Engineering Journey

1.1 Design

Our first design consisted of a colorimetric assay using gold nanoparticles and spectrophotometry to detect low levels of the target antigens. These antigens were biomarkers for ovarian cancer stem cells CD133 and CD117. In this initial iteration, we spoke to an expert in microfluidics at Concordia University, Dr. Shi, and he gave us feedback that said that it would be more accessible and efficient to create a lateral flow assay instead of the colorimetric assay we had in mind. Through this discussion and further research, we found that there are ways to make a sensitive lateral flow assay. Additionally, we decided to detect CD44 instead of CD133 because CD44 and CD117 often appear concurrently in ovarian cancer.

Gold nanoparticles come in many sizes and have different binding properties. Therefore, we did research on which kind of gold nanoparticles should be used in our assay. At first, we discussed using 20 nm gold nanoparticles for the colorimetric assay, but when we switched our design to a lateral flow assay we decided to use 40 nm gold nanoparticles, since this is the standard. Upon further research, the team discovered two types of bonding interactions between gold nanoparticles and antibodies: passive and covalent. Covalent bonds are formed between carboxylated gold nanoparticles while passive conjugation occurs between standard citrate-stabilized gold nanoparticles.

Although covalent bonding to carboxylated gold nanoparticles is reportedly more effective, these types of gold nanoparticles are much more expensive to purchase. Since one of the goals of our project is sustainability, we decided to experiment on both types of gold nanoparticles. Additionally, there is no current protocol for the conjugation of CD117 or CD44 to either type of gold nanoparticle, so our experiments can contribute to the creation of protocols that include these antibodies. We used a standard pH optimization protocol to find the optimal pH for conjugating CD117 to citrate-stabilized gold nanoparticles. Secondly, we used the Cytodiagnostics protocol as a template to create the functionalization-conjugation protocol of CD117 to 40nm gold nanoparticles.

In parallel, we decided to work on in silico modeling of monovalent and multivalent CAR T-cell therapy targeting the same antigens that would be detected (CD44 and CD117). More information on this can be found in the dry lab section.

1.2 Build

We created a procedure for the passive conjugation of CD117 and CD44 to standard gold nanoparticles based on the cytodiagnostics procedure “Adsorption of Proteins to Gold Nanoparticles” (1). We first ran our experiments with CD117 antibodies, but the procedure for optimization can be used for CD44 as well. In order to create a procedure for the conjugation of standard citrate stabilized gold nanoparticles with CD117, we needed to run a pH optimization procedure with varying pH levels. The experimental procedure that we used can be found here: (link to experiments tab).

1.3 Test

Since we had a larger quantity of IgG antibodies that were intended for use on the control dot of the lateral flow assay, at the suggestion of our mentors, we first ran the optimization procedure by conjugating IgG antibodies to the standard gold nanoparticles. For this, we tested the efficiency of 4 different pHs using 3 different buffers: MES at pH 5.5, Sodium phosphate 0.1M at pH 6.5 & 7.5, borate buffer 0.1 at pH 8.5.

1.4 Learn

To evaluate the functionalized gold nanoparticles with IgG in MES buffer pH 5.5, the test was performed on a lateral flow test strip as a reverse test with anti-CD117 anti-human in mouse at the control line and IgG anti-rat in goat at the test line. The evaluation was successful as two red dots which presented the functionalized gold nanoparticles bound to the IgG anti-rat in goats and anti-CD117 anti-human in mice. A picture is included in the results page (insert link to results page).

2.1 Design and Build

Knowing that antibodies tend to behave similarly, we used this information to optimize the pH of standard gold nanoparticles conjugated to CD117 antibodies. We edited the protocol for the pH optimization procedure to be more detailed and fit our experimental factors. Our goal was to repeat the procedure using CD117 at pH 5.5 in the MES buffer.

2.2 Test

We ran another experiment to verify that 5.5 in MES buffer was the optimal pH for the conjugation of CD117 to citrate-stabilized gold nanoparticles. The solution came back as purple which means that the antibodies were not successfully conjugated.

2.3 Learn

The optimal pH and buffer for the conjugation of IgG to 40nm citrate-stabilized gold nanoparticles is not the same as the optimal pH of CD117.

3.1 Design

Instead of running more tests right away, we decided to experiment with the conjugation of the carboxylated gold nanoparticles. Although carboxylated gold nanoparticles are more expensive, covalent conjugation is reported as a more effective method by creating stronger bonds between the gold nanoparticles and antibodies. This influenced our decision to preserve the amount of CD117 to run tests with the carboxylated gold nanoparticles first before returning to the experiment on conjugating standard gold nanoparticles.

3.2 Build

We built our protocol based on the Cytodiagnostics protocol for functionalizing and conjugating 40 nm gold nanoparticles (2). We ran the experiments on both IgG and CD117 antibodies in order to observe whether one antibody gave a different result from another.

3.3 Test

We went through the procedure of functionalizing and conjugating the carboxylated gold nanoparticles with IgG and CD117. The first conjugate solutions were low in volume to perform UV-vis spectroscopy so we performed a dot blot assay to test whether functionalization/conjugation was successful, as suggested by the company that sells the gold nanoparticles, Cytodiagnostics. This gave a negative result, demonstrating that either the functionalization was unsuccessful or neither the CD117 or IgG antibodies were conjugated to the gold nanoparticles.

3.4 Learn

We decided to perform the same experiment but make a sufficient quantity antibody-gold nanoparticle solution in order to perform UV-vis spectroscopy. The dot blot assay is a long procedure with multiple steps so it was unclear which step might have caused an issue. Therefore, we learned that using UV-vis spectroscopy was more suitable for testing purposes.

4.1 Design

Since the immuno-blot dot assay contains multiple steps, it is easier to make errors that lead to negative results and difficult to know where the results come from. Therefore, we adjusted our protocol so that the verification was done using UV-vis spectroscopy to observe whether there was a peak wavelength shift. We also included multiple incubation times in our design to find the optimal incubation time for CD117.

4.2 Build

The method is as follows: we measure the peak wavelength of the unconjugated gold nanoparticles and do the same for the conjugated gold nanoparticles, within the 450 to 700 range.

4.3 Test

The conjugated AuNP-CD117Ab were incubated for either 3h or 4h. They both displayed a peak wavelength of 535nm, which is a shift from the 530nm peak wavelength of 40nm carboxylated gold nanoparticles.

4.4 Learn

The shift in peak wavelength indicates that either incubation time is sufficient for the conjugation process. With this information, we edited our conjugation procedure. Seeing as three hours of incubation time is shorter than four hours but equally efficient, we altered our functionalization/conjugation protocol to suggest a three hour incubation time.