User:Benjamin Friedel/Notebook/CHEM 471/2015/10/07

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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

  1. AuNP Fiber samples (4) 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.
    1. The samples were then resuspended and combined (gently) in 1 mL of 50mMTris/ 20mM CaCl2 Buffer(pH=8)

Protease Preparation

  1. 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 53.517µM.
    1. Through M1V1= M2V2 the correct volume of protease needed for a final incubation tube concentration of 1uM and final tube volume of 3mL was determined
      1. V1= 56uL
  2. Each sample and blank tube for incubation requires a total volume of 1mL, thus 3mLtotal - 56uL protease - 1mL resuspended AuNP fibers = 1944uL Tris/CaCl buffer.

Incubation Mix Preparation

  1. To the Ocean Optics Cuvette, the 1mL total resuspended fiber samples was added as well as the volume of phosphate buffer as shown above.
    1. Experimental and save parameters were set to leave 2min between scans and to only scan for 2.5 hours. Additionally, the scan ranged from 190.96nm-891.81nm.
    2. To begin the experiment, the lower save file button and then the play button were clicked, creating an immediate first save file.
  2. Then, immediately after the second save file was generated after 2min, 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.


Figure 1

Figure 1 above shows the absorbance values for each scan after the first 2min at 530nm are shown as time progressed. This is because AuNP fibers were previously determined to have an absorbance max at 530nm, so their breakdown into solution upon digestion of AuNP fibers by alpha-chymotrypsin was monitored. This figure shows that before the protease was added, the fibers have a stronger absorbance. After protease addition the quick decrease in absorbance that reached a lower limit around 0.8 suggests that the fibers are degraded into constituents that do not absorb at 530nm and that this occurs relatively quickly and comprehensively.



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