Three basic components lay the foundation of our fungal diagnostic device. The DNA extraction microfluidic chip, the concentrator chip and the fluorescence detection platform. The diagnosis process begins with the microfluidic chip that extracts DNA from the lysed cells through surface chemistry. In order to improve the accuracy of the diagnosis, a concentrated mix of the DNA is produced using the microfluidic concentrator chip. Finally, the fluorescence detection module detects the presence of fungal pathogens in the DNA sample.
The operation of the microfluidic chip can be observed in the Engineeringtab.
Building
First, a 12mm x 8mm piece of hydrophilic paper is cut out and loaded onto the Silhouette Cameo cutter. Using the Autoblade setting, the designed channel is carved into the hydrophilic paper. The channel path is then removed using a knife and discarded. The top layer of protective film covering the hydrophilic paper is also removed and discarded, exposing its sticky side. A similarly sized piece of hydrophobic film is wiped clean and pressed against the hydrophilic paper’s stick side firmly to remove air bubbles. Using a puncher, holes are created at the entry and exit site of the channel. These sites are then wiped thoroughly with Isopropyl alcohol to enable the glue to stick to the hydrophobic film. Using a hot glue gun, a cut pipette tip is glued at the entry site of the chip. A syringe tip for attachment to the pump is glued to the exit site.
Figure 1: DNA Extraction using the microfluidic chip
Cleaning
After the glue has dried, the bottom layer of protective film covering the hydrophilic paper is also removed and discarded. The chip and another piece of the clean hydrophobic film are placed into a Plasma Purification Unit to be cleaned for 10 minutes. After cleaning, the hydrophobic film is pressed against the sticky side of the hydrophilic paper tightly to join them together. This removes any external impurities from the chip that can lead to contamination in experiments.
Functionalization
After cleaning the chip, a 4:1 solution of DNA-free water and APTES stock is prepared. 300µl of this solution is then passed at 100 µl/min through the chip, but not allowed to flow out. The microfluidic chip was then incubated for 60 minutes at 65 C. After this, the APTES stock solution was rinsed out using 900 µl of distilled DNA-free water flowing at 300 µl/min. Functionalization served the purpose of improving the binding capability of DNA to the inner walls of the chip.
Working Procedure
The main function of the microfluidic chip is the extraction and purification of the target DNA from the lysed bacterial cells. The initial input into the chip is a mixture of dead bacterial cells and exposed DNA. The DNA is attracted to the walls of the functionalized chip through the surface chemistry, while the other impurities are not. This allows us to flush out these impurities, then extract the target DNA which will be sent to the concentrator chip to amplify the concentration of the target DNA. This extracted DNA is then used for either quantitative or qualitative analysis.
The second component of our hardware setup is the concentrator chip. It functions to concentrate the DNA sample mix electrokinetically such that the resultant DNA concentration becomes easier to detect. When the DNA sample is passed through the concentrator chip, there occurs a mass transfer of DNA molecules onto capture probes. The capture probe array and the sample reservoir are separated by a special PEDOT:PSS membrane. The DNA is diffused into the capture probes through the membrane. This process is accelerated by the fact that our concentrator chip does the transfer electrokinetically which has a higher efficiency compared to other similar DNA concentration techniques. The concentrator chip in our model allows for a concentration factor of roughly (300-1,000) fold after 15 minutes for DNA samples from an initial sample concentration ranging from 1 to 100 nM.
Figure 2: A schematic diagram of a microfluidic concentrator chip.
The final step in the diagnosis process is the fluorescence detection whereby the DNA sample is excited and the emitted fluorescence is used to ascertain the status of the DNA sample. The following diagram illustrates our approach to the fluorescence detection component.
Figure 3: Fluorescence detection schematic
Excitation
The DNA sample was mixed with FAM fluorescent dye which exhibits peak emission fluorescence when excited with light having wavelength of 495 nm. However, 495 nm wavelength corresponds to blue/cyan color which diminishes the character of green fluorescence emission. Therefore, we used Ultraviolet light emitted through UV LEDs for excitation purposes instead. The emission fluorescence due to excitation is in the range of 520 - 530 nm that falls in the range for green color.
Dichroic filter
In order to make our prototype as accurate as possible and reduce possible sources of interference from external light sources, we added a dichroic filter after the DNA sample to allow only light with wavelength of 530 nm to pass through. Brightline 530/43 single-band bandpass filter was used for this purpose in the experiment. It ensured that the light falling on the sensor only originates from the sample excitation, that is, if it gets excited at all.
Figure 4: Brightspace Basic dichroic filter
RGB sensor
For the sensor, we used TCS RGB 34735. It is a light sensor that can detect the Red, Green and Blue composition in the incident light. We used the Green and the Blue readings to calibrate our statistical model for the device.
Figure 5: TCS RGB 34735 light sensor
Microcontroller
The light sensor was connected to the microcontroller Arduino Nano. The microcontroller used information collected from the light sensor and after conducting the statistical analysis, decided on the status of the sample. The statistical model used to differentiate between the positive and negative samples used a threshold intensity value of green light emission from the sample. A threshold intensity value was established after conducting the experiment using 10 positive and 10 control samples and observing the intensity of the emitted green light.
