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

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 Revision as of 09:05, 13 August 2013 (view source) (Autocreate 2013/08/13 Entry for User:Allison_K._Alix/Notebook/Thesis_Research)← Previous diff Current revision (11:17, 14 August 2013) (view source) (→Data) (17 intermediate revisions not shown.) Line 6: Line 6: | colspan="2"| | colspan="2"| - ==Entry title== + ==Scaled up Silica-Coating Procedure== - * Insert content here... + + 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 [http://pubs.acs.org/doi/full/10.1021/la7019846 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 [http://pubs.acs.org/doi/full/10.1021/ac0600555 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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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

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)

Data

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.