User:Benjamin Friedel/Notebook/CHEM 471/2015/12/02

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Objective

The objective for today is to measure the conductivity, UV-vis spectra, and ICP gold concentrations of the 24 hours incubated AuNP fiber samples that began incubation on 12/01/15.

Protocol

Conductivity Conductivity measurements were recorded at room temperature with the Orion VersaStar Advanced Electrochemistry Meter using an Orion 012005MD Conductivity Cell (USA). After incubation at 37˚C the lysozyme AuNP fiber reaction mixes were centrifuged for 1min at 12,000rpm. Then the conductivity probe was directly introduced to the falcon tube containing the samples and blank without disturbing or contacting the fibers annealed to the walls. For the AuNP fiber synthesis supernatant, the supernatant was drawn off of the reaction vials and tested directly in the falcon tube in which they were stored.

UV-Vis Ocean Optics absorbance spectrophotometry was used again to measure the absorbance of AuNP fiber samples that had been incubated in varying concentrations of alpha-chymotrypsin for 24 hours. 15 AuNP fiber samples were spun down at 300 RPM for 10 minutes and the supernatant was pipetted off. The samples were resuspended in an amount of Tris buffer and alpha-chymotrypsin that would give a 3mL sample with a concentration of protease of either 0M (labeled the supernatant), 1µM, 100nM, 10nM, or 1nM. Triplicates of each concentration were measured. The supernatant from the AuNP fibers that had just been synthesized was collected and designated as the blank.


ICP ICP samples were prepared for testing using the incubated α-chymotrypsin fiber samples of varying α-chymotrypsin concentrations, having three trials for each concentration. The samples were centrifuged for 5 minutes to remove any fiber fragments from solution. 3mL of supernatant from these tubes were transferred to a 20mL Falcon tube, including the blank (no α-chymotrypsin) and the supernatant from before incubation. These samples were then run through the ICP at the preset parameters done by Dr. Hartings to test for the gold ion concentration. Between each runs, the ICP was cleaned by running through deionized water.

Data

Conductivity

Figure 1

Figure 1 shows conductivity measurements in uS/cm for AuNP fibers samples incubated for 24 hours at 0nM, 1nM, 10nM, 100nM and 1uM alpha-chymotrypsin. Additionally, this includes conductivity readings from the supernatant of the AuNP fiber synthesis reaction mix that was drawn off prior to incubation. The standard error for each data point is shown as all but the 0nM blank are averaged from 3-4 measurements. These error bars are very small in the context of overall readings.

Figure 2

Figure 2 above shows the raw conductivity measurements as well as analyzed data taken from the protease incubated samples, non protease incubated blank and AuNP synthesis reaction mix supernatant. For protease concentrations with multiple replicates the average and standard error of the mean for each group was calculated and is shown above.

UV-Vis

Figure 3

The above figure shows the absorbance of the AuNP fiber fragments that were in solution after a 24 hour incubation in varying concentrations of alpha-chymotrypsin. It plots absorbance as a function of the wavelength of incident light; each line represents a different concentration of alpha-chymotrypsin. Since multiple trials for each concentration were run, the absorbance was corrected by first averaging the absorbance at each wavelength for these trials and then subtracting a blank from these averages. The blank was AuNP fibers that had been incubated for 24 hours without alpha-chymotrypsin. The 0nM samples were supernatant from AuNP fibers that had just been formed. These were not incubated.


Figure 4

The above figure plots absorbance of AuNP as a function of alpha-chymotrypsin concentration at 530nm. 530nm was specifically selected because this wavelength of light is the wavelength at which AuNP absorb most strongly; thus, absorbance at this wavelength was due to the AuNP that had been released into solution as the AuNP fibers were degraded. A line of best fit is shown on the graph.


ICP

Figure 5

Figure 5 shows a graphical representation of the ICP data collected, with the average gold ion concentration in comparison to protease concentration. Error bars show the standard deviations of the gold ion concentration averages between sample of the same concentration.


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