Objective
Ocean Optics Spectrophotometry was used to measure absorbance of an AuNP fibers sample over time as it was incubated with the protease, alpha-chymotrypsin. This was performed to track how alpha-chymotrypsin digests lysozyme based AuNP fibers.
Protocol
AuNP Fiber Sample Preparation
- AuNP Fiber samples (6) synthesized by Dr. Hartings were centrifuged at 300rpm for 10min. Afterwards, the supernatant was gently removed as to not disturb the fibers and release them into solution.
- The samples were then resuspended and combined (gently) in 1 mL of 50mMTris/ 20mM CaCl2 Buffer(pH=8)
Protease Preparation
- 1mL stock solution of alpha-chymotrypsin was made by adding 1mL of 50mM phosphate buffer (pH=8) to dry protease for a final concentration of 35.15625µM.
- Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 100nM and final tube volume of 3mL was determined
- V1= .0853uL
- A 1/200 dilution of this initial protease stock was performed to increase the necessary volume so that it could be more accurately pipetted. The necessary volume of protease from 1/200 dilution was 17.1uL.
- Each sample and blank tube for incubation requires a total volume of 1mL, thus 3mLtotal - 17.1uL protease - 1mL resuspended AuNP fibers = 1982.9uL Tris/CaCl buffer.
Incubation Mix Preparation
- To the Ocean Optics Cuvette, the 1mL total resuspended fiber samples was added as well as the volume of phosphate buffer as shown above.
- Experimental and save parameters were set to leave 20min between scans and to only scan for 24 hours with an integration time of 1ms an averaging over 100 scans. Additionally, the scan ranged from 190.96nm-891.81nm.
- To begin the experiment, the lower save file button and then the play button were clicked, creating an immediate first save file.
- Then, immediately after the second save file was generated after 20min, the volume of protease (alpha chymotrypsin) as shown above was added to the sample.
Data
Data Processing
In this experiment, the blank or control data set is considered to be the first data point collected after the initial data point collection. To correct for this blank data (without protease), the absorbance values at each wavelength were subtracted from the absorbance data for each 2 minute measurement after addition of protease (alpha chymotrypsin). Then, to correct for instrumental noise, the absorbance values for the last 60nm for each 2min scan were averaged individually and subtracted from that individual scan.
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