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).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.
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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
Introduction
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 1.1, 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:
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Chamber 1 is oval.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.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,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 Figure 1.3. At the same time, we also designed a verification experiment for the light transmission of the chip(can be found in demonstrate section )
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Working Process
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.
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
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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 1.5.1)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 used the PCR tube filled with E. coli bacteria to simulate the chip inside environment and re-measured the growth curve of E. Coli inside Microfluidic chip(Figure 1.5.2). Comparing these two similarities growth curves, we basically confirm the hypoxic environment inhibits the growth of E. coil in the chip.
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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 to the curves(Figure 1.6), 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.
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And also,the dry powder we got is shown in Figure 1.6.1 and Figure 1.6.2
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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.
Conclusion
The experimental results have shown:
①:The chip's cultivating performance is worse than centrifuge tube although it can meet our basic requirments on short-term cultivation.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.
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 1.7)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.
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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 Experimental results
We got the following dates(Figure 1.8).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.
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Conclusion
At 37 μl / min injection conditions, the chip does not produce bubbles and clogging, but there will be a small amount of beads being washed into the downstream chamber. How to eliminate this part of error led by these magnetic beads of is still need to considerate as the improvement of the project.
Reference
[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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