Device Prototype

Our team decided that in order to ensure that this system is physically feasible that we would aim to produce a working physical prototype. In this process it was imperative that the end-user was considered and that the materials used made our system economically feasible and compatible with the biological system. Through collaboration with the transformers, detectors and our human practices subteams we were able to manufacture a working prototype as the first stage of development.

Process

(Edit)***Integrate creation paragraph below into this section*** Our device is portable and user friendly. A drop of blood from a patient first needs to go through sample preparation to extract target DNA out of the sample. A sample with extracted DNA then flows into the Polymerase Chain Reaction (PCR) chamber where target DNA is amplified via isothermal PCR. Reaction takes a couple of hours to complete and the sample is manually pushed out of the PCR chamber into separate chambers with B. subtilis. In each of the chambers, B. subtilis is engineered to detect a specific disease allowing for multidiagnosis.

Economic feasability - Two-Tiered Model

Economic feasibility is of the utmost importance when dealing with diagnostics in the developing world and therefore our team chose to tackle this issue using a two-tiered development phase. An inexpensive model would utilize the basics of the colourmetric system in which a simple colour change would indicate presence of the specified disease. This signal is easily visible and readable and requires very little technology other than a chamber for Polymerase Chain Reaction (PCR) to occurs and consequential separation of the sample into separate detection wells. Additionally in order to ensure that there is development potential should this modular system expand to other industries a completely digitized device was also explored. The colourmetric signal of the reporter is picked up by a colour sensor that detects the reflected light of this signal, converts it into a voltage and is output to an LCD screen. This exercises the potential of being able to send results of a test directly to a patient's file with bluetooth use.

Manufacturing

The goal of producing a physical prototype brought different perspectives of the best way to produce such a device. Through expert consultation it was determined that microfluidics would increase the price greatly and would not be ideal for our device, however using polytetrafluoroethylene tubing would alleviate this issue and prevent cells from adhering to the inner walls of the device. Both options of 3D printing and machine manufacturing were explored, however it was determined that manufacturing the device would allow us to have direct control of the use of polytetrafluoroethylene for both the chambers and the transport tubing. In collaboration with a mechanical engineering student William Gill from the Schulich School of Engineering Machine Shop - a design was created and analyzed and produced from the desired materials.

Fluid Flow Analysis and Angle Optimization

In our initial design, most of the fluid would flow into the outside chambers from the PCR chamber. In order to make sure the fluid flows equally into the three chambers, fluid flow analysis was performed. On optimal angle for the tubings was found to be 20 degrees.

Figure 1: Fluid flow analysis for different angles between the tubings and the fluid flow in the PCR chamber

The design on the device was then changed to have chambers located symmetrically around the chamber. That allows us to add more chambers if necessary and cut the tubing distance. The fluid flow analysis on the new design with three chambers was also performed.

Figure 2: Fluid flow analysis for the new design with three chambers around the PCR chamber

Colour Sensor

For digitalized version of our device, we are using ColorPal Parallax colour and light sensor to detect color change in bacteria culture in each of the three chambers. Parallax sensor uses its’ LED lights to shine red, green, and blue at the sample. Then the sensor measures the reflected light and outputs the voltage proportional to all the light that it sees. The Arduino Code then converts the sensor values to red, green, and blue components and outputs it to the screen (or LCD display). Since our prototype has three different chambers with bacteria, the initial plan with the ColorPal Parallax colour and light sensor was to measure the color in all three chambers at once. In order to achieve that, we would need to have three different colors for the chambers. In theory, we expected to see noticeable change in red, green, and blue components when just one of the colors was present versus two different colors present at a time (co-infection). Figure 1 shows the set up that we used to test the colour sensor with three chambers. However, the results were inconsistent. We had little control over the system and the sensitivity of the sensor goes down in this set up. For example, if one of the stripes got contaminated with RFP, we would not be able to tell using the color sensor.

Figure 2: Color sensor testing set up: three stripes within 1 cm range imitating the three chambers. Four test squares clockwise: 3 blank; 1 red and 2 blank; 1 red, 1 blue, and 1 blank; 1 blue and 2 blank.

After initial testing, we decided to measure each chamber individually. The sensor was a lot better at detecting one color at a time, even if it was rather faint. Initially, the code for the color sensor was referenced the from Parallax website, but was thoroughly modified to suit our specific purposes. The sensor needs to output five consistent measurements before the color is outputted. Once the color is changed, the sensor detects new measurements and outputs the new color after 5 consistent measurements.

Images of Machined Device (Design 1)

Design Optimization and New Desing

Our first design was analyzed and optimized to make it cheaper, more compact, and allow for the addition of extra chambers. The main change to the design is the location of B. subtilis chambers. The three chambers are now located symmetrically (120° angle between the chambers) around the PCR chamber. First of all, it allows us to use shorter pieces of tubing reducing the cost of the device and making it more compact. Secondly, this design allows us to easily add more chambers if needed. As long as the chambers are symmetrical around the PCR chamber, the fluid would flow equally into each chamber, so fluid flow analysis would not be necessary.