Sample Prep/Reaction Medium Pathways
@Yaman Garg
The list of all the sample prep options with the feasible ones marked with green and the ones we will not proceed with for now/unpreferred marked red can be found on the engineering options miro board. Starting from all the available options for Sample Prep/DNA Extraction, the team has met to make the decisions summarized below:
- Sonication and Electrical lysis were marked as unpreferred for now as the team realized that 1. developing these methods would be too time-consuming since they have not been used in integrated POC devices or have been utilized for commercial POC sample prep solutions, 2. the integration of these methods into the final device would also be hard, and 3. the other methods available are very much more developed, hence should be prioritized.
- On-chip bead-beating was marked as unpreferred for now as the method would also be very time consuming to develop, optimize, and integrate than the other bead beating solutions, and would also be quite complicated to prototype, test, and manufacture. The use of microfluidic chip technology for the method is also unpreferred since these chips have been reported to have frequent issues such as air bubbles etc. Meeting with Prof. Mohammad Qasaimeh
- Whatman FTA Paper was put in the lowest priority in the feasible sample prep methods as 1. The method takes 30 minutes which is quite long as compared to other available options, 2. The PCR efficiencies reported have been as low as 66%, 3. The method is unflexible in the sense that the extracted DNA in the FTA paper cannot be separated from it and all the reagents have to be passed through the paper for the amplification and detection process, 4. Can only be used for fluorescence
- Brainstorming about Lysis Buffer + Silica Membrane option, and looking at implementations such as Biomeme's M1 Sample Prep and Zhao et al. 2019 's Universal Syringe, we realize the engineering problem behind this option is: "passing (multiple times) a fluid through a filter and doing it for multiple fluids". A solution which we decided on, for now, was creating a device - sort of a Mini-Opentrons- that uses a revolver like a cartridge with multiplex capabilities (think of multiple rings) that can be moved to align chambers with the syringe + silica filter and a syringe attached to silica filter (like Biomeme M1) that can be automated to move up and down and draw fluid in and out to perform steps. After sample prep, the amplification and CRISPR step can be done by another syringe not attached to a filter. Finally, detection can be done by either fluorescence or LFA using the fluid moving capability of the second syringe.
- Thinking about sample prep with Bead beating and Lysis Buffer + Paramagnetic particles (PMPs), and taking options such as OmniLyse, and mobiNAAT (Magnetic movement + Smartphone based fluoroscence + electrical heating) we realized that the engineering problem with the implementation of these options is "to move the fluid from amplification (RPA/LAMP) stage to the CRISPR process and further to the LFA strip in case of such". Multiple solutions were suggested for tackling this problem"
- Mini Opentrons - Using the a moving syringe system described above that can move the amplified DNA to CRISPR chamber
- Vertical piercing solution for RPA -> CRISPR barrier - We use the omnilyse sharp tip or the swab or any such object to pierce a horizontal membrane separating the vertical amplification and CRISPR chamber
- Using magnets as piercing agents for a membrane barrier between RPA -> CRISPR- Using a sharp piercing magnetic ring that can be moved using a permanent magnet outside the chamber.
- Using magnet as gates forming barrier between RPA -> CRISPR:
- A widening tube approach - The magnetic bottom surface of the amplification chamber is lowered using a permanent magnet outside until the wider CRISPR chamber is reached where this barrier is effectively diminished.
- A Sliding Gate Approach - The magnetic bottom surface of the amplification chamber is slid inside a cavity using another magnet to effectively open the gate between CRISPR and amplification chamber.
- An Oil based Approach- The magnetic bottom surface of the amplification chamber has cavities through which oil can be passed. The oil is filled between the amplification and CRISPR reagents and when the magnetic barrier is lowered (using another magnet outside), the oil forces the CRISPR and amplified DNA to mix up.
- PMP based methods for transport from RPA -> CRISPR as well - Need to be researched further but one suggestion is to change the solution properties of amplified DNA to re-bind DNA with PMPs and use a permanent magnet to transport them to CRISPR chamber where the DNA is unbounded from the PMPs.