Hardware
1. Background
1.1 Initial stage of the project:
The current COVID-19 detection platforms have problems such as cumbersome operation, large body size, they need to be equipped with professional operators, and are limited to use in central laboratories. Therefore, a visual detection hardware device that is simple to operate and easy to carry and can be used in communities, rural hospitals and homes is very necessary. In the initial design of the project, the biological part was designed to jointly detect the nucleic acid and protein markers of the novel coronavirus. The centrifugal chip plans to integrate all the steps of the novel coronavirus detection into the chip, and realize the following three functions at the same time: pre-processing of the sample to be tested, signal amplification of the target molecule, and signal visualization detection. Thus, the original centrifugal chip module can be divided into a sample pre-processing area, an isothermal amplification area, and a signal detection and transduction area based on the specific biosensing function implemented. Meanwhile, for the application scenarios of instant inspection, we further design:
1) Parallel multi-channel detection method in sectors;
2) Manual "whistle" type liquid path driving method and microvalve-based flow control method;
3) Reagent consumables Processing method;
4) Temperature control and detection system adapted to the downstream mobile phone support module.
Reagent consumables required are pre-processed in the chip in advance, and those for different targets are stored or embedded in different sectors. When it is finally delivered to the client for use, the client only need to add samples, and perform simple operations in accordance with the established process to achieve the release of liquid reagents, the dissolution of solid reagents, and the use of consumables without additional reagents or consumables.
Initially, the project envisages that after users use the sample, they will first go to the sample pre-processing part. In the pre-processing part of the sample, for the protein to be tested in the sample, our project plans to complete the pre-processing steps of cell lysis and aptamer replacement before sample injection. After cell lysis, the aptamer replacement system for N protein or S protein can be used to process the protein signal into nucleic acid signal. For the processing of nucleic acid signals, in the early stage of our project design, we considered extracting and purifying them by magnetic bead method. The fine separation of magnetically responsive particles using the movement of particles induced by a magnetic field, that is, magnetophoresis, is an effective technology developed in recent years to selectively separate cells and high molecular weight nucleic acids. The target biomolecules labeled with magnetic particles separated by the magnetic separation device can be directly used for subsequent analysis and testing.
In the signal detection part, the initial plan of the project is to use external equipment to build and design a fluorescence detection unit based on the mobile phone platform. In terms of fluorescence detection method, we plan to adopt oblique incidence fluorescence detection method which is conducive to miniaturization. We use SOLIDWORKS for 3D modeling, design a box that can be equipped with a microfluidic chip and a mobile phone holder that matches different mobile phone models, and subsequently could design an adjustable mobile phone holder to match different mobile phone models. Moreover, in addition to using the LED light source that comes with the mobile phone, an LED light source using a button battery is added as the light source to the mobile phone holder to ensure the stability of the light source.
Finally, it is planned to use 3D printing technology to complete the model. The figure below shows a mobile phone holder that matches a single model of mobile phone.
In the drive design of the centrifugal chip, the initial plan of the project is to adopt a whistle-type centrifugal chip design scheme based on the "whistle principle". Punch holes on both sides of the chip shell and fix the two bungee cords, then pull the bungee cord by external force to switch between "winding" and "unfolding", which can pull the centrifugal disc chip to produce a certain rotation speed and centrifugal force (speed can reach 125,000 rpm, and the equivalent centrifugal force is 30,000 g)[1], so as to achieve hydraulic drive, In addition, microvalves are set at bayonet ports in different areas to ensure the directional movement of the fluid and make the fluid stay and work in a specific area.
1.2 Final design of the project:
In the final experimental stage of the project, we conducted experimental verification on the manual whistle driving method adopted in the initial plan, and found that the driving method based on the whistle principle has problems such as poor stability, easy leakage, and structural instability. Under the conditions of manual centrifugation, this project is finally implemented to drive the microfluidic chip through a fidget spinner[2], which can control the centrifugal drive more stably.
For the sample pre-treatment area planned to be built at the beginning of the project, if we need to perform multiple reactions in the microfluidic chip and build multiple chambers, it will be difficult to control the liquid path, and we need to add microvalves and other structures for control. Considering the streamlining of the microfluidic chip structure and the ease of controlling the liquid path, this project was finally implemented as the two steps of the cell lysis step and the conversion of protein signals into nucleic acid signals before the microfluidic chip injection.
In the extraction of nucleic acid, the magnetic bead method mainly includes four steps of lysis, binding, washing and elution. Through literature research, we found that the operation steps of the magnetic bead method in the microfluidic chip are more complicated, so the magnetic bead method has higher requirements for liquid path control and reaction process control. In the current experiment, the efficiency of nucleic acid extraction by the magnetic bead method in the chip is not high. And at the same time, through experiments conducted in the microfluidic chip, the amplified signal of the nucleic acid signal that does not pass the magnetic bead method is detected, we find that it can already meet the needs of detection. Our project is finally implemented as direct amplification of unpurified nucleic acid signals.
Corresponding to the changes in the above design ideas, we only need to consider the two functions of the pre-processing of the sample to be tested in the structural design of the centrifugal chip, the signal amplification of the target molecule and the signal visualization detection. Therefore, the chip we designed mainly contains nucleic acid The two major structures of the amplification chamber and the test strip chamber, as well as the injection port and microchannels.
The design of the amplification chamber is relatively simple, and it only needs to cooperate with the temperature control module of the mobile phone to provide a suitable temperature for the chamber. After the nucleic acid signal is amplified by the RCA and HCR technology in the previous part, the biological signal needs to be converted into a visual light signal. Therefore, the main function of the test strip is to use colloidal gold technology and lateral tomography technology to realize the energy conversion path, so as to perform more accurate detection of experimental results.
In the detection part, the mobile phone holder and detection light path designed in the first version of the project have problems such as difficulty in experimental verification and unstable position parameters of different mobile phone cameras. Resulting from the need of portable equipment, we retain the colloidal gold technology for testing, upload the photos taken to the mini program, analyze the R value of colloidal gold, and compare with the standard positive value to get the final result.
2. Project overview
In our project, a microfluidic chip driven by a fidget spinner is used as a reaction carrier to realize the biological part. The manufacturing material is polymethyl methacrylate (PMMA), and the chip is assembled by hot-key bonding method. The overall frame of our microfluidic chip can be approximately regarded as a rectangle, the turning radius is slightly smaller than the average length of the adult hand gap, and there is a rotating shaft composed of a roller bearing and a gasket in the center of the chip. By holding the gasket in the center and rotating the chip with the other hand, the centrifugal force generated by the rotation can be used to drive the liquid path of the microfluidic chip.
According to the biological part of the detection process, this project decided to perform sample pre-processing steps such as cell lysis, release of contents, and protein signal conversion outside the chip. On the basis of the microfluidic chip, we successively realize the isothermal amplification of nucleic acid in the isothermal amplification area, and realize the visual signal conversion based on the lateral chromatography test strip in the signal detection area. Finally, the mobile phone support module optically detects the signal on the test strip, and performs subsequent signal processing. Specifically, on the test strip, our project adopts a signal conversion method based on colloidal gold technology, and compares the colloidal gold degree with the standard positive result through the color reaction to obtain the reaction result.
The temperature control module is mainly composed of a single-chip microcomputer, a ceramic heater and a thermistor. The combination of these three can meet our temperature requirements for HCR and RCA reactions.
In the optical signal visual detection part, we use the MINA framework to build a WeChat mini program to take pictures and upload the colloidal gold test strips, as well as reading the colloidal gold image with the software, and fitting the relationship between the image characteristics and the concentration of the test object, and finally get the virus test results.
3. Project specific design
3.1 Material:
According to the definition of George Whitesides, microfluidic technology is a technology that uses channels of tens to hundreds of microns in size to process or manipulate a small amount of fluid. On this micro-scale, the surface characteristics of the equipment will be extremely magnified, which undoubtedly may give it unique functions and cause problems that will not be encountered on the macro-scale.[3] In addition, the difference of each material also determines the physical characteristics, biological characteristics and processing methods of this microfluidic device. Therefore, we need to pay special attention to the materials used to manufacture microfluidic chips and compare their properties and preparation methods.
Common microfluidic chip materials include inorganic materials silicon and quartz, organic polymer materials polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA) and cloth, etc. Their respective processing methods and physical properties are shown in Table 1.
| Material | Light transmittance | Toughness | Permeability | processing method |
|---|---|---|---|---|
| Fabric base | Poor | High | Medium | Weaving, wax transfer, cutting, photolithography, etc. |
| Silicon | Good | Low | Poor | Lithography and etching, etc. |
| PMMA | Good | Low | Poor | Injection molding, mold hot pressing and CNC milling, etc. |
| PDMS | Good | High | Good | Injection molding, mold hot pressing and CNC milling, etc. Hot pressing, injection molding, molding, laser cutting, 3D printing, etc. |
In this year's project, we make a microfluidic chip for rapid detection of COVID-19. In order to meet the epidemic prevention requirements, the chip is designed to disposable. At the same time, since we choose colloidal gold detection as the detection method, we have high requirements on the light transmittance of the chip material. In this context, our microfluidic chip materials will choose PMMA with low prices and simple manufacturing methods. While making full use of its advantages such as good light permeability, strong bio-friendliness, simple manufacturing and low cost, it avoids the defects of being easily contaminated by reagents, difficult to clean, and low reusability.
3.2 Chip production:
As a material that is widely used in industry, the processing and manufacturing process of PMMA has been mature, and its processing method is simple and easy and the micro-manufacturing cost is low.
According to the centrifugal chip layer we designed, we can obtain 1-4 layers of different structures. In order to ensure the cleanliness of the chamber after the chip is bonded, the surface is cleaned with absolute ethanol and deionized water in sequence. After cleaning, wipe off the remaining water stains with lens cleaning paper, place the chip layers in the glass plate in order, fix each layer of the chip with solid micro pins, cover another layer of glass plate and clamp it. Place it in a 170°C environment and heat it for 10 minutes, take out the fixed micro pins and heat it for 5 minutes to complete the bonding of the chip to obtain the required microfluidic chip. Finally, place the rolling bearing in the center of the chip to complete the centrifugal chip processing.
3.3 Chip structure:
Based on the designed biological part, the centrifugal chip module needs to integrate the following functions: target molecule signal amplification and signal visual detection. Therefore, the centrifugal chip module can be divided into an isothermal amplification zone and a signal detection and transduction zone according to the specific biosensing function realized. For the sample pretreatment of the microfluidic chip, this project decided to take the steps of cell lysis, release of contents, and protein signal conversion outside the chip. In the nucleic acid signal amplification part, HCR and RCA amplification technology are adopted, the pre-processed sample is added to RNase and proteinase K, and the mixed solution is directly injected into the RPA amplification chamber through a pipette for amplification.
The overall construction frame of the disc chip is a disc-like disc, and the manufacturing material is made of polymethyl methacrylate (PMMA). The center of the disc is composed of a roller bearing and its central solid shaft and a gasket. The driving method is similar to a fingertip top. The microfluidic chip is rotated by holding the gasket and pushing with the other hand. The chip uses the kinetic energy transmitted by the pusher and converts it into centrifugal force to drive the sample into the next chamber.
Our project is based on solidworks software to design the chip structure, as shown in the figure below. The chip is designed with a micro-reaction chamber, a micro-channel and a hole, and contains two symmetrical detection modules, which can simultaneously detect two independent samples. The micro device is composed of four layers of 2 mm PMMA boards, which are connected as a whole by means of hot-key bonding method. The first layer contains the sample inlet, the second layer contains part of the nucleic acid amplification chamber and test strip chamber and microchannels, the third layer contains part of the nucleic acid amplification chamber and test strip chamber, and the fourth layer is only Test strip chamber. The sequence of the liquid path is the injection port, the nucleic acid amplification chamber, and the test strip chamber. The radius of the nucleic acid amplification chamber is about 5mm. In the nucleic acid amplification chamber, the sample is subjected to isothermal amplification at a constant temperature (provided by the mobile phone module device). At the same time, during the actual use of the RPA nucleic acid amplification area, surface modification treatment is also required to prevent the reaction mixture from directly contacting the inner surface of the channel, avoiding the surface adsorption of biological/chemical particles, and preventing microfluidic interference. Inhibition of nucleic acid amplification and carryover contamination. The size of the test strip chamber is 3.9mm*59mm, which is used to store the test strip for subsequent testing operations.
After the sample is added, hot melt glue or sealing film is used to seal the chip injection port, and the sample is injected into the nucleic acid amplification chamber, and isothermal amplification is performed in the mobile phone module. After the amplification is completed, the microfluidic chip is turned upside down and rotated, and the sample will be driven by centrifugal force to flow into the test strip chamber for the next step of detection. By controlling the amount of sample entering the microfluidic chip, the height of the sample chamber after adding the reaction system will not reach the second layer of microchannels, so the sample will not enter the test strip area in advance. Due to the pseudo-centrifugal effect, the test strip will be subjected to gravity and centrifugal force away from the axis. At the same time, because the chamber size is slightly smaller than the test strip by 0.2mm, the test strip will be stuck inside to ensure that the test strip will not fall.
Based on the previous model construction, we have manufactured a prototype of a centrifugal microfluidic chip. In this chip, we did not install a spacer, but directly held the solid center shaft to rotate. Preliminary experiments have shown that the chip can maintain a basically stable rotation and speed when the central shaft is held in hand.
3.4 Test strip detection area
After the nucleic acid signal is amplified by the previous RCA and HCR technology, the biological signal needs to be converted into a visual light signal. Therefore, the main function of the signal detection and transduction area in the microfluidic chip is to use lateral chromatography test strip technology and colloidal gold technology to realize the transduction path, so as to perform more accurate detection of the experimental results.
Lateral chromatography technology uses a large-pore microporous membrane (NC membrane, nitrocellulose membrane) as a carrier to fix specific antigens, antibodies or nucleic acid probes on the NC membrane. When the sample to be tested is added to the sample pad at one end of the test strip, it moves laterally through capillary action, and a specific immune reaction occurs with the colloidal gold on the binding pad. Then it moves to the NC membrane, being captured by the antigen, antibody or nucleic acid probe immobilized on the NC membrane surface, and gathers on the detection belt. By visually observing the density of the light reflection signal of the nitrocellulose surface marker (colloidal gold), intuitive color results can be obtained. Other unbound markers cross the detection zone and flow into the absorbent pad to achieve the purpose of automatic separation. After the microfluidic chip drives the pre-processed sample to the isothermal amplification zone for amplification, the detection system needs to extract the information from the processed sample and convert it into the final result[4].
This project designed a detection method based on lateral flow chromatography test strips. The method process includes the following three steps: sample preparation, immune response, and colloidal gold detection. We need to add the sample to be tested to the lateral flow test paper, capture the sample to be tested on the test line of the lateral flow test paper through immunochromatographic reaction, and finally diagnose whether the sample has novel coronavirus through color development.
Colloidal gold-based immunochromatographic assay (GICA) is a solid-phase labeling immunoassay technology that organically combines colloidal gold labeling technology, immunodetection technology and chromatography analysis technology. It has the characteristics of simplicity, time saving, less sample usage, and easy interpretation of results. It is a lateral immunochromatographic method that uses colloidal gold as a tracer to determine the target qualitatively or quantitatively. The most suitable colloidal gold particle size for immunoassay is 20-40nm, and the colloidal gold in this particle size range is ruby color. It is negatively charged under alkaline conditions and can bind to the protein through electrostatic and hydrophobic interaction, and does not affect the activity of the conjugate. During detection, the sample is added to the sample pad at the end of the test paper and moved forward by capillary action. The analyte in the sample interacts with the immune colloidal gold on the binding pad and forms a complex. The complex moves to the detection line, specifically binds to the antibody (or antigen) on it, and is retained, and aggregates on the detection line. When the complex accumulates to a certain amount, it shows a red color characteristic of colloidal gold. The immunocolloidal gold that is not bound to the analyte is not captured by the detection line, and migrates to the quality control line and binds to the antibody on it to show red, achieving the purpose of separating from the complex, and indicating that the test paper is working properly.[5]
The standard lateral flow chromatography test paper includes five parts, namely sample pad, conjugate pad, reaction membrane (usually nitrocellulose membrane), absorption pad and back plate, as shown in the figure below, while the chromatography test The paper strip is mainly composed of sample pad, NC film, absorbent pad, and back plate. The assembly diagram is shown in the figure:[6]
In the design and operation process, this project first mixes the positive sample, the loading buffer and the colloidal gold solution that have been subjected to the HCR reaction in a 37°C incubator in advance. The volumes of the three solutions are 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. Then rotate the centrifugal chip to make the mixed solution enter the test strip area, and cover the entire test strip under capillary action. Observe the condition of the C line and T line of the test strip. The strips of the test strip in the figure below are C line and T line from left to right, and double strips appear to prove a positive result.
Repeat the above steps to react the blank control group without positive samples, and get the negative band results as shown in the figure below:
3.5 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.
3.6 Mobile phone interaction module
For colloidal gold detection, after taking a photo with a mobile phone, we analyze the R value after the image is collected and compare it with the positive R value to determine the result of the test.
Through the establishment of the small program, we have completed the following functions: reading the colloidal gold, fitting the relationship between image features and solution concentration, outputting the image, and outputting the predicted concentration.
Software interactive program module: Based on the development of WeChat mini program , extract features such as image color (RGB), and use the least square method to perform linear fitting to obtain the relationship between features and concentration. Least squares method is a mathematical optimization technique. It finds the best function match of the data by minimizing the sum of squares of errors. The least square method can be used to easily obtain unknown data, and minimize the sum of squares of errors between the obtained data and the actual data. [7]
Software interaction process: the mobile phone shoots multiple images, the mini program reads the images, manually enters the solution concentration, image processing, fitting the relationship between the image characteristics and the solution concentration, the new image is input in the mini program, and the predicted concentration is output.The following are the specific functions of WeChat mini program.
1. Enter the concentration value, click Upload Image to upload the picture, click Color Feature1 to extract the color features, the user can upload at least 2 samples up to 5 samples;
2. Upload a new image to extract new features, use the least square method to fit and output the predicted concentration.
3.7 Innovation
1. It innovatively combines the biological detection process with the design of the hardware chip, skillfully USES the centrifugal driving force constructed by the chip and the internal liquid path evaluation model to complete the lateral flow chromatograph test strip detection, which greatly improves the detection efficiency and accuracy and provides a more convenient and fast novel Coronavirus instant detection method.
2. The microfluidic chip adopts the fidget spinner type liquid path driving mode, which reduces the dependence on large analytical instruments and improves the universality.
3. The combination of WeChat small programs can greatly improve work efficiency, and the realization of software interaction can be more convenient and fast.
4. References
[1]Bhamla, M. S. et al. Hand-powered ultralow-cost paper centrifuge. Nat. Biomed. Eng. 1, 0009 (2017).
[2]Michael, I. , Kim, D. , Gulenko, O. , Kumar, S. , & Cho, Y. K. . (2020). A fidget spinner for the point-of-care diagnosis of urinary tract infection. Nature Biomedical Engineering, 4(6), 1-10.
[3]Ren Kangning,Zhou Jianhua,Wu Hongkai. Materials for microfluidic chip fabrication.[J]. Accounts of chemical research,2013,46(11).
[4] Yang Yongliang. Microfluidic chip and detection device for CD4 cell counting [D]. Beijing University of Chemical Technology, 2016.
[5] Man Yan. Study on highly sensitive and rapid detection of protein based on microfluidic chip [D]. Beijing Institute of Technology, 2015.
[6]. Beijing University of Chemical Technology, 2016.
[7] Zhang Ying, Feng Hai, Wu Qiuping, Tan Zeng, he Jiabei. Research progress of colloidal gold immunochromatography [J]. Advances in Modern Biomedicine, 2015 J 15 (12): 2349-2351.
Created By Beijing Institute of Technology