Centrifugal microfluidic-based detection chip and application thereof
By designing a detection chip based on centrifugal microfluidics, combining a flow channel layer and a substrate layer, the automated addition and incubation of samples and reagents is achieved, solving the problem of long processing time in traditional PRNTs and making it suitable for multiplex immunoassays.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTHERN UNIVERSITY OF SCIENCE AND TECHNOLOGY
- Filing Date
- 2022-11-18
- Publication Date
- 2026-06-09
Smart Images
Figure CN116078447B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of microfluidic chip technology, and in particular to detection chips based on centrifugal microfluidics and their applications. Background Technology
[0002] The plaque reduction neutralization test (PRNT) is an experimental method that uses viral plaque technology to determine the neutralizing titer based on the serum volume that reduces the number of plaques by 50%. It has become the gold standard for determining viral neutralization efficacy after vaccination in recent years. Traditional PRNT uses fluorescence-based neutralization assays or spurious virus neutralization assays for detection. However, these methods are time-consuming and require operation in a biosafety level 3 laboratory, which significantly limits the application of PRNT in large-scale serological diagnostics and vaccine evaluation. Microfluidic chip technology provides a universal platform for developing multiplex immunoassays. Compared to commercial ELISA kits, chips require fewer samples and reagents, have lower detection limits, and shorter detection times. Therefore, it is necessary to provide a microfluidic chip that can replace traditional PRNT assays and automate detection. Summary of the Invention
[0003] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a detection chip based on centrifugal microfluidics, which can replace the traditional PRNT experiment and complete automated detection.
[0004] A first aspect of this application provides a detection chip based on centrifugal microfluidics, the detection chip comprising: sequentially arranged in a vertical direction:
[0005] The flow channel layer is equipped with a sample chamber, a reagent chamber, and a reaction flow channel. The sample chamber is used to load the sample solution, and the reagent chamber is used to load the reagent. The sample chamber and the reagent chamber are respectively connected to the reaction flow channel. The sample chamber and the reagent chamber can deliver the sample solution and the reagent to the reaction flow channel at different rotation speeds.
[0006] The base layer has a first surface adjacent to the channel layer, on which a trap is fixed and forms a strip. The strip at least partially overlaps with the reaction channel in the vertical direction to form a detection zone. The trap is used to capture a target in the sample solution.
[0007] The detection chip according to the embodiments of this application has at least the following beneficial effects:
[0008] This detection chip combines centrifugal microfluidics with detection technology. It coats the substrate with the captured material and adds the sample and reagent into the reaction channel by controlling the rotation speed. This completes the steps of adding the sample and reagent and incubating in the detection zone, improving the automation of the detection. At the same time, it uses the detection zone formed by the strip and the reaction channel to detect the sample, which can better replace the traditional PRNT experiment.
[0009] In some embodiments of this application, the strips and reaction channels overlap at least partially in the vertical direction to form detection points, and an array of several detection points is arranged to form a detection area.
[0010] In some embodiments of this application, the strip is a straight strip or an arc strip.
[0011] In some embodiments of this application, a first channel is further provided on the flow channel layer, through which the sample chamber and the reagent chamber are connected to the reaction flow channel, and the first channel has a first hydrophobic inner wall.
[0012] In some embodiments of this application, the flow channel layer is further provided with a second channel and a waste liquid pool. The reaction flow channel is connected to the waste liquid pool through the second channel, and the second channel has a second hydrophobic inner wall. Utilizing the first and second hydrophobic inner walls, the volume of the sample liquid and / or reagent in the reaction flow channel can change with the rotational speed.
[0013] In some embodiments of this application, the first hydrophobic inner wall and the second hydrophobic inner wall are superhydrophobic inner walls.
[0014] In some embodiments of this application, the superhydrophobic inner wall is the inner wall modified with a superhydrophobic coating.
[0015] In some embodiments of this application, the capture substance is selected from at least one of antigen, antibody, hapten, ligand, receptor, protein, polypeptide, and oligonucleotide.
[0016] In some embodiments of this application, the target is selected from at least one of antigen, antibody, hapten, ligand, receptor, protein, polypeptide, polynucleotide, polysaccharide, lipid, lipopolysaccharide, lipoprotein, glycoprotein, steroid, anion, cation, virus, bacteria, and cell.
[0017] In some embodiments of this application, the reagent chamber includes a buffer chamber and a detection liquid chamber, which are respectively connected to a reaction channel. The buffer chamber is used to load a buffer solution, and the detection liquid chamber is used to load a detection liquid. The detection liquid contains a detectable marker that can bind to a target captured in the detection area.
[0018] A second aspect of this application also provides a detection system comprising the aforementioned detection chip.
[0019] In some embodiments of this application, the detection system further includes a detection module for detecting the detection area.
[0020] A third aspect of this application also provides a method for detection using the aforementioned detection chip, the method comprising the following steps:
[0021] Load the sample solution into the sample chamber and the reagents into the reagent chamber;
[0022] Adjust the rotation speed so that the sample solution and reagent chamber flow through the reaction channel to the detection zone, and determine the concentration of the target in the sample solution based on the target captured in the detection zone.
[0023] In some embodiments of this application, adjusting the rotation speed so that the sample solution and reagent chamber flow through the detection zone via the reaction channel, and determining the concentration of the target in the sample solution based on the target captured in the detection zone includes:
[0024] S1: Adjust the rotation speed to allow the sample solution to flow through the reaction channel and into the detection zone for incubation, allowing the traps in the detection zone to capture the target;
[0025] S2: Adjust the rotation speed again to allow the reagent to flow through the reaction channel and through the detection zone, rinsing away any unreacted sample solution;
[0026] S3: Determine the concentration of the target in the sample solution based on the target captured in the detection area.
[0027] In some embodiments of this application, the reagents include a variety of reagents, and after rinsing away unreacted sample solution, incubation is also included to allow the reagents to react with the complex formed by the capture and the target.
[0028] In some embodiments of this application, determining the concentration of the target in the sample solution includes qualitatively determining whether the sample solution contains the target (i.e., whether the concentration is 0) and quantitatively determining the concentration of the target in the sample solution.
[0029] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description
[0030] Figure 1 This is a side view of the substrate layer of a detection chip in one embodiment of this application.
[0031] Figure 2 This is a schematic diagram of the flow channel layer of the detection chip in one embodiment of this application.
[0032] Figure 3 This is a schematic diagram of a detection chip formed by combining a base layer and a flow channel layer in one embodiment of this application.
[0033] Figure 4 This is a schematic diagram of the template layer for preparing the detection chip in one embodiment of this application.
[0034] Figure 5 This is a schematic diagram illustrating the principle of antibody preparation and detection using a sandwich method in one embodiment of this application.
[0035] Figure 6 This is a schematic diagram illustrating the principle of preparation and detection when the detection chip detects neutralizing antibodies using a competitive method in one embodiment of this application.
[0036] Figure 7 These are photographs taken under different states during a simulation experiment of the detection chip in one embodiment of this application.
[0037] Figure 8 This is the result of the detection chip detecting antibody RBD in one embodiment of this application.
[0038] Figure 9 This is a schematic diagram of the detection chip in another embodiment of this application.
[0039] Reference numerals: base layer 100, first surface 101, strip 110, trap 111, flow channel layer 200, center 201, sample chamber 211, reagent chamber 212, first reagent chamber 212a, second reagent chamber 212b, first channel 221, main channel 221a, first branch channel 221b, second branch channel 221c, third branch channel 221d, second channel 222, reaction flow channel 230, waste liquid pool 240, detection area 300, detection point 310. Detailed Implementation
[0040] The following will clearly and completely describe the concept and technical effects of this application in conjunction with embodiments, so as to fully understand the purpose, features and effects of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are all within the scope of protection of this application.
[0041] The embodiments of this application are described in detail below. The described embodiments are exemplary and are only used to explain this application, and should not be construed as limiting this application.
[0042] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number, and "approximately" means within the range of ±20%, 10%, 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, 0.1%, etc. of the stated number. The use of "first" and "second" is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.
[0043] In the description of this application, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.
[0044] refer to Figure 1 The image shows a side view of the substrate layer of a detection chip in some embodiments of this application. The detection chip includes a substrate layer 100 with a first surface 101. Captures 111 are fixed on the first surface 101. Multiple microscopically, the captures 111 aggregate and arrange on the first surface 101 to form macroscopically, stripes 110. In some specific embodiments, the captures 111 are fixed on the first surface 101 by methods including, but not limited to, physical adsorption (e.g., through at least one non-covalent form such as polar interaction, hydrophobic interaction, electrostatic interaction, etc.), covalent coupling (e.g., reaction of amino and carboxyl groups, using coupling agents such as EDC, CMC, ATPES, glutaraldehyde, etc.), thiol coupling, click chemistry, glycosyl fixation (e.g., sodium periodate oxidation, sodium borite linkage), Staudinger reaction, Diels-Alder reaction, natural chemical linkage, affinity interaction (e.g., streptavidin-biotin), etc.
[0045] refer to Figure 2This diagram illustrates a flow channel layer of the detection chip in some embodiments of this application. The detection chip further includes a flow channel layer 200, on which a sample chamber 211, a reagent chamber 212, and a reaction flow channel 230 are provided. The sample chamber 211 is used to load sample solution, and the reagent chamber 212 is used to load reagents. Both the sample chamber 211 and the reagent chamber 212 are connected to the reaction flow channel 230. In some embodiments, the sample chamber 211 and the reagent chamber 212 are at unequal distances from the center 201 of the detection chip. Under the combined action of centrifugal force and capillary force, when a certain rotational speed condition is met, the sample solution flows out of the sample chamber 211 and into the reaction flow channel 230; when another rotational speed condition is met, the reagent flows out of the reagent chamber 212 and into the reaction flow channel 230. Thus, by controlling the rotational speed, the sample solution and reagents can reach the reaction flow channel 230 in a certain order at different rotational speeds, thereby automatically reacting or completing pre-treatment or post-treatment operations.
[0046] refer to Figure 3 This illustration shows a schematic diagram of a complete detection chip formed by sequentially arranging a base layer and a flow channel layer in the vertical direction in some embodiments of this application. When the base layer 100 and the flow channel layer 200 form the detection chip, the base layer 100 is adjacent to the flow channel layer 200 with its first surface 101. The reaction flow channel 230 and the strip 110 overlap at least partially in the vertical direction, forming a detection area 300. In some specific embodiments, the strip 110 and the reaction flow channel 230 overlap to form detection points 310, and there are multiple strips 110 and / or reaction flow channels 230, so that several detection points 310 are arranged in an array to form the entire detection area 300. In some specific embodiments, the array of detection points 310 formed by the strip 110 and the reaction flow channel 230 can be a rectangular array or a ring (fan-shaped) array. When a rectangular array is used, both the strip 110 and the reaction channel 230 are straight lines and perpendicular to each other on a plane perpendicular to the vertical direction. The resulting detection points 310 can be densely arranged to form a smaller detection area, which is more advantageous for the collection of subsequent detection results. However, when a ring (fan-shaped) array is used, the strip 110 can be arc-shaped, and the distribution of the resulting detection points 310 on the channel layer is more dispersed. The advantage is that it can ensure that the strips in the detection area can be parallel units.
[0047] Combination Figure 2 and Figure 3In some specific embodiments, a first channel 221 is also provided on the flow channel layer. The sample chamber 211 and the reagent chamber 212 are respectively connected to the reaction flow channel 230 through the first channel 221. The first channel 221 acts as a "valve" or "pump" to regulate whether the sample liquid or reagent in the sample chamber 211 and reagent chamber 212 enters the reaction flow channel 230. Taking the sample chamber 211 as an example, when the detection chip is stationary or the rotation speed does not meet the requirements, the capillary force at the connection between the first channel 221 and the sample chamber 211 is large while the centrifugal force is insufficient, and the sample liquid is left in the sample chamber 211, and the "valve" is closed. When the rotation speed increases, the valve opens, and the sample liquid is "pumped out" of the sample chamber 211 by the centrifugal force and enters the reaction flow channel 230 through the first channel 221. Specifically, the first channel 221 has a first hydrophobic inner wall, which can regulate the capillary force through its hydrophobic effect, thereby regulating the threshold rotation speed and its accuracy for valve opening and closing. In addition, the valve opening and closing speed threshold is mainly adjusted by the distance between the corresponding chamber and the center of the channel, as well as the size of the channel. The size can be understood at least as the cross-sectional area of the sample liquid or reagent flowing in the channel, determined by the width and height of the channel cross-section.
[0048] In some embodiments, the flow channel layer is further provided with a second channel 222 and a waste liquid pool 240. The reaction flow channel 230 is connected to the waste liquid pool 240 through the second channel 222, which has a second hydrophobic inner wall. Similar to the first channel 221, the second channel 222 acts as a "valve" or "pump" to control the sample liquid or reagent flowing into the reaction flow channel 230 to remain in the detection area for reaction, pretreatment, or post-treatment, or to enter the waste liquid pool 240 after the reaction or treatment is completed. The second hydrophobic inner wall can ensure that a sufficient amount of sample liquid or reagent is retained in the reaction flow channel 230, so that it can incubate and combine with the captured material fixed on the substrate layer. At the same time, through the combination of the first and second hydrophobic inner walls, the volume of sample liquid and / or reagent in the reaction flow channel 230 can be precisely controlled in each step of the reaction, and can be changed accordingly with different rotation speeds. For example, after the sample solution enters the reaction channel 230 and completes incubation, the rotation speed is increased to send the reagent in the reagent chamber 212 into the reaction channel 230. This flushes away the incubated sample solution while leaving a sufficient amount for subsequent incubation and bonding. The first and second hydrophobic inner walls are only used to mark the inner walls of different channels and do not directly constitute a limitation on the hydrophobicity of the inner walls. They can be reasonably set according to the specific reaction principle and conditions.
[0049] The hydrophobicity refers to the affinity or repulsion of the inner walls of the first channel 221 and the second channel 222 for sample solutions and reagents. It can be characterized by two parameters: the contact angle and the sliding angle when the sample solution and reagent come into contact with the inner walls of the first channel 221 and the second channel 222. Hydrophobicity means that the contact angle of the sample solution and reagent on the inner wall is greater than 90 degrees. In some preferred embodiments, the inner walls of the first channel 221 and the second channel 222 are superhydrophobic inner walls, meaning that the contact angle between the sample solution and reagent and the inner wall is greater than 150 degrees while the sliding angle is less than 10 degrees. Superhydrophobic inner walls have excellent performance; the capillary force at the interface between the sample solution and reagent and the inner wall is sufficiently large while the frictional resistance is extremely small, thus allowing for more precise setting of the threshold rotation speed. Furthermore, the superhydrophobic inner walls require contact angles greater than 155 degrees, greater than 160 degrees, and greater than 165 degrees; and sliding angles less than 9 degrees, 8 degrees, 7 degrees, 6 degrees, and 5 degrees. When the sample solution and reagent are aqueous or oil-based solutions / dispersions, the hydrophobic / superhydrophobic inner wall can be correspondingly set as a hydrophobic / superhydrophobic inner wall or an oleophobic / superoleophobic inner wall. The following explanation uses a superhydrophobic inner wall as an example.
[0050] Factors determining the superhydrophobicity of the inner wall include the surface roughness and chemical composition of the material. Therefore, it can be achieved through at least one of the following methods: modifying the inner wall with a low surface energy material or constructing a micro / nano rough structure on the inner wall. Based on the above principles, superhydrophobic inner walls can be obtained through methods such as template methods, etching methods, phase separation methods, chemical vapor deposition, electrospinning, layer-by-layer assembly methods, sol-gel methods, electrochemical deposition methods, and solution immersion methods. The superhydrophobic inner walls obtained by the above methods have high control precision and small error with the theoretically calculated centrifugation speed threshold. Therefore, when multiple samples and / or reagents need to be added to a detection effluent, more stages can be set within a limited centrifugation speed range, and the speed requirement can be lower.
[0051] In some specific embodiments, the superhydrophobic inner wall can be achieved by modifying the inner wall with a superhydrophobic coating. Specifically, the superhydrophobic coating can be applied using a coating agent comprising polymer and micro / nano particles through a processing technique. The processing method can at least involve coating the inner wall with the superhydrophobic agent and then drying it. Further, the diameter D50 of the micro / nano particles is 1 nm to 1 μm, 1 nm to 100 nm, 5 nm to 50 nm, or 5 nm to 20 nm, for example, 1 nm, 2 nm, 3 nm, 5 nm, 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, or 1 μm. Preferably, in some embodiments, the diameter D50 of the micro / nano particles is approximately 15 nm. In some specific embodiments, the micro / nano particles can be at least one of inorganic micro / nano particles, organic micro / nano particles, hybrid micro / nano particles, or metallic micro / nano particles. Inorganic micro / nano particles include at least one of the following: carbon, calcium carbonate, silicon dioxide, titanium dioxide, alumina, zinc oxide, aluminosilicate, aluminum hydroxide, zinc phosphate, aluminum phosphate, zinc sulfate, and barium sulfate. Organic micro / nano particles include at least one of the following: polyethylene, polyvinyl chloride, polystyrene, polypropylene, and polycarbonate. Hybrid micro / nano particles include at least one of the following: MOF (such as ZIF, UiO, MIL), COF, and PCN. Metallic micro / nano particles include at least one of the following: iron, copper, and zinc. In some specific embodiments, the polymer is a low surface energy polymer, and the superhydrophobic properties of the inner wall are achieved by the combined action of the low surface energy polymer and the micro / nano particles. Low surface energy polymers include, but are not limited to, low surface energy organosilicon resins (such as polysiloxanes), fluorocarbon resins, fluorosilicone resins, and epoxy resins, among which polydimethylsiloxane (PDMS) is the most commonly used. Low surface energy refers to a surface energy below 100 mN / m, further subdivided into below 50 mN / m, 25 mN / m, 22 mN / m, and 20 mN / m. Due to the poor compatibility between micro / nano particles and polymers, to avoid agglomeration affecting stability, hydrophobic modification of the micro / nano particles can be performed to further improve the interfacial properties between them and the polymer. Specific hydrophobic modification methods include, but are not limited to, the use of coupling agents, such as silane coupling agents: 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PFDTES), octadecyltrichlorosilane (OTS), aminopropyltriethoxysilane (APTES), fluorinated poly(hexafluoromethacrylate-glycidyl methacrylate) polymer (PFG), hexadecyltrimethoxysilane (HDTMS), tetraethoxysilane (TEOS), ethoxytrimethylsilane (TMES), KH-550, KH-560, KH-570, etc.The mass-to-volume ratio of micro / nano particles to coupling agent varies depending on the specific type. In some specific embodiments, the mass-to-volume ratio is 1g micro / nano particles: 0.1-1ml coupling agent, further it can be 1g micro / nano particles: 0.3-0.6ml coupling agent, even further it is 1g silica micro / nano particles: 0.3-0.6ml coupling agent, and even further it is 1g silica micro / nano particles: 0.3-0.6ml PFDTES. In some specific embodiments, the mass ratio of micro / nano particles to polymer is 1:(0.1-10), further it is 1:(0.5-5), 1:(0.8-3), 1:(1-2), 2:3. Furthermore, it is understandable that hydrophobicity and oleophobicity, as well as superhydrophobicity and superoleophobicity, are not completely opposite. One can choose a double-hydrophobic or superdouble-hydrophobic inner wall, that is, a liquid-repellent or superliquid-repellent coating that is resistant to both aqueous and oily sample solutions. For example, at least a superdouble-hydrophobic coating can be prepared using a coating reagent containing an aqueous solution of aluminum phosphate and silica micro / nanoparticles modified with fluorosilane.
[0052] In the detection chip of this application, the target to be detected can be any optional biomolecule, small molecule, ion, organism, etc., such as at least one of antigens, antibodies or fragments thereof, haptens, ligands, receptors, proteins, peptides, polynucleotides, polysaccharides, lipids, lipopolysaccharides, lipoproteins, glycoproteins, steroids, anions, cations, viruses, bacteria, cells, etc. Depending on the target, the trap used to capture the target can be at least one of antigens, antibodies or fragments thereof, haptens, ligands, receptors, proteins, peptides, polynucleotides, etc. Furthermore, a suitable method can be selected to immobilize the trap onto the substrate layer.
[0053] In some specific embodiments, after the traps immobilized on the substrate layer capture the target to form a complex, a detectable label needs to be bound to the complex to qualitatively or quantitatively analyze the amount of the target in the sample solution. Detectable labels include, but are not limited to, at least one of colored substances, fluorescent substances, luminescent substances (e.g., chemiluminescent substances, bioluminescent substances), radioactive substances, and enzymes. In some embodiments, a detectable label is modified onto a analyte capable of specifically binding to the target to achieve the binding of the detectable label to the target. In some specific embodiments, the binding of the analyte to the target can be achieved on a detection chip. For example, multiple reagent chambers are provided, at least one of which is used to load a solution containing the analyte. After the sample solution reacts with the traps, the solution containing the analyte is fed into the detection zone for reaction by adjusting the rotation speed.
[0054] In some specific embodiments, the detectable label does not directly have a detection effect; instead, it needs to exhibit color after a colorimetric reaction to be detectable. For example, horseradish peroxidase typically requires a colorimetric reaction with a chromogenic substrate before detection. Therefore, the reaction between the detectable label and the chromogenic substrate can also be implemented on the detection chip. For instance, multiple reagent chambers are provided, at least one of which is used to hold a solution containing the chromogenic substrate. After the reaction between the detectable label and the complex is completed, the solution containing the chromogenic substrate is introduced into the detection area by adjusting the rotation speed for the colorimetric reaction. It is understandable that due to limitations such as the size of the detection chip and the rotation speed thresholds for different samples and reagents, in some embodiments, the detection chip cannot have too many rotation speed levels. In this case, the colorimetric reaction can also be initiated by introducing the substrate solution from the outside after the reaction between the detectable label and the complex is complete.
[0055] In some specific implementations, according to the processing sequence of pretreatment, reaction, posttreatment, and cleaning, the threshold required for the corresponding sample or reagent to flow from the first channel to the reaction channel gradually increases.
[0056] refer to Figure 2 and 3 In some embodiments, reagent chamber 212 includes a first reagent chamber 212a and a second reagent chamber 212b. The first reagent chamber 212a is used to load reagents containing detectable labels, and the second reagent chamber 212b is used to load buffer solution to wash away unbound detectable labels and other reactants in the detection zone. Therefore, the rotation speed thresholds of sample chamber 211, first reagent chamber 212a, and second reagent chamber 212b gradually increase, causing the sample, reagents containing detectable labels, and buffer solution to be sequentially fed into the detection zone for reaction or washing as the rotation speed gradually increases.
[0057] In some specific embodiments, the materials of the substrate layer and the flow channel layer can be independently selected from at least one of polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), polycarbonate (PC), polystyrene (PS), epoxy resin, glass, silicon wafer, etc., preferably inert, non-toxic, and optically sound PDMS material. For the flow channel layer, corresponding sample chambers, reagent chambers, and reaction channels can be formed on the above materials using at least one of photolithography, soft photolithography, etching, etc. In some specific embodiments, the flow channel layer and the substrate layer can be bonded together to form a detection chip using at least one of the following bonding processes: thermoforming, laser bonding, adhesive bonding, solvent bonding, surface modification bonding, plasma-assisted bonding, etc. For materials such as PDMS, plasma bombardment can be used to open silicon-oxygen bonds to form hydroxyl groups, and a condensation reaction can occur at the interface to complete the bonding. The solvent used for solvent bonding is usually an organic solvent, including specific solvents such as acetone, ethanol, methanol, isopropanol, acetonitrile, etc. The adhesive used for bonding can be a liquid adhesive, a solid film adhesive, or a specific functional adhesive, including photosensitive or heat-sensitive liquid adhesives. Specific types of adhesives include, but are not limited to, at least one of epoxy resin, polyurethane, polystyrene, polyacrylate, and ethylene-vinyl acetate copolymer. It is understood that, to avoid affecting the fixation of the traps on the substrate, other non-bonding methods can also be used to bond the two together.
[0058] This application also provides a detection system including the aforementioned detection chip. In some specific embodiments, the detection system further includes a detection module capable of detecting the reaction results of the detection area on the detection chip. In some specific embodiments, the detection module can also output the detection results.
[0059] The above implementation methods will be described below with reference to specific examples.
[0060] Example 1
[0061] This embodiment provides a detection chip that uses antigen proteins as traps to detect antibodies. The preparation process is as follows:
[0062] 1. Preparation of superhydrophobic reagents
[0063] (1) Add 1g of silica nanoparticles (D50 of about 15nm) to a 50mL centrifuge tube containing 40mL of toluene, and sonicate for 30 minutes with an ultrasonic instrument at a power of 300W.
[0064] (2) After ultrasonic treatment, 0.6 mL of 1H,1H,2H,2H-perfluorodecyltriethoxysilane (PFDTES) was added and magnetically stirred at room temperature for 48 hours. The silane-modified silica nanoparticles were washed with anhydrous ethanol and dried.
[0065] (3) The silane-modified silica nanoparticles obtained in step (2) are mixed with polydimethylsiloxane (PDMS) at a mass ratio of 2:3 and added to 10 mL of ethyl acetate. The mixture is ultrasonically treated with an ultrasonic instrument at a power of 300 W for 30 minutes and magnetically stirred at room temperature for 1 hour to obtain a superhydrophobic reagent.
[0066] 2. Fabrication of the detection chip
[0067] 2.1 Preparation of antigen protein bands
[0068] (1) The first surface of the PDMS substrate layer was treated with oxygen plasma for 1 min, and then combined with the PDMS template layer. The template layer has the following characteristics: Figure 4 The pattern shown has a groove running vertically through it.
[0069] (2) Prepare a 10% aminopropyltriethoxysilane (APTES)-ethanol solution, add it to the groove, let it stand at room temperature for 30 minutes, wash with anhydrous ethanol to remove excess silane reagent, and put it in an 80℃ oven to dry for 2 hours.
[0070] (3) After the chip cools down, an antigen protein solution is added to the groove. After standing at room temperature for half an hour, the antigen protein molecules are fixed to the first side of the PDMS substrate, thereby forming parallel antigen protein bands on the first side.
[0071] 2.2 Assembly of the detection chip
[0072] Remove the template layer from the PDMS base layer and bond it to the flow channel layer.
[0073] Flow channel layer such as Figure 2As shown, it has a virtual center 201 for representing the relative positions of other structures, and is provided with at least 5 sets of independent sample chambers 211, reagent chambers 212 and reaction channels 230, wherein the reagent chambers 212 include a first reagent chamber 212a and a second reagent chamber 212b. Sample chamber 211, first reagent chamber 212a, and second reagent chamber 212b are connected to reaction channel 230 via a first channel 221. The first channel 221 has a circumferential main channel 221a. For a portion of sample chamber 211, first reagent chamber 212a, and second reagent chamber 212b, the first channel 221 also has radially branched channels 221b, second branched channels 221c, and third branched channels 221d. Sample chamber 211, first reagent chamber 212a, and second reagent chamber 212b are connected to reaction channel 230 via the first branched channels 221b, second branched channels 221c, and third branched channels 221d (if present) that converge into the main channel 221a. Sample chamber 211 is used to load sample solution, first reagent chamber 212a is used to load HRP-labeled secondary antibody solution, and second reagent chamber 212b is used to load PBST buffer. The sizes of the first branch channels 221b, second branch channels 221c, and third branch channels 221d corresponding to the same set of sample chambers 211, first reagent chamber 212a, and second reagent chamber 212b gradually decrease, while the size of the main channel 221a gradually decreases or remains constant along the direction of solution flow. The sample chambers 211, first reagent chamber 212a, and second reagent chamber 212b control their threshold rotation speeds to 300 rpm, 450 rpm, and 750 rpm respectively by controlling the sizes of the first branch channels 221b, second branch channels 221c, third branch channels 221d, or main channel 221a connected to them, as well as their distances from the center 201. Once the corresponding threshold rotation speed is exceeded, the solution loaded inside flows out. To avoid the bonding process affecting the antigen protein fixed on the base layer, the flow channel layer and the base layer are directly bonded together by adhesion. The reaction flow channel 230 and the antigen protein strip 110 are arranged perpendicularly to each other, interlacing to form detection points 310. Since there are multiple reaction flow channels 230 and strips 110, these multiple detection points 310 are arranged in an array to form a detection area 300. A waste liquid tank 240 is also provided downstream of the reaction flow channel 230 along the solution flow direction. The reaction flow channel 230 and the waste liquid tank 240 are connected by a second channel 222. The size of the second channel 222 is smaller than that of the first channel 221.
[0074] The prepared superhydrophobic reagent is added to the first channel 221 and the second channel 222. After drying, a superhydrophobic coating is formed on the inner walls of the first channel 221 and the second channel 222, giving them superhydrophobic inner walls. The first channel 221, modified with a superhydrophobic coating, acts as a valve to control the sequential flow of sample liquid and reagent in the sample chamber 211 and reagent chamber 212. The second channel 222, also modified with a superhydrophobic coating, acts as a secondary valve to precisely control the reagent flow, flushing the sample liquid away from the detection area and ensuring sufficient reagent remains in the detection area for subsequent incubation.
[0075] The detection chip uses a sandwich method to detect antibodies against RBD. The detection principle is as follows: Figure 5 As shown, a) is a schematic diagram of the preparation and detection process, and b) is the reaction process on the substrate layer. From left to right, firstly, the receptor-binding domain (RBD) of the antigen viral spike protein is immobilized on the substrate layer using a silane coupling agent (APTES). Then, after the substrate layer and the flow channel layer are bonded together, the non-specific binding sites of the RBD are blocked beforehand with bovine serum albumin (BSA). When the sample solution containing antibodies in the sample chamber is fed into the detection zone under controlled rotation speed, the antibodies specifically bind to the RBD and are captured. Next, the HRP-secondary antibody solution in the first reagent chamber is fed into the detection zone. Some of the HRP-secondary antibody binds to the antibody bound to the RBD, while some free HRP-secondary antibody remains. The buffer solution is then pumped in by adjusting the rotation speed to wash away the free HRP-secondary antibody. Then, chemiluminescent substrate solution can be added to the detection point array in the detection zone to detect the chemiluminescent signal. Simultaneously, the corresponding antibody concentration is further calculated based on the obtained chemiluminescent signal intensity. At this point, the higher the antibody concentration, the more secondary antibody binds to it using the sandwich method, and the higher the signal intensity.
[0076] refer to Figure 6 If a competitive method is used to detect neutralizing antibodies using this detection chip, the principle is as follows: Figure 6The diagram illustrates the preparation and detection process, as well as the reaction process on the substrate layer. From left to right, the receptor-binding domain (RBD) of the antigen viral spike protein is first immobilized on the substrate layer using a silane coupling agent (APTES). After the substrate layer and the flow channel layer are bonded together, the non-specific binding sites of the RBD are pre-blocked with bovine serum albumin (BSA). When the sample solution containing neutralizing antibodies is fed into the detection zone under controlled rotation speed, the neutralizing antibodies specifically bind to and are captured by the RBD. Then, the HRP-ACE2 solution from the first reagent chamber is fed into the detection zone. HRP-ACE2 competitively binds to the RBD with the neutralizing antibodies. Buffer solution is then pumped in by adjusting the rotation speed to wash away free HRP-ACE2. Chemiluminescent substrate solution is then added to the detection point array in the detection zone to detect the chemiluminescent signal. Simultaneously, the concentration of the corresponding neutralizing antibody is calculated based on the obtained chemiluminescent signal intensity. At this point, the higher the concentration of neutralizing antibody, the fewer HRP-ACE2 molecules can bind to the RBD, and the lower the signal intensity.
[0077] Example 2
[0078] A simulation experiment was conducted using the centrifugal microfluidic chip described in Example 1, with reference to... Figure 7 The chip is pre-loaded with HRP-labeled secondary antibody (red dye) and PBST buffer (green dye). The sample (blue dye) is added to the sample chamber. The chip is then rotated for 10 seconds at 300 rpm using a custom rotor. The sample breaks through the first channel of the microvalve and enters the detection array formed by the reaction channels and RBD bands in the detection area. After 30 minutes of incubation, the rotation speed is increased to 450 rpm. The HRP-labeled secondary antibody (red dye) breaks through the first channel of the microvalve and enters the detection array formed by the reaction channels and RBD bands in the detection area. After another 30 minutes of incubation, the rotation speed is increased to 750 rpm. The PBST buffer (green dye) breaks through the first channel of the microvalve and enters the detection array formed by the reaction channels and RBD bands in the detection area, washing away excess HRP-labeled secondary antibody.
[0079] Example 3
[0080] Standard gradient solutions of antibodies were prepared with concentration gradients of 1.56, 6.25, 25, and 100 ng / mL. After completing chip assembly and detection according to the steps in Examples 1 and 2, chemiluminescent substrate solution was added to the detection area and the detection results were captured using a camera. A standard curve was plotted based on the detected chemiluminescent signal intensity values.
[0081] The results are as follows Figure 8 As shown in the figure, a represents the photographic result, and b represents the standard curve. The figure demonstrates that this detection chip can accurately and conveniently detect samples.
[0082] Example 4
[0083] This embodiment provides a detection chip, for reference... Figure 9 It has a center 201 and is provided with at least 5 independent sample chambers 211, reagent chambers 212 and reaction channels 230. The reagent chambers 212 include a first reagent chamber 212a and a second reagent chamber 212b. The sample chambers 211, the first reagent chamber 212a and the second reagent chamber 212b are connected to the reaction channels 230 through a first channel 221. The first channel 221 has a circumferential main channel 221a. For the sample chamber 211, the first reagent chamber 212a, and the second reagent chamber 212b, the first channel 221 also has a radially extending first branch channel 221b, a second branch channel 221c, and a third branch channel 221d. The second branch channels 221c and 221d converge into the main channel 221a, connecting the first reagent chamber 212a and the second reagent chamber 212b to the first channel 221. The main channel 221a is connected to the first branch channel 221b, and the first branch channel 221b is connected to the reaction channel 230. The sample chamber 211 is used to load the sample solution, the first reagent chamber 212a is used to load the HRP-labeled secondary antibody solution, and the second reagent chamber 212b is used to load the PBST buffer. The sample chamber 211, the first reagent chamber 212a, and the second reagent chamber 212b control their threshold rotation speeds to 300 rpm, 450 rpm, and 750 rpm respectively by controlling the size of the first branch channel 221b, the second branch channel 221c, the third branch channel 221d, or the main channel 221a connected to them, as well as their distance from the center 201. Once the corresponding threshold rotation speed is exceeded, the solution loaded inside flows out. A waste liquid tank 240 is also provided downstream of the reaction channel 230 along the solution flow direction, and the reaction channel 230 and the waste liquid tank 240 are connected through a second channel 222. The first channel 221 and the second channel 222 have superhydrophobic inner walls.
[0084] The difference from Example 1 is that the antigen RBD strip on the basal layer is a circumferential arc strip with the center 201 as the center. After the flow channel layer and the basal layer are bonded together, the radial reaction flow channel 230 and the circumferential strip 110 are perpendicular to each other and intersect to form detection points 310. Multiple detection points 310 are arranged to form a fan-shaped detection area.
[0085] Example 5
[0086] This embodiment provides a detection chip, which differs from Embodiment 1 in that the reagent chamber further includes a third reagent chamber. The third reagent chamber is used to load the substrate solution for the colorimetric reaction. The threshold rotation speed of the third reagent chamber is 900 rpm. After the threshold is exceeded, the substrate solution is pumped out from the third reagent chamber to the detection area.
[0087] Example 6
[0088] This embodiment provides a detection chip, which differs from Embodiment 1 in that multiple capture bands are formed by different antigen RBDs fixed on the base layer.
[0089] In summary, the centrifugal microfluidic-based detection chip provided in this application utilizes microchannels modified with a superhydrophobic coating as microvalves to control liquid flow, combined with a cross-array structural design. Before detection, antigen proteins are pre-fixed at corresponding positions on the substrate to bind and capture antibody biomarkers in the sample. Then, the microchannels and detection area are superhydrophobically modified and blocked with BSA. HRP-labeled secondary antibody and PBST buffer are pre-loaded into the chamber for sample detection. During detection, only the sample needs to be added. By controlling the centrifugation speed, the sample, HRP-labeled secondary antibody, and PBST buffer are sequentially controlled to pass through the channels in the detection area for incubation and washing. Based on the principle of antigen-antibody immune binding, the biomarkers to be detected in the sample are adsorbed and captured. Finally, a chemiluminescent reaction substrate solution is added to the detection area, releasing a chemiluminescent signal under the catalysis of catalase. The concentration levels of different biomarkers in the sample can be calculated based on the intensity of the chemiluminescent signal in the detection area. Therefore, this scheme can achieve automated simultaneous detection and analysis of multiple biomarkers in multiple samples.
[0090] The present application has been described in detail above with reference to the embodiments. However, the present application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present application. Furthermore, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.
Claims
1. A detection chip based on centrifugal microfluidics, characterized in that, Including those arranged sequentially in the vertical direction: The flow channel layer includes a sample chamber, a reagent chamber, and a reaction channel. The sample chamber is used to load sample solution, and the reagent chamber is used to load reagents. Both the sample chamber and the reagent chamber are connected to the reaction channel, and can respectively deliver the sample solution and the reagents to the reaction channel at different rotation speeds. The flow channel layer also includes a first channel through which the sample chamber and the reagent chamber are connected to the reaction channel. The first channel has a first hydrophobic inner wall. The flow channel layer further includes a second channel and a waste liquid tank, which is located downstream of the reaction channel along the solution flow direction. The reaction channel is connected to the waste liquid pool through the second channel, which has a second hydrophobic inner wall; the first and second hydrophobic inner walls are superhydrophobic inner walls, with a contact angle greater than 150 degrees and a sliding angle less than 10 degrees, and the superhydrophobic inner wall is an inner wall modified with a superhydrophobic coating; the second hydrophobic inner wall can ensure that a sufficient amount of sample liquid or reagent is retained in the reaction channel. Through the combination of the first and second hydrophobic inner walls, the volume of sample liquid and / or reagent in the reaction channel can be precisely controlled in each step of the reaction, so that the volume can change accordingly with different rotation speeds; A base layer having a first surface adjacent to the channel layer, on which a trap is fixed and forms a strip, the strip at least partially overlapping the reaction channel in the vertical direction to form a detection zone, the trap being used to capture a target in the sample solution.
2. The detection chip according to claim 1, characterized in that, The strip and the reaction channel at least partially overlap in the vertical direction to form a detection point, and a plurality of the detection points are arranged in an array to form the detection area.
3. The detection chip according to claim 2, characterized in that, The strip is a straight strip or an arc strip.
4. The detection chip according to any one of claims 1 to 3, characterized in that, The captured substance is selected from at least one of antigen, antibody, hapten, ligand, receptor, protein, polypeptide, and oligonucleotide; or The target is selected from at least one of antigen, antibody, hapten, ligand, receptor, protein, polypeptide, polynucleotide, polysaccharide, lipid, lipopolysaccharide, steroid, anion, cation, virus, and cell.
5. The detection chip according to any one of claims 1 to 3, characterized in that, The reagent chamber includes a buffer chamber and a detection liquid chamber, which are respectively connected to the reaction channel. The buffer chamber is used to load the buffer solution, and the detection liquid chamber is used to load the detection liquid. The detection liquid contains a detectable marker that can bind to a target captured in the detection area.
6. A detection system, characterized in that, Includes the detection chip as described in any one of claims 1 to 5.
7. The detection system according to claim 6, characterized in that, The detection system also includes a detection module, which is used to detect the detection area.
8. A method for detection using the detection chip according to any one of claims 1 to 5, characterized in that, Includes the following steps: Load the sample solution into the sample chamber and the reagents into the reagent chamber; Adjust the rotation speed so that the sample solution and reagent flow through the reaction channel into the detection zone, and determine the concentration of the target in the sample solution based on the target captured in the detection zone.