Proof of Concept
Our project is dedicated to designing an integrated detection system which can be used for the new coronavirus to solve the problems of high detection cost and complex detection process. Based on the biosensing module, the centrifugal chip module and the smart phone module, we can effectively implement the fast, sensitive, portable and low-cost sample-to-answer detection of nucleic acid and protein markers related to coronavirus, which can provide effective coronavirus detection scheme for the places with scarce medical testing resources.
The biosensing module undertakes the function of converting biological signals into optical signals. The target protein to be detected can be obtained by pre-processing the respiratory tract extract of the subject. Through the designed nucleic acid aptamer system, the protein signal can be converted into a nucleic acid signal, and then the nucleic acid signal can be amplified by an isothermal amplification system. Finally, we use colloidal gold chromatography test strip technology to detect the amplified nucleic acid sequence and display the detection results in the form of light signals.
The centrifugal chip module undertakes the function of providing a process carrier for the biosensing module on the micro-scale.Using the "fidget spinner" method, we can manually provide the centrifugal force that drives the fluid in the chip. In the pre-processing area of the chip, we can complete the pre-processing of the sample, and then provide a constant temperature in the isothermal amplification area to achieve nucleic acid signal amplification. In the signal detection and transduction area, we will pre-embed chromatographic test strips for detection to detect and convert the amplified nucleic acid sequence. The mobile phone support module provides the colloidal golden degree detection for the above-mentioned biosensing module. The software designed will implement the human-computer interaction of the terminal, provide the user with the final result delivery, and further serve as an intermediate medium for the extension of the back-end information chain.
According to the workflow, our project first conducts sample pre-processing. This part is mainly carried out by biological methods, including sample collection, cell lysis, protein signal extraction and purification, and nucleic acid aptamer replacement technology.
Based on the inherent resources of the laboratory, in the cell lysis section, we use the lysis solution processing method to extract our detection markers. The four main principles of cell lysate are as follows: 1. Use detergent to destroy lipid bilayer and rupture cells; 2. Dissolve protein; 3. Stabilize the protein through protein denaturation; 4. Inhibit protease activity. When the characteristic protein is extracted and purified, we use the aqueous solution extraction method: In the aqueous solution of the dilute salt buffer system, the protein has better stability and higher solubility. The amount of this solution is usually one to five times the volume of the raw material, and it needs to be stirred evenly during extraction to dissolve the protein. Finally, using the nucleic acid aptamer replacement system, we can replace the obtained N protein signal with a nucleic acid signal through the complementary strand.
The temperature control module can provide the required temperature conditions for nucleic acid amplification. Commonly used PCR amplification has undergone two steps of temperature change, denaturation and annealing, which requires the device to have the ability to quickly raise and lower the temperature in terms of temperature control. In order to meet this requirement, the module needs a larger space to provide a heat dissipation environment for the device, and at the same time needs to be provided with sufficient power. However, because of the project's requirements for miniaturization and portability, we did not use PCR as our amplification method, but adopted HCR and RCA, which have lower requirements for temperature changes, as the amplification method of the target nucleic acid sequence.
Since the optimal amplification temperature required by HCR and RCA is a constant 37°C, we initially considered whether the human body can be used to heat our chip nucleic acid amplification. But considering the biological safety and the stable temperature required for amplification, we gave up this idea.
The temperature control module will be mainly composed of a single-chip microcomputer, ceramic heater and thermistor to complete our temperature requirements for the amplification reaction. We chose a single-chip microcomputer to control the temperature because of its high cost performance, comprehensive functions and strong versatility. Our heating module chooses to use ceramic heating plate production because of its simple structure, low power consumption, fast temperature change and fast temperature compensation. The thermistor is selected for the temperature sensor because it can detect the temperature of about -50°C to 200°C, and its temperature measurement accuracy is between 0.05°C and 1.5°C. In addition, temperature-sensitive resistors also have the advantages of small size, fast response time, and low price.
In this project, the application of Immune colloidal gold technique technology greatly promoted the success of the experiment. In recent years, the advantages of gold-immunochromatography assay(GICA) have been gradually known and widely used in the detection of various samples. These advantages include: (a) Low development cost: simple test paper structure, low cost of consumables and production equipment; relatively easy to manufacture and easy to mass produce; (b) simple and fast to use: GICA method does not require multiple sample additions or washing, complete the test in one step, generally the results can be obtained within 5~15 mi min, and can be tested at the sample collection site; experiment operators rely on few steps, and the results are judged only by eyes or simple optical instruments; (c) relatively stable : The shelf life of GICA test strips is generally 12-24 months, and often does not require refrigeration. The test results can be stored at room temperature for a long time; (d) Wide application range: urine, saliva, whole blood, serum, plasma, body fluids, etc. The sample can be detected with simple or no treatment, and the sample dosage is small. Based on the above characteristics, the GICA method has become a more economical and practical immunochromatographic technique at this stage, which is especially suitable for immediate inspection, large-scale and time-critical inspection and large-area investigation.
Immune colloidal gold technique (GICT) is a labeling immunoassay technology that combines the sensitivity of colloidal gold in the detection with the specificity of the antigen-antibody reaction. It has good performance advantages for the quantitative detection of trace substances. This technology has relatively low requirements for instruments and equipment, and has the characteristics of short detection time and high sensitivity. With the application of chromatography technology to immunological detection, the use of immunochromatographic test strips greatly simplifies the operation process of specific reactions, accelerates the automation process of immune colloidal gold technique equipment, and is more conducive to the promotion and practical application of this project.
The main function of the smartphone in this project is to detect the intensity of the colloidal gold test strip in the centrifugal chip to achieve the visual and quantitative detection of virus signals, so we need to use the MINA framework to build a WeChat mini program. MINA is the internal development code of the official mini program and the alias of the mini program running framework. The WeChat Mini Program is not a technology or a framework. The WeChat Mini Program is an ecosystem. The corresponding ecosystem should be the iOS or Android. The WeChat Mini Programs are very similar to iOS. They are very closed and strictly reviewed. And MINA is a framework of small programs, which provides the interfaces, models and mechanisms required for the operation of small programs. Our moving MINA method builds our WeChat mini program to achieve human-computer interaction.
Created By Beijing Institute of Technology