A centrifugal microfluidic chip, a preparation method and a method for detecting pathogen

Abstract

The application discloses a centrifugal microfluidic chip, a preparation method and a pathogen detection method, and discloses a centrifugal microfluidic chip, which comprises a chip body, seven chambers are arranged on the chip body, a first chamber is used for placing a sample liquid to be detected, a second chamber is used for placing a first biological recognition element modified magnetic bead and a second biological recognition element modified probe, a third chamber is provided with a magnet on a wall surface for magnetically adsorbing the magnetic bead, a fourth chamber is used for placing a cleaning liquid, a fifth chamber is used for placing a probe substrate solution, a sixth chamber is used for collecting the probe substrate solution after reaction with the probe, and a seventh chamber is used for collecting waste liquid; a micropore for sample adding and / or air inlet and / or air outlet is arranged on the top surface of the chip body and corresponds to each chamber, and the micropore corresponding to the fifth chamber is sealed after sample adding. The application further discloses a method for preparing the centrifugal microfluidic chip. The application further discloses a pathogen detection method, which adopts the centrifugal microfluidic chip.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis technology, and specifically relates to a centrifugal microfluidic chip, its preparation method, and a method for pathogen detection. Background Technology

[0002] Pathogens are microorganisms that can cause diseases in animals, plants, or humans, including viruses, bacteria, chlamydia, rickettsia, mycoplasma, spirochetes, and fungi. Pathogens are widespread in nature, with over 400 species capable of infecting humans. Therefore, early screening for pathogens is essential for preventing disease outbreaks. While many effective methods exist for detecting pathogens, such as plate culture, microscopic observation, nucleic acid amplification, and immunoassay, traditional culture methods typically require 2-4 days to complete the test and necessitate specialized biosafety level 2 laboratories, making them unsuitable for handling emergencies and routine monitoring at the grassroots level. While molecular biology methods, such as polymerase chain reaction (PCR), and immunological methods, such as enzyme-linked immunosorbent assay (ELISA), can complete the test in a shorter time (2-4 hours), they require expensive equipment and specialized personnel. Therefore, there is an urgent market need for simple and low-cost pathogen detection equipment.

[0003] Microfluidic chips possess significant small size characteristics. Due to the high surface area-to-volume ratio, strong surface tension, pronounced laminar flow, and strong capillary effect of their internal fluids, microfluidic chips demonstrate immense potential for microbial isolation, detection, and analysis. Furthermore, microfluidic chips offer advantages such as low reagent consumption, high throughput, portability, and facilitating functional module design. This allows for reduced detection times to minutes and reagent volumes to nanoliters or microliters. Microfluidic technology can reduce sample pretreatment steps and requires less operator skill. Integrating sample pretreatment and pathogen analysis steps into microfluidic chips contributes to achieving efficient, high-speed, and automated detection.

[0004] Existing microfluidic chips for pathogen detection include a device for virus screening based on a bowl-shaped centrifugal microfluidic chip, as disclosed in Chinese Patent Publication No. CN115058324A. This device comprises: a clamping plate fitted between a base and a cover plate; a conical chip layer matching the clamping plate and configured as a hollow cone with a microporous wall structure; a bowl-shaped chip formed by the conical chip layer and the clamping plate in a three-dimensional intersecting arrangement; and a motor shaft inserted at the center of the clamping plate for centrifugal mixing of the bowl-shaped chip. The system includes a flat-bottomed circular base with a recessed opening at the top. Inside the base are a central cylinder for inserting a through-hole into the motor, and a positioning pin hole at the edge of the through-hole. The upper circumferential edge of the base has a base groove with a locking mechanism, and the bottom of the base has a concave bottom. Both the positioning pin hole and the base groove are used to position and engage the cover of the clamping plate. The system also includes a liquid inlet channel for the clamping plate, formed by an inlet port, a circular reagent kit, an arc-shaped reaction tank, a circular tube groove, and a grooved liquid guide tube.

[0005] However, the centrifugal microfluidic chip disclosed in the above patent cannot achieve multi-step sequential release of fluid in the microfluidic chip. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention discloses a centrifugal microfluidic chip, its preparation method, and a method for pathogen detection.

[0007] A centrifugal microfluidic chip includes a chip body, wherein the chip body has 7 chambers, namely:

[0008] The first chamber is used to hold the sample solution to be tested.

[0009] The second chamber is used to place magnetic beads modified with the first biometric element and probes modified with the second biometric element. Both the first and second biometric elements are used to bind to the detection target in the sample solution to be tested. Both the magnetic beads modified with the first biometric element and the probes modified with the second biometric element are lyophilized powders. The second chamber is connected to the first chamber by a first siphon channel that allows the liquid in the first chamber to enter the second chamber at a first centrifugation speed.

[0010] The third chamber has magnets on its walls for magnetically attracting the magnetic beads, and the third chamber is connected to the second chamber by a second siphon channel that allows liquid in the second chamber to enter the third chamber at a second centrifugal speed.

[0011] The fourth chamber is used to hold the cleaning solution. A third siphon channel connects the fourth chamber to the third chamber, allowing liquid from the fourth chamber to enter the third chamber at a third centrifugal speed. The third siphon channel has a first vent hole that, at a speed greater than the third centrifugal speed, interrupts the liquid flow within the third siphon channel due to atmospheric pressure.

[0012] The fifth chamber is used to hold the probe substrate solution. A fourth channel connects the fifth and fourth chambers, allowing communication between them after the liquid in the fourth chamber is drained. A fifth siphon channel connects the fifth and third chambers, allowing liquid from the fifth chamber to enter the third chamber at a fourth centrifugal speed. The fifth siphon channel has a second vent that, at centrifugal speeds greater than the fourth, blocks the liquid flow within the siphon channel due to atmospheric pressure.

[0013] The sixth chamber is used to collect the probe substrate solution after the probe has reacted with it.

[0014] The seventh chamber is used to collect waste liquid. A capillary tube is connected to the third chamber, and one end of the capillary tube is connected to a butterfly-shaped channel with two outlet directions. The two outlet directions of the butterfly-shaped channel are respectively connected to the sixth chamber and the seventh chamber. The capillary tube and the butterfly-shaped channel are used to allow the liquid in the third chamber to enter the seventh chamber at the fifth centrifugal speed, or to allow the liquid in the third chamber to enter the sixth chamber at the sixth centrifugal speed. The direction of centrifugal rotation is controlled to distinguish whether the liquid enters the sixth chamber or the seventh chamber.

[0015] The top surface of the chip body is provided with micropores for sample addition and / or air intake and / or exhaust for each chamber. The micropores corresponding to the fifth chamber are sealed after sample addition.

[0016] The first, fifth, and sixth centrifugation speeds are greater than the second centrifugation speed, and the second centrifugation speed is greater than the third and fourth centrifugation speeds.

[0017] Specifically, the wall in the third chamber where the magnet is located can be the top wall and / or the side wall and / or the bottom wall;

[0018] The siphon channel connects various chambers, and liquid only transfers between specific chambers after reaching a certain centrifugal speed. Furthermore, when the centrifugal microfluidic chip's rotation speed does not reach the third centrifugal speed, the cleaning fluid in the fourth chamber cannot enter the third chamber through the third siphon channel. Because the third siphon channel has a first vent, when the centrifugal microfluidic chip's rotation speed exceeds the third centrifugal speed, the liquid flow in the third siphon channel is blocked by atmospheric pressure. In other words, only when the centrifugal microfluidic chip's rotation speed is at the third centrifugal speed can the cleaning fluid in the fourth chamber flow smoothly into the third chamber. This structure allows for the timing of the cleaning fluid entering the third chamber. The machine's precise control allows the cleaning solution to enter the third chamber in batches as needed. The fifth and fourth chambers are connected by a fourth channel, and the micropores in the fifth chamber are sealed after sample addition. When the cleaning solution in the fourth chamber still seals one end of the fourth channel, the probe substrate solution in the fifth chamber cannot enter the third chamber regardless of the speed at which the centrifugal microfluidic chip rotates. Only when the cleaning solution in the fourth chamber can no longer seal one end of the fourth channel, and the centrifugal microfluidic chip rotates at the fourth centrifugal speed, can the probe substrate solution in the fifth chamber enter the third chamber. This structural design ensures that the probe substrate solution enters the third chamber only after the cleaning solution has completed the cleaning process.

[0019] Preferably, the chip body is disc-shaped and has a mounting hole in the center for mounting onto the shaft of the centrifugal motor in a centrifuge during use.

[0020] Preferably, the micropores are all located on the side of the corresponding chamber near the mounting hole.

[0021] Preferably, the first chamber is closer to the mounting hole than the second chamber, the second chamber is closer to the mounting hole than the third chamber, the fourth chamber is closer to the mounting hole than the fifth chamber, the fifth chamber is closer to the mounting hole than the third chamber, and the third chamber is closer to the mounting hole than the sixth and seventh chambers.

[0022] Preferably, the sixth and seventh chambers are located at the same radial position and arranged circumferentially, and the two outlet directions of the butterfly-shaped channel correspond to the adjacent ends of the sixth and seventh chambers, respectively.

[0023] Preferably, each micropore is located in the corresponding chamber on the side near the mounting hole; the first vent is located in the third siphon channel near the mounting hole, and the second vent is located in the fifth siphon channel near the mounting hole.

[0024] Preferably, the first siphon channel, the second siphon channel, the third siphon channel, and the fifth siphon channel are hydrophilic; the fourth channel and the capillary are hydrophobic.

[0025] Specifically, the first, second, third, and fifth siphon channels are hydrophilic modified by hydrophilic reagent treatment, plasma surface treatment, or protein blocking agent treatment to form hydrophilic channels; the fourth channel and capillary are hydrophobic modified by hydrophobic reagent to form hydrophobic channels.

[0026] In addition, the cross-sections of the first siphon channel, the second siphon channel, the third siphon channel, the fifth siphon channel, and the fourth channel are any one of square, rectangle, semicircle, and circle.

[0027] A method for fabricating a centrifugal microfluidic chip includes the following steps:

[0028] (1) Prepare a mold for casting to form the centrifugal microfluidic chip;

[0029] (2) A portion of the raw materials used for casting to form the centrifugal microfluidic chip is poured into the mold for the first step of casting and solidification;

[0030] (3) Place the magnet at the position corresponding to the third chamber;

[0031] (4) Pour the remaining raw materials used to cast the centrifugal microfluidic chip into the mold for the second casting step and solidify, so that the magnet is cast into the top wall of the third chamber, and obtain a centrifugal microfluidic chip with an open bottom surface and no bottom surface in each chamber.

[0032] (5) Take out the product obtained in step (4) and punch holes to form the first vent hole, the second vent hole and each micropore;

[0033] (6) The product obtained in step (5) is mounted on the bottom surface to obtain the centrifugal microfluidic chip.

[0034] Preferably, the bottom surface is made of glass. The product obtained in step (5) and the bottom surface made of glass are plasma modified together and then assembled to form the centrifugal microfluidic chip. The raw materials used to make the centrifugal microfluidic chip can also be any one or a combination of several of the following: organosilicon, glass, quartz, polycarbonate, polymethyl methacrylate, polydimethylsiloxane, and polyethylene terephthalate.

[0035] Preferably, after step (6) is completed, the first siphon channel, the second siphon channel, the third siphon channel and the fifth siphon channel are hydrophilic modified; the fourth channel and the capillary are hydrophobic modified.

[0036] A method for pathogen detection, using the aforementioned centrifugal microfluidic chip, includes the following steps:

[0037] (1) Add the sample solution to be tested to the first chamber, add the magnetic beads modified by the first biometric element and the probe modified by the second biometric element to the second chamber, add the cleaning solution to the fourth chamber, add the probe substrate solution to the fifth chamber, and seal the micropores corresponding to the fifth chamber.

[0038] (2) Centrifuge at the first centrifugation speed so that the supernatant of the sample solution to be tested in the first chamber enters the second chamber through the first siphon channel and mixes and reacts with the magnetic beads modified by the first biometric element and the probe modified by the second biometric element.

[0039] (3) Centrifuge at the second centrifugation speed, so that the reaction liquid after the mixture in the second chamber enters the third chamber through the second siphon channel, and the detection target and probe are directly or indirectly combined with the first biometric element and adsorbed by the magnet through the magnetic beads;

[0040] (4) Centrifuge at the fifth centrifugal speed and control the centrifugal rotation direction so that the unadsorbed liquid in step (3) enters the seventh chamber through the capillary and butterfly-shaped channel;

[0041] (5) Centrifuge at the third centrifugal speed to allow the cleaning solution in the fourth chamber to enter the third chamber through the third siphon channel to clean the substance adsorbed by the magnet;

[0042] (6) Centrifuge at the fifth centrifugal speed and control the centrifugal rotation direction so that the cleaning solution in the third chamber in step (5) enters the seventh chamber through the capillary and butterfly-shaped channel;

[0043] (7) Centrifuge at the fourth centrifugation speed to allow the probe substrate solution in the fifth chamber to enter the third chamber through the fifth siphon channel for the probe substrate to react with the probe.

[0044] (8) Centrifuge at the sixth centrifugation speed and control the centrifugation rotation direction so that the reacted probe substrate solution enters the sixth chamber through the capillary and butterfly-shaped channel;

[0045] (9) Measure the probe substrate solution after the reaction in the sixth chamber to determine the status of the detection target in the sample solution to be tested.

[0046] Specifically, step (2) requires two centrifugations at the first centrifugation speed. When the first centrifugation is performed at the first centrifugation speed, large particles in the sample solution to be tested precipitate. When the second centrifugation is performed at the first centrifugation speed, the supernatant in the sample solution to be tested enters the second chamber from the first chamber through the first siphon channel.

[0047] The sample solution to be tested placed in the first chamber has been homogenized. After centrifugation at the first centrifugal speed, the large particles in the sample solution settle to the bottom of the first chamber under the action of centrifugal force. The connection port between the first siphon channel and the first chamber is located near the middle of the first chamber, ensuring that only the supernatant enters the second chamber through the first siphon channel. After the supernatant flows into the second chamber, the centrifugal microfluidic chip needs to be controlled to perform alternating centrifugation in opposite directions to ensure that the supernatant is fully mixed with the magnetic beads modified by the first biometric element and the probe modified by the second biometric element. Since the sixth and seventh chambers are located in the same radial position but on both sides of the butterfly-shaped channel, the rotation direction of the fifth centrifugal speed and the sixth centrifugal speed are opposite, but the speeds are the same.

[0048] In addition, this pathogen detection method specifically uses biorecognition components such as antibodies, nucleic acid aptamers, and concanavalin A to identify and detect target proteins on the surface of pathogens such as bacteria and viruses; it can also be used to detect certain antigens, such as carcinoembryonic antigen; or extracellular vesicles, bacterial extracellular vesicles, etc.

[0049] Preferably, in step (2), after the supernatant enters the first siphon channel, the rotation speed of the centrifugal microfluidic chip is reduced to 10-100 rpm so that the supernatant fills the first siphon channel;

[0050] In step (4), after all the unadsorbed liquid in step (3) flows into the seventh chamber, the rotation speed of the centrifugal microfluidic chip is reduced to 10-100 rpm so that the cleaning liquid in the fourth chamber fills the third siphon channel before step (5) is performed.

[0051] In step (5), when the cleaning solution is used to clean the substance adsorbed by the magnet, the centrifugal microfluidic chip rotates at 10-100 rpm, and the rotation direction alternates between clockwise and counterclockwise. At this speed, the probe substrate solution fills the fifth siphon channel.

[0052] Compared with the prior art, the advantages of the present invention are:

[0053] Each chamber is connected to the chip body via a siphon channel, a cut-off siphon channel, a capillary, a butterfly-shaped channel, or other channels. The chambers are arranged in an orderly manner. For fluid to transfer between chambers, a specific rotational speed must be achieved under the premise of ventilation. Therefore, by controlling the rotational speed of the centrifugal microfluidic chip and the ventilation status of each chamber, the multi-step sequential release of fluid in the centrifugal microfluidic chip can be realized. In addition, this centrifugal microfluidic chip integrates functions such as sample pretreatment, pathogen capture and labeling, magnetic separation, and cleaning, enabling pathogen detection to be performed with high automation on a centimeter-scale chip, shortening the pathogen detection time and reducing the professional requirements of the detection personnel. Attached Figure Description

[0054] Figure 1 This is a schematic diagram of the structure of the centrifugal microfluidic chip provided by the present invention;

[0055] Figure 2 This is a state diagram of step (1) in embodiment 3 of the present invention;

[0056] Figure 3 This is a state diagram of step (2) in embodiment 3 of the present invention;

[0057] Figure 4 This is a diagram showing the state of the supernatant after it has completely entered the second chamber in step (2) of embodiment 3 of the present invention;

[0058] Figure 5 This is a state diagram of step (3) in embodiment 3 of the present invention;

[0059] Figure 6 This is a diagram showing the state of the reaction solution after it has completely entered the third chamber in step (3) of Example 3 of the present invention;

[0060] Figure 7 This is a state diagram of step (4) in embodiment 3 of the present invention;

[0061] Figure 8 This is a state diagram of step (5) in embodiment 3 of the present invention;

[0062] Figure 9 This is a diagram showing the state of the cleaning fluid after it has completely entered the third chamber in step (5) of embodiment 3 of the present invention;

[0063] Figure 10 This is a diagram showing the state of the cleaning fluid in the third chamber after being drained in step (6) of embodiment 3 of the present invention;

[0064] Figure 11 This is a diagram showing the state of the probe substrate solution after it has completely entered the third chamber in step (7) of embodiment 3 of the present invention;

[0065] Figure 12 This is a state diagram after step (8) is completed in Embodiment 3 of the present invention. Detailed Implementation

[0066] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0067] Example 1

[0068] like Figure 1 As shown, the centrifugal microfluidic chip includes a chip body with seven chambers:

[0069] The first chamber 10 is used to hold the sample solution 12 to be tested.

[0070] The second chamber 20 is used to place magnetic beads modified with the first biometric element and probes modified with the second biometric element. Both the first and second biometric elements are used to bind to the detection target in the sample liquid 12 to be tested. Both the magnetic beads modified with the first biometric element and the probes modified with the second biometric element are lyophilized powders. The second chamber 20 is connected to the first chamber 10 by a first siphon channel 11 that allows the liquid in the first chamber 10 to enter the second chamber 20 at a first centrifugation speed.

[0071] The third chamber 30 has magnets on its walls for magnetically adsorbing magnetic beads. A second siphon channel 21 is connected between the third chamber 30 and the second chamber 20 to allow liquid in the second chamber 20 to enter the third chamber 30 at a second centrifugal speed.

[0072] The fourth chamber 40 is used to hold the cleaning solution 43. A third siphon channel 41 is connected between the fourth chamber 40 and the third chamber 30 to allow liquid in the fourth chamber 40 to enter the third chamber 30 at a third centrifugal speed. The third siphon channel 41 has a first vent 42 that cuts off the liquid flow in the third siphon channel 41 by atmospheric pressure at a speed greater than the third centrifugal speed.

[0073] The fifth chamber 50 is used to hold the probe substrate solution 54. A fourth channel 51 connects the fifth chamber 50 to the fourth chamber 40, allowing communication between the two chambers after the liquid in the fourth chamber 40 is drained. A fifth siphon channel 52 connects the fifth chamber 50 to the third chamber 30, allowing liquid from the fifth chamber 50 to enter the third chamber 30 at a fourth centrifugal speed. The fifth siphon channel 52 has a second vent 53 that, at a centrifugal speed greater than the fourth, prevents the liquid flow within the fifth siphon channel 52 from being cut off by atmospheric pressure.

[0074] The sixth chamber 60 is used to collect the probe substrate solution 61 after reaction with the probe.

[0075] The seventh chamber 70 is used to collect waste liquid 71. A capillary tube 81 is connected to the third chamber 30. One end of the capillary tube 81 is connected to a butterfly-shaped channel 82 with two outlet directions. The two outlet directions of the butterfly-shaped channel 82 are respectively connected to the sixth chamber 60 and the seventh chamber 70. The capillary tube 81 and the butterfly-shaped channel 82 are used to allow the liquid in the third chamber 30 to enter the seventh chamber 70 at the fifth centrifugal speed, or to allow the liquid in the third chamber 30 to enter the sixth chamber 60 at the sixth centrifugal speed. The direction of centrifugal rotation is controlled to distinguish whether the liquid enters the sixth chamber 60 or the seventh chamber 70.

[0076] The top surface of the chip body is provided with micropores 91 for sample addition and / or air intake and / or exhaust for each chamber. The micropores 91 corresponding to the fifth chamber 50 are sealed after sample addition.

[0077] The first, fifth, and sixth centrifugation speeds are greater than the second centrifugation speed, and the second centrifugation speed is greater than the third and fourth centrifugation speeds.

[0078] The chip body is disc-shaped with a mounting hole 92 in the middle for mounting onto the shaft of the centrifugal motor in a centrifuge during use.

[0079] Micro-holes 91 are all located on the side of the corresponding chamber near the mounting hole 92.

[0080] The first chamber 10 is closer to the mounting hole 92 than the second chamber 20, the second chamber 20 is closer to the mounting hole 92 than the third chamber 30, the fourth chamber 40 is closer to the mounting hole 92 than the fifth chamber 50, the fifth chamber 50 is closer to the mounting hole 92 than the third chamber 30, and the third chamber 30 is closer to the mounting hole 92 than the sixth chamber 60 and the seventh chamber 70.

[0081] The sixth chamber 60 and the seventh chamber 70 are located in the same radial position and are arranged circumferentially. The two outlet directions of the butterfly-shaped channel 82 correspond to the adjacent ends of the sixth chamber 60 and the seventh chamber 70, respectively.

[0082] Each micropore 91 is located on the side of the corresponding chamber near the mounting hole 92; the first vent 42 is located in the third siphon channel 41 near the mounting hole 92, and the second vent 53 is located in the fifth siphon channel 52 near the mounting hole 92.

[0083] The first siphon channel 11, the second siphon channel 21, the third siphon channel 41 and the fifth siphon channel 52 are hydrophilic; the fourth channel 51 and the capillary 81 are hydrophobic.

[0084] The first siphon channel 11, the second siphon channel 21, the third siphon channel 41, and the fifth siphon channel 52 are hydrophilic modified by hydrophilic reagent treatment, plasma surface treatment, or protein blocking agent treatment to form hydrophilic channels; the fourth channel 51 and the capillary 81 are hydrophobic modified by hydrophobic reagent to form hydrophobic channels.

[0085] Example 2

[0086] The method for fabricating a centrifugal microfluidic chip includes the following steps:

[0087] (1) Prepare a mold for casting to form a centrifugal microfluidic chip;

[0088] (2) A portion of the raw materials used for casting to form centrifugal microfluidic chips are poured into the mold for the first step of casting and solidification;

[0089] (3) Place a magnet at the position corresponding to the third chamber 30;

[0090] (4) Pour the remaining raw materials used to cast the centrifugal microfluidic chip into the mold for the second casting step and solidify it, so that the magnet is cast into the top wall of the third chamber 30, and obtain a centrifugal microfluidic chip with an open bottom surface and no bottom surface in each chamber.

[0091] (5) Take out the product obtained in step (4) and punch holes to form the first vent 42, the second vent 53 and each micro hole 91;

[0092] (6) The product obtained in step (5) is combined with the glass bottom surface and then plasma modified to obtain a centrifugal microfluidic chip.

[0093] The raw materials used to make centrifugal microfluidic chips are any one or a combination of several of the following: organosilicon, glass, quartz, polycarbonate, polymethyl methacrylate, polydimethylsiloxane, and polyethylene terephthalate.

[0094] After step (6) is completed, the first siphon channel 11, the second siphon channel 21, the third siphon channel 41 and the fifth siphon channel 52 are modified to be hydrophilic; the fourth channel 51 and the capillary 81 are modified to be hydrophobic.

[0095] Example 3

[0096] The method for pathogen detection, using the aforementioned centrifugal microfluidic chip, includes the following steps:

[0097] (1) As Figure 2 As shown, the sample solution 12 to be tested is added to the first chamber 10, the magnetic beads modified with the first biometric element and the probe modified with the second biometric element are added to the second chamber 20, the cleaning solution 43 is added to the fourth chamber 40, the probe substrate solution 54 is added to the fifth chamber 50, and the micropore 91 corresponding to the fifth chamber 50 is sealed.

[0098] (2) Figure 3 , 4 As shown, the sample solution 12 is centrifuged twice at a first centrifugation speed (direction: clockwise, speed: 3000-4000 rpm). During the first centrifugation at the first centrifugation speed, large particles in the sample solution 12 are precipitated. After the supernatant enters the first siphon channel 11, the rotation speed of the centrifugal microfluidic chip is reduced to 10-100 rpm to fill the first siphon channel 11 with supernatant. During the second centrifugation at the first centrifugation speed, the supernatant of the sample solution 12 in the first chamber 10 enters the second chamber 20 through the first siphon channel 11 and mixes and reacts with the magnetic beads modified by the first biometric element and the probe modified by the second biometric element.

[0099] (3) Figure 5 , 6As shown, centrifugation is performed at the second centrifugation speed (direction: clockwise, speed: 800-1200 rpm) so that the reaction liquid 22 after mixing in the second chamber 20 enters the third chamber 30 through the second siphon channel 21. The detection target and probe are directly or indirectly combined with the first biometric element and are adsorbed by the magnet through the magnetic beads.

[0100] (4) Figure 7 As shown, centrifugation is performed at the fifth centrifugation speed (direction: clockwise, speed: 1200-1600 rpm), and the centrifugation rotation direction is controlled so that the unabsorbed liquid in step (3) enters the seventh chamber 70 through the capillary tube 81 and the butterfly-shaped channel 82; after all the unabsorbed liquid in step (3) has flowed into the seventh chamber 70, the rotation speed of the centrifugal microfluidic chip is reduced to 10-100 rpm so that the cleaning liquid 43 in the fourth chamber 40 fills the third siphon channel 41;

[0101] (5) Figure 8 , 9 As shown, centrifugation is performed at the third centrifugation speed (direction: clockwise, speed: 400-800 rpm), allowing the cleaning solution 43 in the fourth chamber 40 to enter the third chamber 30 through the third siphon channel 41 to clean the substance adsorbed by the magnet. When cleaning the substance adsorbed by the magnet with the cleaning solution 43, the rotation speed of the centrifugal microfluidic chip is 10-100 rpm, and the rotation direction is alternating between clockwise and counterclockwise. At this speed, the probe substrate solution 54 fills the fifth siphon channel 52.

[0102] (6) Figure 10 As shown, centrifuge at the fifth centrifugation speed (direction: clockwise, speed: 1200-1600 rpm) and control the centrifugation rotation direction so that the cleaning solution 43 in the third chamber 30 in step (5) enters the seventh chamber 70 through the capillary tube 81 and the butterfly-shaped channel 82;

[0103] (7) Figure 11 As shown, centrifugation is performed at the fourth centrifugation speed (direction: clockwise, speed: 400-800 rpm) to allow the probe substrate solution 54 in the fifth chamber 50 to enter the third chamber 30 through the fifth siphon channel 52 for the probe substrate to react with the probe.

[0104] (8) Figure 12 As shown, centrifuge at the sixth centrifugation speed (direction: counterclockwise, speed: 1200-1600 rpm) and control the centrifugation rotation direction so that the reacted probe substrate solution 61 enters the sixth chamber 60 through the capillary tube 81 and the butterfly-shaped channel 82.

[0105] (9) Measure the probe substrate solution 61 after the reaction in the sixth chamber 60 to determine the status of the detection target in the sample solution 12.

[0106] The sample solution 12 to be tested, placed in the first chamber 10, has been homogenized. After centrifugation at the first centrifugal speed, the large particles in the sample solution 12 settle to the bottom of the first chamber 10 under the action of centrifugal force. The connection port between the first siphon channel 11 and the first chamber 10 is located near the middle of the first chamber 10, ensuring that only the supernatant enters the second chamber 20 through the first siphon channel 11. After the supernatant flows into the second chamber 20, the centrifugal microfluidic chip needs to be controlled to perform alternating centrifugation in opposite directions to ensure that the supernatant is fully mixed with the magnetic beads modified by the first biometric element and the probe modified by the second biometric element. Since the sixth chamber 60 and the seventh chamber 70 are located in the same radial position, but on both sides of the butterfly-shaped channel 82, the fifth centrifugal speed and the sixth centrifugal speed rotate in opposite directions but have the same speed.

Claims

1. A centrifugal microfluidic chip, comprising a chip body, characterized in that, The chip body has 7 chambers, namely: The first chamber is used to hold the sample solution to be tested. The second chamber is used to place magnetic beads modified with the first biometric element and probes modified with the second biometric element. Both the first and second biometric elements are used to bind to the detection target in the sample solution to be tested. Both the magnetic beads modified with the first biometric element and the probes modified with the second biometric element are lyophilized powders. The second chamber is connected to the first chamber by a first siphon channel that allows the liquid in the first chamber to enter the second chamber at a first centrifugation speed. The third chamber has magnets on its walls for magnetically attracting the magnetic beads, and the third chamber is connected to the second chamber by a second siphon channel that allows liquid in the second chamber to enter the third chamber at a second centrifugal speed. The fourth chamber is used to hold the cleaning solution. A third siphon channel connects the fourth chamber to the third chamber, allowing liquid from the fourth chamber to enter the third chamber at a third centrifugal speed. The third siphon channel has a first vent hole that, at a speed greater than the third centrifugal speed, interrupts the liquid flow within the third siphon channel due to atmospheric pressure. The fifth chamber is used to hold the probe substrate solution. A fourth channel connects the fifth and fourth chambers, allowing communication between them after the liquid in the fourth chamber is drained. A fifth siphon channel connects the fifth and third chambers, allowing liquid from the fifth chamber to enter the third chamber at a fourth centrifugal speed. The fifth siphon channel has a second vent that, at centrifugal speeds greater than the fourth, blocks the liquid flow within the siphon channel due to atmospheric pressure. The sixth chamber is used to collect the probe substrate solution after the probe has reacted with it. The seventh chamber is used to collect waste liquid. A capillary tube is connected to the third chamber, and one end of the capillary tube is connected to a butterfly-shaped channel with two outlet directions. The two outlet directions of the butterfly-shaped channel are respectively connected to the sixth chamber and the seventh chamber. The capillary tube and the butterfly-shaped channel are used to allow the liquid in the third chamber to enter the seventh chamber at the fifth centrifugal speed, or to allow the liquid in the third chamber to enter the sixth chamber at the sixth centrifugal speed. The direction of centrifugal rotation is controlled to distinguish whether the liquid enters the sixth chamber or the seventh chamber. The top surface of the chip body is provided with micropores for sample addition and / or air intake and / or exhaust for each chamber. The micropores corresponding to the fifth chamber are sealed after sample addition. The first centrifugation speed, the fifth centrifugation speed and the sixth centrifugation speed are greater than the second centrifugation speed, and the second centrifugation speed is greater than the third centrifugation speed and the fourth centrifugation speed.

2. The centrifugal microfluidic chip according to claim 1, characterized in that, The chip body is disc-shaped and has a mounting hole in the middle for mounting onto the shaft of the centrifugal motor in a centrifuge during use.

3. The centrifugal microfluidic chip according to claim 2, characterized in that, The micropores are all located on the side of the corresponding chamber near the mounting hole.

4. The centrifugal microfluidic chip according to claim 2, characterized in that, The first chamber is closer to the mounting hole than the second chamber, the second chamber is closer to the mounting hole than the third chamber, the fourth chamber is closer to the mounting hole than the fifth chamber, the fifth chamber is closer to the mounting hole than the third chamber, and the third chamber is closer to the mounting hole than the sixth and seventh chambers; the sixth and seventh chambers are located at the same radial position and arranged circumferentially, and the two outlet directions of the butterfly-shaped channel correspond to the mutually close ends of the sixth and seventh chambers, respectively.

5. The centrifugal microfluidic chip according to claim 2, characterized in that, Each micropore is located in the corresponding chamber on the side near the mounting hole; the first vent is located in the third siphon channel near the mounting hole, and the second vent is located in the fifth siphon channel near the mounting hole.

6. The centrifugal microfluidic chip according to claim 1, characterized in that, The first, second, third, and fifth siphon channels are hydrophilic; the fourth channel and the capillary are hydrophobic.

7. A method for preparing the centrifugal microfluidic chip according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Prepare a mold for casting to form the centrifugal microfluidic chip; (2) A portion of the raw materials used for casting to form the centrifugal microfluidic chip is poured into the mold for the first step of casting and solidification; (3) Place the magnet at the position corresponding to the third chamber; (4) Pour the remaining raw materials used to cast the centrifugal microfluidic chip into the mold for the second casting step and solidify, so that the magnet is cast into the top wall of the third chamber, and obtain a centrifugal microfluidic chip with an open bottom surface and no bottom surface in each chamber. (5) Take out the product obtained in step (4) and punch holes to form the first vent hole, the second vent hole and each micropore; (6) The product obtained in step (5) is mounted on the bottom surface to obtain the centrifugal microfluidic chip.

8. The method for fabricating the centrifugal microfluidic chip according to claim 7, characterized in that, The bottom surface is made of glass. The product obtained in step (5) and the bottom surface made of glass are modified by plasma and then assembled to form the centrifugal microfluidic chip. The raw materials used to make the centrifugal microfluidic chip are any one or a combination of several of the following: organosilicon, glass, quartz, polycarbonate, polymethyl methacrylate, polydimethylsiloxane, and polyethylene terephthalate. After step (6) is completed, the first siphon channel, the second siphon channel, the third siphon channel and the fifth siphon channel are hydrophilic modified; the fourth channel and the capillary are hydrophobic modified.

9. A method for detecting pathogens, characterized in that, Using the centrifugal microfluidic chip according to any one of claims 1 to 6 includes the following steps: (1) Add the sample solution to be tested to the first chamber, add the magnetic beads modified by the first biometric element and the probe modified by the second biometric element to the second chamber, add the cleaning solution to the fourth chamber, add the probe substrate solution to the fifth chamber, and seal the micropores corresponding to the fifth chamber. (2) Centrifuge at the first centrifugation speed so that the supernatant of the sample solution to be tested in the first chamber enters the second chamber through the first siphon channel and mixes and reacts with the magnetic beads modified by the first biometric element and the probe modified by the second biometric element. (3) Centrifuge at the second centrifugation speed, so that the reaction liquid after the mixture in the second chamber enters the third chamber through the second siphon channel, and the detection target and probe are directly or indirectly combined with the first biometric element and adsorbed by the magnet through the magnetic beads; (4) Centrifuge at the fifth centrifugal speed and control the centrifugal rotation direction so that the unadsorbed liquid in step (3) enters the seventh chamber through the capillary and butterfly-shaped channel; (5) Centrifuge at the third centrifugal speed to allow the cleaning solution in the fourth chamber to enter the third chamber through the third siphon channel to clean the substance adsorbed by the magnet; (6) Centrifuge at the fifth centrifugal speed and control the centrifugal rotation direction so that the cleaning solution in the third chamber in step (5) enters the seventh chamber through the capillary and butterfly-shaped channel; (7) Centrifuge at the fourth centrifugation speed to allow the probe substrate solution in the fifth chamber to enter the third chamber through the fifth siphon channel for the probe substrate to react with the probe. (8) Centrifuge at the sixth centrifugation speed and control the centrifugation rotation direction so that the reacted probe substrate solution enters the sixth chamber through the capillary and butterfly-shaped channel; (9) Measure the probe substrate solution after the reaction in the sixth chamber to determine the status of the detection target in the sample solution to be tested.

10. The method for pathogen detection according to claim 9, characterized in that, In step (2), after the supernatant enters the first siphon channel, the rotation speed of the centrifugal microfluidic chip is reduced to 10-100 rpm so that the supernatant fills the first siphon channel. In step (4), after all the unadsorbed liquid in step (3) flows into the seventh chamber, the rotation speed of the centrifugal microfluidic chip is reduced to 10-100 rpm so that the cleaning liquid in the fourth chamber fills the third siphon channel before step (5) is performed. In step (5), when the cleaning solution is used to clean the substance adsorbed by the magnet, the centrifugal microfluidic chip rotates at 10-100 rpm, and the rotation direction alternates between clockwise and counterclockwise. At this speed, the probe substrate solution fills the fifth siphon channel.

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