Today's objective is to use Ocean Optics to measure the degradation of AuNP fibers by 1nM alpha-chymotrypsin over 24 hours.
- Ben spun down 6 samples of the AuNP fibers that Dr. Hartings synthesized before class and pipetted off the supernatant. (Spun down at 300 RPM for 10 min.)
- We then suspended the samples in 900µL of Tris Buffer, being very careful not to break the fibers. We added 900µL instead of 1000µL because we were going to use 100µL to wash out the test tubes after we added the fibers to the cuvette. We were then going to add this 100µL to the cuvette to have a total volume of 1000µL of fiber/Tris solution.
- 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 35.15625µM.
- 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 1nM. 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=35.15625µM
V1=volume of alpha-chymotrypsin stock needed
M2=concentration of alpha-chymotrypsin needed in cuvette=0.001µM
V2=total volume in cuvette=3000µL
- Since this volume was so low, we did a 1 in 200 dilution of the protease in Tris buffer. Thus, instead of adding 0.02844µL of the chymotrypsin to the cuvette, we added 200 x 0.0853µL = 17.1µL of the 1 in 200 dilution of the chymotrypsin stock.
- 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 - 17.1µL alpha-chymotrypsin=1982.9µL of Tris buffer
- We then added the AuNP fibers and the Tris buffer to the cuvette and began taking measurements. We used 100µL of Tris buffer to wash out the AuNP fiber tubes and added this 100µL to the cuvette.
- We saved the graph and set the parameters to be:
- Integration time = 1ms
- Average over 100 scans
- 20min between scans
- Stop taking absorbance measurements after 24 hours
- We clicked save, then play
- After the first 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 every 20 min interval of time. 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.
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.