User:Elaine Marie Robbins/Notebook/Capstone/2012/06/19

Concentration of AuNP

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Methods Determine size and concentration of citrate AuNP using UV/Vis. Make AuNP/TS primer complex with approx. 50 primers per AuNP by adding 40.5 μL thiol primers to 5 mL AuNP. Make 4 solutions, each with 100,000 cells: Citrate AuNP/TS primer, incubated Mix 3 μL AuNP/TS primers, 3.5 μL 10 mM tris buffer (pH 7.5), 1 μL dNTP, 16 μL water, and 250 μL diluted cell extract containing 100,000 cells. Incubate at 30°C for 30 min. Add 12.2 μL 100x diluted MB. Incubate at 65°C for 60 min. Citrate AuNP/TS primer, kept at room temperature Mix 3 μL AuNP/TS primers, 3.5 μL 10 mM tris buffer (pH 7.5), 1 μL dNTP, 16 μL water, and 250 μL diluted cell extract containing 100,000 cells. Incubate at room temperature for 30 min. Add 12.2 μL 100x diluted MB. Incubate at 65°C for 60 min. Primers only, incubated Mix 6.5 μL 100x diluted primers, 1 μL dNTP, 16 μL water, and 250 μL diluted cell extract containing 100,000 cells. Incubate at 30°C for 30 min. Add 12.2 μL 100x diluted MB. Incubate at 65°C for 60 min. Primers only, kept at room temperature Mix 6.5 μL 100x diluted primers, 1 μL dNTP, 16 μL water, and 250 μL diluted cell extract containing 100,000 cells. Incubate at room temperature for 30 min. Add 12.2 μL 100x diluted MB. Incubate at 65°C for 60 min. Take fluorescence data for each solution. Repeat, only incubate at 37°C instead of 65°C, don't add water, and add 2.5 μL of PCR buffer to each solution except one. Data Data determined from http://pubs.acs.org/doi/pdfplus/10.1021/ac0702084. Max absorbance 2.954 Wavelength (nm) 523 Absorbance at 450 nm 1.861 Aspr/A450 1.587318646 Approx AuNP diameter (nm) 13 AuNP surface area (nm2) 530.9135 ε450 nm (1/(M*cm)) 1.39*108 AuNP concentration (M) 1.34*10-8 Fluorescence of samples incubated at 65°C: Fluorescence of samples incubated at 37°C: