Team:BIT/Hardware/Device
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 the picture below .
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.
I 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.
1.1 Design:
Temperature control module consists of alumina ceramic, heating film, temperature controller and its support control module composed of four parts. Alumina ceramic is the basis of the microfluidics chip, the microfluidics chip is made of alumina ceramic. The temperature controller controls the heating and stopping of the heater through the temperature controller's temperature sensor. The temperature controller should adjust the desired temperature in advance in the digital display. When used, place the microfluidics chip on the heater. The alumina ceramic allows the microfluidics chip to be heated evenly. Temperature control module base is made of nylon 3D printing, can be a good chimney heating chip and chip, while a certain extent to prevent microfluidics chip heat loss.
1.2 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.
1.3Working 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.
II Peristaltic pump
2.1Design:
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 'Demonstrate').And the pumping time can be set on the serial screen.
2.2Materials:
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.
III Confocal detection of optical path
3.1Design:
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.
3.2Materials:
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'.
3.3Working 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.
Ⅳ 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.Optical part
Introduction
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.
Experiments
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.
1.1.verify high clarity of our chip
1.1.1 Method
Inject 50uL Fluorescent microsphere solution into the chip and detect the optical distribution of fluorescence in the chip. Drip the fluorescent microsphere solution of the same concentration onto the glass slide and detect the optical distribution of fluorescence in the chip. Cover the glass coverslip and detect the optical distribution of fluorescence in the chip.
1.1.2 Experimental results
From A in figure,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.
1.2.verify high light transmission
1.2.1 Method
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 f
1.2.2 Experimental results
From A in figure,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.
1.2.3Discussion
According to result figure ,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.
Futher quantitative analysis by histogram:
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.
2.Temperature control module
Introduction
The purpose of this experiment is to verify that the temperature control module can control the microfluidic chip expression chamber temperature constant at 37 ° C . Because we hope that engineering bacteria can work efficiently, we need to build a temperature control module to provide an optimum growth temperature for E. coli. The fluorescent protein used in the project was expressed by Escherichia coli, and we knew that the optimal growth temperature for E. coli was 37 ° C.
Method
We injected the microfluidic chip into the LB medium and placed it on a temperature control platform for heating. The temperature of the surface of the microfluidic chip was measured by infrared thermometer, and the curve of the surface temperature of the microfluidic chip was plotted over time.
Results
The results show that the temperature module can control the temperature of the microfluidic chip chamber in the range of the optimum growth temperature of E. coli - 37 ° C (± 1 ° C).
3.Peristaltic pump injection testing:
Introduction
we want to verify that we use the peristaltic pump to complete the extraction of air inside the chip,driving the flow of liquid within the chip function
Method
First, we take the microfluidic chip, remove the red solution with a pipette, and then put the pipette tip on the microfluidic chip connector. Then we will make the peristaltic pump connector connected to the microfluidic chip hose,under the negative pressure of the peristaltic pump, the liquid in the pipette tip is injected into the chip.
Results
With the negative pressure of peristaltic pump, the liquid within the chip can be carried out smoothly.
Component Information Table
