CN114660306A - Centrifugal microfluidic chip liquid separation structure and liquid separation method - Google Patents

Abstract

The invention relates to a centrifugal microfluidic chip liquid separation structure and a liquid separation method. In the first centrifugal microfluidic chip separation structure, the separation tank is connected to the first collection tank through the first siphon flow channel, the liquid separation tank is connected to the second collection tank through the second siphon flow channel, and the second collection tank is provided with air holes , the first siphon channel is preferentially filled when the liquid enters the separatory tank. In the second type of centrifugal microfluidic chip separation structure, the separation tank is connected to the first collection tank through the first siphon flow channel, the liquid separation tank is connected to the second collection tank through the second siphon flow channel; the second collection tank is connected to the second collection tank through the flow channel. Connect the first airtight pore groove; both the first siphon flow channel and the second siphon flow channel are hydrophilically modified. The invention can realize the liquid separation of various liquid samples or reagents on the centrifugal microfluidic chip, and can be widely used in the target biomolecule extraction liquid and waste liquid separation in in vitro diagnosis, and sample waste liquid and detection in the process of immune and biochemical detection. liquid separation.

Description

A kind of centrifugal microfluidic chip liquid separation structure and liquid separation method

technical field

The invention relates to the field of medical devices, in particular to a liquid separation structure and a liquid separation method of a centrifugal microfluidic chip.

Background technique

At present, centrifugal microfluidic chips are often used in the field of POCT, and the detection results can be output conveniently and quickly without the operation of professional technicians. It is characterized by integrating microfluidic structures such as a liquid storage tank, a detection tank and a valve on a disc-shaped chip, which drives the flow of microfluidics by centrifugal force, thereby realizing the detection and analysis of samples. Centrifugal microfluidic chips can complete sample pretreatment, mixing, accurate volume quantification and detection. In recent years, centrifugal microfluidic chips have achieved rapid development due to their advantages of integration, multi-parallel detection, high throughput, low cost, automation, etc., and have been widely used in biochemical detection, immunoassay, nucleic acid detection, biomolecule enrichment and food safety, etc.

At present, the centrifugal microfluidic chip liquid separation structure reported in the literature mainly uses two schemes. 1. The chip liquid separation structure uses a Y-shaped flow channel, including one main flow channel and two branch flow channels. When the chip is centrifuged, the chip accelerates the process. The inertial force is generated in the middle and the direction of the inertial force is opposite to the rotation direction of the chip (clockwise and counterclockwise). Purpose; 2. The chip liquid separation structure is also suitable for the Y-shaped flow channel. A trap or an active valve is set on each branch flow channel, and the purpose of liquid separation is achieved by controlling the opening and closing of the valve. However, these two liquid separation schemes have their own problems. Scheme 1 requires a large enough inertial force, and the inertial force lasts for a certain period of time, and the chip needs to be kept in an accelerated state. As a result, the chip needs to be equipped with an ultra-high-speed centrifuge, and when the required separation When the reagent volume of the liquid is large, the liquid separation cannot be completed when the ultracentrifuge reaches the maximum speed. Option 2 requires a valve to be installed on the branch, but the steam trap has the problem of low threshold and instability, resulting in poor repeatability of chip dispensing, while the active valve requires additional accessories on the chip and the instrument used in the chip to make the chip The manufacturing process is more complicated, increasing the cost of using the chip, making it difficult to apply it on a large scale.

SUMMARY OF THE INVENTION

The invention provides a centrifugal microfluidic chip liquid separation structure and a liquid separation method, which can be applied to the separation of target biomolecule extraction liquid and waste liquid in in vitro diagnosis, and various samples and reagents in the immune or molecular detection process enter different tanks for detection in turn .

The first centrifugal microfluidic chip liquid separation structure provided by the present invention includes a liquid separation tank, a first siphon flow channel, a second siphon flow channel, a first collection tank, a second collection tank and an air hole; A siphon flow channel is connected to the first collection tank, and the liquid separation tank is connected to the second collection tank through the second siphon flow channel; the second collection tank is provided with air holes; the first siphon flow channel and the second siphon flow channel are both hydrophilic modified, The hydrophilicity and length of the first siphon flow channel and the second siphon flow channel are set so that when the liquid enters the liquid separation tank, the first siphon flow channel is preferentially filled.

Further, the hydrophilicity of the first siphon flow channel is better than that of the second siphon flow channel, and the length of the second siphon flow channel is longer than that of the first siphon flow channel. The capillary force in the flow channel makes the liquid preferentially fill the first siphon flow channel when it enters the separatory tank.

Further, the first collection tank contains a quantitative pool and an overflow pool, wherein the volume of the quantitative pool is less than the amount of sample added, so that when the reagent enters the first collection tank, the quantitative pool in the first collection tank is preferentially filled, and the excess reagent is into the overflow pool.

Further, relevant reagents for immune or molecular detection can be pre-buried in the separation tank, or relevant filter membranes or screens used for target biomolecule enrichment can be pre-buried; the first collection tank and the second collection tank can be Pre-embedded immunological or molecular detection related reagents.

The present invention also provides a liquid separation method using the above-mentioned first centrifugal microfluidic chip liquid separation structure, comprising the following steps:

1) The chip is centrifuged for the first time, reagent A enters the separatory tank, and after the centrifugation is stopped, due to the hydrophilicity and length settings of the above-mentioned first siphon flow channel and second siphon flow channel, when liquid A enters the separatory tank Fill the first siphon channel first;

2) The chip is centrifuged for the second time, reagent A enters the first collection tank along the first siphon flow channel, and fills the quantitative tank in the tank, and the excess sample enters the overflow tank;

3) The chip is centrifuged for the third time, and reagent B enters the separatory tank. Since the waste liquid quantitative tank connected to the first siphon flow channel is filled with liquid, the air in the middle section of the first siphon flow channel cannot be discharged, and the first siphon flow channel changes. It is a normally closed valve, and the second collection tank connected to the second siphon flow channel has air holes, the second siphon flow channel works normally, and the reagent fills the second siphon flow channel;

4) The chip is centrifuged for the fourth time, and reagent B enters the second collection tank along the second siphon channel.

On the basis of the above-mentioned first liquid separation structure, the present invention also provides a second centrifugal microfluidic chip liquid separation structure, including a liquid separation tank, a first siphon flow channel, a second siphon flow channel, a first collection tank, The second collection tank and the first closed air hole tank; the liquid separator is connected to the first collection tank through the first siphon flow channel, and the liquid separation tank is connected to the second collection tank through the second siphon flow channel; the second collection tank is connected to the third collection tank through the flow channel A closed air hole slot; the first siphon flow channel and the second siphon flow channel are both hydrophilic modified.

Among them, the second collection tank connected with the second siphon flow channel has no air holes. After the liquid enters the liquid separation tank, the second siphon flow channel cannot be filled, but the first siphon flow channel can only be filled, and the second siphon flow channel is normally closed. The function of the valve; the first airtight air hole groove can be penetrated by a needle outside the chip, and when the first airtight air hole groove becomes a air hole after being pierced, the second siphon flow channel can work normally.

Further, the first collection tank contains a quantitative pool and an overflow pool, wherein the volume of the quantitative pool is smaller than the amount of the sample added, so that when the reagent enters the first collection tank, the quantitative pool is preferentially filled, and the excess sample enters the overflow pool.

Further, the relevant reagents for immunity or molecular detection or the relevant filter membranes or screens used for the enrichment of target biomolecules can be pre-embedded in the separation tank, and the first collection tank and the second collection tank can be pre-embedded for immunity. or related reagents for molecular detection.

Further, on the basis of the above-mentioned second type of centrifugal microfluidic chip liquid separation structure, the Nth siphon flow channel, the Nth collection tank, and the N-1st airtight pore tank are correspondingly added, wherein N is an integer greater than or equal to three; One end of the Nth siphon flow channel is connected to the liquid separation tank, the other end is connected to the Nth collection tank, and the Nth collection tank is connected to the N-1th airtight air-tight tank. For example, a third siphon flow channel, a third collection tank, and a second airtight air-tight groove can be added, which can be extended to the liquid separation of three reagents, and so on. Dispensing of three or more reagents.

The present invention also provides a liquid separation method using the above-mentioned second centrifugal microfluidic chip liquid separation structure, comprising the following steps:

1) When the chip is centrifuged for the first time, reagent A enters the separatory tank. After the centrifugation is stopped, since the second collection tank connected to the second siphon channel has no air holes, the second siphon channel is a normally closed valve at this time, and the sample can only be filled. Fill the first siphon channel;

2) Carry out the second centrifugation, reagent A enters the first collection tank along the first siphon flow channel, and fills the quantitative pool in the first collection tank, and the excess sample enters the overflow pool;

3) A needle is used outside the chip to pierce the first airtight air hole groove to form air holes;

4) The chip is centrifuged for the third time, and reagent B enters the separatory tank. Since the waste liquid quantitative pool connected to the first siphon flow channel is filled with liquid, the air in the middle section of the first siphon flow channel cannot be discharged, and the first siphon flow channel changes. A normally closed valve is formed, and one end of the second collection tank connected to the second siphon flow channel forms a stomata due to the puncture of the first airtight air hole slot, the second siphon flow channel works normally, and the reagent fills the second siphon flow channel;

5) The chip is centrifuged for the fourth time, and the reagent enters the second collection tank along the second siphon flow channel.

The centrifugal microfluidic chip liquid separation structure of the present invention has the following advantages:

1. Through the liquid separation structure on the centrifugal microfluidic chip, two or more reagents can be sequentially entered into different collection tanks

2. There will be no cross-contamination between different liquids during the separation process;

3. There is no need to add valves on the chip, and the overall structure is simple;

4. As a functional module, the liquid-separating structure is formed on centrifugal chips with different functional requirements, which can be applied to the separation of target biomolecule extracts and waste liquids in in vitro diagnosis, and various samples and reagents in the process of immunization or molecular detection. slot for inspection.

Description of drawings

Figure 1. Schematic diagram of the first chip dispensing structure.

Figure 2. Schematic diagram of the second chip dispensing structure.

Figure 3. Schematic diagram of the structure of the second chip dispensing structure.

Figure 4. A schematic structural diagram of adding a third siphon channel on the basis of the second chip liquid separation structure.

Figure 5. Schematic representation of sample addition to sample wells.

Figure 6. Schematic of the first centrifugation of the chip.

Figure 7. Schematic of the second centrifugation of the chip.

Figure 8. Reagents are added to the liquid storage tank, and a needle is used outside the chip to puncture the lower polyester film of the airtight air hole groove of the chip to form air holes.

Figure 9. Schematic of the third centrifugation of the chip.

Figure 10. Schematic of the fourth centrifugation of the chip.

Detailed ways

The present invention will be described in further detail below through specific embodiments and accompanying drawings.

FIG. 1 illustrates the liquid separation structure of the first centrifugal microfluidic chip of the present invention, and the chip where the liquid separation structure is located includes an upper chip layer and a lower chip layer that are tightly bonded. The lower layer of the chip can be made of polyester film or other materials. The upper layer of the chip includes at least one detection unit, and each detection unit is provided with a liquid separation structure, and the liquid separation structure includes: a liquid separation tank, a first siphon flow channel, a second siphon flow channel, a first collection tank, and a second collection tank , stomata. The liquid separation tank is connected to the first collection tank through the first siphon flow channel, and the liquid separation tank is connected to the second collection tank through the second siphon flow channel. Air holes are arranged on the second collection tank. Relevant reagents for immunological or molecular detection can be pre-embedded in the separatory tank, or relevant filter membranes or sieves for the enrichment of target biomolecules can be pre-embedded. Reagents related to immunological or molecular detection can be pre-buried in the collection tank. The first siphon flow channel and the second siphon flow channel are both hydrophilic modified. The hydrophilicity and length of the first siphon flow channel and the second siphon flow channel are set so that the first siphon flow channel is preferentially filled when the liquid enters the liquid separator. . In this embodiment, the hydrophilicity of the first siphon flow channel is better than that of the second siphon flow channel, and the length of the second siphon flow channel is longer than that of the first siphon flow channel. The capillary force in the second siphon flow channel makes the liquid preferentially fill the first siphon flow channel when it enters the liquid separator.

In this embodiment, the first collection tank contains a quantitative tank and an overflow tank, wherein the volume of the quantitative tank is smaller than the amount of the sample added, so that when the reagent enters the first collection tank, the quantitative tank in the first collection tank is preferentially filled, and the excess The reagents enter the overflow pool.

Wherein, the liquid separation tank can be connected to a sample tank, a liquid storage tank, etc. through a flow channel for adding samples or reagents, and enters the liquid separation tank through the flow channel.

The steps for realizing liquid separation by adopting the above-mentioned first centrifugal microfluidic chip liquid separation structure are as follows:

1) The chip is centrifuged for the first time, reagent A enters the separatory tank, and after the centrifugation is stopped, due to the hydrophilicity and length settings of the above-mentioned first siphon flow channel and second siphon flow channel, when liquid A enters the separatory tank Fill the first siphon channel first;

2) The chip is centrifuged for the second time, reagent A enters the first collection tank along the first siphon flow channel, and fills the quantitative tank in the tank, and the excess sample enters the overflow tank;

3) The chip is centrifuged for the third time, and reagent B enters the separatory tank. Since the waste liquid quantitative tank connected to the first siphon flow channel is filled with liquid, the air in the middle section of the first siphon flow channel cannot be discharged, and the first siphon flow channel changes. It is a normally closed valve, and the second collection tank connected to the second siphon flow channel has air holes, the second siphon flow channel works normally, and the reagent fills the second siphon flow channel;

4) The chip is centrifuged for the fourth time, and reagent B enters the second collection tank along the second siphon channel.

FIG. 2 illustrates a second centrifugal microfluidic chip liquid separation structure of the present invention, and the chip where the liquid separation structure is located includes a bonded upper chip layer and a lower chip layer. The lower layer of the chip can be made of polyester film or other materials. The upper layer of the chip includes at least one detection unit, and each detection unit is provided with a liquid separation structure, and the liquid separation structure includes:

Separation tank, first siphon flow channel, second siphon flow channel, first collection tank, second collection tank, first airtight air hole tank. The liquid separation tank is connected to the first collection tank through the first siphon flow channel, and the liquid separation tank is connected to the second collection tank through the second siphon flow channel. The second collection tank is connected to the first closed air hole tank through a flow channel. Relevant reagents for immunological or molecular detection can be pre-embedded in the separatory tank, or relevant filter membranes or sieves for the enrichment of target biomolecules can be pre-embedded. Reagents related to immunological or molecular detection can be pre-buried in the collection tank. Both the first siphon flow channel and the second siphon flow channel are hydrophilically modified.

Figure 3 is a schematic diagram of the second centrifugal microfluidic chip liquid separation structure to achieve liquid separation. The second collection tank connected to the second siphon flow channel has no air holes, and the liquid cannot fill the second siphon flow channel after entering the liquid separation tank. Only the first siphon flow channel can be filled, and the second siphon flow channel acts as a normally closed valve; the first airtight air hole slot can be penetrated by acupuncture outside the chip, and when the first airtight air hole slot is pierced, it becomes a stomata , the second siphon channel can work normally.

In this embodiment, the first collection tank contains a quantitative tank and an overflow tank, wherein the volume of the quantitative tank is smaller than the amount of sample added, so that when the reagent enters the first collection tank, the quantitative tank is preferentially filled, and the excess sample enters the overflow tank .

FIG. 4 is a schematic structural diagram of adding a third siphon flow channel on the basis of the second chip liquid separation structure. As shown in the figure, on the basis of the above-mentioned second centrifugal microfluidic chip liquid separation structure, a third siphon flow channel, a third collection tank, and a second airtight air-tight groove are correspondingly added, which can be expanded into the distribution of three reagents. Liquid, and so on, continue to add siphon flow channel, collection tank and airtight pore tank, which can be expanded to the liquid separation of more than three reagents.

The specific liquid separation steps for realizing liquid separation by adopting the above-mentioned second centrifugal microfluidic chip liquid separation structure are as follows:

1) The chip is centrifuged for the first time, and the reagent A pre-added to the sample tank (as shown in Figure 5) enters the separatory tank, as shown in Figure 6, after the centrifugation is stopped, the second collection due to the connection of the second siphon channel There is no air hole in the groove, the second siphon flow channel is a normally closed valve at this time, and the sample can only fill the first siphon flow channel;

2) Carry out the second centrifugation, reagent A enters the first collection tank along the first siphon flow channel, and fills the quantitative tank in the first collection tank, and the excess sample enters the overflow tank, as shown in Figure 7;

3) A needle is used outside the chip to pierce the first airtight air hole groove to form air holes;

4) The chip is centrifuged for the third time, and the reagent B added to the liquid storage tank (as shown in Figure 8) in advance enters the separatory tank, as shown in Figure 9, because the waste liquid quantitative pool connected to the first siphon flow channel has been Filled with liquid, the air in the middle section of the first siphon flow channel cannot be discharged, the first siphon flow channel becomes a normally closed valve, and one end of the second collection tank connected to the second siphon flow channel is formed due to the puncture of the first airtight air hole slot. Air holes, the second siphon flow channel works normally, and the reagent fills the second siphon flow channel;

5) The chip is centrifuged for the fourth time, and the reagent enters the second collection tank along the second siphon flow channel, as shown in Figure 10.

The specific embodiments of the present invention disclosed above are intended to help understand the content of the present invention and implement them accordingly. Those skilled in the art can understand that various substitutions, changes and modifications can be made without departing from the spirit and scope of the present invention. Modifications are possible. The present invention should not be limited to the contents disclosed in the embodiments of this specification, and the protection scope of the present invention shall be subject to the scope defined by the claims.

Claims (10)

1. A centrifugal micro-fluidic chip liquid separation structure is characterized by comprising a liquid separation groove, a first siphon flow channel, a second siphon flow channel, a first collecting groove, a second collecting groove and air holes; the liquid separating tank is connected with the first collecting tank through a first siphon flow passage, and the liquid separating tank is connected with the second collecting tank through a second siphon flow passage; the second collecting tank is provided with air holes; the first siphon runner and the second siphon runner are subjected to hydrophilic modification, and the hydrophilicity and the length of the first siphon runner and the second siphon runner are set so that the first siphon runner is filled with liquid preferentially when the liquid enters the liquid dividing tank.

2. The centrifugal microfluidic chip liquid separation structure of claim 1, wherein the first siphon flow channel has a hydrophilicity better than that of the second siphon flow channel, and the second siphon flow channel has a length longer than that of the first siphon flow channel.

3. The structure of claim 1, wherein the first collection well contains a quantification pool and an overflow pool, wherein the quantification pool has a volume smaller than the sample volume, such that the quantification pool in the first collection well is preferentially filled when the reagent enters the first collection well, and the excess reagent enters the overflow pool.

4. A liquid separation method using the centrifugal microfluidic chip liquid separation structure according to claim 1, comprising the steps of:

1) centrifuging the chip for the first time, enabling the reagent A to enter the liquid separating tank, and filling the first siphon flow channel with the liquid A when the liquid A enters the liquid separating tank after the centrifugation is stopped;

2) centrifuging the chip for the second time, enabling the reagent A to enter the first collecting tank along the first siphon flow channel, filling the quantifying pool in the tank, and enabling the redundant sample to enter the overflow pool;

3) centrifuging the chip for the third time, enabling the reagent B to enter the liquid separating tank, wherein the liquid is filled in the waste liquid quantitative tank connected with the first siphon flow channel, the air cannot be discharged from the middle section of the first siphon flow channel, the first siphon flow channel is changed into a normally-closed valve, the second collecting tank connected with the second siphon flow channel normally works due to the air hole, and the reagent is filled in the second siphon flow channel;

4) the chip is centrifuged for the fourth time, and the reagent B enters the second collecting tank along the second siphon flow passage.

5. A centrifugal micro-fluidic chip liquid separation structure is characterized by comprising a liquid separation groove, a first siphon flow channel, a second siphon flow channel, a first collecting groove, a second collecting groove and a first closed pore groove; the liquid separating tank is connected with the first collecting tank through a first siphon flow channel and is connected with the second collecting tank through a second siphon flow channel; the second collecting tank is connected with the first closed pore tank through a flow passage; the first siphon runner and the second siphon runner are both subjected to hydrophilic modification.

6. The liquid separating structure of the centrifugal microfluidic chip according to claim 5, wherein the first collecting tank contains a quantitative pool and an overflow pool, wherein the quantitative pool has a volume smaller than the sample amount, so that the quantitative pool is preferentially filled when the reagent enters the first collecting tank, and the excess sample enters the overflow pool.

7. The centrifugal microfluidic chip liquid separation structure according to claim 5, further comprising an Nth siphon flow channel, an Nth collection tank, and an N-1 th closed pore tank, wherein N is an integer greater than or equal to three; one end of the Nth siphon flow channel is connected with the liquid dividing groove, the other end of the Nth siphon flow channel is connected with the Nth collecting groove, and the Nth collecting groove is connected with the N-1 th sealed air hole groove.

8. The liquid separation structure of the centrifugal microfluidic chip according to claim 1 or 5, wherein reagents related to immunization or molecular detection are pre-embedded in the liquid separation tank, or reagents related to immunization or molecular detection are pre-embedded in the first collection tank and the second collection tank, or a filter membrane or a screen related to target biomolecule enrichment is pre-embedded in the liquid separation tank.

9. A liquid separation method adopting the centrifugal microfluidic chip liquid separation structure of claim 5 is characterized by comprising the following steps:

1) centrifuging the chip for the first time, enabling the reagent A to enter the liquid separating tank, and after the centrifugation is stopped, because a second collecting tank connected with a second siphon flow channel is not provided with an air hole, the second siphon flow channel is a normally-closed valve at the moment, and the sample can only be filled in the first siphon flow channel;

2) carrying out second centrifugation, enabling the reagent A to enter the first collecting tank along the first siphon flow channel, filling the quantitative pool in the first collecting tank, and enabling the redundant sample to enter the overflow pool;

3) puncturing the first closed air hole groove by using a needle outside the chip to form an air hole;

4) centrifuging the chip for the third time, enabling the reagent B to enter the liquid separating tank, wherein the waste liquid quantitative tank connected with the first siphon flow channel is filled with liquid, the middle section of the first siphon flow channel is air which cannot be discharged, the first siphon flow channel is changed into a normally-closed valve, one end of a second collecting tank connected with the second siphon flow channel is punctured by the first closed air hole groove to form an air hole, the second siphon flow channel normally works, and the reagent is filled in the second siphon flow channel;

5) the chip is centrifuged for the fourth time, and the reagent enters the second collecting tank along the second siphon flow passage.

10. A centrifugal microfluidic chip is characterized by comprising the centrifugal microfluidic chip liquid separation structure of any one of claims 1-3 and 5-8.

Images