An integrated qPCR microfluidic chip structure and its use method

Abstract

The present invention discloses an integrated qPCR microfluidic chip structure and a method for using the same. The microfluidic chip structure comprises a qPCR microfluidic chip body, a hard film and a soft film. The qPCR microfluidic chip body is provided with a sample addition port, a working chamber, a waste liquid chamber, a lysis liquid chamber, an elution liquid chamber and a plurality of cleaning liquid chambers; the sample addition port is provided with a detachable sample rubber plug, and the sample addition port, the lysis liquid chamber, the cleaning liquid chamber, the elution liquid chamber and the waste liquid chamber are respectively connected with the working chamber through a sample flow channel, a lysis liquid flow channel, a cleaning liquid flow channel, an elution liquid flow channel and a waste liquid flow channel; a liquid bag and a puncture structure for puncturing the liquid bag are provided in the lysis liquid chamber, the cleaning liquid chamber and the elution liquid chamber; the present invention realizes the whole process of sample lysis, cleaning, elution and qPCR amplification in one qPCR microfluidic chip, and has the advantages of simple structure, convenient operation and no aerosol pollution; the present invention also provides a method for using the integrated qPCR microfluidic chip, which is convenient for realizing rapid detection of sample nucleic acid.

Description

Technical Field

[0001] The present invention relates to the field of qPCR detection technology based on a microfluidic chip, and specifically to an integrated qPCR microfluidic chip structure; in addition, the present invention also relates to a method for using the integrated qPCR microfluidic chip. Background Art

[0002] Faced with the major challenges of HPV typing and respiratory pathogen detection including COVID-19, it is necessary to complete nucleic acid testing simply and conveniently in a non-PCR laboratory environment in a short period of time to achieve the goal of "sample in - result out". The nucleic acid testing process is mainly divided into three parts: nucleic acid extraction, nucleic acid amplification, and result detection.

[0003] In actual testing, the sample needs to be lysed, washed and eluted. The above steps generally require special equipment and manual operation. In particular, after the sample reacts with the lysis solution, the protein-containing magnetic beads need to be washed multiple times.

[0004] The microfluidic chip in the prior art needs to perform lysis, cleaning and elution of samples in different chambers, which results in a complex structure and low reliability of the microfluidic chip. Summary of the invention

[0005] The purpose of the present invention is to provide an integrated qPCR microfluidic chip structure, which solves the problems of complex structure and low reliability of microfluidic chips in the prior art;

[0006] At the same time, the present invention also provides a method for using an integrated qPCR microfluidic chip, which facilitates the rapid detection of sample nucleic acid.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0008] An integrated qPCR microfluidic chip structure comprises a qPCR microfluidic chip body, a hard film and a soft film, wherein the qPCR microfluidic chip body is provided with a sample addition port, a working chamber, a waste liquid chamber, a lysis liquid chamber, an elution liquid chamber and a plurality of cleaning liquid chambers;

[0009] The sample adding port is provided with a detachable sample rubber plug, and the sample adding port, the lysis liquid chamber, the cleaning liquid chamber, the elution liquid chamber and the waste liquid chamber are connected with the working chamber through the sample flow channel, the lysis liquid flow channel, the cleaning liquid flow channel, the elution liquid flow channel and the waste liquid flow channel respectively; the lysis liquid chamber, the cleaning liquid chamber and the elution liquid chamber are provided with a liquid bag and a puncture structure for puncturing the liquid bag;

[0010] Among them, the working chamber is provided with a sliding seal with piston A and piston B, piston B is located on the left side of piston A, piston A is an active piston, piston B is a passive piston, piston A is used to connect with the driving mechanism in the detection instrument, when the driving mechanism drives piston A to move in the working chamber, piston A can drive piston B to move in the working chamber after contacting piston B, and the side wall of the working chamber forms a magnetic bead particle adsorption surface;

[0011] The qPCR microfluidic chip body is also provided with a number of detection channels, and a breathable membrane is provided at the end of the detection channel; the hard membrane is provided on the qPCR microfluidic chip body and covers the detection channel, and the soft membrane is provided on the qPCR microfluidic chip body and covers the working chamber, waste liquid chamber, lysis liquid chamber, elution liquid chamber and the corresponding channels.

[0012] Among them, the detection flow channel includes a reaction hole main flow channel connected to the working chamber, a plurality of reaction hole liquid inlet flow channels connected to the reaction hole main flow channel, each reaction hole liquid inlet flow channel is connected to a reaction hole exhaust flow channel, and a reaction hole is formed at the connection between the reaction hole liquid inlet flow channel and the reaction hole exhaust flow channel; the air permeable membrane is arranged at the end of the reaction hole exhaust flow channel.

[0013] Further optimization, the waste liquid chamber is arranged on the back side of the qPCR microfluidic chip body, and the lysis liquid chamber, the elution liquid chamber and several cleaning liquid chambers are arranged on the front side of the qPCR microfluidic chip body and are recessed into the waste liquid chamber.

[0014] The puncturing structure is a plurality of puncturing needles arranged at the bottom of the lysis liquid chamber, the elution liquid chamber and a plurality of cleaning liquid chambers.

[0015] The liquid bag is made of aluminum-plastic film or plastic film and is connected with the soft film.

[0016] Among them, the sample flow channel, the lysis solution flow channel, and the cleaning solution flow channel are gathered on the right side of the working chamber.

[0017] Further optimization is performed, a limiting hole connected to the right end of the working chamber is provided on the qPCR microfluidic chip body, and the limiting hole is used for the passage of the driving mechanism.

[0018] Among them, the driving mechanism is detachably connected to the piston A, a connecting hole is provided at the end of the piston A, a plurality of limiting parts are provided on the side wall of the connecting hole, a guiding inclined surface is provided on the limiting part facing the axis of the connecting hole, and the driving mechanism has a clamping part that can cooperate with and disengage from the connecting hole.

[0019] It is further defined that the right end of the working chamber is open and communicated with the outside.

[0020] The present invention discloses a method for using an integrated qPCR microfluidic chip. Before leaving the factory, the initial positions of piston A and piston B need to be adjusted. The initial positions of piston A and piston B are: A1 and B1:

[0021] Piston A is located at the rightmost end of the working chamber, i.e., at A1;

[0022] Piston B is located between the waste liquid flow channel and the eluent flow channel, i.e., at B1. At this time, the sample flow channel, the lysate flow channel, the cleaning liquid flow channel and the waste liquid flow channel are connected to form a working chamber that can complete sample lysis and multiple cleanings.

[0023] The specific steps are as follows:

[0024] Step 1: After adding the sample to the sample adding port, insert the sample rubber plug into the sample adding port, and press the sample rubber plug to allow the sample to enter the working chamber;

[0025] Step 2: The mechanism in the detection instrument presses the soft membrane at the lysis liquid chamber to rupture the liquid bag in the lysis liquid chamber, and the lysis liquid and magnetic beads in the liquid bag enter the working chamber through the lysis liquid flow channel to mix with the sample;

[0026] Step 3: The magnetic attraction mechanism in the detection instrument is turned on to adsorb the protein-adsorbed magnetic beads on the side wall of the working chamber, and then the driving mechanism pushes piston A to move toward piston B. At this time, piston B is still in a stationary state; during the movement of piston A, the lysed waste liquid is discharged into the waste liquid chamber through the waste liquid channel; at this time, the protein-adsorbed magnetic beads will gather on the side wall between piston A and piston B, and piston A is at position A2;

[0027] Step 4: The driving mechanism drives the piston A to return to the position A1, and the squeezing mechanism in the detection instrument squeezes the soft film at the cleaning liquid chamber to rupture the liquid bag in the cleaning liquid chamber. The cleaning liquid in the liquid bag flows through the cleaning liquid flow channel and then enters the working chamber to achieve cleaning of the magnetic beads;

[0028] Step 5: The driving mechanism pushes piston A to move from position A1 to position A2, discharges the waste liquid after cleaning into the waste liquid chamber, and selects the cleaning times according to the needs;

[0029] Step 6: After cleaning, piston A continues to move toward piston B and pushes piston B to the leftmost end of the working chamber, so that piston B is located at position B2, and then piston A returns for a distance and stays at position A3; at this time, the main flow channel of the reaction hole and the eluent flow channel are in a connected state and form an elution chamber;

[0030] Step 7: The squeezing mechanism in the detection instrument presses the soft membrane at the elution liquid cavity, so that the liquid bag at the elution liquid cavity is punctured, and the eluent in the liquid bag enters the elution cavity to elute the magnetic beads;

[0031] Step 8: After elution is completed, the magnetic suction mechanism in the detection instrument opens, and the magnetic beads are adsorbed on the side wall of the elution chamber. Then the driving mechanism pushes piston A toward piston B, and in the process of approaching piston B, the eluted reaction liquid is sent to the main channel of the reaction hole. Finally, piston A is in position A4; the reaction liquid will enter the reaction hole for detection.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention integrates sample lysis, cleaning, elution and reaction, and realizes one-stop operation of qPCR; in actual use, the working chamber is mainly used to realize sample lysis, magnetic bead cleaning and elution; the lysis liquid chamber is used to store the lysis liquid package, the cleaning liquid chamber is used to store the cleaning liquid package, and the elution liquid chamber is used to store the cleaning liquid package; the waste liquid chamber, the lysis liquid chamber, the elution liquid chamber and the cleaning liquid chamber are covered by a soft film, and when the soft film is squeezed, the puncture structure can puncture the liquid package, so that the liquid can enter the working chamber easily;

[0034] At the same time, the present invention slides and installs piston A and piston B in the working chamber, so that the volume of the working chamber can be adjusted, and then by changing the positions of piston A and piston B, a lysis cleaning chamber and a magnetic bead elution chamber can be formed respectively; the sample enters the lysis cleaning chamber through the sample flow channel, and the lysis solution with magnetic beads enters the lysis cleaning chamber through the lysis solution flow channel to mix with the sample to achieve the purpose of lysis. After the lysis is completed, the driving mechanism in the detection instrument drives piston A to move, so that piston A can move toward piston B. At this time, the magnetic attraction mechanism in the detection instrument is opened, so that the magnetic beads with protein are adsorbed on the side wall of the lysis cleaning chamber. During the movement of piston A, the waste liquid after lysis is squeezed into the waste liquid flow channel and then flows into the waste liquid chamber. After the waste liquid is discharged, the driving mechanism drives piston A to reset, and then presses the soft membrane to make the cleaning solution in the cleaning liquid chamber flow into the lysis cleaning chamber to clean the magnetic beads with protein. After the cleaning is completed, the magnetic attraction mechanism in the detection instrument is opened, and the driving mechanism in the detection instrument drives piston A to move again. Liquid discharge operation; after cleaning is completed, the driving mechanism drives piston A to move to piston B, and under the action of piston A, piston A and piston B move synchronously, so that piston B moves to the inlet position of the detection channel, and piston A returns to the set position; then the soft membrane is squeezed to allow the eluent to enter the working chamber from the eluent channel for elution operation. After elution is completed, the driving mechanism drives piston A to move again, squeezes the eluted reaction liquid into the detection channel, and performs PCR amplification and fluorescence detection in the detection channel; the air can be discharged when the sample enters the detection channel through the provided breathable membrane; the present invention realizes the purpose of adjustable working chamber by adjusting the position of piston A and piston B, and realizes mixing, lysis, cleaning and elution of samples by adjusting the position of piston A and piston B. Compared with the prior art in which operations are performed in different chambers of the microfluidic chip, the present invention realizes rapid lysis, cleaning and elution operations in the same area, realizes the purpose of integrated operation, and has the advantages of simple structure and convenient operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 Schematic diagram of the overall structure of the qPCR microfluidic chip of the present invention.

[0037] Figure 2 For the present invention Figure 1 Schematic diagram of the overall structure after removing the soft film, hard film and breathable film.

[0038] Figure 3 For the present invention Figure 2 main view.

[0039] Figure 4 For the present invention Figure 3 Section view of the middle AA plane.

[0040] Figure 5 Schematic diagram of the positional relationship between the puncture structure and the liquid bag of the present invention.

[0041] Figure 6 It is a schematic diagram of the overall structure of the piston A of the present invention.

[0042] Figure 7 Schematic diagram of the internal structure of the piston A of the present invention.

[0043] Figure 8 It is a schematic diagram of the structure of the depression provided on the soft film in the present invention.

[0044] Fig. 9 This is a schematic diagram of the state in which the piston A and the piston B of the present invention are located at the initial positions.

[0045] Fig.10 This is a schematic diagram of the positions of piston A and piston B when discharging lysis liquid or cleaning liquid in the present invention.

[0046] Fig.11 This is a schematic diagram of the positions of piston A and piston B when adding eluent according to the present invention.

[0047] Fig.12 This is a schematic diagram of the positional relationship between piston A and piston B when the eluted reaction solution enters the main channel of the reaction hole in the present invention.

[0048] Reference numerals:

[0049] 101-qPCR microfluidic chip body, 102-piston A, 103-piston B, 104-working chamber, 105-sample flow channel, 106-waste liquid flow channel, 107-elution liquid flow channel, 108-reaction hole main channel, 109-liquid flow channel, 110-lysate flow channel, 111-cleaning liquid flow channel, 112-cleaning liquid chamber, 113-lysate chamber, 114-elution chamber, 115-waste liquid chamber, 116-soft membrane, 117- 7-liquid bag, 118-puncture structure, 119-reaction hole liquid inlet channel, 120-reaction hole, 121-reaction hole exhaust channel, 122-breathable membrane, 123-limiting hole, 124-waste liquid chamber vent, 125-sample rubber plug, 126-sample addition port, 127-dura membrane, 128-connecting hole, 129-limiting part, 130-guide slope, 131-driving mechanism, 132-energy storage chamber, 133-energy storage chamber channel. DETAILED DESCRIPTION

[0050] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0051] In the description of the embodiments of the present invention, it should be understood that the terms "length", "vertical", "horizontal", "top", "bottom", etc. indicating the orientation or positional relationship are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of the present invention.

[0052] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0053] In the embodiments of the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection, an electrical connection, or a communication; it can be a direct connection, or an indirect connection through an intermediate medium, and it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present invention can be understood according to specific circumstances.

[0054] In the embodiments of the present invention, unless otherwise clearly specified and limited, a first feature being "above" or "below" a second feature may include that the first and second features are in direct contact, or may include that the first and second features are not in direct contact but are in contact through another feature between them. Moreover, a first feature being "above", "above" and "above" a second feature includes that the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is higher in level than the second feature. A first feature being "below", "below" and "below" a second feature includes that the first feature is directly above and obliquely above the second feature, or simply indicates that the first feature is lower in level than the second feature.

[0055] The disclosure below provides many different embodiments or examples to implement different structures of the embodiments of the present invention. In order to simplify the disclosure of the embodiments of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the embodiments of the present invention. In addition, the embodiments of the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed.

[0056] The embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0057] Embodiment 1

[0058] See also Figure 1-Figure 12 This embodiment discloses an integrated qPCR microfluidic chip structure, including a qPCR microfluidic chip body 101, a hard film 127 and a soft film 116, and the qPCR microfluidic chip body 101 is provided with a sample addition port 126, a working chamber 104, a waste liquid chamber 115, a lysis liquid chamber 113, an elution liquid chamber 114 and a plurality of cleaning liquid chambers 112;

[0059] The sample adding port 126 is provided with a detachable sample rubber plug 125, and the sample adding port 126, the lysing liquid chamber 113, the cleaning liquid chamber 112, the eluting liquid chamber 114 and the waste liquid chamber 115 are respectively connected with the working chamber 104 through the sample flow channel 105, the lysing liquid flow channel 110, the cleaning liquid flow channel 111, the eluting liquid flow channel 107 and the waste liquid flow channel 106; the lysing liquid chamber 113, the cleaning liquid chamber 112 and the eluting liquid chamber 114 are provided with a liquid bag and a puncture structure 118 for puncturing the liquid bag;

[0060] Among them, the working chamber 104 is provided with a piston A102 and a piston B103 in a sliding seal, the piston B103 is located on the left side of the piston A102, the piston A102 is an active piston, the piston B103 is a passive piston, the piston A102 is used to connect with the driving mechanism 131 in the detection instrument, when the driving mechanism 131 drives the piston A102 to move in the working chamber 104, the piston A102 can drive the piston B103 to move in the working chamber 104 after contacting with the piston B103, and the side wall of the working chamber 104 forms a magnetic bead particle adsorption surface;

[0061] The qPCR microfluidic chip body 101 is also provided with a plurality of detection channels, and a breathable membrane 122 is provided at the end of the detection channel; a hard membrane 127 is provided on the qPCR microfluidic chip body 101 and covers the detection channel, and a soft membrane 116 is provided on the qPCR microfluidic chip body 101 and covers the working chamber 104, the lysis liquid chamber 113, the elution liquid chamber 114 and the corresponding channels.

[0062] The flow channels are specifically a sample flow channel 105, a lysate flow channel 110, a cleaning liquid flow channel 111, an eluent flow channel 107 and a waste liquid flow channel 106;

[0063] The lysis liquid flow channel 110 and a plurality of cleaning liquid flow channels 111 form a liquid flow channel 109 .

[0064] In another embodiment, both piston A 102 and piston B 103 are movable, so that each of them can be individually equipped with a driving mechanism to form an active piston.

[0065] The present invention integrates sample lysis, cleaning, elution and reaction, and realizes one-stop operation of qPCR; in actual use, the working chamber 104 is mainly used to realize sample lysis, magnetic bead cleaning and elution; the lysis liquid chamber 113 is used to store the lysis liquid package, the cleaning liquid chamber 112 is used to store the cleaning liquid package, and the elution liquid chamber 114 is used to store the cleaning liquid package; the soft film 116, the lysis liquid chamber 113, the elution liquid chamber 114 and the cleaning liquid chamber 112 are covered, and when the soft film 116 is squeezed, the puncture structure 118 can puncture the liquid package, so that the liquid can enter the working chamber 104;

[0066] At the same time, the present invention slides and installs piston A102 and piston B103 in the working chamber 104, so that the volume of the working chamber 104 can be adjusted, and then by changing the positions of piston A102 and piston B103, a lysis cleaning chamber and a magnetic bead elution chamber can be formed respectively; the sample enters the lysis cleaning chamber through the sample flow channel 105, and the lysis solution with magnetic beads enters the lysis cleaning chamber through the lysis solution flow channel 110 to mix with the sample to achieve the purpose of lysis. After the lysis is completed, the driving mechanism in the detection instrument drives the piston A102 to move, so that the piston A102 moves toward the direction of the piston B103. At this time, the magnetic attraction mechanism in the detection instrument is opened, so that the magnetic beads with protein are adsorbed on the side wall of the lysis cleaning chamber, and the piston A1 During the movement of piston A102, the waste liquid after lysis is squeezed into the waste liquid flow channel 106 and then flows into the waste liquid chamber 115. After the waste liquid is discharged, the driving mechanism drives piston A102 to reset, and then presses the soft membrane 116 to make the cleaning liquid in the cleaning liquid chamber 112 flow into the lysis cleaning chamber to clean the magnetic beads with protein. After the cleaning is completed, the magnetic suction mechanism in the detection instrument is opened, and the driving mechanism in the detection instrument drives piston A to move and perform the liquid discharge operation again; after the cleaning is completed, the driving mechanism drives piston A102 to move to piston B103. Under the action of piston A102, piston A102 and piston B103 move synchronously, so that piston B103 moves to the inlet position of the detection flow channel, and piston A102 returns to the set position, that is: Figure 8A3 position in the middle; then the soft membrane 116 is squeezed to make the eluent enter the working chamber 104 from the eluent flow channel 107 for elution operation. After the elution is completed, the driving mechanism drives the piston A102 to move again, squeezes the eluted reaction liquid into the detection flow channel, and performs PCR amplification and fluorescence detection in the detection flow channel; the air-permeable membrane 122 is arranged to discharge the air when the sample enters the detection flow channel; the present invention realizes the purpose of adjustable working chamber 104 by adjusting the position of piston A102 and piston B103, and realizes mixing, lysis, cleaning and elution of samples by adjusting the position of piston A102 and piston B103. Compared with the prior art that operates in different chambers of the microfluidic chip, the present invention realizes that rapid lysis, cleaning and elution operations can be performed in the same area; the purpose of integrated operation is achieved, and it has the advantages of simple structure and convenient operation.

[0067] In actual use, the qPCR microfluidic chip body 101 is provided with an air outlet connected to the waste liquid chamber 115 to facilitate the discharge of air when the waste liquid is discharged.

[0068] It should be noted that the lysis solution chamber contains lysis solution and magnetic beads, and the lysis solution can be any lysis solution in the prior art, which will not be described in detail here.

[0069] Among them, the detection channel includes a reaction hole main channel 108 connected to the working chamber 104, and a plurality of reaction hole liquid inlet channels 119 connected to the reaction hole main channel 108. Each reaction hole liquid inlet channel 119 is connected to a reaction hole exhaust channel 121. A reaction hole 120 is formed at the connection between the reaction hole liquid inlet channel 119 and the reaction hole exhaust channel 121; a breathable membrane 122 is arranged at the end of the reaction hole exhaust channel 121.

[0070] In actual use, by setting up a plurality of reaction holes 120, a plurality of small reaction chambers are formed, which can achieve the purpose of detecting multiple items by requiring less reaction liquid and reagent to react.

[0071] Further optimized, the waste liquid chamber 115 is arranged on the back side of the qPCR microfluidic chip body 101 , and the lysis liquid chamber 113 , the elution liquid chamber 114 and a plurality of cleaning liquid chambers 112 are arranged on the front side of the qPCR microfluidic chip body 101 and are recessed into the waste liquid chamber 115 .

[0072] This arrangement enables the closed ends of the lysis liquid chamber 113, the elution liquid chamber 114 and the plurality of cleaning liquid chambers 112 to be located within the waste liquid chamber 115, thereby preventing the lysis liquid chamber 113, the elution liquid chamber 114 and the plurality of cleaning liquid chambers 112 from occupying too much volume, thereby reducing the overall volume of the microfluidic chip and making the qPCR microfluidic chip more compact.

[0073] In another embodiment, the waste liquid chamber 115 , the lysis liquid chamber 113 , the cleaning liquid chamber 112 , and the elution liquid chamber 114 may be arranged on the same side of the microfluidic chip body 101 and covered by the soft film 116 .

[0074] It should be noted that, when the present invention is testing, the driving mechanism 131 in the testing instrument is required to drive the piston A102 to move; the driving mechanism 131 can be a telescopic rod installed in the testing instrument, and the piston A102 is driven by the extension and contraction of the telescopic rod; the driving mechanism 131 only needs to drive the piston A102 to move, and its specific structure is not described one by one here;

[0075] At the same time, it should be made clear that in actual use of the present invention, when performing fluorescence detection on the eluted sample at the reaction well 120, the existing qPCR fluorescence detection can be used, and no further details will be given here.

[0076] At the same time, when squeezing liquids, such as lysis solution, cleaning solution and elution solution into the working chamber 104, it is only necessary to install a corresponding squeezing mechanism inside the instrument to be detected. The squeezing mechanism can be a telescopic rod, such as an electric telescopic rod; of course, in actual use, a cam mechanism can also be used, as long as it can achieve the pressing of the liquid bag, and its specific structure will not be repeated here.

[0077] It should be clarified that the present invention is mainly to achieve the improvement of the qPCR microfluidic chip and does not involve the improvement of the detection instrument. Therefore, the description of the detection instrument in this application is mainly to facilitate those skilled in the art to further understand the present invention and clarify the innovative points of the present invention.

[0078] The puncturing structure 118 is a plurality of puncturing needles arranged at the bottom of the lysis liquid chamber 113 , the elution liquid chamber 114 and the plurality of cleaning liquid chambers 112 .

[0079] In this way, by pressing the soft membrane 116 at the corresponding positions of the lysis liquid chamber 113, the elution liquid chamber 114 and the cleaning liquid chamber 112, the liquid bag can be brought into contact with the puncture needle. After the liquid bag is punctured, the purpose of adding liquid can be achieved, and the liquid in the liquid bag can be added to the working chamber 104 by pressing.

[0080] The liquid bag 117 is made of aluminum-plastic film or plastic film and connected to the soft film 116. During use, it is necessary to ensure that when the soft film 116 is pressed, the liquid bag can move toward the puncture structure and contact the puncture structure.

[0081] That is, the liquid bag is punctured by the puncture needle under the condition of external force pressing the soft membrane, so that the liquid in the liquid bag flows out.

[0082] Further optimized, the sample flow channel 105 , the lysis solution flow channel 110 , and the cleaning solution flow channel 111 are gathered on the right side of the working chamber 104 .

[0083] Among them, a limiting hole 123 connected to the right end of the working chamber 104 is provided on the qPCR microfluidic chip body 101, and the limiting hole 123 is used for the driving mechanism 131 to pass through and separate from the piston A102, that is, to achieve the separation of the driving mechanism 131 from the piston A102 after the test is completed.

[0084] Among them, the driving mechanism 131 is detachably connected to the piston A102, and a connecting hole 128 is provided at the end of the piston A102. A plurality of limiting portions 129 are provided on the side wall of the connecting hole 128. A guiding inclined surface 130 is provided on the limiting portion 129 facing the axis of the connecting hole 128. The driving mechanism 131 has a clamping portion that can cooperate with and disengage from the connecting hole 128.

[0085] In this embodiment, since piston A102 and piston B103 are provided in the working chamber, piston A102 and piston B103 are made of rubber or silicone material, and are connected to piston A102 through a driving mechanism to drive piston A102 to move in the working chamber 104; and the positions of the two pistons (piston A102 and piston B103) are changed by the driving mechanism to form two working chambers for sample lysis and cleaning and protein magnetic bead elution, respectively, and the two working chambers formed are specifically adjusted by the positions of piston A102 and piston B.

[0086] The limiting portion 129 and the piston A102 are an integrated structure and are made of medical-grade rubber or silicone material.

[0087] In this way, it is easy to achieve the coordination and separation of the driving mechanism 131 and the piston A102; a limiting surface will be formed at the right end of the working chamber 104. After the piston A102 moves to the limiting hole 123, the piston A102 stops moving, and the driving mechanism 131 continues to move. After the limiting part 129 is deformed, the locking part and the piston A102 are disengaged.

[0088] Further optimization, in some embodiments, the right end of the working chamber 104 is open and communicated with the outside, so as to facilitate the assembly of the piston A 102 and the piston B 103.

[0089] In order to facilitate those skilled in the art to further understand the present invention, the present invention is further described below in conjunction with the specific use method of the present invention.

[0090] In actual use, the initial positions of piston A102 and piston B103 are: A1 and A2:

[0091] The piston A102 is located at the rightmost end of the working chamber 104, i.e., at A1;

[0092] The piston B103 is located between the waste liquid flow channel 106 and the eluent flow channel 107, i.e., at B1. At this time, the reaction hole main flow channel 108 and the plurality of liquid flow channels 109 are connected to the waste liquid flow channel 106; i.e., the sample flow channel 105, the lysate flow channel 110, and the cleaning liquid flow channel 111 are connected to the waste liquid flow channel 106 and form a working chamber that can complete sample lysis and multiple cleanings.

[0093] The specific steps are as follows:

[0094] Step 1: After adding the sample (sample solution) to the sample adding port 126 , the sample rubber plug 125 is matched with the sample adding port 126 , that is, the sample rubber plug is inserted into the sample adding port, and the sample rubber plug 125 is pressed to allow the sample to enter the working chamber 104 ;

[0095] Step 2: Then, the soft membrane 116 at the lysing liquid chamber 113 is pressed. After the liquid packet in the lysing liquid chamber 113 is broken, the lysing liquid in the liquid packet enters the working chamber 104 through the lysing liquid flow channel 110, so that the lysing liquid and the magnetic beads in the lysing liquid enter the working chamber 104 and mix with the sample;

[0096] Step 3: The magnetic attraction mechanism in the detection instrument is turned on, and the magnetic beads adsorbed with proteins are placed on the side wall of the working chamber 104. The driving mechanism pushes the piston A102 to move toward the piston B103. At this time, the piston B103 is still in a stationary state. During the movement of the piston A102, the waste liquid after lysis is discharged into the waste liquid chamber 115 through the waste liquid channel. At this time, the magnetic beads adsorbed with proteins will gather on the side wall between the piston A102 and the piston B103, and the piston A102 is at the A2 position. After the magnetic attraction mechanism is started, it is mainly used to adsorb the magnetic beads to prevent them from entering the waste liquid chamber. The magnetic attraction mechanism can be an electromagnetic adsorption mechanism, specifically an electromagnet. It can be used to achieve the adsorption of magnetic beads, and its specific mechanism will not be repeated here.

[0097] In actual use, the magnetic attraction mechanism can also be a permanent magnet, which can be adjusted by adjusting the distance between the permanent magnet and the working chamber 104. The specific structure will not be described in detail here.

[0098] Step 4: The piston A102 is reset to the position A1, squeezing the soft membrane 116 at the cleaning liquid chamber 112. After the liquid bag in the cleaning liquid chamber 112 is broken, the cleaning liquid in the liquid bag flows through the cleaning liquid channel 111 and enters the working chamber 104, thereby cleaning the magnetic beads.

[0099] Step 5: The driving mechanism pushes the piston A102 to move from the A1 position to the A2 position, and discharges the cleaned waste liquid into the waste liquid chamber 115. In actual use, the cleaning times can be selected according to the detection needs;

[0100] Step 6: After cleaning, piston A102 continues to move toward piston B103 and pushes piston B103 to the rightmost end of the working chamber 104. Piston A102 pushes piston B103 to move. At this time, piston B103 is located at position B2, and piston A102 returns for a distance and stays at position A3. At this time, the reaction hole main channel 108 and the eluent channel 107 are in a connected state and form an elution chamber.

[0101] In actual use, the return stroke stops at position A3 after a certain distance, and position A3 is the flow channel opening close to the eluent flow channel 107 in the working chamber;

[0102] Step 7: The pressing mechanism in the detection instrument presses the soft membrane 116 at the elution liquid chamber 114, so that the liquid bag at the elution liquid chamber 114 is punctured, and the eluent in the liquid bag enters the elution chamber to elute the magnetic beads; the pressing mechanism in the detection instrument is mainly used to realize the pressing of the soft membrane, and its specific structure is not repeated;

[0103] Step 8: After elution is completed, the magnetic suction mechanism in the detection instrument opens, and the magnetic beads are adsorbed on the side wall of the elution chamber. Then the driving mechanism pushes piston A102 to move toward piston B103. In the process of approaching piston B103, the eluted reaction liquid is sent to the main channel 108 of the reaction hole. Finally, piston A102 is in the A4 position; the reaction liquid will enter the reaction hole 120 for detection, that is, an amplification reaction is performed.

[0104] The present invention specifically discloses an integrated qPCR microfluidic chip structure, which has fewer design components and a simpler and more compact structure. In actual use, its size can be controlled within 80*56*11mm; at the same time, the liquid bag is made of aluminum-plastic film or plastic film to form the liquid bag and the puncture structure 118, which has high reliability, good sealing and controllable volume; the piston A102 and the piston B103 form a variable working chamber with the working chamber 104 to achieve sample mixing, lysis, cleaning and reaction hole 120 filling, the structure is simple and reliable, and the single-side piston A102 movement can drive the piston B103 to move in coordination to achieve the entire extraction process of nucleic acid detection; by arranging a breathable membrane 122 at the end of the detection flow channel, gas can pass through but not liquid or aerosol, ensuring that the gas in the flow channel and the reaction hole 120 is discharged during the filling process, so that the reaction liquid fills the flow channel and the reaction hole 120, and the reaction liquid and the aerosol formed by amplification leak out to avoid pollution.

[0105] Embodiment 2

[0106] This embodiment is further optimized on the basis of the first embodiment. In this embodiment, an energy storage cavity channel 133 connected to the main liquid inlet channel of the reaction hole 120 is provided on the qPCR microfluidic chip body 101, and an energy storage device is provided at the outlet end of the energy storage cavity channel 133 on the qPCR microfluidic chip body 101. The energy storage device includes an elastic diaphragm, and the elastic diaphragm is connected to the qPCR microfluidic chip body 101 to form an energy storage cavity 132.

[0107] The present invention provides an energy storage cavity channel 133 on the qPCR microfluidic chip body 101, which is connected to the main liquid inlet channel of the reaction hole 120, and provides an energy storage device at the outlet end of the energy storage cavity channel 133. When the liquid sample in the working cavity 104 is injected into the main liquid inlet channel of the reaction hole 120, if the amount of injected reaction liquid is too large, the excess reaction liquid can enter the energy storage cavity 132 through the energy storage cavity channel 133. At this time, the reaction liquid will form a bulge at the energy storage cavity 132, thereby collecting the excess reaction liquid and avoiding the situation of bursting. At the same time, when the reaction hole is heated, the reaction liquid expands and increases in volume. At this time, the expanded reaction liquid can also enter the energy storage cavity 132, and the expansion pressure during the heating process can be released. When the reaction hole is cooled, the reaction liquid shrinks and decreases in volume. At this time, the liquid in the energy storage cavity 132 can replenish the reaction hole under the action of elastic force. The energy storage chamber 132 provided in the present invention can ensure that the liquid container will not burst due to overcharging during the filling process, and can also release the expansion pressure during the heating process and the liquid replenishment during the cooling process.

[0108] It should be further clarified that the purpose of the present invention is mainly to solve the technical problems of excessive addition of reaction liquid and expansion of the reaction liquid during heating and cooling, which leads to malfunction of the microfluidic chip.

[0109] A depression is provided on the soft membrane 116 at a position corresponding to the outlet end of the energy storage cavity channel 133 , and the elastic diaphragm is formed at the position where the depression is provided on the soft membrane 116 . After the soft membrane 116 and the hard membrane 127 are connected together, the energy storage cavity 132 is formed at the depression position on the soft membrane 116 .

[0110] That is to say, in this embodiment, there is no need to add an elastic diaphragm, but a depression is set on the soft membrane 116, so that the thickness of the soft membrane 116 located in the depression is thinner, and this part can be identified as an elastic diaphragm. When the sample enters the depression, a bulge can be formed under the action of pressure.

[0111] Among them, a plurality of air outlets are arranged on the hard membrane 127 , and the positions of the air outlets correspond to the ends of the exhaust channels of the reaction holes, and the breathable membrane 122 is bonded to the hard membrane 127 .

[0112] Further optimized, the soft film 116 is a rubber film or a silicone film.

[0113] The hard film 127 is attached to the qPCR microfluidic chip body 101 by pasting or ultrasonic welding.

[0114] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An integrated qPCR microfluidic chip structure, characterized in that: The qPCR microfluidic chip comprises a qPCR microfluidic chip body, a hard film and a soft film. The qPCR microfluidic chip body is provided with a sample addition port, a working chamber, a waste liquid chamber, a lysis liquid chamber, an elution liquid chamber and a plurality of cleaning liquid chambers; The sample adding port is provided with a detachable sample rubber plug, and the sample adding port, the lysis liquid chamber, the cleaning liquid chamber, the elution liquid chamber and the waste liquid chamber are connected with the working chamber through the sample flow channel, the lysis liquid flow channel, the cleaning liquid flow channel, the elution liquid flow channel and the waste liquid flow channel respectively; the lysis liquid chamber, the cleaning liquid chamber and the elution liquid chamber are provided with a liquid bag and a puncture structure for puncturing the liquid bag; Among them, the working chamber is provided with a piston A and a piston B in a sliding seal, and the piston B is located on the left side of the piston A. The piston A is used to connect with the driving mechanism in the detection instrument. When the driving mechanism drives the piston A to move in the working chamber, the piston A contacts the piston B and can drive the piston B to move in the working chamber, and the side wall of the working chamber forms a magnetic bead particle adsorption surface; The qPCR microfluidic chip body is also provided with a plurality of detection flow channels, and a breathable membrane is provided at the end of the detection flow channel; the hard membrane is provided on the qPCR microfluidic chip body and covers the detection flow channel, and the soft membrane is provided on the qPCR microfluidic chip body and covers the working chamber, the lysis liquid chamber, the elution liquid chamber and the corresponding flow channels; The qPCR microfluidic chip body is provided with a limiting hole connected to the right end of the working chamber, and the limiting hole is used for the driving mechanism to pass through and separate from the piston A; The driving mechanism is detachably connected to the piston A. A connecting hole is provided at the end of the piston A. A plurality of limiting parts are provided on the side wall of the connecting hole. A guiding inclined surface is provided on the limiting part facing the axis of the connecting hole. The driving mechanism has a clamping part that can cooperate with and disengage from the connecting hole.

2. The integrated qPCR microfluidic chip structure according to claim 1, characterized in that: The detection flow channel includes a reaction hole main flow channel connected to the working chamber, a plurality of reaction hole liquid inlet flow channels connected to the reaction hole main flow channel, each reaction hole liquid inlet flow channel is connected to a reaction hole exhaust flow channel, and a reaction hole is formed at the connection between the reaction hole liquid inlet flow channel and the reaction hole exhaust flow channel; the air permeable membrane is arranged at the end of the reaction hole exhaust flow channel.

3. The integrated qPCR microfluidic chip structure according to claim 1, characterized in that: The waste liquid chamber is arranged on the back of the qPCR microfluidic chip body, and the lysis liquid chamber, the elution liquid chamber and a plurality of cleaning liquid chambers are arranged on the front of the qPCR microfluidic chip body and are recessed into the waste liquid chamber.

4. The integrated qPCR microfluidic chip structure according to claim 1, characterized in that: The puncturing structure is a plurality of puncturing needles arranged at the bottom of the lysing liquid chamber, the eluting liquid chamber and a plurality of cleaning liquid chambers.

5. The integrated qPCR microfluidic chip structure according to claim 1, characterized in that: The liquid bag is made of aluminum-plastic film or plastic film and is connected with the soft film.

6. The integrated qPCR microfluidic chip structure according to claim 1, characterized in that: The sample flow channel, the lysis solution flow channel, and the cleaning solution flow channel are gathered on the right side of the working chamber.

7. The integrated qPCR microfluidic chip structure according to claim 2, characterized in that: The qPCR microfluidic chip body is provided with an energy storage cavity channel connected to the main channel of the reaction hole. The qPCR microfluidic chip body is provided with an energy storage device at the outlet end of the energy storage cavity channel. The energy storage device includes an elastic diaphragm. The elastic diaphragm is connected to the qPCR microfluidic chip body to form an energy storage cavity.

8. A method for using an integrated qPCR microfluidic chip, characterized in that: The method comprises using the integrated qPCR microfluidic chip structure described in claim 2; Among them, the initial positions of piston A and piston B in the integrated qPCR microfluidic chip structure are: A1 and B1: Piston A is located at the rightmost end of the working chamber, i.e., at A1; Piston B is located between the waste liquid flow channel and the eluent flow channel, i.e., at B1. At this time, the sample flow channel, the lysate flow channel, the cleaning liquid flow channel and the waste liquid flow channel are connected to form a working chamber that can complete sample lysis and multiple cleanings. The specific steps are as follows: Step 1: After adding the sample to the sample inlet, fit the sample rubber plug with the sample inlet, and press the sample rubber plug to allow the sample to enter the working chamber; Step 2: The squeezing mechanism in the detection instrument presses the soft membrane at the lysis liquid chamber. After the liquid bag in the lysis liquid chamber is broken, the lysis liquid and magnetic beads in the liquid bag enter the working chamber through the lysis liquid flow channel and mix with the sample; Step 3: The magnetic attraction mechanism in the detection instrument is turned on, and the magnetic beads adsorbing proteins are placed on the side wall of the working chamber. The driving mechanism pushes piston A to move toward piston B. At this time, piston B is still in a stationary state. During the movement of piston A, the lysed waste liquid is discharged into the waste liquid chamber through the waste liquid channel. At this time, the magnetic beads adsorbing proteins will gather on the side wall between piston A and piston B, and piston A is at position A2. Step 4: Piston A is reset to position A1, squeezing the soft membrane at the cleaning liquid chamber. After the liquid bag in the cleaning liquid chamber is broken, the cleaning liquid in the liquid bag flows through the cleaning liquid flow channel and enters the working chamber to clean the magnetic beads; Step 5: The driving mechanism pushes piston A to move from position A1 to position A2, discharges the waste liquid after cleaning into the waste liquid chamber, and selects the cleaning times according to the needs; Step 6: After cleaning, piston A continues to move toward piston B and pushes piston B to the leftmost end of the working chamber. At this time, piston B is at position B2, and piston A returns for a distance and stops at position A3; at this time, the main flow channel of the reaction hole and the eluent flow channel are in a connected state and form an elution chamber; Step 7: Press the soft membrane at the elution liquid cavity to puncture the liquid bag at the elution liquid cavity, and the elution liquid in the liquid bag enters the elution cavity to elute the magnetic beads; Step 8: After elution is completed, the magnetic suction mechanism in the detection instrument opens, and the magnetic beads are adsorbed on the side wall of the elution chamber. Then the driving mechanism pushes piston A toward piston B, and in the process of approaching piston B, the eluted reaction liquid is sent to the main channel of the reaction hole. Finally, piston A is in position A4; the reaction liquid will enter the reaction hole for detection.

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