User:Madeleine Y. Bee/Notebook/Single Molecule Fluorescence/2013/05/24

Silica-Coating and DNA Attachment Procedures

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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 <br.> ________________________________________________________________________________________________________________________________ <br.> 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.> <br.> ________________________________________________________________________________________________________________________________ <br.> 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) Must be stored at -20°C Moisture sensitive, store in desiccant Manufacturer Instructions 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