Lateral Flow Assay: experimental design
Our procedure introduces a protocol for the functionalization and conjugation of CD117 antibodies to 40 nm carboxylated gold nanoparticles. We also provide a protocol for the conjugation of antibodies to 40 nm citrate-stabilized standard gold nanoparticles. This protocol was refined through experimentation and will allow others to optimize the pH of their experiments.
Our team has also developed a procedure for the building of a lateral flow assay that can be completely done by hand. Typically, the construction of lateral flow assays requires specific, expensive machines to lay on the antibodies and gold nanoparticle-antibody complexes on the respective components. It also requires a specific machine to cut the strips. Our procedure was adapted to be done by hand and is very detailed, meaning that future iGEM teams who also don’t have access to these machines can use our procedure to build their lateral flow assays.
Yeast: primers
In order to build a lateral flow assay, one needs to buy antibodies from a commercial source as well as the antigen to detect. This requires the slaughtering of two different animals. These antibodies are also very expensive to purchase. In addition, we must consider the carbon footprint left by the transportation of the product.
Additionally, in order to test the viability and sensitivity of the lateral flow assay, we need the antigen that it is meant to detect. Once again, this is a protein that must be purchased from an outside source. We must again also consider the carbon footprint that is left by the production and transportation of these antigens.
In our case, the antibodies required are anti-CD117 and anti-CD44. It follows that the antigens required are CD44 protein and CD117 protein. With this said, we have worked on the expression of proteins in yeast to be used in the building of our lateral flow assay. Currently, we are expressing CD44 antigen in S. cerevisiae. There are multiple advantages to using yeast to express proteins, including low cost, high levels of expression both through secretion and intracellular expression. They are also easy to manipulate genetically. Overall, this makes for a sustainable approach to the research and development of lateral flow assays. Our team has built primers for the expression of CD44 in yeast.
In an attempt to develop a new diagnostic tool for ovarian cancer using cancer stem cells as the target, we have developed a lateral flow microfluidic device aimed at the detection of two specific cancer stem cells, CD44 and CD177. When found in combination studies have often indicated a chemotherapy resistant form of ovarian cancer. Our contribution to iGEM in this area is the procedure for the conjugation of CD117 and human IgG antibody to standard citrate-stabilized gold nanoparticles as well as the functionalization and conjugation of CD117 and IgG to carboxylated gold nanoparticles.
Dry Lab: 3D printing CAD design
The hardware model has undergone meticulous development in order to correctly fit all important device components. The ESP32 microprocessor, the LFA, and the IR sensor's safe and secure enclosure were the main priorities.
The hardware model consists of two distinct parts, each of which serves a certain purpose. Strategic apertures were included in the location of the microcontroller and LEDs within the larger container. These apertures are essential for maintaining the correct location of these components while making seamless connections to the required equipment.
The properly placed opening in the larger cage is one standout aspect of the design. This aperture has been positioned carefully to enable precise laser beam targeting of the LFA. Additionally, a clever placement of the IR sensor directly above the region where the laser interacts with the LFA ensures incredibly accurate measurements.
Maintaining a sizable spatial separation between the LFA and the microcontroller was also taken into account. To protect the integrity of the microprocessor and reduce any potential heat-related impacts caused by the laser, this separation is essential.
The IR sensor used is Melexis MLX90614ESF-BCA-000-SP - a 3.3V single zone non-contact infra-red thermometer with high accuracy of 0.5C in a wide temperature range. The sensor is mounted on a breadboard and connected to an ESP32-WROVER microcontroller. We also connect an IR receiver for easy temperature reading using a remote (see figure 3 and figure 4 below).
The code to read from the sensor is simple. We created an Arduino sketch and made use of the Adafruit MLX90614 library. The code reads both the ambient and object temperature from the sensor and outputs to both the Serial Monitor and Serial Plotter, which allows us to track the readings in text and chart forms (figure 2).
