Team:ZJUT China B/Sample processing
Sample Processing
Video 1 Painting of the reactor
We referred to the microfluidic device of Munich 2017 iGEM Team and made some improvements to their device. Unfortunately, due to the impact of the epidemic, we were unable to make this part into a real device.
Our sample processing module also uses the microfluidic chip form, using a screw to advance the syringe to achieve micro-injection and control the liquid. Our microfluidic chip is mainly divided into five areas, mainly made by PDMS. Because it will contact the sample, it must be discarded after each use and handed over to the relevant environmental protection company for disposal.
Figure 1. Schematic diagram of microfluidic chip
Power source-micro syringe
Figure 2. Power source
We changed the propulsion shaft of the syringe to a screw rod, and rotated the nut to move the screw rod down to squeeze out the liquid. The outer side of this nut is a gear, which can be driven by a servomotor. If you stop pushing at this time, the liquid will stay at the farthest end it reaches. The use of a screw rod and a toothed nut increases the accuracy of propelling the liquid. The shape of the syringe is not round but square, as shown in the figure, so that the piston will not rotate and the nut will rotate to advance the screw. Considering the rigidity of the syringe, it needs to be made of plastic.
Figure 3. 3D details
Sample room
Figure 4. Sample chamber
This is a long capsule-like cavity. Its volume is very small. After the liquid passes through, all the air will be taken out due to the capillary phenomenon. This small chamber has an opening to facilitate the addition of samples, and is equipped with a stopper to isolate the air after the sample is added to prevent the leakage of the sample and the entry of external RNase.
Heating and cooling
In this area, the sample will be lysed at a temperature of 90°C, destroying the protein that wraps the nucleic acid fragments, so the nucleic acid fragments are exposed to the outside, which can facilitate subsequent reactions. Because the system we use in the subsequent reaction is freeze-dried protein powder, but the previous area is the heating zone, so if the high-temperature liquid directly contacts the freeze-dried protein powder without cooling, it will easily cause protein denaturation. So we added a cooling zone to this microfluidic chip.
Figure 5. Peltier
We use semiconductor refrigeration fins called peltier which is a heat exchanger to complete heating and cooling. After powering on, it will form a cold end at one end and a hot end at another end. Two wedge-shaped aluminum blocks conduct heat at both ends, thereby transferring heat and cold to the chip. A big advantage of this part of the device is that we can use the characteristics of the semiconductor refrigeration chip to achieve cooling and heating at the same time, and this component is not part of the microfluidic chip, so it can be used multiple times.
Figure 6. Our design
RPA reaction chamber
At this time, the sample has undergone lysis and cooling, and the next step is amplification. The amplification system we choose is RT-RPA-Tx freeze-dried protein powder, which will be activated after being mixed with the reaction solution. It has a very high amplification rate at 37-40°C and can amplify the viral nucleic acid signal very greatly. In order to achieve this constant temperature goal, we use resistance heating. The NTC thermistor can transmit the real-time temperature to the single-chip microcomputer. If the temperature is higher than 38°C, the heating is stopped; otherwise, when the temperature is less than 38°C, the heating process starts. Experiments have proved that our RPA thermostat can fluctuate within a range of ±1℃.
We assemble various components, and the conceptual diagram looks like this: The PDMS microfluidic board will be placed on this device, and the gears of the microfluidic injector can be controlled quantitatively and accurately by the steering gear.
Figure 7-8 3D model
Liquid outlet
In order to reach the next reaction chamber, Cas detection chip, the amplified sample needs to have an output eventually. Through the advancement of the syringe, the liquid is sent to the subsequent chip along the microtube.
See the detecting chip page: please click >>Cas13 Chip<<
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