Paper-based micro-fluidic chip and application thereof

Abstract

The invention discloses a paper-based micro-fluidic chip and application thereof. The paper-based micro-fluidic chip comprises a sample inlet, micro-reaction units, a micro-fluidic chip channel and a waste liquid outlet, wherein the micro-reaction units are connected through the micro-fluidic chip channel; the micro-reaction unit comprises a magnetic bead and a nucleic acid aptamer. According to the invention, the micro-fluidic chip and Mie scattering are combined to establish a novel paper-based micro-fluidic chip rapid detection method for the coronavirus. A hydrophilic cellulose paper-based micro-fluidic chip is pre-loaded in micro-reaction holes of a plurality of micro-fluidic chips to serve as an immunoreaction container, and a polystyrene microsphere-novel coronavirus S1 protein nucleic acid aptamer covalent conjugate covalently coupled in the micro-fluidic reaction holes serves as a chip induction and signal amplification element. The rapid and high-sensitivity detection of the novel coronavirus in a sample can be realized within 1.5-3h without a culture amplification procedure.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a paper-based microfluidic chip and applications thereof. Background Art

[0002] The spike protein (S Protein) is a glycoprotein that extends out of the cell membrane. It is the most important surface protein of COVID-19 and is related to the infectious ability of COVID-19. It is composed of 1255 amino acid residues and may play a key role in the process of virus binding to host cell surface receptors, mediating membrane fusion and entering cells. According to the protein structure and function, it can be divided into two functional units: S1 and S2 protein subunits. S1 can promote the ability of the virus to bind to the host cell receptor. It contains an important C-terminal receptor binding domain (RBD), which is responsible for binding to the receptor.

[0003] In the previous computer model analysis, the spike proteins of the new coronavirus and SARS have low homology, and the amino acid sequence similarity of the two virus spike proteins is only 76.47%. However, some gene regions of the RBD domain of the two viruses have a high degree of homology with SARS. Among the five key sites infected by SARS, one is retained by the new coronavirus, and the remaining four have amino acid substitutions and changes. Four of the five key amino acids in the COVID-19 spike protein that bind to the ACE2 protein have changed, but it maintains the original structural conformation of the SARS virus spike protein and ACE2 protein. This shows that the new coronavirus is still infected and transmitted through the same receptors and mechanisms as SARS.

[0004] In the process of large-scale community screening, screening technology that directly detects the S protein on the surface of COVID-19 by capturing it is particularly important, providing reliable and strong evidence to determine whether a person is infected in a short period of time. At present, the complete gene sequence of COVID-19 has been obtained. By collecting samples of the upper respiratory tract (oropharynx and nasopharynx) and lower respiratory tract (endotracheal suction, sputum or bronchoalveolar lavage) of suspected COVID-19 patients and performing RT-PCR, early diagnosis of patients suspected of COVID-19 infection can be performed. Conventional genetic diagnosis methods are based on reverse transcription PCR and Agrose gel electrophoresis, with low detection sensitivity, and quantitative PCR is expensive, which is not conducive to large-scale early screening, detection, and diagnosis.

[0005] Microfluidic chip is an emerging microfluidic analysis technology platform with the advantages of low cost, simple processing, easy use and carrying. It has great application prospects in application fields such as clinical diagnosis, food quality control and environmental monitoring.

[0006] Although microfluidics has the potential to reduce detection costs, the design and manufacture of some microfluidic chips may require high costs. A fully automated detection system may require complex design and additional hardware support, which may increase the complexity and cost of the system. In addition, microfluidic chips based on nucleic acid detection may need to optimize the efficiency of sample lysis and nucleic acid extraction, which limits the application of this technology. In recent years, the research on paper-based microfluidic chip technology has made rapid progress. Paper is used as a substrate to replace materials such as silicon, glass, and polymers. In the future, it will be deeply integrated with smart phones, open source hardware, and 3D printing technology to expand its functions and move towards the field of POCT. Mainly because of its simple production and low cost, more importantly, hydrophilic cellulose paper is selected to lay microchannels. The capillary action of the cellulose surface can drive the liquid to transfer water samples between the channel and each reaction unit, which can cleverly save the setting of the microfluidic injection driving force device, greatly improving the practicality of microfluidic chip technology. Existing microfluidic systems may require professional operators and complex operating procedures, which are not conducive to rapid deployment and use. The present invention can simplify the operating process and automatic design so that non-professionals can quickly get started. Additionally, the production costs of commercial microfluidic chips can be relatively high, especially when precision machining and special materials are required. The present invention can reduce costs by using more cost-effective materials and manufacturing processes. Summary of the invention

[0007] The object of the present invention is to provide a paper-based microfluidic chip and a rapid detection method for the novel coronavirus S1 protein based on Mie scattered light.

[0008] The technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides a microfluidic chip, comprising an inlet, a microreaction unit, a microfluidic chip channel, and a waste liquid outlet, wherein the microreaction unit is connected through the microfluidic chip channel; the microreaction unit contains magnetic beads and nucleic acid aptamers.

[0010] Preferably, the bottom plate of the micro-reaction unit is hydrophilic cellulose chromatography paper for coupling magnetic beads.

[0011] Preferably, a photoresist layer is provided on the side of the bottom plate of the micro-reaction unit.

[0012] Preferably, the photoresist layer is a negative photoresist layer.

[0013] Preferably, the negative photoresist layer is selected from SU-8 negative photoresist layer.

[0014] Preferably, the nucleic acid aptamer is a nucleic acid aptamer that specifically recognizes the new coronavirus.

[0015] Preferably, the nucleic acid aptamer is a nucleic acid aptamer that specifically recognizes the novel coronavirus S1 protein.

[0016] Preferably, the nucleic acid aptamer comprises a sequence as shown in SEQ ID NO.1: GTCTTGCGGGGCGGCGGG TTGAGAGGA.

[0017] Preferably, the magnetic beads are carboxylated functional magnetic beads.

[0018] Preferably, the carboxylated functional magnetic beads include carboxylated polystyrene magnetic beads.

[0019] Preferably, the particle size of the carboxylated polystyrene magnetic beads is 3 to 8 μm.

[0020] The second aspect of the present invention provides a method for preparing the microfluidic chip according to the first aspect of the present invention, comprising the following steps:

[0021] S1: Inject the magnetic beads through the injection port to fill the microchannel of the microfluidic chip, incubate at 20-30°C for 0.5-1.5h, and use the eluent to elute the uncoupled fixed magnetic beads;

[0022] S2: Inject the nucleic acid aptamer of the new coronavirus S1 protein through the injection port to fill the microchannel of the microfluidic chip, incubate at 32-40°C for 1-3 hours, and use the eluent to elute the uncoupled nucleic acid aptamer.

[0023] Preferably, the elution solution includes but is not limited to PBS.

[0024] The third aspect of the present invention provides the use of the microfluidic chip described in the first aspect of the present invention in detecting the new coronavirus for non-diagnostic purposes or in preparing products for detecting the new coronavirus.

[0025] The fourth aspect of the present invention provides a method for detecting the novel coronavirus for non-diagnostic purposes, comprising the step of using the microfluidic chip described in the first aspect of the present invention to detect a sample to be tested.

[0026] Preferably, the method specifically includes the following steps: injecting the sample to be tested through the injection port to fill the microchannel of the microfluidic chip, incubating at 32-40°C for 1-3 hours, using a Mie scattering detection device to measure the change in scattered light intensity before and after incubation, and calculating the content of the new coronavirus in the sample to be tested.

[0027] Preferably, the specific steps of the Mie scattering detection are: irradiating a light source λ<10*D (D, the linear dimension of the microbead antibody) that satisfies the Mie scattered light formation conditions onto the surface of the microreaction layer, and detecting the change in the intensity of the Mie scattered light before and after incubation at a certain angle θ in the direction of the incident light source, so as to know the content of the new coronavirus in the sample to be tested, thereby achieving the purpose of directly detecting the content of the new coronavirus in the sample.

[0028] A fifth aspect of the present invention provides a product, wherein the product comprises the microfluidic chip described in the first aspect of the present invention.

[0029] Preferably, the product further comprises a Mie scattering detection device.

[0030] The beneficial effects of the present invention are:

[0031] The present invention adopts Mie scattering combined with paper-based microfluidic chip technology to establish a paper-based microfluidic chip rapid detection method for the novel coronavirus S1 protein based on Mie scattered light. By pre-loading a hydrophilic cellulose paper-based microfluidic chip as an immune reaction container in the micro-reaction wells of multiple microfluidic chips, the polystyrene microbeads-nucleic acid aptamer covalently coupled in the microfluidic reaction wells of the novel coronavirus S1 protein are used as chip sensing and signal amplification elements, so as to realize the integration of multiple reactions of the novel coronavirus S1 protein analysis and detection method on a microfluidic chip, and the change in the intensity of the Mie scattered light signal caused by the immune agglutination reaction between the target and the nucleic acid aptamer microbeads of the novel coronavirus S1 protein can realize rapid and highly sensitive detection of the novel coronavirus in the sample within 1.5 to 3 hours without undergoing a culture amplification procedure.

[0032] The paper-based microfluidic chip is combined with Mie scattering technology. The new coronavirus S1 protein on the paper-based microfluidic chip binds to its nucleic acid aptamer. After latex microbead agglutination, the morphological parameters such as the surface particle size change significantly, which is easy to detect. When the latex-sensitized and agglutinated new coronavirus S1 protein nucleic acid aptamer particles are irradiated with a fixed wavelength incident light source of 470-1000nm, the wavelength of the scattered light obtained is not easily affected by the incident wavelength. The precise calculated value that can be obtained by processing the scattered light intensity signal with Mie scattering theory can be easily processed using mature computer technology. Therefore, the establishment of new coronavirus analysis technology by combining Mie scattering with paper-based microfluidic chip technology will definitely have great potential advantages and application development space. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a structural diagram of the paper-based microfluidic chip used for integrated detection.

[0034] Figure 2 This is a partial internal full-process working status diagram of the immune agglutination reaction unit on the paper-based microfluidic chip.

[0035] Figure 3 The cross-sectional side view shows the agglutination reaction of immune substances before and after being irradiated with incident light of a fixed wavelength.

[0036] Figure 4 This is the standard curve for the detection of the new coronavirus.

[0037] Figure numerals: 11. syringe; 12. injection connection hose; 13. injection port; 21. incident light; 22. scattered light; 31. hydrophilic cellulose chromatography paper; 32. SU-8 negative photoresist layer; 41. polystyrene microbeads bound to the S1 protein of the new coronavirus; 42. micro reaction unit (5 mm in diameter, with a branch pipe connected to the main microchannel); 5. main channel of the microfluidic chip; 6. branch channel of the microfluidic chip; 71. waste liquid cup; 72. hose connected to the drain; 73. waste liquid outlet. DETAILED DESCRIPTION

[0038] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0039] Example 1

[0040] This embodiment provides a paper-based microfluidic chip for integrated detection of novel coronavirus, the chip structure is as follows Figure 1 As shown, it includes: 11. syringe; 12. injection connection hose; 13. injection port; 21. incident light; 22. scattered light; 31. hydrophilic cellulose chromatography paper; 32. SU-8 negative photoresist layer; 41. polystyrene microbeads bound to the S1 protein of the new coronavirus; 42. micro reaction unit (diameter 5mm, with a branch pipe connected to the main microchannel); 5. microfluidic chip main channel; 6. microfluidic chip branch channel; 71. waste liquid cup; 72. drainage connection hose; 73. waste liquid outlet.

[0041] This embodiment also provides a method for detecting the novel coronavirus using the above-mentioned paper-based microfluidic chip, comprising the following steps:

[0042] 1. Inject carboxylated polystyrene microbeads with a particle size of 5 μm into the microchannel of the microfluidic chip through the injection port and fill the entire microreaction well, and close the waste liquid outlet valve; since the bottom layer of the microfluidic reaction well is paved with hydrophilic cellulose paper with a surface rich in carboxyl groups, the carboxylated polystyrene microbeads are coupled and fixed in the microfluidic reaction well through the reaction of the coupling agent, and incubate at room temperature of 25°C for 1 hour;

[0043] 2. Elute the uncoupled immobilized beads three times with PBS buffer and continue passing N 2 , blow dry the water in the channels and micropores;

[0044] 3. Inject 10 μg / mL of the aptamer of the novel coronavirus S1 protein into the injection hole and fill the entire micro-reaction hole and channel, and close the waste liquid outlet valve; place the chip in a constant temperature shaking incubator at 37°C for 2 hours;

[0045] 4. Elute the uncoupled aptamers three times with PBS buffer, and continuously introduce nitrogen to dry the water in the channels and micropores;

[0046] 5. Inject the water sample to be tested from the injection hole and fill the entire microchannel and reaction hole, and close the waste liquid outlet valve; place the chip in a constant temperature shaking incubator at 37°C for 2 hours; the new coronavirus S1 protein in the sample fully reacts with the nucleic acid aptamer on the PS microbeads.

[0047] 6. Insert the paper-based microfluidic chip into a handheld Mie scattering detection device to measure the change in scattered light intensity. The scattered light intensity is positively correlated with the amount of S1 protein of the new coronavirus. The virus content can be calculated by substituting the measured light intensity into the detection equation, such as Figure 1 shown.

[0048] The whole process working state of the local immune agglutination reaction unit on the paper-based microfluidic chip is as follows: Figure 2 As shown,

[0049] 1. First, carboxylated polystyrene microbeads with a particle size of 5 μm are evenly distributed and fixedly coupled in the micro-reaction wells;

[0050] 2. Covalently couple the microbeads to the nucleic acid aptamer of the novel coronavirus S1 protein to form a detection unit;

[0051] 3. When encountering the target novel coronavirus S1 protein in the sample, the nucleic acid aptamer on the microspheres will capture the target novel coronavirus S1 protein; the coupled nucleic acid aptamer microbeads suspended and fixed in the reaction unit of the paper-based microfluidic chip will undergo latex agglutination when encountering the novel coronavirus, resulting in significant changes in the morphological parameters of the immune substance, thereby amplifying the presence of the novel coronavirus in the water sample.

[0052] 4. Mie scattered light signal detection: A light source λ<10*D (D, the particle size of the microbead-antibody coupling complex) that meets the Mie scattered light formation conditions is irradiated onto the surface of the above-mentioned immune-reactive substance, and the change in the intensity of the Mie scattered light before and after the immune agglutination reaction is detected at a certain angle θ in the direction of the incident light source. The content of the new coronavirus in the water sample being tested can be obtained, thereby achieving the purpose of directly detecting the content of the new coronavirus in the sample.

[0053] 5. Setting of the incident light λ value: To meet the conditions for forming Mie scattered light, just control and set the incident light λ value. Example: If the incident light λ = 900nm, then D+d (theoretical particle size of the new coronavirus particle)>90nm.

[0054] Example 2

[0055] The SARS-CoV-2 S1 protein (molecular weight 11.6 Kd) with different standard concentration gradients was selected for detection using the method in Example 1. The added SARS-CoV-2 load concentrations were 5, 10, 20, 40, 80, 160, 320 and 640 × 10 7 The copy numbers / mL, converted to S1 protein were 1.73, 3.46, 6.92, 13.84, 27.68, 55.36, 110.72 and 221.44 ng / mL respectively.

[0056] The relative light intensity of Mie scattering measured in each reaction well is It indicates that the relative light intensity values ​​at different concentrations are obtained, and the standard straight line equation is fitted: y = 34.25 + 233.19r = 0.9991, the detection range is: 2.70-173ng / mL, and the method detection limit is: 1.35ng / mL (S / N = 5). Figure 4 shown.

[0057] Example 3

[0058] In order to explore its practical application, a blind sample of environmental water added with the new coronavirus was analyzed. The test results are shown in Table 1, and the relative deviation is within 10%.

[0059] Table 1 Blind sample detection of novel coronavirus

[0060]

[0061] The above specific implementations have been described in detail for the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A microfluidic chip, comprising an inlet, a microreaction unit, a microfluidic chip channel, and a waste liquid outlet, wherein the microreaction unit is connected through the microfluidic chip channel; the microreaction unit contains magnetic beads and nucleic acid aptamers.

2. The microfluidic chip according to claim 1, characterized in that: The bottom plate of the micro-reaction unit is hydrophilic cellulose chromatography paper.

3. The microfluidic chip according to claim 2, characterized in that: A photoresist layer is arranged on the side of the bottom plate of the micro-reaction unit.

4. The microfluidic chip according to claim 3, characterized in that: The photoresist layer is a negative photoresist layer; Preferably, the negative photoresist layer is selected from SU-8 negative photoresist layer.

5. The microfluidic chip according to claim 1, characterized in that: The nucleic acid aptamer is a nucleic acid aptamer that specifically recognizes the new coronavirus; preferably, the nucleic acid aptamer includes a sequence as shown in SEQ ID NO.

1.

6. The microfluidic chip according to claim 1, characterized in that: The magnetic beads are carboxylated functional magnetic beads; Preferably, the carboxylated functional magnetic beads include carboxylated polystyrene magnetic beads; Preferably, the particle size of the carboxylated polystyrene magnetic beads is 3 to 8 μm.

7. The method for preparing the microfluidic chip according to any one of claims 1 to 6, comprising the following steps: S1: Inject the magnetic beads through the injection port to fill the microchannel of the microfluidic chip, incubate at 20-30°C for 0.5-1.5h, and use the eluent to elute the uncoupled fixed magnetic beads; S2: Inject the nucleic acid aptamer of the new coronavirus S1 protein through the injection port to fill the microchannel of the microfluidic chip, incubate at 32-40°C for 1-3 hours, and use the eluent to elute the uncoupled nucleic acid aptamer.

8. Use of the microfluidic chip according to any one of claims 1 to 6 in detecting the novel coronavirus for non-diagnostic purposes or in preparing products for detecting the novel coronavirus.

9. A method for detecting a novel coronavirus for non-diagnostic purposes, comprising the step of using the microfluidic chip according to any one of claims 1 to 6 to detect a sample to be tested; Preferably, the method specifically comprises the following steps: The sample to be tested is injected through the injection port to fill the microchannel of the microfluidic chip, incubated at 32-40°C for 1-3 hours, and the change in scattered light intensity before and after incubation is measured using a Mie scattering detection device to calculate the content of the new coronavirus in the sample to be tested.

10. A product, comprising the microfluidic chip described in the first aspect of the present invention; preferably, the product further comprises a Mie scattering detection device.

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