Today's objective was to run Ocean Optics for AuNP fibers that are being degraded by 100nM alpha-chymotrypsin. From the ocean optics data, we will try to determine the rate at which the chymotrypsin degrades the fibers.
- First, we spun down 4 samples of the AuNP fibers that Dr. Hartings synthesized before class and pipetted off the supernatant. (We spun them down at 300 RPM for 10 min.)
- We then suspended the samples in 1 mL of Tris Buffer, being very careful not to break the fibers.
- The buffer was 50mM Tris and 20mM CaCl2 with pH=8
- Next, we brought up one of our stock volumes of alpha-chymotrypsin to 1mL in the Tris buffer. The final concentration of the alpha-chymotrypsin was 42.188µM.
- We calculated the volume of each component that we needed to add to our cuvette:
- We needed the final volume in the cuvette to be 3mL (including the alpha-chymotrypsin), and we wanted the final concentration of alpha-chymotrypsin in the cuvette to be 100nM. Thus, we did the following calculation to determine how much alpha-chymotrypsin we would need to add to the cuvette:
M1=concentration of alpha-chymotrypsin stock=42.188µM
V1=volume of alpha-chymotrypsin stock needed
M2=concentration of alpha-chymotrypsin needed in cuvette=0.100µM
V2=total volume in cuvette=3000µL
- We needed to add 1mL of the AuNP fiber sample. Thus, the volume of buffer that we would need to add to the cuvette in order to bring the final volume of the cuvette up to 3mL was:
3000µL total -1000µL AuNP fiber sample - 4.74µL alpha-chymotrypsin=1995.26µL of Tris buffer
- We then added the AuNP fibers and the Tris buffer to the cuvette and began taking measurements
- We saved the graph and set the parameters to be 30sec between scans and to stop taking absorbance measurements after 24 hours (after about 2 hours, we changed the parameters to every 20 min)
- We clicked save, then play
- After the second file was created, we added the alpha-chymotrypsin to the sample.
Figure 1: Absorbance of Light by alpha-Chymotrypsin-Degraded AuNP Fiber Samples as a Function of the Wavelength of Incident Light (nm)
The above image shows the absorbance of the AuNP fibers that are being degraded as a function of the wavelength of incident light. Each line on the graph represents a different time period from which the data were taken; the graph shows data for just about every 60 min. The absorbance data were corrected by taking the absorbance measurements from just before the chymotrypsin was added and subtracting those data from every measurement after it. From the graph, it's clear that absorbance increased with time. The highest absorbance peaks seemed to be around 235nm and 530nm.
Figure 2: Absorbance of 520nm Wavelength Light by alpha-Chymotrypsin-Degraded AuNP Fiber Samples as a Function of Degradation Time (min)</center>
The figure above shows the absorbance of the chymotrypsin-degraded AuNP fibers as a function of time. The wavelength of incident light was 530nm.