Team:Concordia/Notebook/Cyborg
Nanoparticle Attachment Laboratory Notebook: Cyborg Method
Day 1:
Purpose: To synthesize conjugated silver nanoparticles and PAH in order to do “cyborg” attachment by finding an effective substitute to a sonication probe.
Sources:
DOI: 10.1039/c4ra15857a
DOI:10.1021/j100214a025
DOI: 10.1021/bp0501423
Protocol:
1.Add 2mL of citrate-capped silver nanoparticles to 10mL of aqueous polyelectrolyte solution (1% PAH)
2.Sonicate for 20 min using the jewelry sonicator set to “high”
-Wrap the sonicator probe with parafilm to coat the metal part - ensure that there is no exposed metal region
-Dip the sonicator probe into the solution in the 15mL conical tube
-Try NOT to have the probe touching the sides of the conical tube
-Close sonicator door PROPERLY - improper closure of sonicator during use can result in hearing damage
-Solution color should be lighter after sonication
3.The nanoparticle solution needs to be stirred for 24h
4.Centrifuge the solution at high (12000rpm) for 2 min
5.Wash 3 times with water
6.Mix 100 μL of E.coli (3.0*107 cells/mL) or yeast cells (2.5*109 cells/mL) with 1mL of 0.45 mg/mL PE-stabilised silver nanoparticles
7.Incubate for 15 min in a low speed shaker
8.Centrifuge at high 2min to separate cells from loose nanoparticles
9.Wash cells with water
Modifications:
To purpose of this day is to experimentally modify the protocol and find an effective alternative for the sonication probe. A sonication probe is available but, due to safety concerns it was deemed a must to find a safer alternative.
Results:
After a few analysis of possible alternatives, it was deemed that a jewelry sonicator will be a safer and efficient alternative to the formation of AgNP-PAH conjugates. After sonicating at high for 20 min the 1% PAH and silver nanoparticle solution turned a semi clear yellow solution.
Day 2:
Purpose:To do “cyborg” attachment of AgNP-PAH conjugates on yeast cells at various volumes of AgNP-PAH conjugates.
Protocol:
1.Add 2mL of citrate-capped silver nanoparticles to 10mL of aqueous polyelectrolyte solution (1% PAH)
2.Sonicate for 20 min using the jewelry sonicator set to “high”
-Wrap the sonicator probe with parafilm to coat the metal part - ensure that there is no exposed metal region
-Dip the sonicator probe into the solution in the 15mL conical tube
-Try NOT to have the probe touching the sides of the conical tube
-Close sonicator door PROPERLY - improper closure of sonicator during use can result in hearing damage
-Solution color should be lighter after sonication
3.The nanoparticle solution needs to be stirred for 24h
4.Centrifuge the solution at high (12000rpm) for 2 min
5.Wash 3 times with water
6.Mix 100 μL of yeast cells (2.5*109 cells/mL) with 1-4mL of PE-stabilised silver nanoparticles
7.Incubate for 15 min in a low speed shaker
8.Centrifuge at high 2min to separate cells from loose nanoparticles
9.Wash cells with water
Results:
Figure 1: 100 μL of yeast cells (2.5*109 cells/mL) with 2mL of AgNP-PAH D-K with controls A-C at 10000X.
Figure 2: 100 μL of yeast cells (2.5*109 cells/mL) with 3mL of AgNP-PAH D-I with controls A-C 10000X.
Figure 3: 100 μL of yeast cells (2.5*109 cells/mL) with 4mL of AgNP-PAH D-I with controls A-C 10000X.
Figure 4: 100 μL of yeast cells (5*109 cells/mL) with 2mL of AgNP-PAH D-K with controls A-C 10000X.
Figure 5: 100 μL of yeast cells (5*109 cells/mL) with 3mL of AgNP-PAH D-L with controls A-C 10000X.
Figure 6: 100 μL of yeast cells (5*109 cells/mL) with 4mL of AgNP-PAH D-K with controls A-C 10000X.
As it can be observed from figures 1-3, there is a significant decrease in the cell number in comparison to the control with vary few of the cells being different to the control to indicate successful cyborg attachment. On the other hand doubling the amount of cells gave more cell samples with celss coated with a brown mass, this is successful cyborg attachment. Thus it is determined by looking at figures 5&6, that 3 to 4 mL of nanoparticles is optimal for cyborg attachment.
Day 3:
Purpose:To do “cyborg” attachment of AgNP-PAH conjugates on yeast cells and use dark field microscopy to see successful cyborg attachment.
Protocol:
1.Add 2mL of citrate-capped silver nanoparticles to 10mL of aqueous polyelectrolyte solution (1% PAH)
2.Sonicate for 20 min using the jewelry sonicator set to “high”
3.The nanoparticle solution needs to be stirred for 24h
4.Centrifuge the solution at high (12000rpm) for 2 min
5.Wash 3 times with water
6.Mix 100 μL of E.coli (3.0*107 cells/mL) or yeast cells (2.5*109 cells/mL) with 4mL of 0.45 mg/mL PE-stabilised silver nanoparticles
7.Incubate for 15 min in a low speed shaker
8.Centrifuge at high 2min to separate cells from loose nanoparticles
9.Wash cells with water
Modifications:
No new modifications were made after the previous protocol, the purpose of this experiments were to find a more effective way to take pictures for more conclusive results of cyborg attachment. For this dark field was considered more effective.
Results:
Figure 7: Darkfield of cyborg attached yeast cells D-G with controls A-C at 200X with various color filters.
As it can be from figure 7 the silver nanoparticles coating and aggregates give off a green fluorescence when portrayed through different color filters. With D being the original and E-G being the different.
