Experiments

Overview

In order to create the lateral flow assay, our team found protocols for the conjugation of citrate-stabilized and carboxylated gold nanoparticles, respectively. We also found protocols for the creation of lateral flow assays (references included in the procedures on the experiments page). Since we don’t have access to the specialized machines required for injecting lines onto the nitrocellulose membrane and cutting the assembled components into strips, we created a fully manual method for the creation of a lateral flow assay. Since our team has only performed experiments on the gold nanoparticles so far, most of the lateral flow assay protocol remains untested. However, it is specific to our experiment in terms of test and control dot placements as well as manual instructions.

We decided to use test & control dots instead of lines for two reasons: in the lab, we found that applying lines of antibody solution manually to the nitrocellulose membrane was ineffective, as the solution spread out instead of forming a line. Secondly, and more importantly, the thermal method to be used for increasing sensitivity requires a dot. This means that the test area on the lateral flow assay must be in the form of a dot.

As for the gold nanoparticles, we used standard pH optimization protocol to find the optimal pH for conjugating CD117 to citrate-stabilized gold nanoparticles. Secondly, we used the Cytodiagnostics protocol for functionalizing/conjugating 40 nm gold nanoparticles as a template to create the functionalization-conjugation protocol of CD117 to 40nm gold nanoparticles.

We are also working on the production of CD 44 antigen in yeast to have an easy-to-make and reproducible antigen where the concentration of the antigen that is expressed on the membrane can be controlled and changed by the promoter. This allows for the simulation of Cancer stem cells at different stages of cancer. Gibson cloning is being used to insert human CD44 into yeast.

Procedures

We truly hope these procedures can help future iGEM teams during their lab adventures.

Previous versions

To see the changes at each iteration, download the previous versions of the procedures below:

Lateral Flow Assay:

• Version 1

Conjugation of standard citrate-stabilized gold nanoparticles:

• Version 2
• Version 1

Functionalization and conjugation of 40 nm carboxylated gold Nanoparticles:

• Version 2
• Version 1