Team:Concordia/Demonstrate/Attachment Results
NANOPARTICLE ATTACHMENT RESULTS:
NANOSHELL ATTACHMENT WITH DIFFERENT SYNTHESIS METHODS
NANOSHELL ATTACHMENT WITH MARTIN SYNTHESIS
NANOSHELL ATTACHMENT WITH BOUGHT NANOPARTICLES
NANOSHELL ATTACHMENT WITH ALOE VERA SYNTHESIZED NANOPARTICLES
NANOSHELL ATTACHMENT USING CDC28 YEAST MUTANT
CYBORG ATTACHMENT WITH TURKEVICH SYNTHESIS
Nanoshell Attachment Dark Field from Synthesized Nanoparticles
Dark Field Microscopy of the Nanoshell Attachment method utilizing different nanoparticle synthesis methods
After confirming the success of nanoparticle attachment through bright-field microscopy, our team searched for a way to add further support to our results. We discovered some studies in which researchers used dark-field microscopy to view nanoparticle attachment to cells. The images above are of spherical gold nanoparticles ranging in size from 1-10 nm attached to Saccharomyces cerevisiae viewed under a dark-field microscope with a camera attachment. The images have been color inverted to better visualize the nanoparticles. The gold nanoparticles appear to glow green under dark-field, this makes it clear which cells are coated in nanoparticles. In the first three images, the nanoparticles used were synthesized from the Martin method and were successfully attached to the yeast cells. In the last image, the nanoparticles were synthesized using garlic extract. These nanoparticles were less successful at attaching to the cells. It is possible that they need to be further purified so there is no plant extract left in the solution to interfere with attachment.
Nanoshell Attachment using Martin Method Nanoparticles
Gold nanoparticles Synthesized using the Martin Mehod attached to yeast cells under different conditions
The second phase of our project involved the attachment of nanoparticles to cells. The images above are of gold nanoparticles ranging in size from 1-10 nm attached to Saccharomyces cerevisiae cells. This followed the synthesis of nanoparticles and confirmation of their size and presence. More details on the synthesis can be found on our synthesis results page. The nanoparticles in the images above were synthesized using the Martin method and then coated with L-cysteine which allowed them to bind to the cell surface of yeast cells. This creates a gold nanoshell that we hope will serve as a form of protection for our yeast cells during the cell battle. To confirm that attachment was successful, the samples were viewed using bright-field microscopy. While individual nanoparticles are not visible through a typical light microscope, large aggregates of the nanoparticle are visible. This attempt at attachment was indeed successful as can be seen by the images above. The dark purple ring surrounding the yeast cells corresponds to our gold nanoshell. When we compare the nanoparticle-coated yeast to control yeast cells, we can see that the dark purple rings are not present in the control. Furthermore, when the cells are viewed in different planes it is clear that the outside of the yeast cells appear dark purple, corresponding to the nanoparticles attached to the surface. The last set of images show what happens if we increase the ratio of nanoparticles-to-cells during the attachment procedure. Some clumping appears in these images.
Nanoshell with Bought Nanoparticles
Bright Field Microscopy of the Nanoshell Method utilizing Cedarlane bought Gold Nanoparticles
Gold nanoparticles were purchased from Cedarlane and used for attachment to yeast cells using the nanoshell method. We felt that it would be important to compare our nanoparticles to industrial nanoparticles. The attachment of these nanoparticles is identical to the ones we synthesized. The dark purple ring surrounding the cells corresponds to the gold nanoshell. There does not seem to be any difference in attachment efficiency between the purchased nanoparticles and the ones we made.
Aloe Vera Attachment Nanoshell Attachment
Bright Field Microscopy of the Nanoshell Method utilizing Gold Nanoparticles synthesized with Aloe vera extract
The aloe vera extract synthesis method was unique because it created a variety of shapes, including nanotriangles. When attached to cells, these shapes may be useful as a “weapon” during the cell battle. The other synthesis methods used in this project create spherical nanoparticles. The images above are of gold nanoparticles synthesized using the aloe vera extract attached to the surface of yeast cells. To attach these nanoparticles, we followed the same procedure as the nanoshell attachment method. These bright-field images indicate that attachment was successful. However, there are several differences between how well these nanoparticles attached compared to the spherical nanoparticles synthesized from the Martin method. First, these nanoparticles attached in larger clumps than the Martin method nanoparticles. In addition to this, the Martin method nanoparticles seemed to have a higher binding efficiency to the yeast cells. The images also show some sort of contamination in these samples. It is most likely components of the plant extract that stayed in the solution despite our efforts to purify these samples through centrifugation. Better purification of the samples may lead to higher attachment efficiency.
Nanoshell Attachment using CDC28 Yeast mutant
Bright Field Microscopy of the Nanoshell Method utilizing CDC28
Our cell battle involves isolating two nanoparticle coated cells through a microfluidics chip and forcing them to come into contact. We had to ensure that the cell that is isolated actually is one of the cells coated in nanoparticles and not a newly divided cell. One of the things that we had to consider was a way to keep our cells from dividing. The solution was one of our biobrick parts which is a CDC28 mutant that prevents yeast budding at the restrictive temperature of 37°C. To be able to use CDC28 in our cell battle, we had to ensure that our attachment methods were compatible with these cells. The images above are of nanoparticles purchased from Cedarlane attached to the surface of CDC28 cells using the Nanoshell method. The images show that attachment was successful, as can be seen from the dark purple rings around the cells. The surfaces of the cells also appear dark purple, corresponding to our gold nanoparticles.
Cyborg Attachment
Bright Field images Cyborg Attachment method utilizing silver Nanoparticles synthesized using the Turkevich Method
The Turkevich synthesis method creates spherical silver nanoparticles. In the images above, nanoparticles synthesized from the Turkevich method were attached to the surface of Saccharomyces cerevisiae cells using the Cyborg attachment method. In this method, we coat our silver nanoparticles with polyallylamine hydrochloride which allows them to bind to the surface of yeast cells. These images are from both bright-field and dark-field microscopy. The images confirm that nanoparticle attachment was successful. We can see large clumps of silver nanoparticles attached to the cells. Unlike the nanoshell method, the nanoparticles do not coat the entire surface of the cell. This should not be a problem during the cell battle because we hope that the clumps of silver nanoparticles will be used as a type of “weapon” and pierce the cell membrane of the opposing cell.
