User:Allison K. Alix/Notebook/Thesis Research/2013/08/13
(→DNA Attachment Procedure 2)
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showing pure AuNP (23.5nM, AuNP coated in silica (5nM) and NH2 functionalized Au@SiO2 ). We assume an even silica coating due to the observed 11nm peak shift of the Au@Si02 .
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Scaled up Silica-Coating Procedure
Original: 1mL AuNP & MHA solution/2μL TES/0.125mL NH4OH/5mL 2-propanol
1) Re-scale to use 9mL AuNP/MHA solution
New: 9mL AuNP&MHA solution/18uL TES/1.125mL NH4OH/45mL 2-propanol
2) Stir solutions for one hour to allow reaction to proceed
3) Centrifuge solutions at 4000 rpm for one hour
4) Re-suspend particles in water
Original concentration of AuNP/MHA solution is 4.7nM
0.767x10^-9moles/L x 1L/1000mL x 36mL = 2.7612 x 10^-11 moles AuNP total
2.7612 x 10^-11 moles AuNP x 6.02 x 10^23 particles/mole = 1.66 x 10^13 total Au nanoparticles
5x10^-9 moles/L x 1L/1000mL x 6.02x10^23 particles/mole = 3.01 x 10^12 particles/mL
1.66 x 10^13 particles / x = 3.01 x10^12 particles/1 mL
x = 5.52mL
Dilute AuNP in 5.52mL water after removing supernantant
5.52mL/24 vials = 230uL per vial.
DNA Attachment Procedure 1
1) Mix 300uL AuNP@SiO2, 30uL APS, 470uL ethanol. Stir for 30 minutes.
2) Centrifuge solution at 9100rpm for 30 second intervals. Rinse 3 times with ethanol and twice with sodium phosphate buffer (SPB), pH=9. Redisperse in 800uL SPB.
3) In a seperate solution, mix 2mg Sulfo-SMCC with 800uL SPB. Vortex to dissolve.
4) Add 800uL Sulfo-SMCC with 800uL Au@SiO2-NH2. Stir for one hour.
5) Centrifuge at 9100rpm, twice with SPB and twice with potassium phosphate buffer (KPB), pH=7.4. Redisperse in 400uL KPB
6) Add 50uL 5uM DNA to 400uL Sulfo-SMCC activated Au@SiO2 solution. Stire for 1 hour
7) Rinse three times with KPB, Redisperse in 150uL KPB.
DNA Attachment Procedure 2
1) Mix 1mL 5nM AuNP@SiO2 with 5uL 1mM APS. Stir for 1hr.
2) Reduce thiol DNA to HS-DNA using DTT. Dissolve 2mg Sulfo-SMCC in 2mL 0.05M KPB. Mix 250uL solution with 250uL 3uM DNA. Mix for 1 hour
3) Centrifuge DNA/Sulfo-SMCC solution for 20 minutes at 13,200rpm. Remove excess Sulfo-SMCC and redisperse DNA in 250uL PBS.
4) Mix 250uL Sulfo-SMCC activated DNA with AuNP@SiO2-NH2 solution. Stir for 2 hours. Centrifuge for 1 hour @ 4000rpm.
5) Resuspend in water (250uL?, 500uL)
Above are spectra showing pure AuNP (23.5nM), AuNP coated in silica (5nM) and NH2 functionalized Au@SiO2 (5nM). We will assume an even silica coating due to the observed 11nm peak shift of the Au@Si02 which is characteristic of silica coated particles.