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Project

Description Design Characterization Results Model Software Hardware

hardware head

Hardware

Hardware is a very large part of our project. However, it is also often the most complex and costly to produce. Therefore, we elected to concentrate our efforts on the most critical part of our design, the microfluidics chip. However, we decided to still thoroughly plan other parts of the hardware, as we considered it critical to have a complete platform for future works on this device.

The sample is first collected, either from the integrated Vortex sampler, of from any other source, and is prepared in 1mL of the liquid buffer. The sample is then split into 8 microtubes, that are then placed into the Covaris sonication apparatus, which will break up the particles and membranes. After this, the microtubes are placed into the cartridge, already prepared with 8 microfluidics chips and the necessary reagents lyophilized, which is then inserted into the A.D.N.

**Figure 1:** Illustration of A.D.N. device design. (Top left) Front view of the device. (Top right) View of the internal components of the device. (Bottom left) Top view of the cartridge with the microfluidics circuits.

Parts of the device:

Outside:

Vortex sampler: A Vortex sampler, which can concentrate large volumes of air into liquid. Sold with the device so sampling can be easily done on site.
Covaris sonicator: A contact-less sonicator that will be either incorporated in the device or installed separately. Will allow to break up particles present in air and break up virus or bacterial membranes, therefore making genetic material more available for purification and detection.
Output screen: A message will be printed if detection is successful or unsuccessful.
Cartridge slot: To insert the organism specific cartridge.
Reagents reservoirs: Reservoirs that hold the necessary solutions for sample treatment. Reservoirs hold enough solutions for multiple cartridges.

Inside:

Pump: Will be used to remove waste from washing steps and help control flow in the chip.
Liquid waste: Where liquid waste will be collected.
Mobile magnetic bar and heater: A mobile heather and magnet that will sequentially be placed under the chips as need be for the extraction steps.
Capillary system: Will carry liquids from microcentrifuge tubes to sample chamber and liquids from reservoirs to chip as need be. Flow will the controlled by peristaltic pumps.
Detection apparatus: Two filters, one to specifically allow for only GFP emission wavelength to reach the photomultiplicator tube, and another filter to remove background level signal. The photomultiplicator tube will be connected to an Arduino logic system that will print a specific message on the display as signal is detected or not.
Reservoirs: Reservoirs that will contain sterile, ddH2O and >99%EtOH for various wash steps.

Cartridge:

Microtube rack: Rack in the cartridge where the microtubes will be inserted after sonication.
Microfluidics chips for detection: 8 microfluidics chips side by side. The initial 1mL sample is split into 8 chips to ease detection and sample treatment.
Depending on the organism of choice, cartridges will range between 20 and 50$

Microfluidics:

The microfluidics chip contains five main chambers and valves to control the flow. In the first chamber, the sample is placed via capillary tubes from the sample collection tubes. Valves are operated using gas pressure to close off the channels when necessary. Once the sample is in the chamber, the first valve opens, and the sample proceeds into the DNA/RNA extraction chamber, which is then closed-off by valves. The nucleic acids will bind to the magnetic beads. Depending on the target sequence, the bead/sample volume ratio can be adjusted to selected for fragments of a certain size. Once the binding is completed, the magnetic bar will raise and trap the magnetic beads at the bottom of the reaction chamber. The waste liquid will be evacuated by the pumps, as the necessary valves open towards the waste chamber. A magnetic bar is lowered, freeing the beads. Beads are washed with ethanol twice. The nucleic acid is finally eluted in sterile water. The now purified nucleic acids are sent to the Loop-Mediated Isothermal Amplification (LAMP) chamber, where the necessary reagents are lyophilized. The metal heater is brought close to the cartridge to start the isothermal amplification of the desired target region. After the reaction (15 to 45 minutes), the amplified sample is conveyed to the final chamber. This chamber contains a lyophilized cell-free expression system, with the ToeHold sequence. When the sample enters the chamber, the expression system components are resuspended and detection can start. Based on experimental data, detection itself will take approximately 4h.

Total detection time is therefore between 5 to 6h. While this is a long time for detection, we are working on improving detection time by increasing ToeHold concentration in the detection chamber and augmenting trigger availability. With sample treatment modification, we hope to reduce detection time to under 2 hours.

The signal will be detected by a photomultiplicator tube, which can detect very faint signals. To avoid false positive, filters will be placed in front of the tube so that only above-background levels are detected. The signal is then treated by a simple Arduino circuit that will output if the sequence of interest, therefore the organism of interest, is detected.

Team:ULaval/Hardware