Team:BIT/HW1
Results
To evaluate if the chip can provide a platform for our project, we conducted a series of experiments.
Ⅰ. Biological part: 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.
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.
Results:
According to Fig.1.1 and Fig.1.2
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.
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(Fig.1.2.1). We decided to use skimmed milk whose quality fraction is 10% to have a try.
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.
Results: 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.
Ⅱ. Fluids part:
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 follows:
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.
Results:
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.
Ⅲ. Optical part:
On the one hand, our chip is the carrier of biological reactions, on the other hand, the chip is also our optical detection platform.The object we are going to detect is GFP and RFP.
The detection principle is using a certain wavelength of light through the chip to stimulate the GFP and RFP in the chamber and detecting the light intensity emitted by GFP and RFP.In this way,we can characterize the amount of fluorescent protein produced by engineering bacteria.Thus the relationship between the reaction of engineering bacteria and the light intensity was established.
So the upper part of the chip will not only through the instrument from the excitation light, but also through the emission light from the engineering bacteria. If the chip has a poor transmittance, both the excitation light and light will be seriously attenuated.Thus it will affect the detection accuracy.
We used NOA81 as the material of our chip. In order to characterize the light transmittance of this material, we used fluorescent microglobulin as the source of fluorescence and observed the fluorescence by fluorescence microscopy to characterize the chip transmittance.
Results:
From A in Figure3.1.1,not only the fluorescence can we detect clearly,but also we can get the position of bubble easily,just like B,only dripping the solution on the glass slide.Compared with C,the blurred detection,We can get that it is high clarity that our chip has the properties of.That’s what we need,high clarify is good for our detection.
a.0.1mol/L fluorescein sodium solution were diluted 7000 times, 10000 times,20000 times,getting solution ①,② and ③.
b.Check their fluorescent values
c.Inject ①,②,③ into the chip and detect the optical distribution of fluorescence in the chip.
Results:
We choose green to do histogram
AVG:it presents the average brightness valueStandard Deviation:The intensity of brightness change
According to Figure 3.2.1,we can find that the chip has very good transparency for the fluorescence.We find that even for 3000 of the fluorescence, a clear capture is also possible.
Here is further quantitative analysis by histogram(Figure 3.2.2):
Comparing A and B,we found that more diluted 3000 times,the standard deviation is reduced by 2.91
Compared with B and C, we found that more diluted 10000 times the standard deviation is reduced by 10.It proves that the material has little to do with its attenuation by it’s linear decay.These have proved that the transparency of our chip is very good.
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