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We envision CascAID as a complete sample-to-answer solution for pathogen detection. Apart from our fluorescence detector “Lightbringer”, we hence had to realize a user friendly environment for the extraction and amplification of target RNA from a patient sample via lysis, followed by a combined reverse transcription, isothermal PCR and transcription step.
We therefore conceived a portable fluidic system featuring a temperature control unit and a chip to conduct all said sample processing steps prior to the readout reaction. By combining all steps on a single, enclosed and disposable chip we reduced the risk of cross-contamination and ensured secure handling of reagents and waste products.
Here, we present an early prototype that addresses these criteria. We created the chip from PDMS via soft lithography, using 3D-printed molds to speed up prototyping cycles compared to photolithography-based methods.
As shown in a different section, fluid flow can be controlled with the pressure supplied by air balloons and a bike tire that powers quake valves. In early tests we used an open source 3D printed peristaltic pump, but we expect that the balloon based solution facilitates scaling down the system, improving portability while also lowering the cost.
Buffer Reservoir
The processing buffer, required for isothermal PCR, comes contained on the disposable chip. It is withheld in a reservoir by magnetic valves, held in place by clamps, pierced into the PDMS, which could later be replaced by normally closed quake valves. For reaction rooms and reservoirs we tried to stick to long channels rather than real chambers to prevent the formation of air bubbles.
Explosive drawing of our PDMS chip.
Sample Uptake
It is convenient to take the patient sample as a swab with absorbent cotton, which can be inserted into a window in the chip that is then closed with a plug. After the chip is connected to the pressure supply, the valves withholding the buffer are opened by removing the clamps. The buffer then flushes the patient sample – including the pathogen – out of the cotton and into the thermolysis chamber.
Labelled PDMS chip.
Thermolysis and Isothermal PCR
Our wetlab experiments showed that thermolysis at 80°C followed by isothermal amplification of the pathogen RNA via Recombinase Polymerase Amplification (RPA) combined with transcription at 37°C provides a reliable and non-hazardous procedure that yields concentrations of target RNA that are detectable by Cas13a. The sample stays in the lysis chamber for 2 minutes and then passes through cooling loops to prevent heat denaturation of the lyophilized protein, which is stored in the RPA chamber. The amplification is conducted for 1.5 hours and finally the processed sample is released onto the paper strip for the readout reaction.
Temperature control unit
We developed “Heatbringer”, our temperature controlling unit, at TECHFEST Munich. For heating the two chambers we used Peltier elements as they were at hand. A probably more appropriate alternative for later prototype versions could be load resistors.
Exploded heater.
We connected the Peltier elements to relays and wrote a temperature control program that we ran on a Raspberry Pie 3, which would turn on the elements until the sensor has measured a certain temperature and turn it off until the temperature drops back to a given value. As a power source, we used a common power bank, making the device portable. This way we were able to create simple, fast heating and low-cost constant temperature chambers for the lysis and isothermal PCR.
Plug plan for our temperature control system.
Outlook
For the final product we plan to strongly scale down our current chip design to reduce reaction volumes, material consumption and increase portability. This requires to adapt techniques for mold production with a better resolution like photolithography and proper choice of a chip material suitable for mass production.
This disposable microfluidic chip would then be loaded with the sample and inserted into a reusable device that integrates fluid control, thermolysis and amplification with the detection unit and conducts all the steps automatically.
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