Engineering
1.Nucleic acid aptamer replacement system applied to sample pretreatment
In this project, a nucleic acid aptamer replacement system is used to design an aptamer replacement system for the N protein of the new coronavirus to convert protein signals into nucleic acid signals. Use biotin-streptavidin to bind magnetic beads on one end of the nucleic acid aptamer, which can be fixed by a magnet, and the aptamer can bind to its complementary chain through the hydrogen bond force of complementary base pairing. The target protein is stored in the aptamer, and the protein will bind to the aptamer. Since the force between the protein and the aptamer is greater than the hydrogen bond force, the complementary chain is released, thereby achieving the purpose of converting the protein signal into a nucleic acid signal. This method is simpler and easier to operate, and the cost is much more considerable, and the detection accuracy is more accurate than only nucleic acid detection, which reduces false — the probability of negative or false positive. Aiming at the binding length of the aptamer and the complementary chain, we designed the complementary chain S1 and S2 in the experiment. The long chain complementary chain S2 reacts well after competing with CKMB. Its length is 25 bp and its sequence is 5' -FAM-CCCAACCCCCTAAAAGCATCCTCCG, this result proves that the nucleic acid aptamer has been successfully obtained.
2.Application of nucleic acid amplification method to signal amplification
In this step, we use RCA and HCR technology to amplify the complementary strands obtained from the previous step of nucleic acid aptamer replacement. We finally use colloidal gold to detect the experimental products. We also successfully carried out the amplification experiment and got the ideal result.
The RCA amplification products obtained in this experiment are as follows: The products of this RCA experiment were characterized by 2% agarose gel electrophoresis. D2000 was used as the marker for this experiment. During agarose gel electrophoresis, the lanes 1-6 from left to right next to D2000 Maker were: blank, sample , Sample, sample, sample, blank, the experimental results are shown in Figure 1. According to the experimental results, we can conclude that because RCA amplified long chains, the bands at the opening of lanes 2-5 can prove the success of this RCA amplification.
Figure 1. RCA experiment result graph
According to the experimental results, we can conclude that there are electrophoresis bands because the long strands of nucleic acid have been amplified from HCR, which proves that the HCR amplification is successful.
Figure 2. HCR experiment result graph
3.Temperature control for stable control of nucleic acid amplification
Our temperature control module can provide our amplification with stable temperature, the commonly used PCR amplification on the temperature change after heating denaturation and annealing of two steps, which requires the device in the temperature control needs to have the ability of rapid heating and cooling, which means you need larger space to provide equipment heat dissipation of the environment, but also has great power to provide our equipment.However, because of our need for miniaturization and portability, we did not adopt PCR as our amplification technology. Instead, we adopted an amplification method with low requirement for temperature change to meet our demand for portability. This amplification method only needs to be amplified at constant temperature.
Since the amplification temperature we needed was constant, we initially considered whether we could use human body to amplify the nucleic acid temperature of our chip. However, considering the biosafety and stable temperature required for amplification, we looked elsewhere.Finally, we chose a single chip microcomputer to control our temperature. The temperature control unit of this project is based on the temperature sensor and automatic control algorithm to achieve a constant temperature of 37℃.Temperature control circuit of the working mechanism is: the use of Pt1000 temperature sensors and form A complete set of temperature measuring circuit temperature signal acquisition temperature control unit, the single-chip computer MSP430 with A/D sampling module, software control algorithm is used to calculate the control quantity, and then through the I/O port output power control MOS tube or load switch on and off, output constant current control hardware circuit, and unit of heating and cooling, heating and rely on simple control PID algorithm, guarantee the stability of temperature.The following picture is the image of temperature data fitting using Matlab after the temperature sensor is used to receive the temperature.
Figure 3. Temperature curve of constant temperature process
Figure 4. Temperature curve during the heating process
4.Application of test strips in optical signal detection
The project uses colloidal gold immunochromatography to carry out accurate control testing of experimental results, the process of which includes the following three steps: sample preparation, immune response and colloidal gold detection. We need to add the pre-treated sample to the specific chamber of the centrifugal chip, make the sample to reach the chamber where the two kinds of side flow test paper are located by rotating the chip and using the centrifugal force, capture the samples to the test line of the side flow test paper respectively through the immune chromatography reaction, and then observe and analyze the color development results of the colloidal gold chromatography strip ,and analyze the colloidal gold images by taking pictures with mobile phones to obtain the comparison results of the red chromaticity in RGB with the standard positive chromaticity. The following is the experimental operation:
① Mix the positive sample, the loading buffer solution and the colloidal gold solution that have been subjected to the HCR reaction in a 37 ℃ incubator in advance. The volume of the three solutions is 15 μL, 70 μL and 15 μL, totaling 100 μL. Inject the mixed solution into the chip chamber, and place the test strip with the capture probe embedded in the specific area;
② Rotate the centrifugal chip to make the mixed liquid enter the test strip area, and the liquid soaks the entire test strip under capillary action. Observe the condition of the C line and T line of the test strip. In Figure 5, the strips of the test strip from left to right are the C line and T line respectively, and double strips appear to prove a positive result;
Figure 5. Process of mixing liquid injection and flow into test strip
Figure 6. Positive test strip result
③ Repeat the above steps to react the blank control group without positive samples to obtain the negative band result as shown in Figure 7.
Figure 7. Negative test strip result
5.Fidget spinner is used in centrifugal chip drive
Centrifugal force drive is a unique technology in microfluidic driving technology, and its system uses the centrifugal force generated by the chip to make circular motion as the driving force of the liquid flow. The flow velocity can be adjusted and controlled by changing the rotation speed of the chip and designing different channel configurations.
The special feature of our centrifugal microfluidic chip is that it does not use a motor, but uses a pushing hand to drive the microfluidic chip to rotate, so as to construct a simulated gravity field, that is, the centrifugal field, and the flow is controlled by the volume force acting on the material in the centrifugal microfluidic chip, so as to get rid of the complicated laboratory equipment. Centrifugal microfluidic chip can be designed as a closed system, which means that the centrifugal microfluidic chip can realize the functions of pre-storage, release, mixing and reaction of reagents or samples, which greatly enhances the convenience of the chip. One of the characteristics of centrifugal force drive is that the power is applied to all the liquid flows on the chip. The advantage of centrifugal force is that it can provide the driving force of all the structural units on the chip at the same time, which is of great significance for the high-throughput analysis of microfluidic chips; Due to the characteristics of centrifugal motion, centrifugal microfluidic chips usually build flow channels and chambers on a CD-shaped disk, and the radian of each part needs to be adjusted or designed according to the rotation center. In the centrifugal microfluidic chip, the volume force can be controlled by controlling the rotation speed and angular acceleration, and driven by the volume force, the liquid in the flow channel will flow by itself, and because there is no other liquid flowing in, the centrifugal microfluidic chip needs to open a hole to let the air enter the original space of the substitute liquid, so as to avoid the vacuum force restricting the liquid flow. Therefore, unlike the continuous liquid flow in other microfluidic chips, the flow in centrifugal microfluidic chips is mostly gas-liquid two-phase flow.
Based on the previous model construction, we have produced a centrifugal microfluidic chip prototype, as shown in the figure below. Instead of installing gaskets in this chip, we rotate directly with a solid center shaft. Preliminary tests show that the chip can maintain a basically stable rotation and speed with the handheld center shaft. The following is the experimental operation:
①In order to simulate the fluid flow during the actual rotation of the centrifugal chip, we injected 100μL of pigment into the amplification chamber of the chip, and placed a test strip in the chip test strip area in advance. It is evaluated by visually observing the pigment flow. Figure 8 shows that the pigment can be injected normally without entering the test strip area;
Figure 8. Pigment injection
②Turn the chip upside down to deposit the pigment in the amplification chamber on the other side of the chip chamber, and at the same time turn the centrifugal chip to rotate around the central axis. The liquid in the amplification chamber enters the test strip chamber under the action of centrifugal force, and covers the entire test strip under the capillary action of the test strip. By observing the liquid flow during the rotation process and the pigment diffusion of the test strip, it can be proved that the centrifugal force provided by the toggle chip can drive the liquid in the amplification chamber into the detection area of the test strip.
Figure 9. Actual driving situation
6.Application of mini program development to the detection of optical results
Through the construction of the Mini Program, we have completed the following functions: reading images, fitting image features and solution concentration, output image, output predictive concentration.
Software interaction module: based on the development of WeChat Mini Program, extract image color (RGB) and other features, use the least squares method to linearly fit to obtain the relationship between feature and concentration.
The least squares method (also known as the least square method) is a mathematical optimization technology. It matches the best function of finding data by minimizing the square of the error. The least squares method can be used to easily find unknown data and minimize the sum of the error between the obtained data and the actual data.
Software interaction process: the mobile phone takes multiple images, reads the image in the small program, manually enters the solution concentration, image processing, fittings the relationship between the image characteristics and the solution concentration, inputs new images in the small program, and outputs and predicts the concentration.
Figure 10. WeChat mini program operation interface
Description of WeChat Mini Program:
1.Enter the concentration value and click Upload Image to upload the picture. Click Color Feature1 to extract color features, you can upload at least 2 to 5 samples;
2.Upload a new image to extract new features, use the least square method to fit and output the predicted concentration.
Created By Beijing Institute of Technology