Centrifugal micro-fluidic chip and chemiluminescence immunoassay method

Abstract

The invention discloses a centrifugal micro-fluidic chip and a chemiluminescence immunodetection method, and relates to the technical field of micro-fluidic chips, specifically, the centrifugal micro-fluidic chip comprises a chip body of a rotary structure and a plurality of groups of detection units arranged in the chip body in an annular array; the detection unit is sequentially provided with a sample storage cavity, a reaction cavity and a waste liquid cavity from the near end to the far end of the rotation center of the chip body, the sample storage cavity is communicated with the reaction cavity through a main channel, the main channel is arranged in the radial direction of the chip body, and the reaction cavity is communicated with the waste liquid cavity through a hydrophilic siphon valve; according to the centrifugal micro-fluidic chip and the chemiluminescence immunoassay method, based on passive valve integration schemes of different triggering types, the risk of interference of all valves is reduced, control over the chip is more accurate and simpler, repeatability is better, the chip does not depend on external structure intervention, fluid driving and control are achieved only through the internal valves, and the chip is simple in structure, convenient to operate and high in practicability. And the method has considerable advantages in the aspect of realizing automation.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and more specifically, to a centrifugal microfluidic chip. In addition, 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, etc. Among them, chemiluminescence immunoassay has the characteristics of simple operation, high sensitivity, and stable results, which 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. 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 analysis workflow. The detection platform uses a simply configured photon counter, which 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, it is challenging to integrate multiple analytical steps based on immunomagnetic bead manipulation into centrifugal microfluidic chips, which limits the application of centrifugal microfluidics in chemiluminescence detection.

[0004] In the prior art, 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] At present, the commonly used solution for integrating immune response steps is to set multiple valves on the centrifugal microfluidic chip structure to allow continuous release of fluids in a controlled manner in time and space. For active valves such as laser-irradiated iron wax microvalve (LIFM), although it is very easy to control multiple microvalves, it inevitably increases the complexity of the system, which is one of the disadvantages for POCT applications. For centrifugal microfluidic chips using passive valves, most studies focus on using the same trigger type valve to achieve sequential control of multiple liquids, such as the integration of multiple capillary valves, serial hydrophilic siphon valves, multiple centrifugal-pneumatic valves, or the recently proposed Euler force-driven siphon valve. Although these technologies make full use of the characteristics of passive valves and can realize the opening of valves on demand, the phenomenon of overlapping valve opening conditions is still inevitable. For valves of the same trigger type, their driving principles are the same and only differ in critical conditions, which increases the difficulty of integration.

[0006] In summary, how to provide a centrifugal microfluidic chip and chemiluminescent 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, which is 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 to realize automation of the detection process, shorten the time required for detection, and effectively ensure 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, comprising a chip body of a rotary structure and a plurality of detection units arranged in a ring array in 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 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;

[0012] The detection unit further comprises 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 a capillary valve, and the secondary storage chambers are connected to the main channel and / or the reaction chamber via a siphon valve driven by Euler force;

[0013] The sample storage chamber, the reaction chamber, the primary storage chamber and the secondary storage chamber are respectively connected to each other with liquid inlet holes at one end close to the rotation center of the chip body, and 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 with air vents 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 connecting 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 sheet and a base sheet bonded by ultrasonic bonding, and a fixing hole is provided at the rotation center of the cover sheet and the base sheet;

[0018] The cover sheet is provided with the liquid inlet hole 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 sheet and the base sheet 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 disk is provided with the liquid inlet hole and the vent hole 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 penetrate the plate;

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

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

[0026] The upper surface of the fourth layer of the 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 liquid 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 increased sequentially, so that the capillary valves corresponding to the main channel and the primary storage chamber are turned on sequentially, and then the sample, the antibody 1-magnetic beads and the antibody 2-acridinium ester are sequentially injected into the reaction chamber; so that the antibody 1-magnetic beads and the antibody 2-acridinium ester are combined 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 turned on, thereby allowing the cleaning liquid to flow into the reaction chamber to clean the immune complex to remove unbound substances;

[0035] Controlling the addition of a magnetic field below the reaction chamber to aggregate the immune complexes, and controlling the stopping of the addition 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 be turned on, 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 value comprises:

[0040] Build 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 of photons to the host computer for output.

[0043] Preferably, the rotation speed of the chip body is increased sequentially so that the capillary valves corresponding to the main channel and the primary storage chamber are turned on sequentially, and then the sample, the antibody 1-magnetic beads and the antibody 2-acridinium ester are sequentially injected into the reaction chamber; so that the antibody 1-magnetic beads and the antibody 2-acridinium ester are combined 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 oscillated 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 turned on, 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 that the cleaning liquid and the immune complex are vibrated and mixed.

[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 chambers through valves of different forms. The corresponding valves and storage chambers can be connected to the reaction chambers under specific conditions, 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 into separate chambers respectively. During the reaction, they are gradually added into the reaction chamber, which effectively avoids the loss of freedom of magnetic beads while retaining the advantages of liquid phase reaction.

[0052] 2. During the process, various reagents are released sequentially, with less human 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

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

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

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

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

[0058] FIG5 is a schematic diagram of the structure of the fourth disk of the specific chip body provided by the present invention;

[0059] FIG6 is an exploded schematic diagram of a four-layer disc of a specific chip body provided by the present invention;

[0060] FIG7 is a schematic diagram of a specific chemiluminescent immunoassay method provided by the present invention;

[0061] FIG8 is a curve diagram of the rotation speed of the chip body in a specific chemiluminescent immunoassay process provided by the present invention;

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

[0063] Figure 1 - In Figure 9:

[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 liquid storage chamber; 502. Pre-excitation liquid 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 hole; 12. Vent hole; 13. Fixing hole; 14. Shutter; 15. Photon counting probe; 16. Counting unit; 17. Host computer. DETAILED DESCRIPTION

[0065] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work 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 a passive valve integration solution of different trigger types to reduce the risk of interference between valves. The chip control 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.

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

[0068] Please refer to Figure 1 - Figure 6, a centrifugal microfluidic chip, comprising a chip body with a rotating 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 rotation center of the chip body. The sample storage chamber 1 and the reaction chamber 2 are connected through a main channel 7, and 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;

[0070] The detection unit further includes a plurality of groups of primary storage chambers 4 and secondary storage chambers 5, wherein the primary storage chamber 4 is connected to the main channel 7 and / or the reaction chamber 2 via a capillary valve 6, and the secondary storage chamber 5 is connected to the main channel 7 and / or the reaction chamber 2 via an Euler force driven siphon valve 8;

[0071] 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 respectively 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 respectively provided with a vent hole 12.

[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 arranged on the chip body, that is, four groups of sample detection can be performed simultaneously, saving detection time;

[0073] The detection unit is integrated with a sample storage chamber 1 for storing initial samples, a primary storage chamber 4 for storing antibodies, and a secondary storage chamber 5 for storing cleaning solution and / or pre-excitation solution, so that the samples can be mixed in the chip body without mixing them first and then filling them into the chip body, thus ensuring 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 by a radially arranged main channel 7, so as to ensure that when the chip body rotates, the initial sample in the sample storage chamber 1 can be firstly filled into the reaction chamber 2;

[0075] The primary storage chamber 4 is connected to the reaction chamber 2 by a capillary valve 6. When the rotation speed of the chip body reaches the critical condition of the capillary valve 6, the capillary valve 6 is automatically turned on, so that the antibodies in the primary storage chamber 4 can be sequentially added to the reaction chamber 2 and react sequentially with the sample filled therein;

[0076] The secondary storage chamber 5 is connected to the reaction chamber 2 by using the Euler force driven siphon valve 8. When the rotation 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 the pre-excitation liquid is added 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, the capillary valve 6, and the Euler force-driven siphon valve 8 are different, the conduction order of the three valves can be controlled by controlling the rotation speed and rotation acceleration of the chip body, and the conduction conditions do not overlap, effectively avoiding any two valves from being turned on at the same time, thereby ensuring the accuracy of the order of various reagent injections, and the rotation speed and rotation acceleration of the chip body are easier to control, which is convenient for realizing the automation of detection;

[0078] At the same time, 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, respectively, so as to facilitate the filling of samples, antibodies, cleaning liquid and pre-excitation liquid in the initial stage and avoid the overflow of the liquid from the liquid inlet 11 during the centrifugation process;

[0079] Moreover, ventilation holes 12 are respectively 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 close to the rotation center of the chip body, so that when the liquid flows into the corresponding cavity, the original air in the corresponding cavity can be discharged to maintain the internal pressure balance and prevent 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 not equal;

[0081] By setting a plurality of groups of primary storage cavities 4 with different gyration radii, the gyration radii of the corresponding capillary valves 6 are also different. The 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 the capillary valves 6 with different gyration radii correspond to different chip body rotation speeds. That is, the conduction sequence of the primary storage cavities 4 with different gyration radii can be controlled by controlling the rotation speed of the chip body, so as to realize 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 acceleration of the chip body rotation, 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. 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 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 sheet is provided with a liquid inlet hole 11 and a vent hole 12 that penetrate through;

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

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

[0090] The fixing hole 13 is used to 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 ensured.

[0093] In some embodiments, Figure 2 - As shown in FIG6 , 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 disks;

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

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

[0097] As shown in FIG6 , the overall processing difficulty can be reduced by layering the disc. For example, laser engraving using PMMA sheets can help improve processing speed and accuracy, and different layers of discs can be bonded using pressure-sensitive adhesives to ensure the sealing of the contact surface.

[0098] In 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 engraved is selected to be 2mm.

[0099] In order to facilitate the connection between the chip body and the driving device, fixing holes 13 with different diameters can be opened in the centers of different layers of disks, such as a fixing hole 13 with a diameter of 8 mm is opened in the centers of the first, second and third layers of disks, and a fixing hole 13 with a diameter of 6 mm is opened in the center of the fourth layer of disks, so as to facilitate the connection and fixation between the chip body and the driving 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 diameter of the other vent holes 12 is designed to be 1mm.

[0101] In some embodiments, the lower surface of the first disc is laser engraved with the upper 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 disk, so as to further reduce the structural design difficulty of the second and third layers of the disk 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] The method is shown in Figures 7 to 9 and includes:

[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 increased sequentially, so that the capillary valves 6 corresponding to the main channel 7 and the primary storage chamber 4 are turned on sequentially, and then the sample, antibody 1-magnetic beads and antibody 2-acridinium ester are injected into the reaction chamber 2 sequentially; so that the antibody 1-magnetic beads and antibody 2-acridinium ester are combined with 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 be turned on, 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 stop of adding 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, until the pre-excitation liquid completely enters the reaction chamber 2, and the control chip body stops rotating;

[0116] The excitation liquid is injected into the reaction chamber 2, and the chemiluminescence value is detected in situ.

[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 provided with a first capillary valve 601 and a second capillary valve 602 respectively;

[0119] 50 μL of Ab1-beads and 50 μL of Ab2-acridinium ester are 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 are injected into the washing 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, in situ detection of chemiluminescent values ​​comprises:

[0125] Build a darkroom;

[0126] A photon counting probe 15 is arranged directly 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] As shown in FIG9 , in a dark room, it is convenient to accurately count the photons in the reaction chamber 2 , and a photon counting probe 15 is used for counting to ensure the efficiency and accuracy of 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 the 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 opened 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 increased sequentially, so that the capillary valves 6 corresponding to the main channel 7 and the primary storage chamber 4 are turned on sequentially, and then the sample, antibody 1-magnetic beads and antibody 2-acridinium ester are sequentially injected into the reaction chamber 2; so that the antibody 1-magnetic beads and antibody 2-acridinium ester are combined with the sample to form an immune complex, including:

[0131] After the sample, antibody 1-magnetic beads and 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, antibody 2-acridinium ester and the sample are oscillated 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 turned on, thereby allowing the cleaning liquid to flow to 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 that the cleaning liquid and the immune complex are shaken and mixed.

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

[0135] Fill each chamber with medicine through the liquid inlet hole 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, but the first capillary valve 601 and the second capillary valve 602 do not reach the critical speed corresponding to the conduction condition, so they are not conducted;

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

[0138] The rotation speed of the chip body is accelerated to 1600 RPM. At this time, the second capillary valve 602 reaches the critical speed condition, and 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 reagents are mixed by inertial force. Ab1 and Ab2 begin to combine with the markers in the sample to form immune complexes.

[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 vertex 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 driven at a 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, and 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, and then the chip body remains stationary to trigger the hydrophilic siphon valve 9, and the cleaning waste liquid 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 top 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 liquid 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, the 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 chemiluminescent 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 ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified in a number of ways, 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 rotating structure and a plurality of detection units arranged in a ring array in 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 via a main channel (7), the main channel (7) is arranged along the radial direction of the chip body, and the reaction chamber (2) and the waste liquid chamber (3) are connected via 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 respectively connected to each other and provided 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 and provided with a vent hole (12) at one end close to the rotation center of the chip body.

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: The secondary storage chambers (5) are at least two groups, and at least two groups of the 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 the 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 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; 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: The gap between the cover sheet and the base sheet around the detection unit is surrounded by bonding ribs.

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 ventilation 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 penetrate the plate; All the chambers of the detection unit that penetrate 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 disk is engraved with the upper end structure of the capillary valve (6) by laser; The upper surface of the fourth layer of the disc is engraved with the lower end structure of the capillary valve (6) by laser.

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 a 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 sequentially opened, and the sample, the antibody 1-magnetic beads and the antibody 2-acridinium ester are sequentially injected into the reaction chamber (2); 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, so that the Euler force-driven siphon valve (8) corresponding to the cleaning liquid is turned on, 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 stop of the addition of the magnetic field when the hydrophilic siphon valve (9) is turned on; Controlling the chip body 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 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 be turned on, 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 value comprises: Build a darkroom; A photon counting probe (15) is arranged 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 sequentially opened, thereby allowing the sample, the antibody 1-magnetic beads and the antibody 2-acridinium ester to be sequentially injected into the reaction chamber (2); 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 oscillated 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 change continuously so that the cleaning liquid and the immune complex are vibrated and mixed.

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