A centrifugal microfluidic chip and chemiluminescence immunoassay method

Abstract

The present invention discloses a centrifugal microfluidic chip and a chemiluminescent immunoassay method, which relate to the technical field of microfluidic chips. Specifically, the chip comprises a chip body with a rotating structure and a plurality of detection units arranged in a ring array in the chip body; the detection units are sequentially provided with a sample storage chamber, a reaction chamber and a waste liquid chamber from the proximal end to the distal end of the rotation center of the chip body, the sample storage chamber and the reaction chamber are connected through a main channel, the main channel is arranged along the radial direction of the chip body, and the reaction chamber and the waste liquid chamber are connected through a hydrophilic siphon valve; the centrifugal microfluidic chip and the chemiluminescent immunoassay method are based on a passive valve integration scheme of different trigger types, which reduces the risk of interference between valves, makes the control of the chip more precise and simple, and has better repeatability, and the chip does not rely on external structure intervention, but only relies on internal valves to achieve fluid drive and control, which has considerable advantages in achieving automation.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic chip technology, and more specifically, to a centrifugal microfluidic chip. Furthermore, the present invention also relates to a chemiluminescent immunoassay method applied to the centrifugal microfluidic chip. Background Art

[0002] Commonly used immunoassay methods include fluorescence, absorbance, chemiluminescence, and the like. Among them, chemiluminescence immunoassay has the characteristics of simple operation, high sensitivity, and stable results, and is of great significance for the early diagnosis of many diseases. Traditional immunoassay instruments are large, expensive, and require professional maintenance, which limits their application in areas with limited medical resources. Therefore, the development of simpler and cheaper detection methods is of vital importance for the early diagnosis of diseases, and the highly integrated centrifugal microfluidic platform is an ideal choice for efficient and fully automated marker detection. The drive of the fluid on the centrifugal microfluidic platform only requires one motor, without the need for redundant external instruments, which greatly improves the integration, miniaturization, and automation of the analytical workflow. The detection platform uses a simply configured photon counter, does not rely on complex design and optical path alignment, and is very suitable for use in point-of-care testing (POCT) equipment.

[0003] However, integrating multiple analytical steps based on immunomagnetic bead manipulation into centrifugal microfluidic chips is challenging, which limits the application of centrifugal microfluidics in chemiluminescence detection.

[0004] In the existing technology, samples are pre-mixed outside the chip and then loaded onto the chip for testing, which has limitations in the integrity and automation of the chemiluminescence detection step; or the centrifugal disk used requires the capture magnetic beads to be pre-placed and fixed in the U-shaped detection area. This method loses the freedom of the magnetic beads and the advantages of liquid-phase reaction to a certain extent.

[0005] Currently, a common approach to integrating immune response steps involves placing multiple valves on a centrifugal microfluidic chip structure to allow for continuous fluid release with temporal and spatial control. While active valves, such as laser-irradiated wax-iron microvalves (LIFMs), can easily control multiple microvalves, this inevitably increases system complexity, a disadvantage for point-of-care (POCT) applications. In contrast, in centrifugal microfluidic chips employing passive valves, most research focuses on sequentially manipulating multiple liquids using the same trigger-type valves, such as the integration of multiple capillary valves, serially connected hydrophilic siphon valves, multiple centrifugal-pneumatic valves, or the recently proposed Euler force-actuated siphon valve. While these technologies leverage the properties of passive valves and enable on-demand valve opening, overlapping valve opening conditions are unavoidable. Valves of the same trigger type share the same actuation principle and differ only in critical conditions, which complicates integration.

[0006] In summary, how to provide a centrifugal microfluidic chip and chemiluminescence immunoassay method that can avoid automation limitations during the detection process, avoid losing the freedom of magnetic beads and the advantages of liquid-phase reactions, while not increasing the complexity of the system and avoiding overlapping valve opening conditions, is a problem that currently needs to be solved by technical personnel in this field. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a centrifugal microfluidic chip based on a passive valve integration solution of different trigger types to reduce the risk of interference between valves. The control of the chip is more precise and simple, with better repeatability. The chip does not rely on external structure intervention, but only relies on internal valves to achieve fluid drive and control, which has considerable advantages in achieving automation.

[0008] Another object of the present invention is to provide a chemiluminescent immunoassay method applied to the above-mentioned centrifugal microfluidic chip, which realizes the automation of the detection process, shortens the time required for detection, and effectively ensures the accuracy of the detection results.

[0009] In order to achieve the above object, the present invention provides the following technical solutions:

[0010] A centrifugal microfluidic chip comprises a chip body with a rotary structure and a plurality of detection units arranged in a ring array within the chip body;

[0011] The detection unit is provided with a sample storage chamber, a reaction chamber, and a waste liquid chamber in sequence from the proximal end to the distal end of the rotation center of the chip body. The sample storage chamber and the reaction chamber are connected by a main channel, and the main channel is arranged along the radial direction of the chip body. The reaction chamber and the waste liquid chamber are connected by a hydrophilic siphon valve.

[0012] The detection unit further includes a plurality of groups of primary storage chambers and secondary storage chambers, wherein the primary storage chambers are connected to the main channel and / or the reaction chamber via capillary valves, and the secondary storage chambers are connected to the main channel and / or the reaction chamber via Euler force-driven siphon valves;

[0013] The sample storage chamber, the reaction chamber, the primary storage chamber and the secondary storage chamber are respectively connected to each other and provided with liquid inlet holes at one end close to the rotation center of the chip body; the sample storage chamber, the reaction chamber, the primary storage chamber, the secondary storage chamber and the waste liquid chamber are respectively connected to each other and provided with ventilation holes at one end close to the rotation center of the chip body.

[0014] Preferably, there are at least two groups of the first-level storage chambers, and the gyration radii of at least two groups of the first-level storage chambers are unequal.

[0015] Preferably, there are at least two groups of secondary storage chambers, and at least two groups of secondary storage chambers are symmetrically arranged on both sides of the main channel, and the Euler force-driven siphon valves used to connect the two groups of secondary storage chambers are symmetrically arranged.

[0016] Preferably, a third capillary valve is connected in series in the descending channel of the connection portion between the hydrophilic siphon valve and the waste liquid chamber.

[0017] Preferably, the chip body comprises a cover plate and a base plate bonded by ultrasonic bonding, and fixing holes are provided at the rotation centers of the cover plate and the base plate;

[0018] The cover plate is provided with the liquid inlet and the vent hole penetrating therethrough;

[0019] All chambers, channels and valve bodies of the detection unit are arranged on the substrate.

[0020] Preferably, a bonding rib is formed in the gap between the cover plate and the base plate around the detection unit.

[0021] Preferably, the chip body comprises four layers of stacked discs adhered by pressure-sensitive adhesive, and a fixing hole is provided at the rotation center of each layer of the discs;

[0022] The first layer of the disc is provided with the liquid inlet and the vent holes penetrating the plate;

[0023] The second layer of the disc is provided with all the chambers, channels and valve bodies of the detection unit that pass through the plate;

[0024] All the chambers of the detection unit that pass through the plate are arranged on the third layer of the disc.

[0025] Preferably, the lower surface of the first layer of the disc is laser engraved with the upper end structure of the capillary valve;

[0026] The upper surface of the fourth disk is engraved with the lower end structure of the capillary valve by laser.

[0027] A chemiluminescent immunoassay method, applied to any one of the centrifugal microfluidic chips described above, comprising:

[0028] injecting a sample into the sample storage cavity;

[0029] injecting antibody 1-magnetic beads into at least one group of the primary storage chambers;

[0030] injecting antibody 2-acridinium ester into at least one group of the primary storage chambers;

[0031] injecting cleaning fluid into at least one group of the secondary storage chambers;

[0032] injecting a pre-excitation liquid into at least one group of the secondary storage chambers;

[0033] The rotation speed of the chip body is sequentially increased to open the capillary valves corresponding to the main channel and the primary storage chamber in sequence, thereby sequentially injecting the sample, the antibody 1-magnetic beads, and the antibody 2-acridinium ester into the reaction chamber; so that the antibody 1-magnetic beads and the antibody 2-acridinium ester combine with the sample to form an immune complex;

[0034] Controlling the chip body to rotate at a set acceleration, so that the Euler force corresponding to the cleaning liquid drives the siphon valve to be opened, thereby allowing the cleaning liquid to flow into the reaction chamber to clean the immune complex and remove unbound substances;

[0035] Controlling the addition of a magnetic field below the reaction chamber to aggregate the immune complexes, and controlling the cessation of the magnetic field when the hydrophilic siphon valve is turned on;

[0036] Controlling the chip body to rotate so that the waste liquid in the reaction chamber flows into the waste liquid chamber, until all the waste liquid enters the waste liquid chamber, and controlling the chip body to stop rotating;

[0037] Controlling the chip body to rotate at another set acceleration so that the Euler force corresponding to the pre-excitation liquid drives the siphon valve to conduct, thereby allowing the pre-excitation liquid to flow into the reaction chamber to react with the immune complex until the pre-excitation liquid completely enters the reaction chamber, and controlling the chip body to stop rotating;

[0038] An excitation solution is injected into the reaction chamber, and the chemiluminescence value is detected in situ.

[0039] Preferably, the in situ detection of chemiluminescence values comprises:

[0040] Building a darkroom;

[0041] A photon counting probe is arranged directly below the reaction chamber, and a shutter is installed between the reaction chamber and the photon counting probe;

[0042] The photon counting probe is controlled to read the number of photons in the reaction chamber and transmit the number to the host computer for output.

[0043] Preferably, the rotation speed of the chip body is increased sequentially to sequentially open the capillary valves corresponding to the main channel and the primary storage chamber, thereby sequentially injecting the sample, the antibody 1-magnetic beads, and the antibody 2-acridinium ester into the reaction chamber; so that the antibody 1-magnetic beads and the antibody 2-acridinium ester combine with the sample to form an immune complex, comprising:

[0044] When the sample, the antibody 1-magnetic beads and the antibody 2-acridinium ester are sequentially injected into the reaction chamber, the rotation speed of the chip body is controlled to continuously change so that the antibody 1-magnetic beads, the antibody 2-acridinium ester and the sample are shaken and mixed in the reaction chamber.

[0045] Preferably, controlling the chip body to rotate at a set acceleration so that the Euler force corresponding to the cleaning liquid drives the siphon valve to be opened, thereby allowing the cleaning liquid to flow into the reaction chamber to clean the immune complex and remove unbound substances, including:

[0046] After the cleaning liquid flows into the reaction chamber, the rotation speed of the chip body is controlled to change continuously so as to allow the cleaning liquid and the immune complex to vibrate and mix.

[0047] Compared with the prior art, the centrifugal microfluidic chip provided by the present invention has at least the following beneficial effects:

[0048] 1. The chip body integrates multi-level storage chambers, which are connected to the reaction chamber through different forms of valves. Specific conditions can be used to connect the corresponding valves and storage chambers with the reaction chamber, thereby achieving automatic mixing of samples and improving the automation execution capability of the entire detection process.

[0049] 2. Different storage chambers are connected to the reaction chamber using different valves, specifically capillary valves, Euler force-driven siphon valves, and hydrophilic siphon valves. The conduction conditions of the above three valves are different, so when controlling their conduction, there is little mutual interference, which helps to simplify the structural design of the chip body.

[0050] The chemiluminescent immunoassay method provided by the present invention is applied to the above-mentioned centrifugal microfluidic chip and has at least the following beneficial effects compared with the prior art:

[0051] 1. The sample, antibody, cleaning solution and pre-excitation solution are added to separate chambers respectively. During the reaction, they are gradually added to the reaction chamber, which effectively avoids the loss of the freedom of the magnetic beads while retaining the advantages of liquid phase reaction.

[0052] 2. During the process, various reagents are released sequentially, with less manual intervention, high repeatability, and short detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0054] Figure 1 This is a schematic structural diagram of a specific detection unit provided by the present invention;

[0055] Figure 2 This is a schematic structural diagram of the first layer of the disc of the specific chip body provided by the present invention;

[0056] Figure 3 This is a schematic structural diagram of the second disk of the chip body provided by the present invention;

[0057] Figure 4 This is a schematic structural diagram of the third disk of the chip body provided by the present invention;

[0058] Figure 5 This is a schematic structural diagram of the fourth disk layer of the chip body provided by the present invention;

[0059] Figure 6 This is an exploded schematic diagram of a four-layer disc of a specific chip body provided by the present invention;

[0060] Figure 7 This is a schematic diagram of the specific chemiluminescent immunoassay method provided by the present invention;

[0061] Figure 8 This is a graph showing the rotation speed of the chip body during the specific chemiluminescent immunoassay process provided by the present invention;

[0062] Figure 9 This is a schematic diagram of the specific process of in-situ detection of chemiluminescence values provided by the present invention.

[0063] Figures 1-9 middle:

[0064] 1. Sample storage chamber; 2. Reaction chamber; 3. Waste liquid chamber; 4. Primary storage chamber; 401. Magnetic bead storage chamber; 402. Acridinium ester storage chamber; 5. Secondary storage chamber; 501. Cleaning solution storage chamber; 502. Pre-excitation solution storage chamber; 6. Capillary valve; 601. First capillary valve; 602. Second capillary valve; 7. Main channel; 8. Euler force-driven siphon valve; 801. First Euler force-driven siphon valve; 802. Second Euler force-driven siphon valve; 9. Hydrophilic siphon valve; 10. Third capillary valve; 11. Liquid inlet; 12. Vent hole; 13. Fixing hole; 14. Shutter; 15. Photon counting probe; 16. Counting unit; 17. Host computer. DETAILED DESCRIPTION

[0065] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0066] The core of the present invention is to provide a centrifugal microfluidic chip based on an integrated solution of passive valves of different trigger types, which reduces the risk of interference between valves. The chip control is more precise and simple, with better repeatability. The chip does not rely on external structural intervention, but only relies on internal valves to achieve fluid drive and control, which has considerable advantages in achieving automation.

[0067] Another core of the present invention is to provide a chemiluminescent immunoassay method applied to the above-mentioned centrifugal microfluidic chip, which realizes the automation of the detection process, shortens the time required for detection, and effectively ensures the accuracy of the detection results.

[0068] Please refer to Figures 1-6 , a centrifugal microfluidic chip, comprising a chip body of a rotary structure and a plurality of detection units arranged in a ring array in the chip body;

[0069] The detection unit is provided with a sample storage chamber 1, a reaction chamber 2, and a waste liquid chamber 3 in sequence from the proximal end to the distal end of the chip body's rotation center. The sample storage chamber 1 and the reaction chamber 2 are connected by a main channel 7, which is arranged along the radial direction of the chip body. The reaction chamber 2 and the waste liquid chamber 3 are connected by a hydrophilic siphon valve 9.

[0070] The detection unit also includes several groups of primary storage chambers 4 and secondary storage chambers 5. The primary storage chambers 4 are connected to the main channel 7 and / or the reaction chamber 2 through capillary valves 6, and the secondary storage chambers 5 are connected to the main channel 7 and / or the reaction chamber 2 through Euler force-driven siphon valves 8.

[0071] The sample storage chamber 1, the reaction chamber 2, the primary storage chamber 4 and the secondary storage chamber 5 are respectively connected to each other with a liquid inlet hole 11 at one end close to the rotation center of the chip body, and the sample storage chamber 1, the reaction chamber 2, the primary storage chamber 4, the secondary storage chamber 5 and the waste liquid chamber 3 are respectively connected to each other with a vent hole 12 at one end close to the rotation center of the chip body.

[0072] Several groups of detection units are arranged in a circular array in the chip body, such as Figure 3 As shown, four groups of detection units are set on the chip body, which means that four groups of samples can be tested at the same time, saving detection time;

[0073] The detection unit integrates a sample storage chamber 1 for storing the initial sample, a primary storage chamber 4 for storing antibodies, and a secondary storage chamber 5 for storing cleaning solution and / or pre-excitation solution. This allows the samples to be mixed within the chip body, eliminating the need for pre-mixing before adding them to the chip body. This ensures the freedom of the magnetic beads and the advantages of liquid-phase reaction during the detection process.

[0074] At the same time, the sample storage chamber 1 is directly connected to the reaction chamber 2 through a radially arranged main channel 7, ensuring that when the chip body rotates, the initial sample in the sample storage chamber 1 can be firstly added to the reaction chamber 2;

[0075] The primary storage chamber 4 is connected to the reaction chamber 2 via a capillary valve 6. When the rotational speed of the chip reaches the critical condition for the capillary valve 6 to open, the capillary valve 6 automatically opens, allowing the antibodies in the primary storage chamber 4 to be sequentially added to the reaction chamber 2 and react sequentially with the sample added therein.

[0076] The secondary storage chamber 5 is connected to the reaction chamber 2 by an Euler force-driven siphon valve 8. When the rotational acceleration of the chip body reaches the critical condition for the Euler force-driven siphon valve 8 to be turned on, the Euler force-driven siphon valve 8 is turned on, and the cleaning liquid or pre-excitation liquid is injected into the reaction chamber 2 to react sequentially with the substances in the reaction chamber 2.

[0077] Since the critical conditions for the conduction of the main channel 7, capillary valve 6, and Euler force-driven siphon valve 8 are different, the conduction order of the three valves can be controlled by controlling the rotation speed and rotational acceleration of the chip body. The conduction conditions do not overlap, effectively preventing any two valves from being conducted at the same time, thus ensuring the accuracy of the injection sequence of various reagents. In addition, the rotation speed and rotational acceleration of the chip body are relatively easy to control, facilitating the automation of detection.

[0078] At the same time, liquid inlet holes 11 are provided in communication with each other at one end of the sample storage chamber 1, the reaction chamber 2, the primary storage chamber 4, and the secondary storage chamber 5, which are close to the rotation center of the chip body. This facilitates the addition of samples, antibodies, cleaning solutions, and pre-stimulation solutions in the initial stage, and prevents these liquids from overflowing from the liquid inlet holes 11 during the centrifugation process.

[0079] Moreover, ventilation holes 12 are provided in communication with each other at one end of the sample storage chamber 1, the reaction chamber 2, the primary storage chamber 4, the secondary storage chamber 5 and the waste liquid chamber 3 near the rotation center of the chip body, so that when the liquid flows into the corresponding cavity, the original air in the corresponding cavity is discharged, thereby maintaining the internal pressure balance and preventing the liquid from overflowing through the ventilation holes 12 during the centrifugation process.

[0080] In some embodiments, there are at least two groups of primary storage chambers 4, and the gyration radii of at least two groups of primary storage chambers 4 are unequal;

[0081] By providing multiple groups of primary storage cavities 4 with different gyration radii, the corresponding capillary valves 6 also have different gyration radii. Capillary valves 6 with different gyration radii can obtain different linear speeds under the premise of the same chip body rotation speed. That is, the conduction conditions of capillary valves 6 with different gyration radii correspond to different chip body rotation speeds. That is, by controlling the rotation speed of the chip body, the conduction order of primary storage cavities 4 with different gyration radii can be controlled, thereby realizing the sequential injection of different antibodies into the reaction chamber 2 and sequential reaction with the sample.

[0082] In some embodiments, there are at least two groups of secondary storage chambers 5, and at least two groups of secondary storage chambers 5 are symmetrically arranged on both sides of the main channel 7, and the Euler force-driven siphon valves 8 for connecting the two groups of secondary storage chambers 5 are symmetrically arranged;

[0083] The symmetrically arranged Euler force drives the siphon valve 8 so that the acceleration direction corresponding to the conduction condition is opposite. That is, by controlling the direction of the chip body rotation acceleration, the conduction sequence of the Euler force driven siphon valve 8 is controlled, and the conduction sequence of different secondary storage chambers 5 and reaction chambers 2 is controlled.

[0084] In some embodiments, a third capillary valve 10 is connected in series in the descending channel of the connection portion between the hydrophilic siphon valve 9 and the waste liquid chamber 3;

[0085] The hydrophilic siphon valve 9 is mainly controlled by hydrophilicity. In order to prevent the hydrophilic siphon valve 9 from being turned on too early, a third capillary valve 10 is installed in its descending channel to inhibit it, thereby preventing the hydrophilic siphon valve 9 from being turned on when the chip body rotates at a low speed.

[0086] In some embodiments, the chip body includes a cover sheet and a base sheet bonded by ultrasonic bonding, and a fixing hole 13 is provided at the rotation center of the cover sheet and the base sheet;

[0087] The cover is provided with a liquid inlet hole 11 and a vent hole 12;

[0088] All chambers, channels and valve bodies of the detection unit are arranged on the substrate;

[0089] A two-layer cover plate design is used to engrave all the chambers, channels, and valve bodies of the detection unit on the substrate and shield them with a cover plate. A liquid inlet 11 and a vent 12 are provided on the cover plate to complete liquid filling and air pressure balance in the chamber. The overall chip body has a relatively simple structure and is easy to process and manufacture.

[0090] The fixing hole 13 is used to conveniently fix the chip body to its rotation driving device.

[0091] In some embodiments, a bonding rib is provided in the gap between the cover sheet and the base sheet around the detection unit;

[0092] By providing adhesive ribs around the periphery of the detection unit, the sealing performance around the detection unit is further improved, mutual interference of materials between different detection units is avoided, and the accuracy of the detection results is guaranteed.

[0093] In some embodiments, as Figure 2-Figure 6 As shown, the chip body comprises four layers of stacked discs adhered by pressure-sensitive adhesive, and a fixing hole 13 is provided at the rotation center of each layer of discs;

[0094] like Figure 2 As shown, the first layer of the disc is provided with a liquid inlet 11 and a vent 12 that penetrate the plate;

[0095] like Figure 3 As shown, the second disc is provided with all the chambers, channels and valve bodies of the detection unit that penetrate the plate;

[0096] like Figure 4 As shown, the third disc is provided with all the chambers of the detection unit that penetrates the plate;

[0097] like Figure 6 As shown, the overall processing difficulty is reduced by layering the disc. For example, using PMMA sheets for laser engraving helps improve processing speed and accuracy, and different layers of discs are bonded with pressure-sensitive adhesive to ensure the sealing of the contact surface.

[0098] During the design, in order to ensure that the chamber has sufficient capacity, the thickness of the plates of different layers of discs can be selected to be different. For example, the thickness of the plates of the first, second and fourth layers of discs is selected to be 0.5mm, while the thickness of the plate of the third layer of disc with the chamber is selected to be 2mm.

[0099] To facilitate the connection between the chip body and the drive device, fixing holes 13 of different diameters can be opened in the centers of different layers of disks. For example, fixing holes 13 with a diameter of 8 mm can be opened in the centers of the first, second, and third layers of disks, and a fixing hole 13 with a diameter of 6 mm can be opened in the center of the fourth layer of disks, thereby facilitating the connection and fixation between the chip body and the drive device.

[0100] At the same time, when designing the liquid inlet 11, the diameter is uniformly 1mm. When designing the vent hole 12, considering that more liquid needs to be added to the waste liquid chamber 3, the diameter of the vent hole 12 of the waste liquid chamber 3 is designed to be 1.5mm, and the diameters of the other vent holes 12 are designed to be 1mm.

[0101] In some embodiments, the lower surface of the first disc is laser engraved with the upper end structure of the capillary valve 6;

[0102] The upper surface of the fourth disc is laser engraved with the lower end structure of the capillary valve 6;

[0103] Considering the space occupied by the capillary valve 6, the corresponding structure of the capillary valve 6 is engraved at the corresponding positions of the first and fourth layers of the disc, further reducing the structural design difficulty of the second and third layers of the disc and the overall processing difficulty.

[0104] In addition to the centrifugal microfluidic chip disclosed in each of the above embodiments, the present invention also provides a chemiluminescent immunoassay method applied to the above centrifugal microfluidic chip;

[0105] Methods such as Figure 7-Figure 9 As shown, including:

[0106] Injecting a sample into the sample storage chamber 1;

[0107] Injecting antibody 1-magnetic beads into at least one set of primary storage chambers 4;

[0108] injecting antibody 2-acridinium ester into at least one set of primary storage chambers 4;

[0109] injecting cleaning liquid into at least one set of secondary storage chambers 5;

[0110] Injecting pre-excitation liquid into at least one set of secondary storage chambers 5;

[0111] The rotation speed of the chip body is gradually increased to open the capillary valves 6 corresponding to the main channel 7 and the primary storage chamber 4, thereby allowing the sample, antibody 1-magnetic beads, and antibody 2-acridinium ester to be injected into the reaction chamber 2 in sequence; so that the antibody 1-magnetic beads and antibody 2-acridinium ester bind to the sample to form an immune complex;

[0112] The chip body is controlled to rotate at a set acceleration, so that the Euler force corresponding to the cleaning liquid drives the siphon valve 8 to conduct, thereby allowing the cleaning liquid to flow into the reaction chamber 2 to clean the immune complex and remove unbound substances;

[0113] Controlling the addition of a magnetic field below the reaction chamber 2 to aggregate the immune complexes, and controlling the termination of the magnetic field when the hydrophilic siphon valve 9 is turned on;

[0114] The chip body is controlled to rotate so that the waste liquid in the reaction chamber 2 flows into the waste liquid chamber 3 until all the waste liquid enters the waste liquid chamber 3, and the chip body is controlled to stop rotating;

[0115] The control chip body is accelerated to rotate at another set acceleration, so that the Euler force corresponding to the pre-excitation liquid drives the siphon valve 8 to conduct, thereby allowing the pre-excitation liquid to flow into the reaction chamber 2 to react with the immune complex. When the pre-excitation liquid completely enters the reaction chamber 2, the control chip body stops rotating;

[0116] Inject the excitation solution into the reaction chamber 2 and perform in-situ detection of the chemiluminescence value.

[0117] like Figure 1 As shown, the primary storage chamber 4 includes a magnetic bead storage chamber 401 with a large gyration radius and an acridinium ester storage chamber 402 with a small gyration radius;

[0118] The magnetic bead storage chamber 401 and the acridinium ester storage chamber 402 with a small gyration radius are respectively provided with a first capillary valve 601 and a second capillary valve 602;

[0119] 50 μL of Ab1-beads and 50 μL of Ab2-acridinium ester were injected into the magnetic bead storage chamber 401 and the acridinium ester storage chamber 402 with a small gyration radius, respectively;

[0120] The secondary storage chamber 5 includes a cleaning liquid storage chamber 501 located on the clockwise side of the main channel 7 and a pre-excitation liquid storage chamber 502 located on the counterclockwise side of the main channel 7;

[0121] The cleaning liquid storage chamber 501 and the pre-excitation liquid storage chamber 502 are symmetrically provided with a first Euler force driven siphon valve 801 and a second Euler force driven siphon valve 802;

[0122] 100 μL washing buffer and 70 μL pre-trigger solution were injected into the cleaning solution storage chamber 501 and the pre-trigger solution storage chamber 502 respectively;

[0123] Among them, Ab1-beads is antibody 1-magnetic beads, Ab2-acridinium ester is antibody 2-acridinium ester, washing buffer is washing solution, and pre-trigger solution is pre-trigger solution.

[0124] In some embodiments, detecting chemiluminescence values in situ comprises:

[0125] Building a darkroom;

[0126] A photon counting probe 15 is set just below the reaction chamber 2, and a shutter 14 is installed between the reaction chamber 2 and the photon counting probe 15;

[0127] Control the photon counting probe 15 to read the number of photons in the reaction chamber 2 and transmit it to the host computer 17 for output;

[0128] like Figure 9 As shown, in a dark room, it is convenient to accurately count the photons in the reaction chamber 2, and the photon counting probe 15 is used for counting to ensure the efficiency and accuracy of the counting;

[0129] At the same time, when designing the darkroom, a shutter 14 is used to separate the photon counting probe 15 and the reaction chamber 2 to further block ambient light and protect the detection window. During detection, an excitation liquid is added to the reaction chamber 2, and the photons generated in the reaction chamber 2 are transmitted to the photon counting probe 15 through the open shutter 14. After being processed by the counting unit 16, the signal is converted into a digital signal and transmitted to the host computer 17 for output.

[0130] In some embodiments, the rotation speed of the chip body is sequentially increased to sequentially open the capillary valves 6 corresponding to the main channel 7 and the primary storage chamber 4, thereby sequentially injecting the sample, antibody 1-magnetic beads, and antibody 2-acridinium ester into the reaction chamber 2; so that the antibody 1-magnetic beads and antibody 2-acridinium ester combine with the sample to form an immune complex, including:

[0131] After the sample, antibody 1-magnetic beads and antibody 2-acridinium ester are injected into the reaction chamber 2 in sequence, the rotation speed of the chip body is controlled to continuously change so that the antibody 1-magnetic beads, antibody 2-acridinium ester and the sample are shaken and mixed in the reaction chamber 2.

[0132] In some embodiments, the chip body is controlled to rotate at a set acceleration so that the Euler force corresponding to the cleaning liquid drives the siphon valve 8 to be opened, thereby allowing the cleaning liquid to flow into the reaction chamber 2 to clean the immune complex and remove unbound substances, including:

[0133] After the cleaning liquid flows into the reaction chamber 2, the rotation speed of the chip body is controlled to change continuously so as to vibrate and mix the cleaning liquid and the immune complex.

[0134] like Figure 7 and Figure 8 As shown in Figure 2, the specific process of chemiluminescent immunoassay is as follows:

[0135] Fill each chamber with medicine through the liquid inlet 11;

[0136] The chip body is rotated to a speed of 600 RPM. At this time, the sample in the sample storage chamber 1 flows into the reaction chamber 2. However, the first capillary valve 601 and the second capillary valve 602 have not reached the critical speed corresponding to the conduction condition, so they are not conductive.

[0137] The chip body speed is accelerated to 1200 RPM. At this time, the first capillary valve 601 reaches the critical speed condition, and the Ab1-beads break through the first capillary valve 601 and enter the reaction chamber 2.

[0138] The chip body speed is accelerated to 1600 RPM. At this time, the second capillary valve 602 reaches the critical speed condition, and the Ab2-acridinium ester is also driven into the reaction chamber 2;

[0139] By continuously changing the rotation speed of the chip body, the liquid in the reaction chamber 2 is repeatedly oscillated, and the inertial force is used to complete the mixing of the reagents. Ab1 and Ab2 begin to combine with the markers in the sample to form an immune complex;

[0140] A magnetic field is added to the bottom of the reaction chamber 2, and the immune complexes are gathered under the attraction of the magnetic force. Then the chip body stops rotating, and the hydrophilic siphon valve 9 is triggered. The reagent waste liquid in the reaction chamber 2 breaks through the siphon apex under the action of capillary force.

[0141] The chip body is accelerated to 1600 RPM and the waste liquid is continuously discharged into the waste liquid chamber 3;

[0142] The chip body stops rotating and is then accelerated at the set acceleration. Under the action of the Euler force, the cleaning liquid breaks through the top of the first Euler force-driven siphon valve 801 and then flows into the reaction chamber 2 at a high speed. The chip is repeatedly shaken again to complete the cleaning of the immune complex and remove unbound substances.

[0143] A magnetic field is added to allow the immune complex to settle. The chip body then remains stationary, triggering the hydrophilic siphon valve 9, and the cleaning wastewater begins to flow to the top of the hydrophilic siphon valve 9.

[0144] The chip body is accelerated to 1600 RPM and the waste liquid is continuously discharged into the waste liquid chamber 3;

[0145] The chip body is accelerated in the reverse direction at a set acceleration, and the pre-excitation liquid breaks through the apex of the second Euler force-driven siphon valve 802, and then flows into the reaction chamber 2 at a high speed to react with the acridinium ester modified on the immune complex;

[0146] An injection needle is used to add an excitation solution into the reaction chamber 2 through the liquid inlet 11 of the reaction chamber 2, and the chemiluminescence value is detected in situ to complete the entire chemiluminescence immunoassay process.

[0147] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0148] It is worth noting that as a universal solution for magnetic beads and fluid control, this chip can be further expanded to applications such as cell sorting and extraction, as well as nucleic acid extraction, purification, amplification and detection. The method is similar to the principle of chemiluminescence immunoassay and will not be repeated here.

[0149] The centrifugal microfluidic chip and chemiluminescent immunoassay method provided by the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A centrifugal microfluidic chip, characterized in that: A chip body comprising a rotary structure and a plurality of detection units arranged in a ring array within the chip body; The detection unit is provided with a sample storage chamber (1), a reaction chamber (2) and a waste liquid chamber (3) in sequence from the proximal end to the distal end of the rotation center of the chip body. The sample storage chamber (1) and the reaction chamber (2) are connected through a main channel (7). The main channel (7) is arranged along the radial direction of the chip body. The reaction chamber (2) and the waste liquid chamber (3) are connected through a hydrophilic siphon valve (9). The detection unit further comprises a plurality of groups of primary storage chambers (4) and secondary storage chambers (5), wherein the primary storage chambers (4) are connected to the main channel (7) and / or the reaction chamber (2) via a capillary valve (6), and the secondary storage chambers (5) are connected to the main channel (7) and / or the reaction chamber (2) via an Euler force-driven siphon valve (8); The sample storage chamber (1), the reaction chamber (2), the primary storage chamber (4) and the secondary storage chamber (5) are connected to each other at one end close to the rotation center of the chip body and are provided with a liquid inlet hole (11); the sample storage chamber (1), the reaction chamber (2), the primary storage chamber (4), the secondary storage chamber (5) and the waste liquid chamber (3) are connected to each other at one end close to the rotation center of the chip body and are provided with a vent hole (12).

2. The centrifugal microfluidic chip according to claim 1, characterized in that: There are at least two groups of the first-level storage chambers (4), and the gyration radii of at least two groups of the first-level storage chambers (4) are unequal.

3. The centrifugal microfluidic chip according to claim 1, characterized in that: There are at least two groups of secondary storage chambers (5), and at least two groups of secondary storage chambers (5) are symmetrically arranged on both sides of the main channel (7), and the Euler force-driven siphon valves (8) for connecting the two groups of secondary storage chambers (5) are symmetrically arranged.

4. The centrifugal microfluidic chip according to claim 1, characterized in that: A third capillary valve (10) is connected in series in the descending channel at the connection portion between the hydrophilic siphon valve (9) and the waste liquid chamber (3).

5. The centrifugal microfluidic chip according to any one of claims 1 to 4, characterized in that: The chip body comprises a cover plate and a base plate bonded by ultrasonic bonding, and a fixing hole (13) is provided at the rotation center of the cover plate and the base plate; The cover plate is provided with the liquid inlet hole (11) and the vent hole (12) penetrating therethrough; All chambers, channels and valve bodies of the detection unit are arranged on the substrate.

6. The centrifugal microfluidic chip according to claim 5, characterized in that: Adhesive ribs are arranged in the gap between the cover plate and the base plate around the detection unit.

7. The centrifugal microfluidic chip according to any one of claims 1 to 4, characterized in that: The chip body comprises four layers of stacked disks adhered by pressure-sensitive adhesive, and a fixing hole (13) is provided at the rotation center of each layer of the disks; The first layer of the circular disc is provided with the liquid inlet hole (11) and the vent hole (12) penetrating the plate; The second layer of the disc is provided with all the chambers, channels and valve bodies of the detection unit that pass through the plate; All the chambers of the detection unit that pass through the plate are arranged on the third layer of the disc.

8. The centrifugal microfluidic chip according to claim 7, characterized in that: The lower surface of the first layer of the disc is laser engraved with the upper end structure of the capillary valve (6); The upper surface of the fourth layer of the disc is laser engraved with the lower end structure of the capillary valve (6).

9. A chemiluminescent immunoassay method, applied to the centrifugal microfluidic chip according to any one of claims 1 to 8, characterized in that: The method comprises: Injecting a sample into the sample storage chamber (1); Injecting antibody 1-magnetic beads into at least one group of the primary storage chambers (4); Injecting antibody 2-acridinium ester into at least one group of the primary storage chambers (4); injecting cleaning liquid into at least one group of the secondary storage chambers (5); Injecting pre-excitation liquid into at least one group of the secondary storage chambers (5); The rotation speed of the chip body is increased sequentially, so that the capillary valves (6) corresponding to the main channel (7) and the primary storage chamber (4) are opened sequentially, and the sample, the antibody 1-magnetic beads, and the antibody 2-acridinium ester are injected into the reaction chamber (2) in sequence; so that the antibody 1-magnetic beads and the antibody 2-acridinium ester are combined with the sample to form an immune complex; Controlling the chip body to rotate at a set acceleration to open the Euler force-driven siphon valve (8) corresponding to the cleaning liquid, thereby allowing the cleaning liquid to flow into the reaction chamber (2) to clean the immune complex and remove unbound substances; Controlling the addition of a magnetic field below the reaction chamber (2) to aggregate the immune complexes, and controlling the cessation of the addition of the magnetic field when the hydrophilic siphon valve (9) is turned on; Controlling the rotation of the chip body so that the waste liquid in the reaction chamber (2) flows into the waste liquid chamber (3) until all the waste liquid enters the waste liquid chamber (3), and controlling the chip body to stop rotating; Controlling the chip body to rotate at another set acceleration, so that the Euler force corresponding to the pre-excitation liquid drives the siphon valve (8) to conduct, thereby allowing the pre-excitation liquid to flow into the reaction chamber (2) to react with the immune complex until the pre-excitation liquid completely enters the reaction chamber (2), and controlling the chip body to stop rotating; An excitation liquid is injected into the reaction chamber (2), and the chemiluminescence value is detected in situ.

10. The chemiluminescent immunoassay method according to claim 9, characterized in that: The in situ detection of chemiluminescence values comprises: Building a darkroom; A photon counting probe (15) is provided directly below the reaction chamber (2), and a shutter (14) is installed between the reaction chamber (2) and the photon counting probe (15); The photon counting probe (15) is controlled to read the number of photons in the reaction chamber (2) and transmit the number to the host computer (17) for output.

11. The chemiluminescent immunoassay method according to claim 9, characterized in that: The rotation speed of the chip body is increased sequentially so that the capillary valves (6) corresponding to the main channel (7) and the primary storage chamber (4) are opened sequentially, thereby allowing the sample, the antibody 1-magnetic beads, and the antibody 2-acridinium ester to be injected into the reaction chamber (2) in sequence; so that the antibody 1-magnetic beads and the antibody 2-acridinium ester are combined with the sample to form an immune complex, comprising: When the sample, the antibody 1-magnetic beads and the antibody 2-acridinium ester are sequentially injected into the reaction chamber (2), the rotation speed of the chip body is controlled to continuously change so that the antibody 1-magnetic beads, the antibody 2-acridinium ester and the sample are shaken and mixed in the reaction chamber (2).

12. The chemiluminescent immunoassay method according to claim 9, characterized in that: Controlling the chip body to rotate at a set acceleration to open the Euler force-driven siphon valve (8) corresponding to the cleaning liquid, thereby allowing the cleaning liquid to flow into the reaction chamber (2), cleaning the immune complex, and removing unbound substances, including: After the cleaning liquid flows into the reaction chamber (2), the rotation speed of the chip body is controlled to continuously change so that the cleaning liquid and the immune complex are vibrated and mixed.

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