNPs will be calculated using Beer's Law.<br.> The supernatants contain such a significant amount of nanoparticles because we were unsure of how long to centrifuge the samples. By trial and error, we believe the best settings are approximately 11 minutes at 10000 rpm, or until the nanoparticles are visibly at the bottom of the tube and the solution has lost most pink color, characteristic of dispersed gold nanoparticles. If 11 minutes is visibly insufficient, repeat at 10000 rpm for two minutes at a time.<br.>

MHA Day One Procedure

Originally outlined on 05/24/2013, updated on 07/12/20130<br.>

  • 0.0144g MHA in 5mL 2-propanol, combine with 5mL AuNPs
    • Note: MHA is neither known or unknown to be soluble in water, but is definitely soluble in 2-propanol which is also the solvent for the second part of the silica-coating procedure
  • Stir overnight



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