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<br.>
and Synthesis and Self-Assembly of Au@SiO2 Core−Shell Colloids<br.>
Reaction
SiO4C8H20(aq)+AuNPs(aq*) (NH4OH/C3H8O)→ AuNPs@SiO2(aq*)+O2C8H20(aq**)<br.>
'*' denotes colloidal system<br.>
'**' denotes dissolution in solvent, not water<br.>
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
- 1mL 9.4nM, 10nm AuNP/no stabilizer/10μL TES/0.125mL NH4OH/5mL propanol
- 1mL 9.4nM, 10nm AuNP/no stabilizer/20μL TES/0.125mL NH4OH/5mL propanol
- 1mL 9.4nM, 10nm AuNP/no stabilizer/40μL TES/0.125mL NH4OH/5mL propanol
- 1mL 9.4nM, 10nm AuNP/MHA/5μL TES/0.125mL NH4OH/5mL propanol <br.>
- 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)<br.>
V=3.17mL of 29.67nM AuNP in 6.83mL filtered H2O <br.>
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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*)<br.>
'*'denotes colloidal system
Activating the thiol DNA with Sulfo-SMCC
S-DNA(aq)+C16H17N2O9SNa(aq*)(/KP buffer)→SSMCC activated-S-DNA(aq*)<br.>
'*'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***)<br.>
'*'denotes dissolution in buffer, not water<br.>
'**'denotes colloidal system<br.>
'***'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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