Sieve-through vertical flow system for particle-based bioassays
A measurement system and particle technology, applied in biological testing, laboratory containers, measuring devices, etc., can solve the problems of low yield, high level of legacy pollution, low detection sensitivity, etc., to improve performance and reduce legacy pollution Effect
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Embodiment 1
[0070] Example 1: Device
[0071] device prototype
[0072] The sieving platform consists of a reaction unit and an absorbent pad ( figure 1 ). The reaction cell was designed using SOLIDWORKS (Villacoublay Cedex, France, Dassault Systèmes) and prototyped using a Stratasys 3D printer (Rehovot, Israel, Stratasys) ( figure 1 a and 1b). The reaction unit consists of a reaction chamber and two support columns. A piece of polycarbonate membrane with micron-scale pores was glued to form the bottom of the reaction chamber ( figure 1 c). A variety of porous membranes of various pore sizes are commercially available (Sigma-Aldrich, Missouri, USA) ). Two support columns keep the membrane in suspension so that the membrane does not come into contact with any surface. Use CO 2 Absorbent pads (Ahlstrom Filtration, Helsinki, Finland) were cut to size by a laser cutter (Epilog Laser, CO, USA). The absorbent pad is kept separate from the reaction chamber and is only in contact with ...
Embodiment 2
[0078] Example 2: Using a device to separate high-purity DNA
[0079] DNA extraction
[0080] Human genomic DNA (gDNA) was extracted using the Qiagen Biosprint 15 blood kit (Qiagen, Venlo, Netherlands). All reagents were prepared according to the manufacturer's instructions. First mix 400 ng of human gDNA (Promega, WI, USA) in 20 μL of water with 20 μL of buffer AL, 20 μL of isopropanol, and 2 μL of magnetic particles. The mixture was incubated in a reaction unit with a membrane with 3-μm pores for 10 minutes at room temperature. After incubation, remove liquid by placing the reaction unit on an absorbent pad. Waste liquid will flow through the porous membrane and be absorbed by the absorbent pad. The washing process is accomplished by adding wash buffer to the reaction chamber and then using an absorbent pad to allow the spent wash buffer to be removed across the membrane. Wash the pellet once with 50 µL of buffer AW1 and twice with 50 µL of buffer AW2. Finally, 20 μL o...
Embodiment 3
[0098] Example 3: Isolation of mRNA
[0099] Using poly(T)-conjugated magnetic particles (Thermo Fisher Scientific, Massachusetts, USA, mRNA DIRECT TM kit) to isolate mRNA. Incubate the cell pellet with 50 µL of lysis buffer and 10 µL of oligo(dT)25-functionalized magnetic particles. After incubation, poly(A)-tailed mRNAs are hybridized to poly(T)-conjugated magnetic particles. Waste is removed through the membrane using an absorbent pad. Next, the particles were washed four times with 50 μL of Wash Buffer A and once with 50 μL of Wash Buffer B, which was removed by adding the buffer to the reaction chamber and passing it through the membrane with an absorbent pad. Isolated mRNA was eluted in 10 μL of elution buffer at 70°C. The eluate is collected by pressurizing the chamber with a syringe, which forces the solution through the membrane into the container.
[0100] For comparison, we also performed mRNA isolation in microcentrifuge tubes according to the manufacturer's...
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