Difference between revisions of "Team:BIT/Design"

Design

This year's project, using microfluidic chip as a platform, equipped with genetic engineering ideas and molecular cloning means, ultimately achieve the purpose that using biosensors for biomarker detection.

Our chip consists of two chambers:

Chamber 1 is oval(long axis length:18mm, short axis length:6mm). In our design, the function of the chamber is to hold our magnetic beads (fixed with magnets) with the aptamer and provide a place for the sample to bind to the aptamer so that the the complementary strands with lysine can get detached . Our intention to design the chamber as an oval is that the elongated structure can reduce the bubble generated by the difference in flow rate between the cavity wall and the middle stream.

Chamber 2 is round( diameter:12mm) Within this chamber ,there are gel pillars(diameter: 0.5mm). The contents of the chamber are engineering bacteria frozen into dry powdery and trypsin. After the reaction in the chamber is carried out for a certain period of time, the mixed reaction of the culture medium and the reaction liquid flows from the upstream into the chamber under the negative pressure generated by the peristaltic pump

First of all, we select aptamers AP273, which has best affinity for AFP. We modified a biotin at the end of its 5’ end, which has affinity for streptavidin. Because of the presence of streptavidin-modified beads, aptamer has been locked firmly on beads. At the same time, a complementary chain was synthesized at its protein binding site. We modified an amino group at the 3’ end of the complementary chain which can be combined with lysine protected by BOC anhydride to link lysine to the complementary chain. Because Van der Waals forces between AFP and aptamers is stronger than hydrogen bond, due to the action of magnet lying at the bottom of the reaction system, aptamers are separated from complementary chain, one at the bottom, the other in the supernatant. At this point, with the action of trypsin, lysine can be separated from the complementary chain, and start his adventure.

In the second cavity, there is a lysine deficient E.coli which can hardly grow without the addition of lysine. Meanwhile, we transformed a plasmid with fluorescent gene into the E.coli ΔLysA. When the E.coli ΔLysA has received the lysine signal released from the complementary chain, it begins to grow with the expression of the fluorescent protein. In this way, we can transform the lysine signal to the fluorescence signal. Consider that the lysine concentration could be weak, we decided to use a strong promoter(BBa_J23100) and a cyclic amplifier system to enhance the signal, and a dual fluorescence system to improve the sensitivity.

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