User:Allison K. Alix/Notebook/Thesis Research/2013/08/13

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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

Calculations

Original concentration of AuNP/MHA solution is 4.7nM

4.7nM(9mL)=55.143mL(x)

x= 0.767nM

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

  • We want a 5nM solution

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

Adapted from Silica Coated, Au/Ag Striped Nanowires for Bioanalysis

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

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

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)

Data

Image:Pic_of_spec.JPG‎

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.



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