Difference between revisions of "Team:BIT/HW3"

Ⅰ.Microfluidic chip

 We chose the microfluidic chip because it has three significant advantages in the application:
  1.The tiny volume of the microfluidic chip allows the liquid reaction system to be miniaturized and integrated. It can use a small amount of liquid and space to achieve complex biochemical reactions;
  2.Reactions on the microfluidic chip can be quantitative analysis and detection;
  3. Microfluidic chip can be combined with external devices to make biochemical reactions become automated and intelligent.
  Based on the characteristics of microfluidic chip, more and more people use microfluidic chip as the platform of biochemical detection and POCT [1]. For example, there has been a microfluidic platform which can detect C-reactive protein in 2008,and the limit of detection is 2.6 ng/ml.Besides,it can be produced in large quantities[2].Similarly, in the case of hepatotoxicity assessment, a POCT method based on microfluidic systems overcomes the shortcomings of long processing times and high levels of personnel in traditional methods[3].It is worth mentioning that there already have mature POCT equipments based on microfluidic platform in the market.It can be seen that the microfluidic platform not only has irreplaceable performance advantages, but also has certain marketization potential.

Design:

An important feature of microfluidic chips is that they are capable of artificially designing liquid lines and reaction chambers. Our project involves three reaction processes: the separation of the aptamer and the complementary strand on the magnetic beads, the separation of the complementary strand from the small molecule, and the biochemical reaction of the small molecule and the engineered bacteria. According to the needs of the project, we designed the two reaction chambers as shown, and used the peristaltic pump, the magnet plate and the heating plate as supporting auxiliary equipment.

 

As can be seen from Fig1, 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 (see the equipment section), the following reaction occurs:
①: Freeze-dried engineering bacteria’s recovery with the help of culture medium and constant temperature provided by the heating plate
②: Trypsin catalyzes cracking of DNA chain,lysine on the complementary strand falls into free small molecule
③: Lysine goes into the engineering bacteria, biochemical reaction within the engineering bacteria began.

We can use the design of hardware to detect the reaction generated fluorescence generated in reaction③, and we can draw a conclusion through analyzing and processing.Besides,the material of the chip is an important factor affecting the detection of fluorescence.For the material of chip,we choose NOA 81.

In normal conditions it’s in liquid state.It will solidify with exposure of 320nm to 380nm wavelength UV light We made the chip using the material NOA 81. The material is normally liquid, and when subjected to UV light from the 320nm to 380nm band, the material is quickly cured to form a solid with good hardness and toughness. [4] According to this characteristic, we can use soft lithography method to achieve the production of the chip . The reason why we used NOA81 as the material of our chip is not just it’s UV curing characteristics, the light transmittance of this material is also taken into consideration. The transmittance provided by the manufacturer is shown in Fig 5. At the same time, we also designed a verification experiment for the light transmission of the chip(can be found in demonstrate section )

Briefly,our chip went through following process between fabrication and finishing detection:

①:Injecting magnetic beads to chamber 1 from Inlet 1,then freeze-drying the liquid.
②:Injecting engineering bacteria to Chamber 2 from Inlet 2;then freeze-drying the liquid.Then the chip is ready to use.
③:When needing detection,taking the chip,connecting the peristaltic pump and injecting a volume of sample and culture medium mixture from Input 3.
④:After the reaction is complete, the peristaltic pump pumps the reaction solution into the chamber with negative pressure. The biochemical reaction takes place and enters the subsequent detection process

Demonstrate:

①:The freeze-drying and recovery of engineering bacteria can be finished in the chip.And the engineering bacteria needs to keep a relatively high activity after recovery process.
②:The engineering bacteria can survive in the chip for the short period between recovery and finishing a detection.
③:Verify the flow of liquid in the microfluidic chip at the best injection rate.
④:Verify the adsorption of magnets and bead - aptamer complexes
⑤:verify high clarity and high light transmission of our chip

   

1.Biological part

In our project, the chip is the carrier of biological reactions, its most basic function is to provide a growth environment for engineering bacteria. In order to be used smoothly as a biological carrier in our project, the chip needs to meet the following conditions:
①:The freeze-drying and recovery of engineering bacteria can be finished in the chip.And the engineering bacteria needs to keep a relatively high activity after recovery process.
②:The engineering bacteria can survive in the chip for the short period between recovery and finishing a detection.

 

1.1Experiment about investigating the Growth and Metabolism status’s change of E.coli inside and outside the Microfluidic Chip.

According to the growth curve of E. Coli inside Microfluidic chip,Fig 7, the growth of E. coil was somewhat inhibited. But in the short time, the inhibition is weak. And our project aimed at detecting quickly, so the affinity of E. coil with our chip can meet our projects’ requirements. By analyzing the process and principles of the experiment, we think maybe the hypoxic environment in the chip inhibits the growth of E. coil in the chip. So, we use the PCR tube filled with E. coli bacteria to simulate the chip inside environment and re-measure the growth curve of E. Coli inside Microfluidic chip. Compare these two similarities growth curves, we basically confirm the hypoxic environment inhibits the growth of E. coil in the chip.

 

   

1.2 E. coli freeze drying experiment.

To achieve fast and convenient detection on chip, we designed a chamber on microfluidic chip to cultivate bacteria. However, it brought new problem-the storage and transportation of bacteria. Based on the question, we came up with an idea. Make the bacteria into dry powder with the method of vacuum freeze drying. An then, when we need to use the chip, just inject some medium, recover the bacteria and it can work for you.
From room temperature to low temperature, ice crystals may generate, and then puncture bacteria cells. So we need to find a reagent to protect bacteria. At the beginning, we choose glycerol. Soon, we found glycerol is too sticky too let the powder out. We decided to use skimmed milk whose quality fraction is 10% to have a try.