Team:BIT/HW4
Device
We divided the instrument into eight functional modules: microfluidic chip, confocal detection of optical path, power board, structure, serial screen, microcontroller, temperature control module, peristaltic pump.Their size and position in the instrument can be seen in Fig 2.
The power supply provides the rated voltage for each power unit. The structure printed by 3D printer is used for fixing individual components.The microcontroller is responsible for controlling of the work of the various components,passing information to the serial port, processing the data of the detection.The serial screen is responsible for displaying the test results, and providing the human-computer interaction platform.The microfluidic chip is used as a platform for the entire reaction, and the detection is also done directly on it.Temperature control module and peristaltic pump assist microfluidic chip to achieve it’s function.The confocal detection of optical path is used to detect the fluorescence intensity.Here we will introduce their function one by one.
II Temperature control module
Temperature control module provide constant temperature environment in the chip for two reasons:1.E.coil need constant temperature environment when they restore activity from freeze-dried state;2.E. coli have the best growth and metabolism state in constant temperature environment.
2.1 Design:
Temperature control module consists of four parts, namely: alumina ceramic, heating piece, temperature sensor module and it’s supporting control module.The alumina ceramic is the base of the chip, and the chip is connected to it. The rest of the temperature control board is located on the instrument, and the chip is placed on it when used.The alumina ceramic allow the chips to heat evenly.The control module control the work of the heater piece.The temperature sensor provide temperature information of microfluidic chip to the microcontroller at real-time.
2.2 Material:
Alumina ceramic piece’s thermal conductivity is 23W/(m^2*K), and the size is 45 * 30 mm. Heating piece’s rated voltage is 5V, the power is 10W, and the size is 40 x 40mm. Temperature sensor’s accuracy is 0.1 ° C.
2.3Working Process:
Its working process is very simple:When the chip temperature is higher than 37 degrees, the control module control the heating piece to stop heating;when the temperature is higher than 37 degrees, the heating piece works again.
III Peristaltic pump
3.1Design:
The injection and the flow of liquid in the chip is achieved through the negative pressure produced by the peristaltic pump.Peristaltic pump suck the air inside the chip, so that the air pressure inside the chip will be less than atmospheric pressure.That’s how the negative pressure is produced.Under the negative pressure,the liquid will flows into the chip.What’s more,the flow rate can be determined by controlling the pumping speed of the peristaltic pump, and the liquid inflow volume can be controlled by controlling the pumping time.Through the experiment we found that the best speed of liquid flow is 37uL/min(Experiment can be found in project page).And the pumping time can be set on the serial screen.
2.3Working Process:
This year we choose peristaltic pump of model S100-2B + TH10B.This type of peristaltic pump has long service life and is small in size and cheap in price.
Ⅳ Confocal detection of optical path
2.3Working Process:
In order to detect the intensity of GFP and RFP that produced by E.coli, we designed a dual optical path confocal fluorescence detection device.This is our optical path diagram, we used two high-power LED, four filters, five convergence lens, three long-shaped two-color lens and two photoelectric sensor OPT101(The detailed information of the parts are listed in the component information table).
2.3Working Process:
This year we chose GFPmut3b,its excitation peak is 501nm, and emission peak is 511nm. And we also choose mRFP1, its excitation peak is 584nm, and emission peak is 607nm.Based on their information and the experimental data we obtained, we chose light sources and other optical components. Such as light source’s excitation peaks are 490nm and 585nm.For further details on other optical components, you can see at the component information table.
2.3Working Process:
First, the LED emits a laser with a peak of 490nm.Through the reflection of the dichroic mirror and the focus of the converging lens, the laser will forces on chamber II.The GFP in E. coli will be stimulated to produce green fluorescence with a peak of 511 nm.Fluorescence will eventually focus on OPT101.Similarly, red fluorescence will be detected by OPT101 in the same process.Through the photoelectric sensor OPT101, the optical signal will becom1e electrical signal, then it will be converted into digital signals through AD acquisition board.After that,it will be passed to the micro controller for analysis.
Ⅴ Operation
As shown in figure,while detecting,a user mainly need to finish sampling,injecting and connecting process.The specific reacting and optical detecting process is automatic,which can be operated simply on serial screen.Also,we designed concise UI on serial screen for users:
As can be seen above,our UI offered brief guidance to users,making sure users can easily finish a detection.
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.
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