Methods
Summary
The core of dPCR is how to partition the qPCR solution into tens of thousands of separated spaces to PCR individually. Mainly there are two ways and both ways look terrible for home use.
Droplet digital PCR (ddPCR) divides solution with oils to make nanoliter-sized oil droplets using pumps and microfluidic plates like this video. So the whole system tends to be complicated. Watch how Bio-Rad's ddPCR works.
Chip-based digital PCR use a microfluidic chip with a micrometer pockets like this $1000 non-reusable Thermofisher's Microfluidic Array Plate. I got a quote for a prototype and this is the reply.
"We use LIGA if the geometries of microfluidics are very small gap between the channels is less than 0.2mm or radiuses needed less than 0.1mm. Then the tool cost is 15-20k EUR. To produce prototypes with such small geometries we can prototype on glass. The minimal cost is 1-5k EUR for a few prototypes."
So we choose PCB solder stencil for chip-based digital PCR. It's easy to order custom stencils online. For example, JLCPCB makes your own for only $7 within 24 hours. As the IC chip gets smaller, the minimum diameter of a hole for a stencil gets smaller, currently 1mil=25.4μm. That's enough for dPCR chip. Stencil is made from stainless steel so we think it can be reusable.
The resolution of microscopes for PCB workers also gets higher. A pixel of this one is 1.55x1.55μm and autofocus 60FPS at 4K resolution. That is enough fast to track with a deep learning model. Watch Brian's epic video and now 1mil=25.4μm is quite a big for you.
PCR, XY stage, and all software are easy for us. So we think that we can make dPCR by combining them. We already applied this deep learning model with YOLOv8 and ByteTrack algorithm to moving droplet and it perfectly worked. Check Lab Note or latest slide to see the whole image diagram.
Challenges
The heat test of qPCR solution in stencil holes is the biggest challenge for us. Temperature changes from room temperature to 98 degrees Celsius. To prevent evaporation, it might be necessary to put natural oil or cover glass onto the stencil. We hope we don't need to control air pressure while sealing but we will make some tools if needed.
Pre Analysis Plan
The color of the qPCR solution drastically changes to fluorescent yellow if the target DNA is included in the pocket. It's clear to see in the screenshot of the microscope camera. so the sensitive photodiode and amplification circuit which we made for qNinja are unnecessary for dNinja. We first use color dye and fluorescein sodium to make several solutions and count them all on a single stencil. After its success, we will order quantified custom DNA and check the calculated concentration matches.
Protocols
This project has not yet shared any protocols.