User:Allison K. Alix/Notebook/Thesis Research/2013/08/13: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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5.52mL/24 vials = 230uL per vial. | 5.52mL/24 vials = 230uL per vial. | ||
==DNA Attachment== | ==DNA Attachment Procedure 1== | ||
1) Mix 300uL AuNP@SiO2 | 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. | |||
Latest revision as of 23:10, 26 September 2017
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Scaled up Silica-Coating ProcedureOriginal: 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 CalculationsOriginal 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
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 1Adapted 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 2Adapted 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
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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