User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/05/24

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Silica-Coating and DNA Attachment Procedures Main project page
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May 24, 2013

Write out procedures for synthesizing Silica-coating AuNPs and attaching to S-DNA

Preparing Silica-Coated AuNP

from A General Method To Coat Colloidal Particles with Silica done by Allison Alix

Chemicals Needed

  • TES (tetraethyl orthosilicate)
  • PVP (Poly(vinylpyrrolidone))
  • Ethanol
  • Acetone
  • Ammonia (29.3 wt % NH3 in water)
  • Hydrofluoric acid

Procedure

Part One: Adsorption of PVP

  • Calculate the amount of PVP to provide the colloids with 60 PVP molecules per nm2
  • Dissolve the PVP in water by ultrasonication for 15 minutes
  • Combine the calculated amount of PVP with AuNPs and stir at 600 rpm for 24 hours at room temperature
  • Centrifuge solution and remove supernatant
  • Re-disperse the sediment in a solution of ammonia in ethanol

Part Two: Growth of the Silica Shell

  • Add an amount TES solution (10 vol % in ethanol) depending on the desired thickness of the silica shell, while stirring at 600 rpm
  • Stir reaction mixture for 12 hours

Calculations


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Preparing Silica-Coated AuNP

from Gold and Silica-Coated Gold Nanparticles as Thermographic Labels for DNA Detection
and Synthesis and Self-Assembly of Au@SiO2 Core−Shell Colloids

Reaction

SiO4C8H20(aq)+AuNPs(aq*) (NH4OH/C3H8O)→ AuNPs@SiO2(aq*)+O2C8H20(aq**)
'*' denotes colloidal system
'**' denotes dissolution in solvent, not water

Chemicals Needed

  • ~80μL TES (tetraethyl orthosilicate)
  • ~1mL NH4OH
  • ~25mL 2-propanol
  • 5μL MHA (mercaptohexadecanoic acid)

Procedure

  • Add appropriate volumes of AuNPs to 2-propanol, then, under constant stirring add the ammonium hydroxide and TES
  1. 1mL 9.4nM, 10nm AuNP/no stabilizer/10μL TES/0.125mL NH4OH/5mL propanol
  2. 1mL 9.4nM, 10nm AuNP/no stabilizer/20μL TES/0.125mL NH4OH/5mL propanol
  3. 1mL 9.4nM, 10nm AuNP/no stabilizer/40μL TES/0.125mL NH4OH/5mL propanol
  4. 1mL 9.4nM, 10nm AuNP/MHA/5μL TES/0.125mL NH4OH/5mL propanol
    • Attach MHA (for stability):
      • Add 5mL of 10mM MHA to 5mL of AuNPs, stir overnight
  • Stir solutions for one hour to allow reaction to proceed
  • Centrifuge solutions at 4000 rpm for one hour
  • Re-suspend particles in water to a final concentration of 5nM

Calculations

  • Make 9.4nM, 10nm AuNPs from 29.67nM, 10nm AuNPs synthesized by Allison Alix 5/10/13

(9.4nM)(10mL)=(29.67nM)(V)
V=3.17mL of 29.67nM AuNP in 6.83mL filtered H2O


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Modifying Silica-Coated AuNP with thiol DNA

from Gold and Silica-Coated Gold Nanparticles as Thermographic Labels for DNA Detection

Reactions

Fuctionalizing the AuNPs@SiO2 with NH2

AuNPs@SiO2(aq*)+H2N(CH2)3Si(OCH3)3(l)→AuNPs@SiO2-NH2(aq*)
'*'denotes colloidal system

Activating the thiol DNA with Sulfo-SMCC

S-DNA(aq)+C16H17N2O9SNa(aq*)(/KP buffer)→SSMCC activated-S-DNA(aq*)
'*'denotes dissolution in buffer, not water

Reacting the SSMCC-activated DNA with the NH2-functionalized, silica-coated AuNPs

SSMCC activated-S-DNA(aq*)+AuNPs@SiO2-NH2(aq**)→DNA-S-SSMCC-NH-AuNPs@SiO2(aq***)
'*'denotes dissolution in buffer, not water
'**'denotes colloidal system
'***'denotes colloidal system/dissolution in buffer

Chemicals Needed

  • 5μL, 1mM APS ((3-aminopropyl)trimethoxysilane)
  • 1mg SSMCC (sulfosuccinimidyl-4-N-maleimidomethyl cyclohexane-1-carboxylate)
  • 0.05M KH2PO4
  • 0.05M K2HPO4

→ ~2mL 0.05M KP buffer, pH 7.4

Procedure

  • Combine 1mL of 5nM silica-coated AuNP with 5μL of APS ((3-aminopropyl)trimethoxysilane), 1mM, for one hour
  • Separate reaction: 1.5mL of thiol DNA (3μM) reacted with 1mg of SSMCC (sulfosuccinimidyl-4-N-maleimidomethyl cyclohexane-1-carboxylate) in 1.5mL of 0.05M KP buffer
    • KP buffer: mix 0.05M solutions of KH2PO4 and KHPO4 until a pH of 7.4 is reached
  • After one hour, separate DNA from excess SSMCC by centrifugation
    • using a 3000 molecular weight cutoff filter; centrifuge at 14000 rpm for 20min
  • Purified, SSMCC-activated DNA is added to NH2 functionalized silica-coated AuNP; stir for two hours
  • Centrifuge solution at 4000 rpm for 1 hour
  • Re-suspend in water to achieve a final concentration of 5nM

Calculations


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