Contribution
① Problems and solutions in the process of our project design (Biology)
Question1:
In the initial stage of the project, we plan to use RPA-Isothermal amplification technology to amplify the multi nucleic acid signals obtained in the sample pre-processing stage for subsequent detection, including the extraction and purification of 2019-nCoV nucleic acid signal (nucleic acid detection) and the complementary chain signal (protein detection) of 2019-nCoV characteristic protein through aptamer replacement. However, in the actual experiment, we found that the length of complementary chain obtained by aptamer replacement in protein detection is very short, far less than the length of primer required by RPA, which shows that our project design has flaws.
Solution:
After investigation, we decided to replace RPA technology with HCR technology and RCA technology. On the basis of retaining the advantages of RPA technology, such as isothermal, rapid, efficient and convenient, the two technologies can greatly expand long and short chain nucleic acid fragments in a short period of time, which is very consistent with the needs of our project. Therefore, we initially decided to use RCA technology to amplify complementary chain in protein detection and HCR technology to amplify nucleic acid signal in nucleic acid detection.
Question2:
In the initial project, one of the important reasons for us to use RPA technology is to use RPA to amplify the nucleic acid signal in the Sherlock technology used in the nucleic acid signal detection and energy exchange part. However, due to the change of nucleic acid amplification method in the first problem, our Sherlock technology has been greatly affected, and we have no previous experimental accumulation of this technology, so the experimental operation is also very difficult.
Solution:
After careful consideration, in order to cope with the change of nucleic acid amplification mode in question 1, we decided to delete the Sherlock technology of nucleic acid signal detection and energy exchange, and directly use the lateral chromatography strip to detect our amplification results. In this way, we can avoid the huge risk that Sherlock technology may bring, and also can reduce the experimental cost and unnecessary waste of human and financial resources.
Question3:
In the middle and later stage of the project implementation, we found that the nucleic acid detection pathway in the project has been well studied in the world, so there is no outstanding innovation point, which cannot reflect the progress of the project.
Solution:
Since there are no outstanding innovations in the nucleic acid detection pathway, we think it is unnecessary to repeat the mature experiments. At the same time, in order to save time and cost, we finally decided to delete the nucleic acid detection pathway to highlight the protein detection pathway in the project. Different from the nucleic acid detection pathway, aptamer is used to convert the non amplified protein signal into the amplified nucleic acid signal, which greatly improves the accuracy of detection and breaks the traditional protein detection method based on antigen antibody binding. Finally, we decided to use HCR and RCA technology to amplify the protein detection pathway, and use two kinds of lateral chromatography strips (colloidal gold, fluorescent microspheres) to detect our amplification results. We hope to compare and analyze the results through 2 * 2 nucleic acid amplification detection mode to obtain more accurate detection results.
② Problems and solutions during project design (hardware)
Question1:
In the early stage of our project, the design of microfluidic chip tends to use the pulling whistle driven centrifugal chip based on the "pulling whistle principle". The spring rope is pulled by external force to switch between the "winding" and "unfolding" states, and the centrifugal disc chip is pulled to produce certain rotation speed and centrifugal force. However, after our later model construction and experimental verification, the centrifugal chip driven by pulling whistle has a considerable defect, that is, it cannot guarantee the horizontal balance of the chip during centrifugation, and it is very unstable in the process of rotation, which will lead to tremor and leakage.
Solution:
We improved the original design of pulling whistle to a microfluidic chip driven by fidget spinner. The centrifugal force generated by the rotation of fidget spinner can also drive the flow of liquid well. Because of the stable orbit of the shaft, it can guarantee our stability in the horizontal direction.
Question2:
According to our original design, all our biological treatment parts will be integrated in the chamber of the microfluidic chip. However, considering the simplification of the microfluidic chip structure and meeting the detection needs of POCT, we should try to avoid using manual or motor-driven micro valves, but this means that the reactions in the microfluidic chips may interfere with each other during multiple driving.
Solution:
Because we cannot solve the problem of the multiple driving of microfluidic chip in a short time, we plan to do split cell and convert protein signal into nucleic acid signal these two steps before the injection of microfluidic chip.
Question3:
The paramagnetic particle method mainly includes four steps: pyrolysis, combination, washing and elution, and the conditions of each step are stringent. After literature research, we found that the operation steps of paramagnetic particle method in microfluidic chip is complex, and it is difficult to realize in the microfluidic chip in this project.
Solution:
At present, the efficiency of nucleic acid extraction by paramagnetic particle method in chip is not high. At the same time, through the experiment in microfluidic chip, we find that the amplified signal without paramagnetic particle method can meet the needs of detection, so the step of nucleic acid signal extraction can be deleted.
Based on the above research, this project changes to conduct the sample pre-processing process for protein signal such as aptamer replacement before sample’s injection after cell structure destruction. In the pretreatment process of reagent consumables, we only embed the strip in the centrifuge chip, and other reaction steps are planned to be completed before injection. In the final delivery to the user, the user needs to carry out the sample pre-processing process.
Question4:
In our design, the power supply is directly from the mobile phones to the external devices. However, after our research and analysis, it is impossible for mobile phones to directly power external devices or units. For example, the micro USB interface is common in mobile phones, however, according to international standards, it is impossible to supply power from the mobile phone to other devices. If the power supply to the external equipment is strengthened through the earphone jack of mobile phone, only small current and small voltage can be output. At present , 3.5mm headphone interface has been gradually replaced. To sum up, it is not easy to use the mobile phone to charge the device in reverse.
Solution:
In order to solve the above problems, we design two schemes: the first scheme is to use the external power bank to supply power, which is not only easy to get, but also easy to carry and use. The design of the external device is also very simple, only need a USB interface to connect the charging device. Another design method is to select a battery that can power the device and design it on the same device as other modules. The battery can choose 18650 lithium battery which is commonly used in the market.
③ Microfluidic chip and related content production protocol
Synopsis of instruments:
The instruments used by the iGEM-BIT team in the 2020 project will be consist of our own designed fingertip gyro-driven microfluidic chips, lateral flow tomographic strips, temperature control modules, optical sensing modules, and intelligent external machine frame structures. The goal of this project is to design a palm-held laboratory capable of detecting 2019-nCoV with high accuracy and throughput.
In the process of the project development, we designed and completed the following experiments:
1. Fabrication of photoresist chip.
2. Fabrication of PMMA chip.
3. Test strip
Experimental display:
1. Fabrication of photoresist chips:
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Objective:
The basic model of the microfluidic chip based on fingertip gyroscope was fabricated using the photoresist method under laboratory conditions, and the preliminary design of the chip was verified.
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Design:
In the experiment, we use a positive photoresist, which can dissolve in the developing solution when the exposed part undergoes photochemical reaction under the condition of ultraviolet irradiation, while the unexposed part is insoluble in the developing solution and remains on the substrate. The same pattern on the mask will be copied to the substrate to complete the fabrication of the designed chamber and channel.
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Steps:
(1) The mask is cut and cleaned (firstly, it is cleaned with tap water, sprayed with detergent, cleaned with detergent, then cleaned with deionized water, and finally dried with a blower), which is convenient for subsequent experiments.
(2) The underlying structure of the photogel chip:
① Use a little photogel at the diagonal point of the mask frame, and solidify it with ultraviolet light to make the mask frame solidify stably on the glass slide. Add about 0.2 ml of photogel into the mask frame, cover with the mask, and push the photogel under the mask evenly with tweezers to fill the mask frame, and use the tweezers to drive away the bubbles under the mask. According to the thickness of the photogel tiles, the photogel is fixed by irradiating them with ultraviolet light for 25s-30s.
②After the photocoagulation is solidified, gently pick up a corner with tweezers to observe whether the photocoagulation has solidified on the surface of the glass sheet. If there is no change of angle to observe, if it has solidified on the surface of the glass sheet, then tear off the mask of the surface quickly with tweezers.
③Drop about 0.2 ml of photogel on the solidified surface again, and lay the mask on the track so that there are no bubbles between the mask and the underlying photogel. Special attention should be paid to aligning the mask tracks of each layer. The photogel was fixed by irradiation with ultraviolet light for 10s-15s.
④After photocoagulation, gently pick up a corner and observe whether the photocoagulant is solidified on the surface of the underlying photocoagulant. If it has solidified, gently tear off the mask with tweezers. The process of tearing off the mask should not be too rapid and should be gentle.
(3) Use a little photogel at the diagonal point of the mask, fix the mask frame on the chromium plate, and fix the photogel with ultraviolet light. Repeat the above steps to fix the second mask frame on the original basis. After fixing the mask frame, point about 0.5 ml of photogel, cover it with a mask, try to make no bubbles between the mask and the photogel, and tear the mask off the chrome plate slowly with ultraviolet light for 30 s.
(4) Clean and dry the mask and glass plate with the photogel lotion, align and bond them together under the microscope, irradiate them with ultraviolet light for about 30 seconds, and tear off the mask quickly after confirming that the two have bonded. Carefully observe to determine whether the chip channel is smooth and whether the chamber track is blocked.
(5) Intubation: put the gasket made into the intubation of the chip, insert the thin tube that has roughened the orifice into the chip, fix it with photogel, and illuminate it with ultraviolet light for 5 s. Repeat the above steps to intubate each tube in turn.
(6) Edge sealing: the edges of the chip were sealed sequentially with photogel and illuminated with ultraviolet light for 30 s. Finally, place a 50 degree oven overnight.
2. Bonding of PMMA Chips
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Objective:
The purchased processed PMMA chips were bonded together to complete the final assembly of the microfluidic chips.
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Design：
As a thermoplastic, PMMA has a low melting point property. So we can realize chip bonding of each layer of PMMA chip at high temperature.
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Experimental materials:
Processed layered PMMA chips, absolute ethanol, distilled water, Petri dishes, muffle stoves, glass plates, pins, clips.
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Steps:
(1) Remove the surface membrane of PMMA chip, rinse the residual impurities on the surface with absolute ethanol and distilled water, and place them on the Petri dish to dry.
(2) Adjust the muffle furnace temperature to 170 C and keep the temperature for 1 h.
(3) Place the chip on the glass plate in turn, insert a micro pin at the specific socket where the chip has been processed to help the chip bond, align the back cover with another glass plate and tighten it with a clamp, and heat it for 6 minutes before removing it.
(4) After the glass plate is naturally cooled, remove the micro pin in the chip, and then heat it for another 10 minutes to remove it to complete the chip bonding.
3. Preparation and assembly of colloidal gold strips
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Objective:
Preparation of colloidal gold strips for fluorescence signal detection.
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Experimental materials:
Apt-21 solution, TE buffer, phosphate buffer, streptavidin, ultrafiltration tube, poly-A solution, syringe, shaker, PVC plate, etc.
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Steps:
(1) Solution preparation for test line:
1OD Apt-21 solution was taken, centrifuged at 12,000 rpm for 30 s at 4℃, and prepared into 100μM storage solution using TE buffer.
1 mg streptavidin was dissolved in 1 mL phosphate buffer.
42μL of 1 mg/L streptavidin was mixed with 26 μL of Apt-21 solution and placed at room temperature for 3 h.
The mixture was centrifuged at 12,000 rpm for 10 min at 4℃ in a 30 KD ultrafiltration tube, and the lower solution was discarded.
Then invert the ultrafiltration tube, centrifuge 1000g at 44℃ for 30 min to obtain the solution, the total volume is about 30 μL.
(2) Solution configuration for quality control line
1OD Apt-21 solution was taken, centrifuged at 12,000 rpm for 30 s at 4℃, and prepared into 100μM storage solution using TE buffer.
42μL of 1 mg/L streptavidin was mixed with 26 μL of poly-A solution and placed at room temperature for 3 h.
The mixture was centrifuged at 12,000 rpm for 10 min at 4℃ in a 30 KD ultrafiltration tube, and the lower solution was discarded.
Then invert the ultrafiltration tube, centrifuge 1000g at 44℃ for 30 min to obtain the solution, the total volume is about 30 μL.
(3) NC film streaking operation
Cut NC film about 20 cm long and fix it on the sample dispenser;
Open the instrument, carry out initialization operation, and then set parameters to determine the values of X and Y. First extract air with syringe, then set the liquid capacity, then extract air once, adjust the distance between scribe needle and NC film, click RUN button;
25 μL of Apt-2-avidin solution with a concentration of 5 OD/30 μL was taken and the scribed operation was carried out at a speed of 1 μL /cm.
25 μL of poly-A-avidin solution with a concentration of 0.05 OD/30 μL was taken and the scribed operation was carried out at a speed of 1 μL /cm.
Place in self-sealing bag and add appropriate amount of desiccant overnight.
(4) Treatment of sample pad
Cut 4 mm width sample pad;
The cut sample pad is packed in a self-sealing bag, and a proper amount of sample pad treatment buffer is added to make the sample pad completely immersed;
The self-sealed bags were placed on a shaker and incubated for 30 min.
Wash the sample pad with PBS solution until it is free of foam, dry it for future use.(5) Assembly of test strips
Place the PVC board on the workbench and attach the NC film to the center of the PVC board.
The sample pad was placed on one side of the PVC board and overlapped with the NC film for about 2 mm;
The absorbent pad was placed on the other side of the PVC plate and overlapped with the NC film for about 2 mm and stored at room temperature after cutting.
④ Research on the popular science environment of the current universal platform
Team BIT write a research report to investigate the popular science work environment of Weibo and bilibili, two major platforms with a large number of users in China. Through this research report, other iGEM teams can understand which platform is more suitable for science popularization work, so as to help each team achieve better results. And we cooperated with team SCUT-China to conduct research in this area.
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Created By Beijing Institute of Technology