Abstract

 It can be seen from the previous introduction that the detection of biomarkers requires two reactions and one detection:The reaction of biomarkers and aptamer,the change of fluorescent protein’s concentration in E.coli,and the detection of fluorescence intensity .That means we need an instrument which can both provide platform for reactions and detect the intensity of the fluorescent protein.Unfortunately we did not find an suitable instrument for our project,so we decided to design one by ourselves(The structure can be seen in Figure 1.1).By reviewing literatures, we decided to use microfluidic chips.Microfluidic chip is a microchip that integrates man-made micro-pipelines and can controls the flow of liquid inside it.Microfluidic chip is a perfect platform for biochemical reactions and fluorescence detection,and it can be combined with external devices.In the case of microfluidic chip, we designed other functional modules such as optical detection module, temperature control module,so that the whole detection process can be completed on only one instrument. Microfluidic chip is important for our project, because it significantly reduces the time and cost of whole detection.Next I will introduce the design and performance testing of every functional modules. We hope that our design can provide reference for other teams.

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 in Figure 2.1, and used the peristaltic pump, the magnet plate and the heating plate as supporting auxiliary equipment. As can be seen from Figure, 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.

Materials

 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,then freeze-drying the liquid.
  ②:Injecting engineering bacteria to Chamber 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.Biological part

Introduction

 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.

 

Experiments

 In order to characterize the biological properties of the chip, we conducted a series of experiments on the basis of measurement of OD600 and the fluorescence intensity:

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

1.1.1 Experimental purpose

 Our project aims to achieve fast detection of biomarks on the chip, and we need to use E.coil in the detection process.That means we need verify whether the affinity of E. coil with our chip can meet our requirements. And we designed an experiment to cultivate E. coil to verify this problem.

1.1.2 Method

 First, we selected E.coli that was imported into BBa_K876070 plasmid as the strain of the experiment.Second,we cleaned the chip with anhydrous ethanol and PBS as preprocessing, to make the chip environment more conducive to microbial growth.Third,we injected E. coli into chamber II, put the chip in 37 degree incubator, and measured its OD:600 every two hours, and last draw its growth curve.

1.1.3 Experimental results

 According to the growth curve (Figure 1.1)of E. Coli inside Microfluidic chip, 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.

1.1.4Discussion

 To improve the hypoxic environment in the chip, let the strain of the experiment grow better, we have some ideas. And we are still studying it, here to discuss with you.
  ①: Transform microchip structure
Leave some vent hole on one side of the chamber, adhere a pellicle which has good air permeability and waterproofness. This will help chamber take gas exchange with air, and solve the hypoxic problem.
  ②: Add oxygen carrier
Process microchip with some oxygen carrier like hemoglobin, some of them will remain in the inner surface of chamber. They will release some oxgyen when the environment is hypoic. But we don’t know how much oxgyen they will release and whether oxygen carrier will influence E. coil. So, there still need more experiments and study.
  ③: Reserve oxygen in the chamber
When injecting E. coli bacteria, we can reserve some oxygen in chamber. But the chamber is tiny, we must have enough E. coli bacteria to achieve out detection, so there won’t have much room for oxygen. And the gas we reserve maybe will influence other parts of detection, so there still need more experiments and study.

 

1.2 E. coli freeze drying experiment.

1.2.1 Experiment purpose

 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.

1.2.2 Method

 Freeze the suspension of bacteria and protective reagent at -80℃ for an hour. Open the vacuum freeze dryer and push the button of “Compressor” after 5 minutes. Freeze the cold trap of the dryer at -53℃ for an hour. And then, put the pre-frozen suspension into the cold trap. In a vacuum state, the water in suspension will sublime into water vapor leaving the system. Dry powder is left.  We also need an index to evaluate the effect of vacuum freeze drying on bacteria. So we designed such an experiment: we use E coli transformed into BBa_K2305004 as material. After the bacteria was made into dry powder, dissolve the powder with LB medium, resuscitate the bacteria in a shaker of 37℃ and then measure the growth curve. By comparing the curve trend before and after freeze drying, we can make the effect clear.

1.2.3 Experimental results

 According the curves(Figure 1.2), we can include that:   (A).If the skimmed milk is sterilized at 115℃ for 15 minutes, the bacteria can grow better than those untreated.   (B).Though there are some differences between test groups and contrast group, they all have linear area, which we can use in our project. It confirms making bacteria into dry powder is feasible.

1.2.4 Discussion

 We also used the method of dilution coating to measure the viable bacteria cells after 11 hours. But the results are not so well, perhaps the dilution factor is too big.