An efficient and assembleable chemiluminescent microfluidic chip and its application

Abstract

The invention relates to an efficient and assemblable chemiluminescent microfluidic chip and an application thereof. The microfluidic chip comprises: a whole blood sampling port, a magnetic bead and enzyme label sampling port, a cleaning solution and substrate sampling port, a mixing tank, a waste liquid tank, a plasma quantitative tank, a sample waste liquid tank, a blood cell collection tank, a solid phase valve, a detection tank and a magnetic bead collection tank; the whole blood sampling port is connected to the blood cell collection tank, the blood cell collection tank is respectively connected to the mixing tank and the plasma quantitative tank; the other end of the plasma quantitative tank is connected to the sample waste liquid tank; the magnetic bead and enzyme label sampling port, the cleaning solution and substrate sampling port are connected to the mixing tank; the mixing tank is connected to the detection tank; a solid phase valve is arranged in front of the detection tank; a magnetic bead collection tank is arranged behind the detection tank; and the detection tank is connected to the waste liquid tank.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and in particular to a highly efficient and assemblable chemiluminescent microfluidic chip and applications thereof. Background Art

[0002] Centrifugal microfluidic chips refer to chips that use centrifugal force to drive trace amounts of liquid, and use the centrifugal force generated by the centrifugal motor to drive the flow of liquid. Centrifugal microfluidic chips are often used to process batch samples. Under the action of centrifugal force, the liquid can sequentially fill multiple reaction tanks arranged on the same circumference, and can simultaneously perform single-sample multi-item joint testing or multi-sample multi-indicator testing. However, the number of flow channel units in centrifugal microfluidic chips is often limited by the size of the circular structure (the number of reaction tanks is determined by the same circumference). In addition, in order to increase the reaction sensitivity of chemiluminescence detection, magnetic beads are often used as solid phase carriers for capturing antibodies (magnetic beads have a large specific surface area). Controlling the movement of magnetic beads (for mixing or washing steps) requires control with an external magnetic field (electromagnet or permanent magnet), and its control system is relatively complex.

[0003] Therefore, how to realize a centrifugal microfluidic chip with a simple control system and high throughput has become a problem that needs to be solved urgently. Summary of the invention

[0004] To solve the above technical problems, the present invention provides an efficient and assemblable chemiluminescent microfluidic chip and its application, which cleverly uses the combination of centrifugal force and gravity to control the movement of liquid and magnetic beads. Compared with the existing technology, its control system is simpler and more efficient. The chip can detect at least 96 samples at the same time, thereby ensuring that the plasma in the plasma quantitative grooves within different circumferential ranges has the same volume.

[0005] To achieve this object, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides an efficient and assembled chemiluminescent microfluidic chip, which includes: a whole blood sampling port, a magnetic bead and enzyme label sampling port, a cleaning liquid and substrate sampling port, a mixing tank, a waste liquid tank, a plasma quantification tank, a sample waste liquid tank, a blood cell collection tank, a solid phase valve, a detection tank and a magnetic bead collection tank.

[0007] The whole blood sample adding port is connected to the blood cell collection tank, and the blood cell collection tank is respectively connected to the mixing tank and the plasma quantitative tank; the other end of the plasma quantitative tank is connected to the sample waste liquid tank.

[0008] The magnetic bead and enzyme label injection ports, cleaning solution and substrate injection ports are connected to the mixing tank; the mixing tank is connected to the detection tank; a solid phase valve is arranged in front of the detection tank; a magnetic bead collection tank is arranged behind the detection tank; the detection tank is connected to the waste liquid tank.

[0009] The present invention arranges the microfluidic chips in a circle with the central axis of the motor as the center, so that a large number of samples can be detected simultaneously.

[0010] The magnetic beads and enzyme label sample adding port can be used to add magnetic beads and enzyme labels respectively; the cleaning solution and substrate sample adding port can be used to add cleaning solution and substrate respectively.

[0011] Preferably, the whole blood sampling port, the magnetic bead and enzyme label sampling port, and the cleaning solution and substrate sampling port are arranged parallel to each other.

[0012] Preferably, the blood cell collection tank is used to collect blood cells in the lower layer separated due to density difference.

[0013] Preferably, the plasma quantitative tank is used to collect the upper layer of plasma separated due to density difference.

[0014] Preferably, the waste liquid tank is used to collect excess plasma flowing through the plasma quantitative tank.

[0015] Preferably, the volume of the plasma quantitative tank is 3-300 μL, for example, it can be 3 μL, 10 μL, 20 μL, 40 μL, 60 μL, 80 μL, 100 μL, 120 μL, 140 μL, 160 μL, 180 μL, 200 μL, 220 μL, 240 μL, 260 μL, 280 μL or 300 μL, etc.

[0016] Preferably, the solid phase valve is opened by controlling a laser device.

[0017] Preferably, the magnetic bead collection tank is used to collect the settled magnetic beads.

[0018] Preferably, the mixing tank is used for mixing samples, magnetic beads and enzyme labels.

[0019] Preferably, the waste liquid tank is used to collect waste liquid after detection.

[0020] Preferably, the detection groove is used for the reaction between the sample and the substrate.

[0021] In a second aspect, the present invention provides an efficient and assemblable chemiluminescent microfluidic system, comprising: the chemiluminescent microfluidic chip, base plate, motor, laser device and magnetic beads described in the first aspect.

[0022] Preferably, the motor is placed below the base plate.

[0023] Preferably, the microfluidic chip is vertically fixed above the bottom plate with the whole blood sampling port at the top.

[0024] Preferably, the microfluidic chips are arranged in at least three layers around the central axis of the motor; the first layer includes 1-12 microfluidic chips, the second layer includes at least 1-24 microfluidic chips, and the third layer includes at least 1-60 microfluidic chips. The 1-12 may be, for example, 1, 2, 4, 6, 8, 10 or 12; the 1-24 may be, for example, 1, 4, 8, 12, 16, 20 or 24; the 1-60 may be, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60.

[0025] The present invention arranges the microfluidic chips in a circular form, which can form an arrangement of at least three circles. The number of chips in each circle gradually increases from the center to the outside, so that a large number of samples can be processed simultaneously to achieve high-throughput detection.

[0026] In a third aspect, the present invention provides a detection method based on the efficient and assemblable chemiluminescent microfluidic system described in the second aspect, the detection method comprising: injecting the sample into the chip through the whole blood injection port, mixing and extracting by centrifugation and oscillation, then adding cleaning liquid and substrate through the cleaning liquid and substrate injection ports respectively, cleaning and reacting by centrifugation and oscillation, and then performing data detection.

[0027] In the present invention, each step of the operation is controlled only by changing the speed and oscillation frequency, and using gravity to control the movement of the liquid and the magnetic beads, thereby controlling the detection process. The control system of the detection method cleverly combines gravity and centrifugal force. The control system is relatively simple and can efficiently perform batch detection.

[0028] Preferably, the mixed extraction step includes: injecting the sample through the whole blood injection port, separating the blood at a first speed and then collecting blood cells through the blood cell collection tank, collecting plasma through the plasma quantitative tank, and collecting excess plasma flowing through the plasma quantitative tank through the sample waste liquid tank; using a second speed to separate the plasma and transfer it to the mixing tank; adding magnetic beads through the magnetic bead and enzyme label injection port, using a third speed to transfer the magnetic beads to the mixing tank, adding enzyme labels through the magnetic bead and enzyme label injection port, using a fourth speed to transfer the enzyme labels to the mixing tank, and then using a first oscillation for mixing; using a fifth speed to transfer the mixed liquid to the solid phase valve and then opening a laser device to dissolve the solid phase valve, the mixed liquid will enter the detection tank due to gravity, and the magnetic beads will settle in the magnetic bead collection tank due to gravity; using a sixth speed to collect the waste liquid.

[0029] The circumferential radius is the distance between the center of the microfluidic chip axis and the center axis of the motor.

[0030] When the radius position is greater than 10 mm, the relationship between the radius of the circle where the microfluidic chip is located and the separated volume is: R1 / R2∝V1 / V2.

[0031] R1 and R2 are the radii of the circles of different microfluidic chips, and V1 and V2 are the separation volumes of plasma in different microfluidic chips.

[0032] When the second speed is 500-2000 rpm, the relationship between the speed and the separated volume is: (S1 / S2) 2 ∝V1 / V2.

[0033] S1 and S2 are each independently the second rotation speed, and V1 and V2 are the separation volumes of plasma in different microfluidic chips.

[0034] The “∝” mentioned above refers to a proportional relationship.

[0035] In the present invention, in the operation of solution mixing extraction, different centrifugal forces are used to achieve the separation of blood cells and plasma in the blood, and the substances in the plasma are extracted and tested, and enzyme labels are added. The above operations can be achieved only by adjusting the speed, so that the method is suitable for non-technical personnel in this field, which greatly expands the application scenarios of the detection method.

[0036] Preferably, the added amount of the sample is 5-100 μL, for example, 5 μL, 10 μL, 30 μL, 60 μL or 100 μL.

[0037] Preferably, the amount of the magnetic beads added is 10-500 μL, for example, 10 μL, 20 μL, 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL or 500 μL, etc.

[0038] Preferably, the amount of the enzyme label added is 10-500 μL, for example, it can be 10 μL, 20 μL, 50 μL, 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL or 500 μL.

[0039] Preferably, the first speed is 500-10000 rpm, and the time is 5-3600 s. The 500-10000 rpm may be, for example, 500 rpm, 1000 rpm, 3000 rpm, 6000 rpm, 9000 rpm or 10000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0040] Preferably, the second speed is 500-5000 rpm, and the time is 5-3600 s. The 500-5000 rpm may be, for example, 500 rpm, 1000 rpm, 1500 rpm, 2000 rpm, 2500 rpm, 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm or 5000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0041] Preferably, the third speed is 500-5000 rpm, and the time is 5-3600 s. The 500-5000 rpm may be, for example, 500 rpm, 1000 rpm, 2000 rpm, 3000 rpm or 5000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0042] Preferably, the fourth speed is 500-5000 rpm, and the time is 5-3600 s. The 500-5000 rpm may be, for example, 500 rpm, 1000 rpm, 2000 rpm, 3000 rpm or 5000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0043] Preferably, the fifth speed is 500-5000 rpm, and the time is 5-3600 s. The 500-5000 rpm may be, for example, 500 rpm, 1000 rpm, 2000 rpm, 2500 rpm, 3000 rpm, 4000 rpm or 5000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0044] Preferably, the sixth speed is 500-5000 rpm, and the time is 5-3600 s. The 500-5000 rpm may be, for example, 500 rpm, 1000 rpm, 2000 rpm, 3000 rpm or 5000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0045] Preferably, the sedimentation time of the magnetic beads is 30-60 s, for example, it can be 30 s, 35 s, 40 s, 45 s, 50 s, 55 s or 60 s.

[0046] Preferably, the frequency of the first oscillation is 1-20 Hz, the amplitude is 10-360°, and the time is 5-3600 s. The 1-20 Hz may be, for example, 1 Hz, 4 Hz, 8 Hz, 12 Hz, 16 Hz or 20 Hz, etc.; the 10-360° may be, for example, 10°, 50°, 100°, 150°, 200°, 250°, 300°, 330° or 360°, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0047] Preferably, the cleaning and reaction steps include: adding cleaning liquid through the cleaning liquid and substrate injection port, using the seventh rotation speed to transfer the cleaning liquid to the detection tank, using the second oscillation for cleaning, using the eighth rotation speed to collect waste liquid and magnetic beads sedimentation; adding substrate and using the ninth rotation speed to transfer the substrate bed to the detection tank, and mixing with the third oscillation.

[0048] Preferably, the amount of the substrate added is 10-500 μL, for example, 10 μL, 50 μL, 100 μL, 300 μL or 500 μL.

[0049] Preferably, the seventh speed is 500-5000 rpm, and the time is 5-3600 s. The 500-5000 rpm may be, for example, 500 rpm, 1000 rpm, 2000 rpm, 3000 rpm or 5000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0050] Preferably, the eighth speed is 500-5000 rpm, and the time is 5-3600 s. The 500-5000 rpm may be, for example, 500 rpm, 1000 rpm, 2000 rpm, 3000 rpm or 5000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0051] Preferably, the ninth speed is 500-5000 rpm, and the time is 5-3600 s. The 500-5000 rpm may be, for example, 500 rpm, 1000 rpm, 2000 rpm, 3000 rpm or 5000 rpm, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0052] Preferably, the sedimentation time of the magnetic beads is 30-60 s, for example, it can be 30 s, 35 s, 40 s, 45 s, 50 s, 55 s or 60 s.

[0053] Preferably, the frequency of the second oscillation is 1-20 Hz, the amplitude is 10-360°, and the time is 5-3600 s. The 1-20 Hz may be, for example, 1 Hz, 4 Hz, 8 Hz, 12 Hz or 20 Hz, etc.; the 10-360° may be, for example, 10°, 50°, 100°, 150°, 200°, 250°, 300°, 330° or 360°, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s or 3600 s, etc.

[0054] Preferably, the third oscillation has a frequency of 1-20 Hz, an amplitude of 10-360°, and a duration of 5-3600 s. The 1-20 Hz may be, for example, 1 Hz, 4 Hz, 8 Hz, 12 Hz, or 20 Hz, etc. The 10-360° may be, for example, 10°, 50°, 100°, 150°, 200°, 250°, 300°, 330°, or 360°, etc. The 5-3600 s may be, for example, 5 s, 10 s, 100 s, 1000 s, 1500 s, 2000 s, 2500 s, 3000 s, 3500 s, or 3600 s, etc.

[0055] Preferably, the data detection step includes: converting the photon signal into an electronic signal through a photomultiplier tube to obtain a sample concentration value.

[0056] In the present invention, photon signals are converted into electronic signals to obtain the concentration value of the sample. The electronic signal method can accurately and quickly obtain sample information, which is more suitable for high-throughput detection in the present invention.

[0057] In a fourth aspect, the present invention provides an application of the highly efficient and assemblable chemiluminescent microfluidic chip described in the first aspect, the detection method of the highly efficient and assemblable chemiluminescent microfluidic chip described in the second aspect, or the detection method based on the highly efficient and assemblable chemiluminescent microfluidic system described in the third aspect in blood testing.

[0058] Compared with the prior art, the present invention has at least the following beneficial effects:

[0059] 1. The present invention arranges the microfluidic chips in a circular form, which can form an arrangement of at least three circles, and the number of chips gradually increases from the center to the outside of the circle, so that a large number of samples can be processed simultaneously to achieve high-throughput detection.

[0060] 2. In the present invention, the detection process is divided into three steps. Each step is controlled by only changing the speed and oscillation frequency and using gravity to control the movement of the liquid and the magnetic beads, thereby controlling the detection process. The control system of the detection method cleverly combines gravity and centrifugal force. The control system is relatively simple and can efficiently perform batch detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a design diagram of a microfluidic chip.

[0062] Figure 2 It is a schematic diagram of the device of microfluidic chip. DETAILED DESCRIPTION

[0063] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0064] The terms "first", "second", "third", "fourth", etc. (if any) in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0065] like Figure 1The design diagram of the microfluidic chip shown in the figure, 1 is the whole blood sampling port, 2 is the magnetic bead and enzyme label sampling port, 3 is the cleaning solution and substrate sampling port, 4 is the mixing tank, 5 is the waste liquid tank, 6 is the plasma quantification tank, 7 is the sample waste liquid tank, 8 is the blood cell collection tank, 9 is the solid phase valve, 10 is the detection tank and 11 is the magnetic bead collection tank.

[0066] The whole blood sampling port 1, the magnetic bead and enzyme label sampling port 2 and the cleaning liquid and substrate sampling port 3 are arranged side by side and vertically above the motor. The whole blood sampling port 1 is connected to the blood cell collection tank 8, and the blood cell collection tank 8 is respectively connected to the mixing tank 4 and the plasma quantitative tank 6, and the plasma quantitative tank 6 is respectively connected to the sample waste liquid tank 7 and the mixing tank 4. The magnetic bead and enzyme label sampling port 2 and the cleaning liquid and substrate sampling port 3 are respectively connected to the mixing tank 4, and the mixing tank 4 is connected to the detection tank 10, and there is a solid phase valve 9 in front of the detection tank 10, and a magnetic bead collection tank 11 is set behind the detection tank 10. The detection tank 10 is connected to the waste liquid tank 5.

[0067] like Figure 2 As shown, 12 is a microfluidic chip, 13 is a motor and 14 is a base plate. The microfluidic chip 12 is located on the base plate 14 and is placed above the motor 13. The microfluidic chips 12 are arranged in a circle with the central axis of the motor 13 as the center, and three or more circles are arranged on the base plate 14. The first circle includes 12 microfluidic chips, the second circle includes 24 microfluidic chips, and the third circle includes 60 microfluidic chips.

[0068] Example 1

[0069] This embodiment provides a detection method of an efficient and assembled chemiluminescent microfluidic chip for detecting procalcitonin, which specifically comprises the following steps:

[0070] (1) Extraction of procalcitonin from blood

[0071] The procalcitonin detection kit (Suzhou Guoke Junhao, catalog number 1100124012) was used for detection. 50 μL of whole blood sample was added to the microfluidic chip through the whole blood sample port 1, and the microfluidic chip was operated at 5000 rpm for rotation and centrifugation for 60 seconds. The excess whole blood will flow to the sample waste liquid tank 7. The whole blood is separated into upper plasma and lower blood cells due to density difference, and the blood cells are retained in the blood cell collection tank 8. After the plasma separation is completed, the microfluidic chip is operated at 3000 rpm for rotation and centrifugation for 15 seconds, and 15 μL of plasma will be transferred from the plasma quantitative tank 6 to the mixing tank 4. 15μL of magnetic beads were injected into the microfluidic chip through the magnetic beads and enzyme label injection port 2, and the microfluidic chip was controlled to rotate and centrifuge for 10s at 1000rpm. The magnetic beads were transferred to the mixing tank 4. 15μL of enzyme labels were injected into the microfluidic chip through the magnetic beads and enzyme label injection port 2, and the microfluidic chip was controlled to rotate and centrifuge for 10s at 1000rpm. The enzyme labels were transferred to the mixing tank 4. The microfluidic chip was controlled to shake back and forth 60 times at a frequency of 5Hz and an amplitude of 120° for 420s to fully mix the sample, magnetic beads, and enzyme labels.

[0072] After the mixing is completed, the microfluidic chip is controlled to rotate and centrifuge for 15 seconds at 3000 rpm, and the sample mixture will be transferred to the solid phase valve 9. The rotation is stopped and the laser device is turned on to dissolve the solid phase valve 9. The sample mixture will be transferred to the detection tank 10 due to gravity. The magnetic beads will settle in the magnetic bead collection tank 11 due to their high density. After the magnetic beads settle for 30 seconds, the microfluidic chip is controlled to rotate and centrifuge for 20 seconds at 1000 rpm, and the sample waste liquid will be transferred to the waste liquid tank 5.

[0073] (2) Cleaning

[0074] 50 μL of cleaning solution is injected into the microfluidic chip through the cleaning solution and substrate injection port 3, and the microfluidic chip is controlled to rotate and centrifuge for 10 seconds at 1200 rpm, and the cleaning solution will be transferred to the detection tank 10. The microfluidic chip is controlled to shake back and forth 60 times at a frequency of 3 Hz and an amplitude of 120° for 120 seconds to fully clean. After the cleaning is completed, the rotation is stopped, and the magnetic beads are precipitated in the magnetic bead collection tank 11 due to their high density. After the magnetic beads are precipitated for 30 seconds, the microfluidic chip is controlled to rotate and centrifuge for 20 seconds at 1000 rpm, and the sample waste liquid will be transferred to the waste liquid tank 5.

[0075] 30 μL of alkaline phosphatase is injected into the microfluidic chip through the cleaning solution and substrate injection port 3, and the microfluidic chip is controlled to rotate and centrifuge for 10 seconds at 1200 rpm, and the substrate will be transferred to the detection tank 10. The microfluidic chip is controlled to shake back and forth 60 times at a frequency of 3 Hz and an amplitude of 120° for 60 seconds to fully mix the reaction. After the reaction is completed, the rotation is stopped, and the magnetic beads are precipitated in the magnetic bead collection tank 11 due to their high density.

[0076] (3) Data detection

[0077] After the magnetic beads have settled for 30 seconds, the photon signal can be converted into an electronic signal through a photomultiplier tube (PMT), and the sample concentration value is finally obtained through software calculation. The sample concentration is 0.3ng / mL, and the positive standard in the instructions is 0.3ng / mL, which means the result is negative.

[0078] Example 2

[0079] This example is used to explore the whole blood separation effect verification under different circle radii.

[0080] This experiment verifies the whole blood separation efficiency based on different radius positions, that is, different centrifugal forces. The first speed is 5000rpm (acceleration 10000rpm / s), and the centrifuge rotates for 60s; the second speed is 1000rpm (acceleration 5000rpm / s), and the centrifuge rotates for 10s; the chip storage slot is 5mm wide and 5mm deep. The first radius position is 5mm, the second radius position is 10mm, the third radius position is 20mm, and the fourth radius position is 40mm.

[0081] The sample used for the test was a whole blood sample from a 23-year-old woman with a hemolysis ratio of 48%. The amount of whole blood added to each microfluidic chip was 50 μL, the separation speed was 5000 rpm, and the rotation was 60 seconds. A high-speed camera was used to shoot and quantify the blood cell ratio to determine the separation efficiency.

[0082] The results show that: the first radius position is 5 mm, 1.87 μL of plasma is separated; the second radius position is 10 mm, 6.71 μL of plasma is separated; the third radius position is 20 mm, 14.23 μL of plasma is separated; the fourth radius position is 40 mm, 26.21 μL of plasma is separated. From the above four sets of data, we can see that the fourth radius position is 4 times the second radius position (fourth radius / second radius=40mm / 10mm=4), and the separated plasma volume multiple is also close to 4 times (the plasma volume separated by the microfluidic chip at the fourth radius / the plasma volume separated by the microfluidic chip at the second radius=26.21μL / 6.71μL=3.91); the third radius position is 2 times the second radius position (third radius / second radius=20mm / 10mm=4), and the separated plasma volume multiple is also close to 2 times (the plasma volume separated by the microfluidic chip at the third radius / the plasma volume separated by the microfluidic chip at the second radius=14.23μL / 6.71μL=2.12). The second radius position is twice the first radius position (second radius / first radius=10mm / 5mm=2), but the plasma volume multiple separated is not proportional to the multiple of the radius position (plasma volume separated by the microfluidic chip at the second radius / plasma volume separated by the microfluidic chip at the first radius=6.71μL / 1.87μL=2.76). The experimental results show that when the radius position is greater than 10mm, the radius position and separation efficiency show a square relationship.

[0083] Example 3

[0084] This example is used to explore the effect of different speeds on whole blood separation.

[0085] This experiment verifies the efficiency of whole blood separation based on different rotation speeds, i.e. different centrifugal forces. The first speed is 5000rpm, acceleration is 10000rpm / s, and rotation is 60s; the second acceleration is 5000rpm / s, and rotation is 10s; the chip radius position is 10mm, and the chip storage slot width is 5mm and depth is 5mm.

[0086] The sample used for the test was a whole blood sample from a 23-year-old woman with a hemolysis ratio of 48%. The amount of whole blood added to each microfluidic chip was 50 μL, the separation speed was 5000 rpm / s, and the rotation was 60 seconds. A high-speed camera was used to shoot and quantify the blood cell ratio to determine the separation efficiency. In order to explore the effect of different speeds on the whole blood separation effect, the second rotation speed was 500 rpm, 1000 rpm, 2000 rpm and 4000 rpm for experiments.

[0087] Experimental results: When the second rotation speed was 500rpm (S1), 1.62μL of plasma was separated; when the second rotation speed was 1000rpm (S2), 6.71μL of plasma was separated; when the second rotation speed was 2000rpm (S3), 25.98μL of plasma was separated; when the second rotation speed was 4000rpm (S4), 25.72μL of plasma was separated.

[0088] From the above four sets of data, we know that S1 is twice that of S2 (S1 / S2=1000 / 500=2), and the volume of plasma separated is nearly 4 times (the volume of plasma separated when the second rotation speed of the microfluidic chip is S2 / the volume of plasma separated when the second rotation speed of the microfluidic chip is S1=6.71μL / 1.62μL=4.14); S3 is 4 times that of S1 (S3 / S1=2000 / 500=4), and the volume of plasma separated is nearly 16 times (the volume of plasma separated when the second rotation speed of the microfluidic chip is S3 / the volume of plasma separated when the second rotation speed of the microfluidic chip is S1). The second rotation speed of the microfluidic chip is S1, and the separated plasma volume = 25.98μL / 1.62μL=16.03); S4 is 8 times that of S1 (S4 / S1=4000 / 500=8), and the separated plasma volume is close to 16 times (the separated plasma volume of the microfluidic chip at the second rotation speed of S4 / the separated plasma volume of the microfluidic chip at the second rotation speed of S1 = 25.72μL / 1.62μL=15.87). The experimental results show that when the second rotation speed is less than 2000rpm, the rotation speed and separation efficiency show a quadratic relationship.

[0089] In summary, the present invention provides an efficient and assemblable chemiluminescent microfluidic chip and its application, which can cleverly use the combination of centrifugal force and gravity to control the movement of liquid and magnetic beads. Compared with the existing technology, its control system is simpler and more efficient, and the chip can detect at least 96 samples simultaneously.

[0090] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An efficient and assembleable chemiluminescent microfluidic system, characterized in that: The chemiluminescent microfluidic system comprises: a chemiluminescent microfluidic chip, a base plate, a motor, a laser device and magnetic beads; The chemiluminescent microfluidic chip comprises: a whole blood sample loading port (1), a magnetic bead and enzyme label sample loading port (2), a cleaning solution and substrate sample loading port (3), a mixing tank (4), a waste liquid tank (5), a plasma quantitative tank (6), a sample waste liquid tank (7), a blood cell collection tank (8), a solid phase valve (9), a detection tank (10) and a magnetic bead collection tank (11); The microfluidic chip is fixed vertically above the bottom plate with the whole blood sampling port (1) at the top; The microfluidic chips are arranged in at least three layers around the central axis of the motor; the first layer includes 1 to 12 microfluidic chips, the second layer includes at least 1 to 24 microfluidic chips, and the third layer includes at least 1 to 60 microfluidic chips; The solid phase valve (9) is opened by controlling the laser device; The magnetic bead collection tank (11) is used to collect the settled magnetic beads; The blood cell collection tank (8) is used to collect the blood cells in the lower layer separated due to density difference; The plasma quantitative tank (6) is used to collect the upper layer of plasma separated due to density difference; The mixing tank (4) is used for mixing samples, magnetic beads and enzyme labels; The whole blood sample addition port (1) is connected to a blood cell collection tank (8), and the blood cell collection tank (8) is respectively connected to a mixing tank (4) and a plasma quantitative tank (6); the other end of the plasma quantitative tank (6) is connected to a sample waste liquid tank (7); The magnetic bead and enzyme label injection port (2) and the cleaning solution and substrate injection port (3) are connected to a mixing tank (4); the mixing tank (4) is connected to a detection tank (10); a solid phase valve (9) is provided in front of the detection tank (10); a magnetic bead collection tank (11) is provided behind the detection tank (10); and the detection tank (10) is connected to a waste liquid tank (5).

2. The highly efficient and assemblable chemiluminescent microfluidic system according to claim 1, characterized in that: The whole blood sample loading port (1), the magnetic bead and enzyme label sample loading port (2), and the cleaning liquid and substrate sample loading port (3) are arranged parallel to each other; The waste liquid tank (5) is used to collect excess plasma flowing through the plasma quantitative tank; The volume of the plasma quantitative tank (6) is 3-300 μL.

3. The highly efficient and assemblable chemiluminescent microfluidic system according to claim 1, characterized in that: The waste liquid tank (5) is used to collect waste liquid after detection; The detection groove (10) is used for the reaction between the sample and the substrate.

4. The highly efficient and assemblable chemiluminescent microfluidic system according to claim 1, characterized in that: The motor is placed under the bottom plate.

5. A detection method based on the highly efficient and assemblable chemiluminescent microfluidic system according to any one of claims 1 to 4, characterized in that: The detection method comprises: injecting a sample into a chip through a whole blood sample injection port (1), performing mixed extraction by centrifugation and oscillation, respectively adding a cleaning solution and a substrate through a cleaning solution and substrate sample injection port (3), performing cleaning and reaction by centrifugation and oscillation, and then performing data detection.

6. The detection method of the highly efficient and assembled chemiluminescent microfluidic system according to claim 5, characterized in that: The mixed extraction step comprises: injecting a sample through a whole blood sample injection port (1), separating the blood at a first rotation speed, collecting blood cells through a blood cell collection tank (8), collecting plasma through a plasma quantitative tank (6), and collecting excess plasma flowing through the plasma quantitative tank through a sample waste liquid tank (7); separating the plasma and transferring it to a mixing tank (4) at a second rotation speed; adding magnetic beads through a magnetic bead and enzyme label injection port (2), transferring the magnetic beads to the mixing tank (4) at a third rotation speed, adding enzyme labels through a magnetic bead and enzyme label injection port (2), transferring the enzyme labels to the mixing tank (4) at a fourth rotation speed, and then mixing by a first oscillation; transferring the mixed solution to a solid phase valve (9) at a fifth rotation speed, and then opening a laser device to dissolve the solid phase valve (9), so that the mixed solution enters a detection tank (10) due to gravity, and the magnetic beads settle in a magnetic bead collection tank (11) due to gravity; and collecting the waste liquid at a sixth rotation speed; The added amount of the sample is 5-100 μL; The amount of magnetic beads added is 10-500 μL; The amount of enzyme label added is 10-500 μL; The first speed is 500-10000 rpm, and the time is 5-3600 s; The second speed is 500-5000rpm, and the time is 5-3600s; The third speed is 500-5000 rpm, and the time is 5-3600 s; The fourth speed is 500-5000 rpm, and the time is 5-3600 s; The fifth speed is 500-5000 rpm, and the time is 5-3600 s; The sixth speed is 500-5000 rpm, and the time is 5-3600 s; The time for the magnetic beads to settle is 30-60s; The frequency of the first oscillation is 1-20 Hz, the amplitude is 10-360°, and the duration is 5-3600 s.

7. The detection method of the highly efficient and assembled chemiluminescent microfluidic system according to claim 6, characterized in that: The steps of washing and reacting include: adding washing liquid through the washing liquid and substrate injection port (3), transferring the washing liquid to the detection tank (10) at a seventh rotation speed, washing at a second rotation speed, collecting waste liquid and settling magnetic beads at an eighth rotation speed; adding substrate, transferring the substrate bed to the detection tank (10) at a ninth rotation speed, and mixing at a third rotation speed; The amount of the substrate added is 10-500 μL; The seventh speed is 500-5000 rpm, and the time is 5-3600 s; The eighth speed is 500-5000 rpm, and the time is 5-3600 s; The ninth speed is 500-5000 rpm, and the time is 5-3600 s; The time for the magnetic beads to settle is 30-60s, and the time is 5-3600s; The frequency of the second oscillation is 1-20 Hz, the amplitude is 10-360°, and the duration is 5-3600 s; The third oscillation has a frequency of 1-20 Hz, an amplitude of 10-360°, and a duration of 5-3600 s.

8. The detection method of the highly efficient and assembled chemiluminescent microfluidic system according to claim 6, characterized in that: The data detection step includes: converting the photon signal into an electronic signal through a photomultiplier tube to obtain the sample concentration value.

9. Application of the detection method of the efficient assemblable chemiluminescent microfluidic system according to any one of claims 1 to 4 or the efficient assemblable chemiluminescent microfluidic system according to any one of claims 5 to 8 in blood detection.

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