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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 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.

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 )

Working Process

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.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.

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.

2.Fluids part

Introduction

As a microfluidic chip, the assessment of the flow of liquid is a necessary part of characterizing the performance of the chip. For the purposes of this project, the risks that may present during the infusion process include:
①: Blocking may occur in injection process.As a consequence,the injection would be not smooth,which would even lead to chip damage.
②: Bubbles are possibly be produced during injection,which would lead to dead volume increasing and biochemical reaction as well as optical detection process would be impacted consequently.
③: The fast flow rate may make the upstream beads to be washed to the downstream, so that the aptamer’s complementary strand would cleavage and release lysine with absence of the AFP,which would increase the risk of false positive diagnosis
In contrast, in this regard we need to meet the requirements as following:
①: Ability to perform a smooth injection without blocking and bubbles in constant injection rate condition.
②: Magnetic beads would not be washed to the downstream.

Experiments

In order to verify whether the chip can meet the demands,we designed the experiment as following

2.1 Verify the flow of liquid in the microfluidic chip at the best injection rate.

2.1.1 Experiment purpose

At an improper injection rate, a lot of bubbles will be generated when the liquid is injected into the microfluidic chip,which will cause increase of dead volume and decrease of the biological reaction space.There will be a large deviation as a consequence. The purpose of this experiment is to observe and record the flow at a suitable injection rate we have determined.It is proved that it will not produce a large number of bubbles when we inject liquid into microfluidic chip at this rate.

2.1.2 Method

1.Put a certain amount of pigment into the water, to simulate the sample solution
2. Adjust the syringe pump parameters so that the flow rate is 37 μl per minute and connect the syringe and microfluidic chip
3. Start to inject and take a picture

2.1.3Experimental results：

From the experimental results (Figure 2.1)we can see that it will not produce a lot of bubbles when we inject liquid into microfluidic chip at the rate of 37ul/min, that is to say we can ensure the accuracy of the test results.

2.2 Verify the adsorption of magnets and bead - aptamer complexes

2.2.1 Experiment purpose

To verify whether the bead-aptamer complexes will be rushed downstream at 37 μl/minute.

2.2.2 Method

We used a magnetic bead-aptamer complex with a fluorescent complementary strand in this experinment.First we inject magnetic bead suspension into chamber II,then keep injecting water at the speed of 37μl / min for 8 minutes. While injecting water,we used a centrifuge tube to catch out of the liquid from the outlet.After that,we pointed the liquid in the 96-well plate,and detected the fluorescence value under the microplate(The gain is 50).

2.2.3 Experiment

We got the following dates(Figure 2.2).The middle column shows the fluorescence intensity of the liquid from the outlet.Other columns represent fluorescence intensity of control group:

You can see that the experimental group data is not much higher than the blank control group data, but still has a small increase,indicating that the adsorption capacity of the magnet can not completely absorb the beads, but the adsorption capacity is very strong.

Conclusion

The experimental results have shown:
①:The chip can ensure the normal growth of bacteria in a short time, but its long-term cultivating performance is not so good.According to our analysis,it may be due to the hypoxia environment in the chip.
②:The hypoxic environment does have a great impact on the growth of bacteria
③:Freeze-drying and recovery process of engineering bacteria can be completed
④:The bacteria remains relatively high activity after recovery
In summary, as biological reaction carrier, the chip can be qualified in the short term bacterial culture,so the chip can meet our basic requirements.But the performance of long-term cultivating is poor.According to our analysis,it may be due to the internal hypoxic environment of the chip.Experiments have shown as proof that the hypoxic environment does have a great impact on the growth of bacteria.
At the same time, although we did not carry out the freeze-drying experiments in the chip, but the experimental results proved that the idea of freeze-dried engineering bacteria is feasible.

Refernce

[1]:Mazher-Iqbal Mohammed and Marc P. Y. Desmulliez,Lab Chip, 2011,11, 569-595
[2]:C. Jonsson, M. Aronsson, G. Rundstrom, C. Pettersson, I. Medel- Hartvig, J. Bakker, E. Martinsson, B. Liedberg, B. MacCraith, O. Ohman and J. Melin, Lab Chip, 2008, 8, 1191–1197
[3]:Geok Soon Lim,Joseph S. Chang, Zhang Lei,Ruige Wu, Zhiping Wang, Kemi Cui and Stephen Wong,Lab Chip, 2015,15, 4032-4043
[4]:http://www.norlandprod.com/adhesives/noa%2081.html

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@@ Line 186: / Line 186: @@
 <h4>2.2.1 Experiment purpose</h4>
 <p>To verify whether the bead-aptamer complexes will be rushed downstream at 37 μl/minute.</p>
 <h4>2.2.2 Method</h4>
-<p>We got the following dates(Figure 2.2).The middle column shows the fluorescence intensity of the liquid from the outlet.Other columns represent fluorescence intensity of control group:</p>
-<p>You can see that the experimental group data is not much higher than the blank control group data, but still has a small increase,indicating that the adsorption capacity of the magnet can not completely absorb the beads, but the adsorption capacity is very strong.</p>
 <p>We used a magnetic bead-aptamer complex with a fluorescent complementary strand in this experinment.First we inject magnetic bead suspension into chamber II,then keep injecting water at the speed of 37μl / min for 8 minutes. While injecting water,we used a centrifuge tube to catch out of the liquid from the outlet.After that,we pointed the liquid in the 96-well plate,and detected the fluorescence value under the microplate(The gain is 50).</p>
 <h4>2.2.3 Experiment</h4>
+<p>We got the following dates(Figure 2.2).The middle column shows the fluorescence intensity of the liquid from the outlet.Other columns represent fluorescence intensity of control group:</p>
+<p>You can see that the experimental group data is not much higher than the blank control group data, but still has a small increase,indicating that the adsorption capacity of the magnet can not completely absorb the beads, but the adsorption capacity is very strong.</p>
   <h3>Conclusion</h3>