Centrifugal microfluidic chip

Abstract

The present invention discloses a centrifugal microfluidic chip, the detection chip has a rotation center, and the detection chip includes at least one detection unit that can independently perform detection, the detection unit includes a mixed cultivation tank for providing a reaction space, a spring is fixedly arranged below the outside of the detection chip, and the free end of the spring is connected to a magnet; when the detection chip does not rotate, the magnet is outside the working area; when the detection chip rotates around the rotation center at a certain speed, the magnet completely covers the working area. The present invention operates the magnetic particles in the mixed cultivation tank by designing a mobile magnetic field, and during the flushing process, an attraction is generated on the magnetic particles in the mixed cultivation tank, so that the magnetic particles can be kept in the mixed cultivation tank and not washed away; the present invention pre-seals solid reagents and liquid reagents in the chip, and only needs to inject the sample once to automatically complete the complex sample addition and reaction steps.

Description

Technical Field

[0001] The invention relates to the technical field of microfluidic chips, and in particular to a centrifugal microfluidic chip. Background Art

[0002] In order to simplify the lengthy and complicated defects of traditional detection steps, microfluidic chip technology is often combined for detection (such as biochemical detection, immune detection, molecular detection, etc.). However, there are still many difficulties that cannot be overcome in microfluidic detection chips. (i) The sample addition steps are complicated (the detection process often requires multiple sample addition steps, such as: samples, quality control products / calibrators, diluents, cleaning solutions, multiple reagents, etc.). (ii) In the prior art, antibodies / antigens that need to participate in the reaction are often connected to the surface of magnetic particles to react with the samples in the reaction tank. In the cleaning step, in order to prevent the magnetic particles from being washed out of the reaction tank, the commonly used solution is: a magnetic field is generated in the reaction tank area through a magnetic field generating device, and the magnetic field is controlled to be added to the reaction tank during the cleaning stage to retain the magnetic particles in the reaction tank through magnetic force. In other stages, such as the mixing stage, the magnetic field needs to be removed. The disadvantages of this solution are: it is necessary to increase the step of controlling the presence or absence of the magnetic field, which increases the control operation of the reaction process; because it is necessary to control the addition or removal of the magnetic field, usually either a moving mechanism is used to control the movement of the magnetic field or an electrically controlled magnetic field generating device is used, which will lead to increased equipment complexity and cost.

[0003] Therefore, how to design the structure of the microfluidic chip to solve at least one of the above problems in order to integrate the traditional lengthy and complicated detection scheme into a simple, fast, and industrial mass production-suitable detection scheme is an important topic. Summary of the invention

[0004] In order to solve at least one of the above technical problems, the present invention provides a centrifugal microfluidic chip.

[0005] The technical solution adopted by the present invention is: a centrifugal microfluidic chip, the detection chip has a rotation center, and the detection chip includes at least one detection unit that can independently perform detection, the detection unit includes a mixed cultivation tank for providing a reaction space, a spring is fixedly arranged below the outside of the detection chip, and the free end of the spring is connected to a magnet;

[0006] Wherein, when the detection chip is not rotating, there is no overlapping area between the projection of the magnet and the mixed cultivation groove directly below along the thickness direction of the detection chip; when the detection chip rotates at a certain speed around the rotation center, the magnet compresses or stretches the free end of the spring due to the centrifugal effect, so that the projection area of ​​the mixed cultivation groove directly below can be completely covered by the projection area of ​​the magnet along the thickness direction of the detection chip;

[0007] Wherein, the extension line of the spring in the compression or extension direction passes through the rotation center.

[0008] Preferably, the fixed end of the spring is far away from the rotation center, and the free end of the spring and the magnet are close to the rotation center;

[0009] When the detection chip does not rotate, the spring is in a natural state, and the magnet on the free end of the spring is directed toward the rotation center and passes over the area directly below the mixed cultivation tank along the thickness direction of the detection chip;

[0010] When the detection chip rotates, the spring is compressed, and the magnet moves to the area directly below the mixed cultivation trough in the direction away from the rotation center, and the projection area of ​​the mixed cultivation trough directly below the detection chip along the thickness direction of the detection chip is completely covered by the projection area of ​​the magnet along the thickness direction of the detection chip.

[0011] Preferably, the fixed end of the spring is close to the rotation center, and the free end of the spring and the magnet are far away from the rotation center;

[0012] When the detection chip does not rotate, the spring is in a natural state, and the magnet on the free end of the spring is located in an area between the rotation center and the projection area of ​​the mixed culture tank directly below the detection chip in the thickness direction;

[0013] When the detection chip rotates, the spring is stretched, and the magnet moves to the area directly below the mixed cultivation trough in the direction away from the rotation center, and the projection area of ​​the mixed cultivation trough directly below the detection chip along the thickness direction is completely covered by the projection area of ​​the magnet along the thickness direction of the detection chip.

[0014] Preferably, the detection unit also includes a whole blood storage tank, a whole blood injection hole connected to the whole blood storage tank for injecting a blood sample to be tested, a plasma serum quantification tank connected to the whole blood storage tank through a whole blood transmission channel, a plasma serum transmission channel connecting the plasma serum quantification tank to the mixed incubation tank, a waste liquid tank connected to the mixed incubation tank through a waste liquid transmission channel, and a vent connected to the waste liquid tank.

[0015] Preferably, the whole blood transmission channel is connected to a residual blood cell storage tank at a position upstream of the quantitative tank;

[0016] An overflow port is arranged on one side of the plasma serum quantitative tank close to the rotation center, and the overflow port is connected to a plasma serum waste liquid tank.

[0017] Preferably, the whole blood transmission channel comprises a radial segment channel connected to the outlet end of the whole blood storage tank and a circumferential segment channel connected to the end of the radial segment channel, the end of the circumferential segment channel is connected to the plasma serum quantitative tank, and the plasma serum quantitative tank is located on a side of the circumferential segment channel close to the rotation center;

[0018] The remaining blood cell storage groove is communicated with the middle part of the circumferential section channel, and the remaining blood cell storage groove is located on a side of the circumferential section channel away from the rotation center.

[0019] Preferably, a microfluidic valve is provided on the waste liquid transmission channel connected to the outlet end of the mixed cultivation tank;

[0020] The plasma serum transmission channel has a first inverted U-shaped section, and the waste liquid transmission channel has a second inverted U-shaped section.

[0021] Preferably, a solid reagent storage tank storing N different solid reagents is provided in the mixed culture tank;

[0022] The solid reagent includes first, second, ..., and Nth solid reagents, each of which is coated with a protective film on the outside. The first solid reagent is coated with a first protective film on the outside, and the second solid reagent is coated with a first protective film and a second protective film in sequence from the outside to the inside. By analogy, the Nth solid reagent is coated with a first protective film, a second protective film, ..., and an Nth protective film in sequence from the outside to the inside.

[0023] The first to Nth protective films can all be dissolved in the mixed incubation tank under the action of the dissolving reagent provided by the detection unit, so that the protective films coated on the outside of the first to Nth solid reagents have different dissolution times.

[0024] Preferably, it further comprises at least one dissolving reagent storage tank and at least one reaction reagent storage tank communicated with the mixed incubation tank;

[0025] The first to Nth protection films are formed of the same material that can be dissolved by the dissolving reagent stored in the dissolving reagent storage tank.

[0026] Preferably, the liquid reagents in the dissolving reagent storage tank and the reaction reagent storage tank are sealed and packaged in pre-sealed bags, and the pre-sealed bags can be partially ruptured under the action of external force to release the liquid reagents therein and provide them to the mixed incubation tank.

[0027] Preferably, the body of the detection chip comprises a bottom plate, an upper cover and a microfluidic channel opened on the bottom plate, and the upper cover is sealed and buckled on the bottom plate, so that the microfluidic channel between the bottom plate and the upper cover forms the detection unit;

[0028] The upper cover above the liquid reagent storage tank is provided with a puncture hole, and the pre-sealed bag can be punctured through the puncture hole by an external puncturing device to release the liquid reagent therein.

[0029] The present invention at least includes the following beneficial effects:

[0030] 1. The centrifugal microfluidic chip of the present invention operates the magnetic particles in the mixed cultivation tank by designing a mobile magnetic field. During the flushing process, the magnetic particles in the mixed cultivation tank are attracted, so that the magnetic particles can be kept in the mixed cultivation tank and not washed away; the whole process is automatically matched according to whether the rotation condition is required during the reaction process, and no additional control operation of the entry and withdrawal of the magnetic field is required, that is, during flushing, the detection chip needs to be rotated, and the magnetic field can be added to the working area by rotation to attract the magnetic particles; when mixing, the magnetic field is not required, and the detection chip does not need to rotate, and the magnet can automatically withdraw from the working area;

[0031] 2. The present invention pre-seales solid reagents and liquid reagents in the chip, and only requires one injection of the sample to automatically complete the complex sample addition and reaction steps;

[0032] 3. The present invention only requires a small amount of sample (tens of microliters) for detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic diagram of the structure of a centrifugal microfluidic chip in the general concept of the present invention;

[0034] Figure 2 A schematic diagram of scheme 1 of a mobile magnetic field in the general concept of the present invention;

[0035] Figure 3 A schematic diagram of scheme 2 of a mobile magnetic field in the general concept of the present invention;

[0036] Figure 4 is a schematic structural diagram of a detection unit in Example 1 of the present invention;

[0037] Figure 5 Schematic diagram of the structure of the centrifugal microfluidic chip in Example 1 of the present invention;

[0038] Figure 6 This is a schematic diagram of the principle of Example 2 of the present invention.

[0039] Description of reference numerals:

[0040] 1—whole blood injection hole; 2—whole blood storage tank; 3—whole blood transmission channel; 4—residual blood cell storage tank; 5—plasma serum waste liquid tank; 6—plasma serum quantitative tank; 7—plasma serum transmission channel; 8—dissolving reagent storage tank; 9—first reaction reagent storage tank; 10—second reaction reagent storage tank; 11—mixing incubation tank; 12—solid reagent storage tank; 13—microfluidic valve; 14—waste liquid transmission channel; 15—waste liquid tank; 16—vent; 70—first inverted U-shaped section; 100—detection unit; 101—body of the detection chip; 102—rotation center; 110—spring; 111—magnet; 112—working area; 140—second inverted U-shaped section. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0042] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0043] like Figure 1 As shown, a centrifugal microfluidic chip of this embodiment has a rotation center 102, and the detection chip includes at least one detection unit 100 that can perform detection independently, the detection unit 100 includes a mixed culture tank 11 for providing a reaction space, and a spring 110 is fixedly arranged below the outside of the detection chip, and a magnet 111 is connected to the free end of the spring 110;

[0044] When the detection chip is not rotating, there is no overlapping area between the projection of the magnet 111 and the mixed cultivation tank 11 directly below along the thickness direction of the detection chip; when the detection chip rotates at a certain speed around the rotation center 102, the magnet 111 compresses or stretches the free end of the spring 110 due to the centrifugal effect, so that the projection area of ​​the mixed cultivation tank 11 directly below (called the working area 112, at this time the magnet 111 can generate a magnetic force on the magnetic particles in the mixed cultivation tank 11 to attract the magnetic particles) can be completely covered by the projection area of ​​the magnet 111 along the thickness direction of the detection chip;

[0045] The extension line of the spring 110 in the compression or extension direction passes through the rotation center 102 .

[0046] The sample and related reagents react in the mixed incubation tank 11. Magnetic particles are usually used as carriers in the reaction. The reagents are adsorbed on the magnetic particles and the magnetic particles are manipulated by an external magnetic field. Generally speaking, during the mixing stage, the chip does not rotate, and the reagents on the magnetic particles react with the samples in the mixed incubation tank 11. At this time, an external magnetic field is not required; when entering the flushing stage, an external magnetic field needs to be introduced to manipulate the magnetic particles through the external magnetic field so that the magnetic particles remain in the mixed incubation tank 11 to prevent the magnetic particles from being carried away by the flushing liquid. In current conventional technology, a device that can generate a magnetic field is usually used to generate a magnetic field in the mixed incubation tank 11 area, and the presence or absence of the magnetic field is controlled by switching or turning the power on and off. Its defects include: 1. Additional equipment, resulting in increased costs and external auxiliary equipment; 2. Additional operational steps to control the presence or absence of the magnetic field.

[0047] In the present invention, the above-mentioned problem can be solved by designing a mobile magnetic field. The main idea of ​​the present invention is: when the mixed cultivation tank 11 needs a magnetic field, the magnet 111 automatically enters the lower area (working area 112) of the mixed cultivation tank 11; when the magnetic field needs to be removed, the magnet 111 automatically withdraws from the working area 112). The present invention is realized by designing a mobile magnetic field structure scheme in which the spring 110 connects the magnet 111. The whole process is automatically matched according to whether the reaction itself requires a rotation condition, and no additional control operation of the magnetic field is required. That is, during flushing, the detection chip needs to be rotated, and the magnetic field can be added to the lower area (working area 112) of the mixed cultivation tank 11 by rotation; when mixing, no magnetic field is needed, and the detection chip does not need to be rotated. At this time, the magnet 111 can automatically withdraw from the working area 112, so that the magnetic field in the working area 112 is removed.

[0048] The scheme of the present invention is specifically as follows: a fixed spring 110 is arranged below the chip in the thickness direction, and the free end of the spring 110 is fixedly connected to the magnet 111. The fixed end of the spring 110 can be directly connected to the chip or connected to the work table below the chip for providing rotation. It is only necessary to ensure that the spring 110 and the magnet 111 can rotate together with the chip. The expansion and contraction direction of the spring 110 must be the same as the radial direction of the chip rotation circle, that is, the two ends of the spring 110, the magnet 111, and the rotation center 102 are in a straight line. When the detection chip rotates around the rotation center 102 at a certain speed, the magnet 111 compresses or stretches the free end of the spring 110 due to the centrifugal effect, so that the magnet 111 enters the working area 112, so the magnet 111 can enter from the end far from the rotation center 102, and can also enter from the end near the rotation center 102, so at least the following two optional schemes are included.

[0049] Reference Figure 2 In one embodiment, the mobile magnetic field selection scheme 1 is specifically:

[0050] The fixed end of the spring 110 is far away from the rotation center 102, and the free end of the spring 110 and the magnet 111 are close to the rotation center 102;

[0051] When the detection chip does not rotate, the spring 110 is in a natural state, and the magnet 111 on the free end of the spring 110 passes over the working area 112 toward the rotation center 102; that is, there is no overlapping area between the magnet 111 and the working area 112;

[0052] When the detection chip rotates, the spring 110 is compressed, and the magnet 111 moves in the direction away from the rotation center 102 to the area directly below the mixed cultivation tank 11, that is, enters the working area 112, and the projection area of ​​the mixed cultivation tank 11 directly below the detection chip along the thickness direction is completely covered by the projection area of ​​the magnet 111 along the thickness direction of the detection chip, that is, the size of the magnet 111 can completely cover the working area 112.

[0053] Reference Figure 3 , In another embodiment, the mobile magnetic field selection scheme 2 is specifically: the fixed end of the spring 110 is close to the rotation center 102, and the free end of the spring 110 and the magnet 111 are far away from the rotation center 102;

[0054] When the detection chip does not rotate, the spring 110 is in a natural state, and the magnet 111 on the free end of the spring 110 is located between the rotation center 102 and the projection area of ​​the mixed culture tank 11 directly below the detection chip in the thickness direction; that is, the magnet 111 is between the rotation center 102 and the working area 112;

[0055] When the detection chip rotates, the spring 110 is stretched, and the magnet 111 moves in the direction away from the rotation center 102 to the area directly below the mixed cultivation tank 11, that is, enters the working area 112, and the projection area of ​​the mixed cultivation tank 11 directly below the detection chip along the thickness direction of the detection chip is completely covered by the projection area of ​​the magnet 111 along the thickness direction of the detection chip, that is, the size of the magnet 111 can completely cover the working area 112.

[0056] The above is the overall concept of the present invention, and further embodiments are provided below on the basis of the overall concept to illustrate the present invention in more detail.

[0057] Example 1

[0058] Reference Figure 4-5 The detection unit 100 also includes a whole blood storage tank 2, a whole blood injection hole 1 connected to the whole blood storage tank 2 for injecting a blood sample to be tested, a plasma serum quantitative tank 6 connected to the whole blood storage tank 2 through a whole blood transmission channel 3, a plasma serum transmission channel 7 connecting the plasma serum quantitative tank 6 to the mixing incubation tank 11, a waste liquid tank 15 connected to the mixing incubation tank 11 through a waste liquid transmission channel 14, and a vent 16 connected to the waste liquid tank 15.

[0059] Furthermore, the position on the whole blood transmission channel 3 upstream of the quantitative tank is connected to the remaining blood cell storage tank 4;

[0060] An overflow port is provided on one side of the plasma serum quantitative tank 6 close to the rotation center 102 , and the overflow port is connected to the plasma serum waste liquid tank 5 .

[0061] The whole blood transmission channel 3 includes a radial segment channel connected to the outlet end of the whole blood storage tank 2 and a circumferential segment channel connected to the end of the radial segment channel, the end of the circumferential segment channel is connected to the plasma serum quantitative tank 6, and the plasma serum quantitative tank 6 is located on the side of the circumferential segment channel close to the rotation center 102;

[0062] The remaining blood cell storage groove 4 is connected to the middle part of the circumferential section channel, and the remaining blood cell storage groove 4 is located on the side of the circumferential section channel away from the rotation center 102.

[0063] Furthermore, a microfluidic valve 13 is provided on the waste liquid transmission channel 14 connected to the outlet end of the mixed cultivation tank 11; the microfluidic valve 13 is a normally closed valve, and the microfluidic valve 13 can have various shapes, and can be a capillary valve, and when a predetermined pressure is applied, the capillary valve is passively opened; or the microfluidic valve 13 is a valve that is actively operated by receiving electrical power or energy from the outside through an operating signal.

[0064] The plasma serum transmission channel 7 has a first inverted U-shaped section 70, and the waste liquid transmission channel 14 has a second inverted U-shaped section 140. The first inverted U-shaped section 70 and the second inverted U-shaped section 140 are both slender, and provide the following functions: when the rotation speed of the chip body reaches a certain value, the liquid can pass through; that is, through the cooperation of siphon action and centrifugal action, the liquid is allowed to pass only when the rotation speed of the chip body reaches a certain value.

[0065] In this embodiment, the chip body has at least 8 detection units 100, and can perform at least 8 item detections simultaneously.

[0066] Example 2

[0067] As a further improvement, in this embodiment, a solid reagent storage tank 12 storing N different solid reagents is provided in the mixed culture tank 11; wherein, the solid reagent storage tank 12 can also store a variety of insoluble microspheres, such as magnetic immune microspheres.

[0068] The solid reagent includes first, second, ..., and Nth solid reagents, all of which are coated with protective films on the outside. The first solid reagent is coated with a first protective film on the outside, and the second solid reagent is coated with a first protective film and a second protective film in sequence from the outside to the inside. By analogy, the Nth solid reagent is coated with a first protective film, a second protective film, ..., and an Nth protective film in sequence from the outside to the inside.

[0069] The first to Nth protective films can all be dissolved in the mixed incubation tank 11 under the action of the dissolving reagent provided by the detection unit 100, so that the protective films coated on the outside of the first to Nth solid reagents have different dissolution times.

[0070] The highly efficient microfluidic detection chip further preferably includes at least one dissolving reagent storage tank 8 and at least one reaction reagent storage tank connected to the mixing and incubation tank 11;

[0071] The first to Nth protection films are made of the same material that can be dissolved by the dissolving reagent stored in the dissolving reagent storage tank 8 .

[0072] The liquid reagents in the dissolving reagent storage tank 8 and the reaction reagent storage tank are sealed and packaged in pre-sealed bags. The pre-sealed bags can be partially ruptured under the action of external force to release the liquid reagents therein and provide them to the mixing and incubation tank 11.

[0073] The chip of the present invention is mainly used for blood sample detection. During the detection process, a variety of solid reagents are usually required to react with samples in sequence (liquid reagents also need to be added during the process). In the traditional scheme, they are added in sequence during the reaction process, which is relatively cumbersome. In the present invention, the solid reagent is pre-installed in the solid reagent storage tank 12, and the liquid reagent is pre-packaged in the dissolving reagent storage tank 8 and the reaction reagent storage tank. Only one-time addition is required. During the reaction process, the pre-sealed bag is ruptured by operation to release the liquid reagent in sequence as required; the solid reagent has different dissolution times due to the design of the protective film, so that it can also react in sequence as required. Specific examples will be further explained in the following embodiments.

[0074] Among them, the protective film is designed to be used to coat solid reagents on the one hand to achieve the molding and packaging of solid reagents, and on the other hand, more importantly, to make different solid reagents have the required dissolution time. All protective films are made of the same material and are designed to be able to dissolve the dissolved reagents pre-packaged in the chip in a certain period of time. Obviously, the thickness of the protective film is positively correlated with the dissolution time, so different dissolution times can be obtained by controlling different protective film thicknesses and / or the number of protective film layers. Therefore, for the chip of the present invention, the relevant reaction reagents can be added at one time, and can react in sequence according to the design requirements. The above is the overall concept of the present invention. For a more specific design scheme for the protective film, the present invention also provides the following two specific embodiments.

[0075] First of all, it is important to understand that when the reagents in the reaction are solid reagents, the process in the reaction is: the solid reagent is dissolved first, and then the reaction is carried out after it is completely dissolved. Therefore, the time required for a single reaction includes the time for the solid reagent to dissolve plus the time for the reaction.

[0076] In this embodiment, the design scheme is: after the previous reaction is completed, the next reaction starts (that is, the protective film outside the solid reagent participating in the next reaction is completely dissolved, and the solid reagent can enter the solution system to start the reaction), and several reactions are carried out completely in series.

[0077] For ease of understanding, relevant symbols and formulas are introduced below for description.

[0078] Assume that a total of N reactions need to be carried out in sequence: F1, F2, ..., F n ;

[0079] The solid reagents participating in N reactions are the first, second, ..., Nth solid reagents in sequence; the first solid reagent is coated with a first protective film on the outside, the second solid reagent is coated with a first protective film and a second protective film on the outside, and so on, the Nth solid reagent is coated with a first protective film, a second protective film, ..., an Nth protective film on the outside; all protective films can be dissolved by a dissolving reagent within a certain period of time;

[0080] The reaction process requires the first to Nth solid reagents to dissolve and react in sequence, that is, when the N-1th reaction is completed, all the protective films on the outside of the Nth solid reagent (the first, second, ..., N-1th, Nth protective films) are completely dissolved, so that the Nth solid reagent body can begin to dissolve and enter the reaction system to react;

[0081] The total time T1 required for the first reaction F1 is the dissolution time tm1 of the first protective film plus the reaction time tz1 of the first solid reagent body, that is, T1 = tm1 + tz1;

[0082] The total time T2 required for the second reaction F2 is the dissolution time tm1 of the first protective film and the dissolution time tm2 of the second protective film plus the reaction time tz2 of the first solid reagent body, that is, T2 = tm1 + tm2 + tz2; ...

[0083] Reaction F n-1 The total time required is T N-1 is the sum of the dissolution time of the first to N-1th protective films: (tm1+tm2+...+tm N-1 ) plus the reaction time tz of the N-1 solid reagent body N-1 , that is, TN-1 =(tm1+tm2+...+tm N-1 )+tz N-1 ;

[0084] Nth Reaction F n The total time required is T N is the sum of the dissolution time of the first to Nth protective films: (tm1+tm2+...+tm N-1 +tm N ) plus the reaction time tz of the Nth solid reagent body N , that is, T N =(tm1+tm2+...+tm N-1 +tm N )+tz N ;reference Figure 6 ;

[0085] Satisfies the following relationship: tm1+tm2+...+tm N-1 +tm N =T N-1 =(tm1+tm2+...+tm N-1 )+tz N-1 ;

[0086] That is tm N =tz N-1 , that is, the dissolution time of the innermost protective film of the solid reagent in the latter reaction is exactly the same as the reaction time of the solid reagent body in the previous reaction, so by controlling the thickness of each protective film, the required reaction time of the protective film can be achieved. For example, for the first and second reactions, it satisfies: tm1+tm2=T1=tm1+tz1, tm2=tz1.

[0087] Total reaction time T=tm1+tz1+tz2+...+tz N-1 +tz N Compared with the conventional method, it is equivalent to only adding one more tm1 (dissolution time of the first protective film), but the reaction reagents are added at one time, which reduces the operation steps and complexity.

[0088] For example, in one embodiment, there are three reactions A, B, and C that need to be performed in sequence, and the reaction time of each reaction is 5 minutes, 10 minutes, and 15 minutes, respectively. In the three reactions, the solid reagents involved are solid reagent 1, solid reagent 2, and solid reagent 3, respectively;

[0089] The solid reagent 1 has a first protective film on the outside, the dissolution time of the first protective film is 1 minute, and the total reaction time of the solid reagent 1 is 4 minutes;

[0090] The solid reagent 2 has a first protective film and a second protective film from the outside to the inside. The dissolution time of the first protective film is 1 minute, so it is necessary to design the dissolution time of the second protective film to be 4 minutes, that is, the total reaction time of the solid reagent 2 is 5 minutes;

[0091] The solid reagent 3 has a first protective film, a second protective film, and a third protective film from the outside to the inside. The dissolution time of the first protective film is 1 minute, and the dissolution time of the second protective film is 4 minutes. It is necessary to design the dissolution time of the third protective film to be 5 minutes; that is, the total reaction time of the solid reagent 3 is 5 minutes.

[0092] During the reaction, solid reagent 1, solid reagent 2, and solid reagent 3 are pre-installed in the solid reagent storage tank 12 of the chip body. When the dissolved reagent immerses all the solid reagents, the first protective films on the outside of solid reagent 1, solid reagent 2, and solid reagent 3 start to react at the same time, and all the first protective films are completely dissolved after 1 minute; the solid reagent 1 body starts to react with the solution system (if a liquid reagent is needed at this time, the liquid reagent stored in the reaction reagent storage tank can be allowed to enter the solution system), and reaction A is completed after 4 minutes; at this time, the second protective film on the outside of solid reagent 2 and solid reagent 3 is completely dissolved (if a liquid reagent is needed at this time, the liquid reagent stored in the reaction reagent storage tank can be allowed to enter the solution system), the solid reagent 2 body starts to react with the solution system, and after 5 minutes, reaction B is completed; at this time, the third protective film on the outside of solid reagent 3 is completely dissolved (if a liquid reagent is needed at this time, the liquid reagent stored in the reaction reagent storage tank can be allowed to enter the solution system), the solid reagent 3 body starts to react with the solution system, and reaction C is completed after 5 minutes. The total time for the three reactions is 1+5+10+15=31 minutes, which is only 1 minute longer than the original time.

[0093] For example, in an optional scheme, for the above-mentioned protective film, the material is trehalose film, the dissolving agent is deionized water, the thickness of the first protective film is 0.01-0.05 mm, and the dissolution time is 1 minute; the thickness of the second protective film is 4 times that of the first protective film, and the dissolution time is 4 minutes; the thickness of the third protective film is 5 times that of the first protective film, and the dissolution time is 5 minutes.

[0094] In a preferred embodiment, the body 101 of the detection chip includes a bottom plate, an upper cover and a microfluidic channel opened on the bottom plate, and the upper cover is sealed and buckled on the bottom plate, so that the microfluidic channel between the bottom plate and the upper cover forms a detection unit 100;

[0095] A puncture hole is provided on the upper cover above the liquid reagent storage tank, and the pre-sealed bag can be punctured through the puncture hole by an external puncturing device to release the liquid reagent therein.

[0096] Of course, in other embodiments, the rupture of the pre-sealed bag can also be achieved by adopting an external squeezing force. For example, no puncture hole is provided, and the upper part of the liquid reagent storage tank is squeezed by an squeezing device, so that the pre-sealed bag is ruptured to release the liquid reagent therein.

[0097] In this embodiment, a dissolving reagent storage tank 8 and two reaction reagent storage tanks are provided on the chip body. The dissolving reagent storage tank 8 stores pre-packaged diluent (i.e., dissolving reagent), and the two reaction reagent storage tanks respectively store pre-packaged first liquid reagent and second liquid reagent.

[0098] A specific operation step of the chip of the present invention is provided below:

[0099] 1. A whole blood sample (venous blood, peripheral blood) is injected from the whole blood injection port 1 and stored in the whole blood storage tank. The microfluidic chip is controlled to rotate around its rotation center 102 at a first speed (first acceleration). The whole blood sample is transmitted to the remaining blood cell storage tank 4 and the plasma serum waste liquid tank 5 through the whole blood transmission channel, and most of the blood cells are stored in the remaining blood cell storage tank 4.

[0100] 2. The microfluidic chip is controlled to rotate at a second rotation speed (second acceleration), and the excess plasma / serum is transferred to the plasma / serum waste liquid tank 5.

[0101] 3. The microfluidic chip is controlled to rotate at a third rotation speed (third acceleration), and a certain amount of plasma / serum is transferred to the mixed culture tank 11 through the plasma-serum transfer channel.

[0102] The diluent (i.e., the dissolving reagent), the first liquid reagent, and the second liquid reagent are pre-packaged in the dissolving reagent storage tank 8, the first reaction reagent storage tank 9, and the second reaction reagent storage tank 10, respectively. The liquid reagent pre-packages can be punctured in sequence by an external spike device, and the liquid reagents can be released in sequence in conjunction with the centrifugal speed (release order: diluent, first liquid reagent, second liquid reagent); solid reagents are pre-packaged in the solid reagent storage tank, including magnetic immune microspheres (antibodies are linked to the surface of the microspheres), freeze-dried reagent beads, etc., and the reaction can be combined with the oscillation conditions (amplitude, frequency) to control the microfluidic chip for oscillation mixing, and a variety of solid reagent beads (here, a variety of freeze-dried reagent beads) can be dissolved in sequence and incubated / reacted with the sample. It should be understood that in addition to N kinds of soluble solid reagents (i.e., freeze-dried reagent beads), the solid reagent storage tank also includes magnetic immune microspheres, where the reagents that need to be dissolved are a variety of freeze-dried reagent beads, and the same is true below.

[0103] 4. Operate the external spike device to pass through the puncture hole, puncture the pre-sealed bag of the diluent, release the diluent, and operate the microfluidic chip to rotate at the fourth speed (fourth acceleration), so that the diluent can be transferred to the mixed cultivation tank 11. At this time, the liquid in the mixed cultivation tank 11 can be retained in the mixed cultivation tank 11 due to the blocking effect of the microfluidic valve 13. The various freeze-dried reagent beads that need to be dissolved can be dissolved in sequence according to the above scheme; if other reagents need to be added during the reaction, the pre-sealed bag containing the corresponding reagents can be punctured by operating the external spike device. Of course, during the reaction process, a variety of oscillation conditions (including set amplitude, frequency, operation time, etc.) can also be configured to operate the microfluidic chip for oscillation mixing to assist the reaction, so that the freeze-dried reagent beads react with the sample in sequence. The reagents are added sequentially, which greatly simplifies the operation.

[0104] 5. Control the microfluidic chip to rotate at the fifth speed (fifth acceleration) to allow the mixed liquid to break through the microfluidic valve 13. Since the liquid pressure is greater than the microfluidic valve 13, the liquid will be transferred from the mixing incubation tank 11 (12) to the waste liquid tank 15 through the liquid transfer channel. In the mixing incubation tank 11, the reagent freeze-dried beads are dissolved to react (the dissolution can be promoted by controlling the microfluidic chip to oscillate). The magnetic immune microspheres therein are controlled by an external mobile magnetic field (refer to the figure, mobile magnetic field selection scheme 1 in this embodiment) to avoid the loss of magnetic immune microspheres during the cleaning step. Specifically: 1. During the mixing stage, the chip is controlled by oscillating back and forth. The chip will not rotate continuously, and the magnet 111 will stay at a high position (such as Figure 1 As shown, the magnet 111 is located on the side of the working area 112 near the rotation center 102), and will not attract the magnetic immune microspheres; 2. During the flushing stage, the centrifugal speed is used to drive the liquid to the waste liquid tank 15. At this time, the chip rotates continuously, and the magnet 111 is driven by the centrifugal force to compress the spring 110 and stay at a low position (the magnet 111 completely covers the working area 112), which can attract the magnetic immune microspheres to the mixed culture tank 11 and will not be flushed to the waste liquid tank 15.

[0105] 6. Operate the external spike device to pierce the pre-sealed bag of the first liquid reagent, and operate the microfluidic chip to rotate at the sixth speed (sixth acceleration), so that the first liquid reagent can be transferred to the mixing and incubation tank 11. At this time, the liquid in the mixing and incubation tank 11 can be retained in the mixing and incubation tank 11 due to the blocking effect of the microfluidic valve 13. During the reaction process, certain oscillation conditions can be provided to operate the microfluidic chip for oscillation mixing. After the reaction is completed, operate the microfluidic chip to rotate at the seventh speed (seventh acceleration) to break through the microfluidic valve 13 after mixing. Since the liquid pressure is greater than the microfluidic valve 13, the liquid will be transferred from the mixing and incubation tank 11 to the waste liquid tank 15 through the liquid transfer channel. Similarly, the magnetic immune microspheres are controlled by an external mobile magnetic field (refer to the figure, the mobile magnetic field selection scheme 1 in this embodiment), which can avoid the loss of magnetic immune microspheres in the washing step.

[0106] 7. Operate the external spike device to pierce the pre-sealed bag of the second liquid reagent, and operate the microfluidic chip to rotate at the eighth speed (eighth acceleration), so that the second liquid reagent can be transferred to the mixing and incubation tank 11. At this time, the liquid in the mixing and incubation tank 11 can be retained in the mixing and incubation tank 11 due to the blocking effect of the microfluidic valve 13. During the reaction process, certain oscillation conditions can be provided to operate the microfluidic chip to oscillate and mix. After the reaction is completed, it can be detected through an external detection system (such as optical, fluorescent, chemiluminescent equipment, etc.).

[0107] Example 3

[0108] Provided is an application example of the chip of the present invention for chemiluminescent detection of hepatitis A virus antibodies.

[0109] The steps are:

[0110] 1. 50 μl of venous whole blood (male sample, hematocrit ratio of 69%, i.e., 31% plasma and 69% blood cells) with sodium citrate anticoagulant was injected into the whole blood storage tank through the whole blood injection hole 1, and the disc chip was manipulated to rotate for 60 seconds at 3,000 RPM (acceleration of 10,000 RPM / s), so that the whole blood sample was transferred to the remaining blood cell storage tank 4 and the plasma serum quantitative tank 6 (quantified to 10 μl) through the whole blood transfer channel, and most of the blood cells were stored in the remaining blood cell storage tank 4. The disc chip was manipulated to rotate for 10 seconds at 1,200 RPM (acceleration of 3,400 RPM / s), and the excess plasma was transferred to the plasma serum waste liquid tank 5. The disc chip was manipulated to rotate for 5 seconds at 600 RPM (acceleration of 1,250 RPM / s), and 10 μl of plasma was transferred to the mixed culture tank 11 through the plasma / serum transfer channel.

[0111] The diluent (dissolving reagent), the cleaning solution (the first liquid reagent), and the substrate (the second liquid reagent) are pre-packaged in the dissolving reagent storage tank 8, the first liquid reagent storage tank, and the second liquid reagent storage tank, respectively. The magnetic immune microspheres (superparamagnetic microparticles coated with streptavidin), the HAV antigen / lyophilized reagent beads, and the anti-HAVAg antibody / lyophilized reagent beads are pre-packaged in the solid reagent storage tank.

[0112] 2. Operate the external spike device to puncture the pre-packaged diluent, and operate the disc chip to rotate for 5 seconds at 2,500RPM (acceleration of 3,000RPM / s), which can transfer the diluent to the mixing culture tank 11. At this time, the liquid in the mixing culture tank 11 can be retained in the mixing culture tank 11 due to the blocking effect of the microfluidic valve 13. Operate the microfluidic chip to oscillate and mix 15 times with an amplitude of 370 degrees and a frequency of 10Hz, which can dissolve the HAV antigen / lyophilized reagent beads and react with the sample for 2 minutes; operate the microfluidic chip to oscillate and mix 15 times with an amplitude of 250 degrees and a frequency of 8Hz, and react the magnetic immune microspheres with the pre-liquid for 3 minutes; operate the microfluidic chip to oscillate and mix 20 times with an amplitude of 135 degrees and a frequency of 5Hz, which can dissolve the anti-HAVAg antibody / lyophilized reagent beads and react with the pre-liquid for 3 minutes. The disk chip is rotated at 3,500 RPM (acceleration of 5,250 RPM / s) for 5 seconds. Since the liquid pressure of the mixed liquid is greater than the microfluidic valve 13, it will break through the microfluidic valve 13 and be transferred to the waste liquid tank 15 through the liquid transfer channel. The magnetic immunomicrospheres can be controlled by an external mobile magnetic field to avoid losing the magnetic immunomicrospheres during the washing step.

[0113] 3. Operate the external spike device to puncture the pre-packaged cleaning liquid, and rotate the disc chip at 2,500RPM (acceleration of 3,000RPM / s) for 5 seconds to transfer the cleaning liquid to the mixing culture tank 11. At this time, the liquid in the mixing culture tank 11 can be retained in the mixing culture tank 11 due to the blocking effect of the microfluidic valve 13. Operate the microfluidic chip with an amplitude of 180 degrees and a frequency of 15Hz to oscillate and mix 20 times. After the cleaning is completed, operate the disc chip to rotate at 3,500RPM (acceleration of 5,250RPM / s) for 5 seconds. The waste liquid after cleaning will break through the microfluidic valve 13 and be transferred to the waste liquid tank 15 through the liquid transfer channel because the liquid pressure is greater than the microfluidic valve 13. The magnetic immune microspheres can be controlled by an external mobile magnetic field to avoid losing the magnetic immune microspheres during the cleaning step.

[0114] 4. Operate the external spike device to pierce the substrate pre-package, and operate the disc chip to rotate for 5 seconds at 2,500RPM (acceleration of 3,000RPM / s), and the substrate can be transferred to the mixing culture tank 11. At this time, the liquid in the mixing culture tank 11 can be retained in the mixing culture tank 11 due to the blocking effect of the microfluidic valve 13. Operate the microfluidic chip to oscillate and mix 20 times with an amplitude of 180 degrees and a frequency of 15Hz. After 300 seconds of reaction, it can be detected. The luminescence value is 7,318RLU. Substituting it into the reaction curve, the concentration value is 31.2IU / L, which is not much different from the value of 29.8IU / L ​​measured by the Roche Elecsys2010 immunoassay analyzer. Both are judged to be hepatitis A virus infection or past infection or the presence of antibodies after injection of hepatitis A vaccine.

[0115] Example 4 Mixing efficiency analysis

[0116] The microfluidic chip was manipulated to oscillate back and forth to achieve the function of rapid mixing. Experimental parameters: amplitude (300 degrees, 200 degrees, 100 degrees), frequency (15Hz, 12Hz, 8Hz), mixing tank depth (800μm, 600μm, 400μm); fixed parameters: the working liquid volume was 50 microliters in total, including 25 microliters of colorless deionized water (viscosity 1cP) and 25 microliters of colored glycerol (viscosity 5cP), and the number of oscillations was fixed at 5 times; experimental analysis: using a high-speed camera and image analysis software, the mixing efficiency was analyzed by pictures (0% means no mixing, 100% means complete mixing).

[0117] (Experiment 1), fixed frequency 15Hz, mixing tank depth 800μm, oscillation times 5 times, control the microfluidic chip with amplitude 300 degrees, 200 degrees, and 100 degrees for oscillation mixing, and take screenshots and analyze them with a high-speed camera. Experimental data: The microfluidic chip is controlled with an amplitude of 300 degrees for oscillation mixing, and the mixing efficiency is 98%; the microfluidic chip is controlled with an amplitude of 200 degrees for oscillation mixing, and the mixing efficiency is 45%; the microfluidic chip is controlled with an amplitude of 100 degrees for oscillation mixing, and the mixing efficiency is 12%. Experimental results: The larger the amplitude, the better the mixing efficiency. In addition, the amplitude and mixing efficiency show a quadratic proportional relationship.

[0118] (Experiment 2), fixed amplitude of 300 degrees, mixing tank depth of 800μm, oscillation times 5 times, control the microfluidic chip for oscillation mixing at frequencies of 15Hz, 12Hz, and 8Hz respectively, and take screenshots and analyze them with a high-speed camera. Experimental data: The mixing efficiency of the microfluidic chip for oscillation mixing at a frequency of 15Hz is 98%; the mixing efficiency of the microfluidic chip for oscillation mixing at a frequency of 12Hz is 51%; the mixing efficiency of the microfluidic chip for oscillation mixing at a frequency of 8Hz is 15%. Experimental results: The greater the frequency, the better the mixing efficiency. In addition, the frequency and mixing efficiency are in a cubic proportional relationship.

[0119] (Experiment 3), fixed amplitude of 300 degrees, fixed frequency of 15Hz, oscillation times 5 times, oscillation mixing test was performed at mixing tank depths of 800μm, 600μm, and 400μm, respectively, and screenshots were taken and analyzed by high-speed camera. Experimental data: When the mixing tank depth is 800μm, the mixing efficiency is 98%; when the mixing tank depth is 600μm, the mixing efficiency is 42%; when the mixing tank depth is 400μm, the mixing efficiency is 13%. Experimental results: The deeper the mixing tank depth, the better the mixing efficiency. In addition, the mixing tank depth and mixing efficiency show a cubic proportional relationship.

[0120] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A centrifugal microfluidic chip, the microfluidic chip having a rotation center, and the microfluidic chip comprising at least one detection unit for independent detection, the detection unit comprising a mixing culture tank for providing a reaction space, characterized in that: A spring is fixedly arranged below the exterior of the microfluidic chip, and a magnet is connected to the free end of the spring; Wherein, when the microfluidic chip is not rotating, the projection of the magnet and the mixed cultivation groove directly below along the thickness direction of the microfluidic chip has no overlapping area; when the microfluidic chip rotates around the rotation center at a certain speed, the magnet compresses or stretches the free end of the spring due to the centrifugal effect, so that the projection area of ​​the mixed cultivation groove directly below can be completely covered by the projection area of ​​the magnet along the thickness direction of the microfluidic chip; Wherein, the extension line of the spring in the compression or extension direction passes through the rotation center.

2. The centrifugal microfluidic chip according to claim 1, characterized in that: The fixed end of the spring is far away from the rotation center, and the free end of the spring and the magnet are close to the rotation center; When the microfluidic chip is not rotating, the spring is in a natural state, and the magnet on the free end of the spring is directed toward the rotation center and passes over the area directly below the mixed culture tank along the thickness direction of the microfluidic chip; When the microfluidic chip rotates, the spring is compressed, and the magnet moves to the area directly below the mixed incubation tank in the direction away from the rotation center, and the projection area of ​​the mixed incubation tank directly below the microfluidic chip along the thickness direction is completely covered by the projection area of ​​the magnet along the thickness direction of the microfluidic chip.

3. The centrifugal microfluidic chip according to claim 1, characterized in that: The fixed end of the spring is close to the rotation center, and the free end of the spring and the magnet are far away from the rotation center; When the microfluidic chip does not rotate, the spring is in a natural state, and the magnet on the free end of the spring is in an area between the rotation center and the projection area of ​​the mixed culture tank directly below the microfluidic chip in the thickness direction; When the microfluidic chip rotates, the spring is stretched, and the magnet moves to the area directly below the mixed incubation tank in the direction away from the rotation center, and the projection area of ​​the mixed incubation tank directly below the microfluidic chip along the thickness direction is completely covered by the projection area of ​​the magnet along the thickness direction of the microfluidic chip.

4. The centrifugal microfluidic chip according to claim 2, characterized in that: The detection unit also includes a whole blood storage tank, a whole blood injection hole connected to the whole blood storage tank for injecting a blood sample to be tested, a plasma serum quantification tank connected to the whole blood storage tank through a whole blood transmission channel, a plasma serum transmission channel connecting the plasma serum quantification tank to the mixed incubation tank, a waste liquid tank connected to the mixed incubation tank through a waste liquid transmission channel, and a vent connected to the waste liquid tank.

5. The centrifugal microfluidic chip according to claim 4, characterized in that: A position on the whole blood transmission channel upstream of the quantitative tank is connected to a residual blood cell storage tank; An overflow port is arranged on one side of the plasma serum quantitative tank close to the rotation center, and the overflow port is connected to a plasma serum waste liquid tank.

6. The centrifugal microfluidic chip according to claim 5, characterized in that: The whole blood transmission channel includes a radial segment channel connected to the outlet end of the whole blood storage tank and a circumferential segment channel connected to the end of the radial segment channel, the end of the circumferential segment channel is connected to the plasma serum quantitative tank, and the plasma serum quantitative tank is located on a side of the circumferential segment channel close to the rotation center; The remaining blood cell storage groove is communicated with the middle part of the circumferential section channel, and the remaining blood cell storage groove is located on a side of the circumferential section channel away from the rotation center.

7. The centrifugal microfluidic chip according to claim 6, characterized in that: A microfluidic valve is provided on the waste liquid transmission channel connected to the outlet end of the mixed cultivation tank; The plasma serum transmission channel has a first inverted U-shaped section, and the waste liquid transmission channel has a second inverted U-shaped section.

8. The centrifugal microfluidic chip according to any one of claims 1 to 7, characterized in that: The mixed culture tank is provided with a solid reagent storage tank storing N different solid reagents; The solid reagent includes a first solid reagent, a second solid reagent to an Nth solid reagent, all of which are coated with a protective film on the outside. The first solid reagent is coated with a first protective film on the outside, and the second solid reagent is coated with a first protective film and a second protective film in sequence from the outside to the inside. By analogy, the Nth solid reagent is coated with a first protective film, a second protective film to an Nth protective film in sequence from the outside to the inside. The first to Nth protective films can all be dissolved in the mixed incubation tank under the action of the dissolving reagent provided by the detection unit, so that the protective films coated on the outside of the first to Nth solid reagents have different dissolution times.

9. The centrifugal microfluidic chip according to claim 8, characterized in that: Also included is at least one dissolving reagent storage tank and at least one reaction reagent storage tank communicated with the mixed incubation tank; The first to Nth protective films are made of the same material that can be dissolved by the dissolving reagent stored in the dissolving reagent storage tank.

10. The centrifugal microfluidic chip according to claim 9, characterized in that: The liquid reagents in the dissolving reagent storage tank and the reaction reagent storage tank are sealed and packaged in pre-sealed bags, and the pre-sealed bags can be partially ruptured under the action of external force to release the liquid reagents therein and provide them to the mixed cultivation tank.

11. The centrifugal microfluidic chip according to claim 10, characterized in that: The body of the microfluidic chip comprises a bottom plate, an upper cover and a microchannel opened on the bottom plate, and the upper cover is sealed and buckled on the bottom plate, so that the microchannel between the bottom plate and the upper cover forms the detection unit; The upper cover above the liquid reagent storage tank is provided with a puncture hole, and the pre-sealed bag can be punctured through the puncture hole by an external puncturing device to release the liquid reagent therein.

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