A centrifugal fully integrated nucleic acid detection microfluidic chip

Abstract

This invention relates to the field of biochemical detection device technology, specifically to a centrifugal fully integrated nucleic acid detection microfluidic chip. Specifically, the microfluidic chip of this invention integrates sample enrichment, sample lysis, nucleic acid extraction, and amplification detection functions. It can precisely control the transfer of multiple fluids using only a rotation speed gradient, without manual intervention or external auxiliary equipment. Furthermore, it employs an innovative design combining a burst valve with a siphon channel and a dual-channel design to split different fluids, overcoming the instability issues that can easily occur with cascaded designs of multi-stage siphon channels, leading to unexpected activation or interruption failures.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection device technology, specifically to a centrifugal fully integrated nucleic acid detection microfluidic chip. Background Technology

[0002] Microfluidics technology, which manipulates micro- and nanofluids through micrometer-level channels to achieve "laboratory on a chip," has achieved commercial breakthroughs in fields such as in vitro diagnostics since its emergence in the 1990s. Among them, centrifugal microfluidic systems, driven by centrifugal force, have become a core direction in the field of nucleic acid detection due to their advantages such as simple processing, controllable cost, and great integration potential.

[0003] Traditional nucleic acid testing relies on multi-step operations in specialized laboratories, which is time-consuming and involves a lot of manual intervention, making it difficult to meet the needs of grassroots and point-of-care testing (POCT). A fully integrated "sample in, result out" solution has become the industry's goal, and centrifugal technology is preferred due to its ability to integrate multiple steps and precisely control flow through rotation speed. To achieve the integration of multiple operational steps, centrifugal microfluidic chips require the use of various microvalves to achieve complex control of multi-stage fluids, among which microvalves formed by siphon channels are the most widely used. Siphon channels achieve directional liquid flow through the dynamic balance of capillary force and centrifugal force. At rest or low speed, the capillary force of the hydrophilic channel dominates, causing the fluid to cross the peak point and fill the channel, thus entering the downstream chamber; at high speed, centrifugal force inhibits liquid flow, preventing premature transfer. In short, siphon channels can precisely control the step-by-step transfer of liquid through rotation speed regulation (such as static filling, high-speed inhibition, and low-speed release), without external force drive, significantly simplifying the design of centrifugal microfluidic chips and reducing costs.

[0004] The core defects of existing technologies are mainly manifested in the following ways: 1. Insufficient integration: Most centrifugal microfluidic chips have a "pseudo-integration" problem—they require manual assistance to complete sample pretreatment or reagent addition, and cannot achieve a fully unattended detection process of "sample in - result out". For example, the microfluidic chip disclosed in Chinese invention patent application CN202211354147.1 does not integrate pretreatment functions such as nucleic acid extraction.

[0005] 2. To achieve multi-step integration, complex flow channels and chamber structures need to be designed. However, existing centrifugal drive methods cannot precisely control the transfer of multiple fluids, often requiring external equipment assistance, resulting in low efficiency and sacrificing the simplicity and reliability of integration. For example, the microfluidic chips disclosed in Chinese invention patent applications CN202211433505.8 and CN202310781753.X require external equipment to press and puncture the capsule structure or heat to activate the paraffin valve.

[0006] 3. Multi-stage siphon channels are prone to instability due to accidental breakage. The reasons are as follows: First, there is no physical barrier between the siphon channel and the fluid, making it susceptible to accidental activation upon contact with the fluid at the inlet. This results in insufficient stability, requiring strict avoidance of unnecessary vibrations to prevent accidental fluid contact. Second, sequential and continuous operation is not possible. The hydrophilicity of the channel's inner wall is easily reduced by scouring or the entry of air bubbles, making it difficult to stably reactivate and transfer subsequent fluids. For example, the microfluidic chip disclosed in Chinese invention patent application CN202210242670.9 has multiple siphon channels, requiring multiple centrifugations to transfer extracted nucleic acids to the detection chamber. This cumbersome operation carries the risk of process breakage.

[0007] 3. The demand for high throughput forces chips to increase the number of chambers and channels, leading to structural complexity that is difficult to reconcile with low-cost chip manufacturing. For example, the microfluidic chip disclosed in Chinese invention patent application CN202410349526.4 has a simple structure and is easy to manufacture, but it can only achieve single-index or a few-index detection, limiting throughput. The high-throughput microfluidic chip disclosed in Chinese invention patent application CN202410313988.0 consists of a 6-layer structure, which is difficult and costly to manufacture, limiting the widespread use of the chip. Summary of the Invention

[0008] Building upon existing technologies, this invention provides a centrifugally integrated microfluidic chip for nucleic acid detection. This microfluidic chip integrates sample enrichment, sample lysis, nucleic acid extraction, and amplification detection functions. The transfer of multiple fluids can be precisely controlled solely by a rotation speed gradient, eliminating the need for manual intervention or external auxiliary equipment. The microfluidic chip of this invention features a simple structure and manufacturing process, resulting in low production costs. Multiple independent nucleic acid detection units can perform parallel multi-sample, multi-index detection, making it particularly suitable for the in vitro diagnostic point-of-care testing (POCT) field.

[0009] Specifically, the microfluidic chip of this invention employs an innovative design that combines a burst valve with a siphon channel and uses a dual-channel system to split different fluids, thereby overcoming the instability problem of accidental activation or interruption failure that easily occurs in the cascaded design of multi-stage siphon channels. Furthermore, it simplifies the flow channel and chamber structure design of the microfluidic chip, thereby reducing chip manufacturing costs, as detailed below: To address the drawback of accidental activation of the siphon channel inlet upon contact with reagents, this invention incorporates a burst valve structure connected in series with the siphon channel, acting as a physical barrier between the siphon channel and the fluid. The burst valve is a passive microfluidic valve that utilizes the capillary force abrupt change caused by the geometric abrupt change in the microchannel to impede liquid flow. The burst valve can only be "opened" when the external driving pressure exceeds a certain critical value (burst pressure threshold). To set a higher burst pressure threshold and improve the controllability and stability of the series-connected siphon channel, the burst valve was optimized. The burst valve adopts a dumbbell-shaped structure with obtuse angles (120°) at both the inlet and outlet, a depth slightly greater than the siphon channel, and a hydrophobic inner wall, thereby increasing the difficulty of spontaneous fluid filling of the burst valve.

[0010] Before using the siphon channel, its inner wall needs to be hydrophilically treated to give it sufficient capillary force for proper functioning. However, when the siphon channel transfers fluid, its inner wall hydrophilicity decreases due to scouring or air bubbles may enter, making it difficult to stably reactivate for transferring the subsequent second fluid. To address this, the sample chamber of the microfluidic chip described in this invention employs a dual-channel structure design, allowing the two fluids to be transferred via two independent siphon channels. To ensure that the siphon channel connecting the sample chamber and the waste chamber does not become ineffective again after transferring the supernatant of the bacterial sample captured by the magnet, it is treated with a hydrophilic reagent diluted 20 times with isopropanol. Therefore, the subsequent nucleic acid to be tested in the sample chamber will be transferred to the detection chamber via the other siphon channel.

[0011] In this regard, the technical solutions of the present invention include, but are not limited to, the following: In some embodiments, the present invention provides a centrifugal microfluidic chip comprising a central through-hole and one or more nucleic acid detection working units, wherein the microfluidic chip is fixed to a rotating shaft through the central through-hole and rotates with the central through-hole as the center to provide centrifugal force to the fluid in the nucleic acid detection working unit; Each nucleic acid testing work unit includes a lysis buffer chamber, a sample chamber, a magnet chamber, a sample waste liquid chamber, a testing chamber, a first siphon channel, a first burst valve, a second siphon channel, a second burst valve, and a third siphon channel; The lysis buffer chamber is used to store nucleic acid lysis buffer; the sample chamber is used to store and enrich the sample to be tested and to lyse and release the nucleic acid of the target microorganism in the sample to be tested; the magnet chamber is used to contain magnetic material to provide a high-intensity magnetic field to the inner wall of the sample chamber near the magnet chamber; the waste liquid chamber is used to collect and store waste liquid; and the detection chamber is used to amplify and detect the nucleic acid in the sample to be tested. The lysis buffer chamber is connected to the sample chamber via a first siphon channel for transferring nucleic acid lysis buffer from the lysis buffer chamber to the sample chamber. A first burst valve is connected in series in the first siphon channel. During the rotation of the microfluidic chip, when the pressure applied by the nucleic acid lysis buffer to the first burst valve is greater than or equal to its required burst pressure threshold, the nucleic acid lysis buffer flows through the first burst valve. The sample chamber is connected to the detection chamber via a second siphon channel to transfer the released nucleic acid template solution from the sample chamber to the detection chamber. A second burst valve is connected in series in the second siphon channel. During the rotation of the microfluidic chip, when the pressure applied by the nucleic acid template solution to the second burst valve is greater than or equal to its required burst pressure threshold, the nucleic acid template solution flows through the second burst valve. The sample chamber is connected to the sample waste liquid chamber via a third siphon channel to completely transfer the waste liquid in the sample chamber to the sample waste liquid chamber. The first rupture valve and the second rupture valve are respectively located near the entrance of the first siphon channel and the second siphon channel, and their inner walls are treated with hydrophobicity. The inner walls of the first, second, and third siphon channels are coated with a hydrophilic substance to give the channels sufficient capillary force for normal conduction. After one liquid transfer, the content of the hydrophilic substance on the inner wall of the channel decreases due to the scouring of the liquid, and the capillary force decreases accordingly, making it difficult to conduct again for a second liquid transfer.

[0012] In some embodiments, when the chip is stationary or under low-speed centrifugation (e.g., at 500-1000 rpm), the liquid in the upstream chamber fills the siphon channel under the action of capillary force, completing the channel opening, and then transfers to the downstream chamber under subsequent low-speed centrifugation (e.g., at 1000-2500 rpm).

[0013] In some embodiments, the first, second, and third siphon channels of the present invention may be interrupted after one liquid transfer and / or due to the introduction of air bubbles, and may be difficult to reconnect for a second liquid transfer.

[0014] In this invention, the siphon channel achieves directional liquid flow through the dynamic balance of capillary force and centrifugal force. At rest or low speed, the capillary force of the hydrophilic channel dominates, causing the fluid to cross the peak point and fill the channel, thus entering the downstream chamber. At high speed, centrifugal force inhibits liquid flow, preventing premature transfer. In summary, the siphon channel can precisely control the stepwise transfer of liquid through speed regulation (e.g., static filling, high-speed inhibition, low-speed release).

[0015] In some embodiments, the shapes of the first rupture valve and the second rupture valve of the present invention are each independently selected from rectangle, triangle, arrow shape or dumbbell shape, and the valve wall at which the virtual extension line of the siphon channel wall at the inlet of the rupture valve points to the inside of the valve and connects with the siphon channel wall is at least 90 degrees, preferably 120 degrees, and the maximum depth of the first rupture valve and the second rupture valve perpendicular to the chip surface is greater than the maximum depth of the first siphon channel and the second siphon channel perpendicular to the chip surface, respectively. And / or, the width and depth of the first siphon channel, the second siphon channel, and the third siphon channel are each independently 100-400 μm, preferably, the width and depth are both 300 μm; And / or, the first siphon channel, the second siphon channel, and the third siphon channel are inverted U-shaped, with their inlet end connected to the upstream chamber and their outlet end connected to the downstream chamber. The siphon apex is closer to the center via of the microfluidic chip than the edge of the upstream chamber. And / or, the second siphon channel and the third siphon channel are located on opposite sides of the sample chamber, such that when the nucleic acid solution is transferred to the detection chamber via the second siphon channel and when the waste liquid is transferred to the sample waste liquid chamber via the third siphon channel, the liquid flow at the inlet of the second and third siphon channels is opposite; And / or, the hydrophilic reagent coated on the inner wall of the third siphon channel has been diluted 20 times with isopropanol to ensure that it will not be re-conducted after a single liquid transfer.

[0016] In some specific implementations, in order to completely transfer the liquid in the chamber via the siphon channel, the inlet of the siphon channel is generally located at the edge of the chamber furthest from the central through-hole; in particular, the first siphon channel is located at the edge of the lysis fluid chamber furthest from the central through-hole, and the second and third siphon channels are located at the edge of the sample chamber furthest from the central through-hole.

[0017] In some specific implementation schemes, the burst valve is generally located near the inlet of the siphon channel. To ensure that the liquid in the chamber can successfully burst the burst valve, it is generally required that, when the chip is stationary, the distance between the liquid surface in the chamber and the central through-hole is less than the distance between the burst valve inlet and outlet and the central through-hole. In particular, when the chip is stationary, the distance between the lysate surface in the lysate chamber and the central through-hole is less than the distance between the first burst valve inlet and outlet and the central through-hole, and in the sample chamber, the distance between the lysate surface and the central through-hole is less than the distance between the second burst valve inlet and outlet and the central through-hole. The impact force that causes the burst valve to burst is the force of centrifugal force driving the fluid in the chamber to impact the valve.

[0018] In some embodiments, both the first rupture valve and the second rupture valve of the present invention are dumbbell-shaped.

[0019] In some specific implementations, the rupture valve is dumbbell-shaped, and at the inlet and outlet of the rupture valve, the virtual extension line of the siphon channel wall pointing into the valve is at an angle of 120 degrees to the rupture valve wall connected to the siphon channel wall.

[0020] In this invention, the length (distance from inlet to outlet) of the burst valve is 1-2 mm, the width is 2-4 mm, and the depth is 150-450 μm.

[0021] In some embodiments, the hydrophilic reagent described in this invention is a commercially available microfluidic chip hydrophilic modification reagent (e.g., the hydrophilic reagent from Wuhan Mesoscopic Biotechnology Co., Ltd., which is used to perform hydrophilic modification through a coating process).

[0022] In some specific implementation schemes, in order to ensure that the third siphon channel connecting the sample chamber and the sample waste chamber is less hydrophilic and less effective after transferring the supernatant of the sample captured by the magnet, it is treated with a hydrophilic reagent diluted 20 times with isopropanol. Therefore, the nucleic acid template to be tested in the sample chamber will be transferred from the second siphon channel to the detection chamber.

[0023] In some specific implementations, when the waste liquid in the sample chamber (i.e., the supernatant after the immunomagnetic beads are captured) comes into contact with the inlet of the third siphon channel, the third siphon channel is activated due to the capillary principle, causing the microfluidic chip to rotate at the second speed to accelerate the transfer of the waste liquid to the sample waste liquid chamber, while the second siphon channel is not activated because the second burst valve connected in series with it is not burst. And / or, when the microfluidic chip rotates at the first speed, the nucleic acid lysis buffer in the lysis buffer chamber can break through the first burst valve, stop centrifugation, and the first siphon channel is activated due to capillary force, so that the microfluidic chip rotates at the second speed to accelerate the transfer of the lysis buffer to the sample chamber, and rotates clockwise and counterclockwise at a third speed less than the second speed to ensure that the target microorganisms bound to the immunomagnetic beads adsorbed on the inner wall of the sample chamber come into full contact with the nucleic acid lysis buffer, thereby completing the lysis of the microorganisms and releasing the nucleic acid.

[0024] And / or, when the microfluidic chip rotates at the first speed, the nucleic acid solution obtained in the sample chamber breaks through the second burst valve, stops centrifugation, and the second siphon channel is activated due to the capillary force principle, so that the microfluidic chip rotates at the second speed to accelerate the transfer of the nucleic acid solution to the detection chamber; When the microfluidic chip rotates at a second or third speed, the pressure exerted by the liquid in the chamber on the burst valve is insufficient to break the first and second burst valves.

[0025] In some implementations, the centrifugal microfluidic chip of the present invention includes two, three, or four nucleic acid detection working units, each of which can independently process one type of sample to be tested, thereby achieving parallel detection.

[0026] In some embodiments, the centrifugal microfluidic chip of the present invention includes two, three, four, five or more nucleic acid detection working units, each capable of independently processing one type of sample to be tested, thus achieving parallel detection. Preferably, the centrifugal microfluidic chip of the present invention includes four nucleic acid detection working units distributed in a centrally symmetrical manner, which can reduce shaking during centrifugation.

[0027] In some implementations, each nucleic acid detection working unit is arranged radially (in the direction of centrifugal force) from the inside out (from the closest to the farthest from the central through-hole) as follows: lysis buffer chamber, sample chamber, magnet chamber, sample waste chamber and detection chamber, or lysis buffer chamber, sample chamber, magnet chamber, detection chamber and sample waste chamber.

[0028] Specifically, in this invention, the larger the volume of the sample chamber, the better, as it can hold more samples and improve the magnetic enrichment effect; the volume of the sample waste liquid chamber should be larger than the volume of the sample chamber to accommodate the waste liquid; the size of the detection chamber determines the size of the subsequent nucleic acid amplification reaction system and affects the amount of nucleic acid amplification reagents required to be embedded inside; the volume of the lysis buffer chamber should be larger than the sum of the volumes of all detection chambers, because after the lysis buffer lyses the bacteria, it is transferred together with the released nucleic acid template to fill the three detection chambers, that is, the volume of the lysis buffer should be larger than the sum of the volumes of all nucleic acid amplification systems.

[0029] In some specific implementations, the volume of the lysis fluid chamber is approximately 200 μL.

[0030] In some specific implementations, the volume of the sample chamber is approximately 900 μL.

[0031] In some specific implementations, the volume of the sample waste chamber is approximately 1000 μL.

[0032] In some specific implementations, there are three or more detection chambers, each with a volume of approximately 30 μL.

[0033] In some embodiments, the centrifugal microfluidic chip of the present invention has a circular disk-shaped structure with a diameter of 100-300 mm, preferably 200 mm; And / or, each nucleic acid detection work unit also includes a detection waste liquid chamber, which is connected to the detection chamber, for storing excess nucleic acid solution flowing out of the detection chamber; And / or, the lysis buffer chamber and the sample chamber are respectively provided with sample loading ports; And / or, the lysis buffer chamber, sample chamber, sample waste chamber and detection waste chamber are all provided with vents to allow the chamber pressure to communicate with atmospheric pressure for pressure balance, and the vents are all located closer to the center through-hole of the microfluidic chip than the edge of the chamber to ensure that reagents do not leak during centrifugation.

[0034] In some embodiments, the centrifugal microfluidic chip of the present invention is assembled from a structural layer and an encapsulation layer. The nucleic acid detection working unit is distributed in the structural layer. The central through hole is composed of a rotating shaft through hole located in the encapsulation layer and a corresponding rotating shaft hole located in the structural layer. Preferably, the encapsulation layer can encapsulate the structural layer by adhesive bonding or hot pressing. And / or, the magnet chamber is a structure in which magnetic material can be installed through.

[0035] In some specific implementations, the encapsulation layer of the centrifugal microfluidic chip of the present invention has magnetic through holes corresponding to the magnetic chamber, so that the magnet can be installed through it.

[0036] In some embodiments, the magnetic material of the present invention is a magnet, preferably an N52 neodymium iron boron permanent magnet; And / or, the sample waste liquid chamber is encapsulated with absorbent material, preferably absorbent cotton; And / or, the detection chamber is pre-embedded with reagents for nucleic acid amplification reactions.

[0037] In some embodiments, the reagents for the nucleic acid amplification reaction of the present invention are selected from reagents for isothermal amplification of nucleic acids such as LAMP, RPA, and RAA.

[0038] In some implementations, the nucleic acid isothermal amplification reagent of the present invention is in the form of liquid, lyophilized powder or lyophilized pellets, preferably lyophilized pellets, for easy pre-embedding and storage.

[0039] In some specific implementations, the centrifugal microfluidic chip of the present invention is disposable. Specifically, the centrifugal microfluidic chip of the present invention is disposable, and to avoid residual bacteria and nucleic acid templates from affecting the detection of subsequent samples, the nucleic acid lysis buffer and nucleic acid amplification reagents pre-packaged in the chip are also disposable.

[0040] In some embodiments, the present invention also provides a rapid nucleic acid detection device comprising the centrifugal microfluidic chip described herein.

[0041] In other embodiments, the present invention provides a nucleic acid detection method, comprising: using the centrifugal microfluidic chip or the nucleic acid detection device described in the present invention.

[0042] In some embodiments, the method of the present invention includes one or more of the following steps: Enrichment of the test sample: The test sample solution captured by immunomagnetic beads in the sample chamber is brought close to the magnetic material so that the immunomagnetic beads bound to the target microorganism are adsorbed onto the inner wall of the chamber, and the waste liquid is brought into contact with the inlet of the third siphon channel. The third siphon channel is opened and activated due to the capillary principle, and is used to transfer the waste liquid (i.e., the supernatant after the immunomagnetic beads are captured) to the sample waste liquid chamber. Preferably, the microfluidic chip is rotated at a second speed to accelerate the transfer of waste liquid to the sample waste liquid chamber, while the second siphon channel is not opened and activated because the second burst valve connected in series with it is not burst. Nucleic acid extraction: The microfluidic chip is rotated at a first speed, causing the nucleic acid lysis buffer in the lysis buffer chamber to break through the first burst valve, stopping centrifugation. The first siphon channel is activated due to capillary force, transferring the lysis buffer to the sample chamber. Preferably, the microfluidic chip is rotated at a second speed to accelerate the transfer of the lysis buffer to the sample chamber. Then, the microfluidic chip is rotated clockwise and counterclockwise at a third speed less than the second speed to ensure that the target microorganisms bound to the immunomagnetic beads adsorbed on the inner wall of the sample chamber come into full contact with the nucleic acid lysis buffer, releasing the nucleic acid from the target microorganisms. Nucleic acid amplification and detection: The microfluidic chip is rotated at a first rotation speed so that the nucleic acid solution obtained in the sample chamber breaks through the second burst valve, stopping centrifugation. The second siphon channel is activated due to capillary force, allowing the nucleic acid solution to be transferred to the detection chamber. Preferably, the microfluidic chip is rotated at a second rotation speed to accelerate the transfer of the nucleic acid solution to the detection chamber. The nucleic acid solution reacts with the pre-embedded nucleic acid amplification reagent in the detection chamber, and then the nucleic acid concentration is detected. When the microfluidic chip rotates at a second or third speed, the pressure exerted by the liquid in the chamber on the burst valve is insufficient to break the first and second burst valves.

[0043] In some embodiments, the first rotational speed of the present invention is 2500~3500 rpm, preferably 3000 rpm; And / or, the second rotational speed is 1000~2500 rpm, preferably 1250-2000 rpm; And / or, the third rotational speed is 500~1000 rpm, preferably 800 rpm.

[0044] The microfluidic chip and detection system containing the present invention address the pain points of existing technologies and achieve multi-dimensional breakthroughs. Its beneficial technical effects include at least the following: 1. The microfluidic chip of the present invention adopts an innovative design that combines a burst valve with a siphon channel and a dual-channel diversion of different fluids, thereby overcoming the instability problem that is prone to unexpected activation or interruption failure in the cascade design of multi-stage siphon channels. The structure and process are simple and the manufacturing cost is low.

[0045] 2. The centrifugal microfluidic chip of the present invention can precisely control the transfer of multiple fluids by means of rotation speed gradient, without the need for manual intervention or external auxiliary equipment, and the four independent nucleic acid detection working units support the parallel detection of multiple indicators, increasing throughput by 1-3 times.

[0046] 3. Traditional fully integrated microfluidic chips generally only include processes such as sample lysis, nucleic acid extraction, amplification and detection. The microfluidic chip of this invention adds a sample enrichment pretreatment process through magnetic separation technology, which improves the integration and truly realizes full automation of "sample in - result out", while improving the sensitivity of nucleic acid detection. Attached Figure Description

[0047] Figure 1 A 3D illustration of a microfluidic chip.

[0048] Figure 2 A 3D illustration of the microfluidic chip packaging layer.

[0049] Figure 3 A 3D illustration showing the structure layers of a microfluidic chip.

[0050] Figure 4 A two-dimensional illustration of the nucleic acid detection working unit of a microfluidic chip.

[0051] Figure 5 This shows a 3D illustration illustrating the design of the burst valve.

[0052] Figure 6 This shows a timing diagram of the gradient centrifugation control process.

[0053] Figure 7 This diagram illustrates the workflow of a microfluidic chip.

[0054] Figure 8 This diagram shows the microbial testing process using a microfluidic chip.

[0055] Figure 9 This diagram illustrates the workflow for bacterial detection based on a microfluidic chip.

[0056] The following are explanations of the labels in the attached diagram: 1-Encapsulation layer; 2-Structural layer; 10-Spindle through hole; 11-Magnet through hole; 20-Spindle hole; 21-Vent hole; 22-Sample dispensing hole; 23-Cycle fluid chamber; 24-Sample chamber; 25-Magnet chamber; 26-Sample waste liquid chamber; 27-Detection waste liquid chamber; 28-Detection chamber; 29-First burst valve; 30-First siphon channel; 31-Second burst valve; 32-Second siphon channel; 33-Third siphon channel. Detailed Implementation

[0057] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0058] Example 1: Centrifugal Fully Integrated Nucleic Acid Detection Microfluidic Chip like Figure 1 The centrifugal fully integrated nucleic acid detection microfluidic chip shown is disc-shaped with a diameter of 200 mm. It is composed of an upper transparent encapsulation layer 1 and a lower structural layer 2, which are tightly bonded together by adhesive. The two rotate around the same centrifugal axis.

[0059] like Figure 2 As shown, the encapsulation layer 1 has a central shaft through hole 10 located at the center for docking with the motor shaft. The encapsulation layer 1 also has four magnet through holes 11 for mounting magnets.

[0060] like Figure 3 As shown, structural layer 2 contains four sets of nucleic acid detection units, each capable of processing one sample independently, enabling parallel detection. Figure 4 As shown, the structure of each unit is distributed radially (in the direction of centrifugal force) from the inside out as follows: lysis fluid chamber 23, sample chamber 24, magnet chamber 25, sample waste liquid chamber 26, detection waste liquid chamber 27 and detection chamber 28.

[0061] The lysis buffer chamber 23 is located at the innermost side and has a volume of about 200 μL. It is used to store nucleic acid lysis buffer and is connected to the sample chamber 24 through the first burst valve 29 and the first siphon channel 30 in series.

[0062] The sample chamber 24, located outside the lysis buffer chamber 23, has a volume of approximately 900 μL and is used to store samples, enrich samples, and release sample nucleic acids. The sample chamber 24 has two outward branch channels: one is connected to the sample waste liquid chamber 26 via the third siphon channel 33, and the other is connected to the detection chamber 28 via the second burst valve 31 and the second siphon channel 32.

[0063] The magnet chamber 25 is located at a specific position outside the sample chamber 24. It is a receiving groove for inserting a permanent magnet from outside the chip (through the magnet through-hole 11 of the encapsulation layer 1) to provide a high-intensity magnetic field to the inner wall area of ​​the sample chamber 24 adjacent to the magnet for sample enrichment.

[0064] The sample waste liquid chamber 26 is located outside the magnet chamber 25, with a volume of approximately 1000 μL. It is pre-encapsulated with absorbent material inside for absorbing and storing waste liquid.

[0065] The detection chamber 28, located on the outermost side, has a volume of approximately 30 μL. It contains pre-embedded lyophilized bulbs for isothermal nucleic acid amplification reactions (such as RPA or LAMP) to amplify the sample nucleic acid and detect signals. The detection chamber 28 is connected via a short channel to the detection waste chamber 27, which stores excess sample nucleic acid reagents.

[0066] Both the lysis buffer chamber 23 and the sample chamber 24 are designed with sample loading holes for reagent injection.

[0067] The lysis buffer chamber 23, sample chamber 24, sample waste liquid chamber 26, and detection waste liquid chamber 27 are all designed with vents to ensure that each chamber is connected to the atmosphere and to avoid negative pressure.

[0068] like Figure 5 As shown, the valve shapes of the first burst valve 29 and the second burst valve 31 can be rectangular, triangular, arrow-shaped, or dumbbell-shaped, with the dumbbell shape being preferred. The expansion angles of the inlet and outlet of the burst valves are designed to be 120° (obtuse angle), and the depth (i.e., the maximum vertical depth from the chip surface) is slightly deeper than the depth of the siphon channel. Furthermore, the inner wall of the valve is hydrophobically treated, increasing the difficulty of spontaneous liquid filling and thus setting a higher burst pressure threshold. Specifically, the hydrophobic treatment steps are as follows: According to the instructions, the purchased hydrophobic modification reagent (manufacturer: Fluo-ST3 hydrophobic reagent from Taichu Technology (Tianjin) Co., Ltd.) is directly coated onto the inner wall of the chip's burst valve, and air-drying or baking forms a thin coating.

[0069] The first siphon channel 30, the second siphon channel 32, and the third siphon channel 33 each have an independent width and depth of 300 μm, and their inner walls are hydrophilically treated to give them strong capillary force. The specific hydrophilic treatment steps are as follows: According to the instructions, the purchased hydrophilic modification reagent (manufacturer: Wuhan Mesoscopic Biotechnology Co., Ltd.) is directly coated onto the inner wall of the siphon channel of the chip, and air-dried or oven-dried to form a thin coating. The third siphon channel 33 is hydrophilically treated using a hydrophilic modification reagent diluted 20 times with isopropanol to ensure that it will not be re-conducted after a single liquid transfer.

[0070] The shaft through hole 10 located in the encapsulation layer 1 corresponds vertically to the shaft hole 20 located in the structural layer 2, so that the two layers are arranged around the same centrifugal axis and are tightly connected.

[0071] The magnet through-hole 11 located in the encapsulation layer 1 corresponds vertically to the magnet chamber 25 located in the structural layer 2, allowing the magnet to be installed through it.

[0072] Example 2: A fully integrated nucleic acid detection method for non-medical diagnostic purposes using centrifugal microfluidic chips. The magnet chamber 25 is equipped with an N52 neodymium iron boron magnet, the sample waste liquid chamber 26 is sealed with absorbent cotton, and the detection chamber 28 is pre-embedded with lyophilized bulbs containing relevant reagents for nucleic acid amplification reaction. Encapsulation layer 1 is a highly viscous transparent pressure-sensitive tape, which is used to encapsulate the structural layer 2 by adhesive bonding. The centrifugal microfluidic chip of this invention can precisely and sequentially trigger the pressure opening of the burst valve and the activation of the siphon channel on the chip by controlling the rotational speed gradients of the centrifugal motor, such as "stop," "low speed," "medium speed," and "high speed," thereby driving different fluids to transfer in different chambers along predetermined paths. Therefore, this microfluidic chip can automatically complete sample enrichment, nucleic acid extraction, nucleic acid amplification, and fluorescence detection without any external pumps, valves, or manual intervention. The specific motor gradient centrifugation control process is as follows: Figure 6 As shown, a positive rotation speed represents counter-clockwise centrifugation, a negative rotation speed represents clockwise centrifugation, and a rotation speed of 0 indicates centrifugation has stopped. The nucleic acid detection workflow of the microfluidic chip under the gradient centrifugation control of this motor is as follows: Figure 7 As shown.

[0073] The specific nucleic acid testing method includes the following steps: 1. Immunomagnetic bead capture: Add 20 μg of immunomagnetic beads to a 500 μL sample containing the pathogen to be tested, mix and incubate at room temperature for 20 min to complete the capture and form an immunomagnetic bead-microbe complex.

[0074] 2. Sample injection, such as Figure 8 As shown in Figure A: The sample containing the immunomagnetic bead microbial complex is injected into the sample chamber 24 of the microfluidic chip through the sample application port 22. 100 μL of nucleic acid lysis buffer is then injected into the lysis buffer chamber 23. The sample application port 22 is then sealed. The microfluidic chip is flipped over and mounted on the motor shaft through the shaft hole 20.

[0075] 3. Sample enrichment, such as Figure 8As shown in Figure B: The microfluidic chip is manually rotated, causing the sample in sample chamber 24 to move closer to magnet chamber 25 and contact the inlet of the third siphon channel 33 connected to sample waste chamber 26. After standing for 90 seconds, the immunomagnetic bead microbial complex in the sample is captured on the inner wall near magnet chamber 25 due to the magnetic field. At the same time, the third siphon channel 33 becomes active due to capillary action. Since the second burst valve 31 connected to detection chamber 28 is not burst, the second siphon channel 32 connected in series is not active. The motor is centrifuged at 1250 rpm clockwise for 30 seconds, and the supernatant in sample chamber 24 is transferred to sample waste chamber 26 via the third siphon channel 33, thus completing the enrichment of the sample. The third siphon channel 33 is interrupted by air bubbles after the supernatant is transferred, and the hydrophilicity of its inner wall is washed away, preventing it from becoming active again.

[0076] 4. Nucleic acid extraction, such as Figure 8 As shown in Figure C: The motor immediately centrifuges at a high speed of 3000 rpm counterclockwise for 3 seconds, causing the nucleic acid lysis buffer in the lysis buffer chamber 23 to break through the first burst valve 29. Centrifugation is stopped for 12 seconds, at which point the first siphon channel 30 is opened due to capillary action. The motor then centrifuges at a low speed of 1250 rpm counterclockwise for 15 seconds, transferring the nucleic acid lysis buffer in the lysis buffer chamber 23 to the sample chamber 24 via the first burst valve 29 and the first siphon channel 30. The motor immediately rotates back and forth at 800 rpm (alternating between counterclockwise and clockwise once per second) for 5 minutes, ensuring thorough mixing and contact between the nucleic acid lysis buffer and the immunomagnetic bead microbial complex fixed to the inner wall of the sample chamber 24, completing the lysis of the microorganisms and releasing the nucleic acid. At this time, the third siphon channel 33 connected to the sample waste liquid chamber 26 cannot be activated again due to decreased hydrophilicity or the presence of air bubbles, and the siphon channel 32 connected to the detection chamber 28 also cannot be activated because the second burst valve 32 connected in series cannot be burst under low-speed centrifugation of 800 rpm.

[0077] 5. Amplification detection, such as Figure 8 As shown in Figure D: After nucleic acid release, the motor centrifuges at 3000 rpm counterclockwise for 3 seconds. The microbial nucleic acid and other reagents released from sample chamber 24 break through the second burst valve 31. Centrifugation is stopped for 42 seconds, at which point the second siphon channel 32 is opened due to capillary action. The motor centrifuges at 2000 rpm counterclockwise for 15 seconds. The nucleic acid and other reagents in sample chamber 24 are transferred via the second burst valve 31 and the second siphon channel 32 and sequentially fill the detection chamber 28. Excess reagents flow into the detection waste liquid chamber 27. The pre-embedded lyophilized reagent bulbs for nucleic acid amplification reaction in detection chamber 28 are dissolved, and the nucleic acid amplification reaction and nucleic acid concentration detection are performed at a specific temperature.

[0078] Example 3: A procedure for rapid on-site detection of bacteria based on a centrifugal microfluidic chip. like Figure 9 As shown, take an appropriate amount of sample, dilute and pretreat it appropriately with buffer solution, and affix a QR code containing sampling information. Add an appropriate amount of immunomagnetic beads to the sample containing bacteria, and then incubate it in a shaking environment for a period of time to achieve effective bacterial capture. Before using the matching portable on-site rapid detection device to carry out the detection work, you need to use the device's barcode scanner to scan the QR code label on the sample to be tested and the microfluidic chip to accurately enter the relevant information into the detection device. After receiving this information, the device will automatically match the pre-set and optimized detection process to ensure that the subsequent detection is carried out according to scientific and accurate steps. Take 500 μL of the sample after the above bacterial capture treatment and 100 μL of nucleic acid lysis buffer, and load them into the sample chamber 24 and lysis buffer chamber 23 of the microfluidic chip, respectively. Seal the sample loading port 22 with tape to prevent sample leakage or external contamination. Then, install and fix the microfluidic chip on the centrifuge tray module of the device and close the top cover of the device. Start the device, and the device will automatically carry out the detection work according to the pre-set detection process.

[0079] During the testing process, gradient centrifugation is first used to sequentially complete key steps such as magnetic separation of bacteria, bacterial lysis, and nucleic acid extraction from the sample. After nucleic acid extraction, the extracted nucleic acid template is transferred to the detection chamber 28 of the microfluidic chip. Subsequently, the temperature of the detection chamber 28 is precisely controlled at 42°C by the temperature control module for isothermal amplification reaction. Simultaneously, a fluorescence excitation light source is activated, and a detection camera continuously acquires fluorescence images of all detection chambers 28 at a frequency of once every 30 seconds to monitor the reaction progress and results in real time. After all sample testing is completed, the device automatically saves relevant data, including fluorescence image test results, sample information, and microfluidic chip information, for subsequent data analysis and traceability. The entire bacterial detection process is highly efficient and fast, and can be completed within 60 minutes. Of this, 25 minutes are used for immunomagnetic capture of bacteria, and approximately 30 minutes are used for subsequent operations such as bacterial concentration detection on the device.

[0080] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A centrifugal microfluidic chip, characterized in that, The microfluidic chip includes a central through-hole and one or more nucleic acid detection working units. The microfluidic chip is fixed to a rotating shaft through the central through-hole and rotates around the central through-hole to provide centrifugal force to the fluid in the nucleic acid detection working unit. Each nucleic acid testing work unit includes a lysis buffer chamber, a sample chamber, a magnet chamber, a sample waste liquid chamber, a testing chamber, a first siphon channel, a first burst valve, a second siphon channel, a second burst valve, and a third siphon channel; The lysis buffer chamber is used to store nucleic acid lysis buffer; the sample chamber is used to store and enrich the sample to be tested and to lyse and release the nucleic acid of the target microorganism in the sample to be tested; the magnet chamber is used to contain magnetic material to provide a high-intensity magnetic field to the inner wall of the sample chamber near the magnet chamber; the waste liquid chamber is used to collect and store waste liquid; and the detection chamber is used to amplify and detect the nucleic acid in the sample to be tested. The lysis buffer chamber is connected to the sample chamber via a first siphon channel for transferring nucleic acid lysis buffer from the lysis buffer chamber to the sample chamber. A first burst valve is connected in series in the first siphon channel. During the rotation of the microfluidic chip, when the pressure applied by the nucleic acid lysis buffer to the first burst valve is greater than or equal to its required burst pressure threshold, the nucleic acid lysis buffer flows through the first burst valve. The sample chamber is connected to the detection chamber via a second siphon channel to transfer the released nucleic acid template solution from the sample chamber to the detection chamber. A second burst valve is connected in series in the second siphon channel. During the rotation of the microfluidic chip, when the pressure applied by the nucleic acid template solution to the second burst valve is greater than or equal to its required burst pressure threshold, the nucleic acid template solution flows through the second burst valve. The sample chamber is connected to the sample waste liquid chamber via a third siphon channel to completely transfer the waste liquid in the sample chamber to the sample waste liquid chamber. The first rupture valve and the second rupture valve are respectively located near the entrance of the first siphon channel and the second siphon channel, and their inner walls are treated with hydrophobicity. The inner walls of the first, second, and third siphon channels are coated with a hydrophilic substance to give the channels sufficient capillary force for normal conduction. After one liquid transfer, the content of the hydrophilic substance on the inner wall of the channel decreases due to the scouring of the liquid, and the capillary force decreases accordingly, making it difficult to conduct again for a second liquid transfer.

2. The centrifugal microfluidic chip according to claim 1, characterized in that, The shapes of the first and second rupture valves are each independently selected from rectangles, triangles, arrows, or dumbbells. The angle between the virtual extension line of the siphon channel wall at the inlet of the rupture valve and the valve wall where the siphon channel wall connects is 90 degrees or more, preferably 120 degrees. The maximum depth of the first and second rupture valves perpendicular to the chip surface is greater than the maximum depth of the first and second siphon channels perpendicular to the chip surface. And / or, the width and depth of the first siphon channel, the second siphon channel, and the third siphon channel are each independently 100-400 μm, preferably, the width and depth are both 300 μm; And / or, the first siphon channel, the second siphon channel, and the third siphon channel are inverted U-shaped, with their inlet end connected to the upstream chamber and their outlet end connected to the downstream chamber. The siphon apex is closer to the center via of the microfluidic chip than the edge of the upstream chamber. And / or, the second siphon channel and the third siphon channel are located on opposite sides of the sample chamber, such that when the nucleic acid solution is transferred to the detection chamber via the second siphon channel and when the waste liquid is transferred to the sample waste liquid chamber via the third siphon channel, the liquid flow at the inlet of the second and third siphon channels is opposite; And / or, the hydrophilic reagent coated on the inner wall of the third siphon channel has been diluted 20 times with isopropanol to ensure that it will not be re-conducted after a single liquid transfer.

3. The centrifugal microfluidic chip according to claim 1, characterized in that, The centrifugal microfluidic chip has a circular disk-shaped structure with a diameter of 100-300 mm, preferably 200 mm. And / or, each nucleic acid detection work unit also includes a detection waste liquid chamber, which is connected to the detection chamber and is used to store excess nucleic acid solution flowing out of the detection chamber; And / or, the lysis buffer chamber and the sample chamber are respectively provided with sample loading ports; And / or, the lysis buffer chamber, sample chamber, sample waste chamber and detection waste chamber are all provided with vents to allow the chamber pressure to communicate with atmospheric pressure for pressure balance, and the vents are all located closer to the center through-hole of the microfluidic chip than the edge of the chamber to ensure that reagents do not leak during centrifugation.

4. The centrifugal microfluidic chip according to any one of claims 1-3, characterized in that, The centrifugal microfluidic chip is assembled from a structural layer and an encapsulation layer. The nucleic acid detection working unit is distributed in the structural layer. The central through hole is composed of a rotating shaft through hole located in the encapsulation layer and a corresponding rotating shaft hole located in the structural layer. Preferably, the encapsulation layer can encapsulate the structural layer by adhesive bonding or hot pressing. And / or, the magnet chamber is a structure in which magnetic material can be installed through.

5. The centrifugal microfluidic chip according to any one of claims 1-4, characterized in that, The magnetic material is a magnet, preferably an N52 neodymium iron boron permanent magnet; And / or, the sample waste liquid chamber is encapsulated with absorbent material, preferably absorbent cotton; And / or, the detection chamber is pre-embedded with reagents for nucleic acid amplification reactions.

6. A rapid nucleic acid detection device, characterized in that, It includes a centrifugal microfluidic chip according to any one of claims 1-5.

7. A nucleic acid detection method, characterized in that, include: Use the centrifugal microfluidic chip according to claim 1 or the nucleic acid detection device according to claim 6.

8. The nucleic acid detection method according to claim 7, characterized in that, It includes one or more of the following steps: Enrichment of the test sample: The test sample solution captured by the immunomagnetic beads in the sample chamber is brought close to the magnetic material so that the immunomagnetic beads bound with the target microorganism are adsorbed onto the inner wall of the chamber, and the waste liquid is brought into contact with the inlet of the third siphon channel. The third siphon channel is opened and activated due to the capillary principle, and is used to transfer the waste liquid to the sample waste liquid chamber. Preferably, the microfluidic chip is rotated at a second speed to accelerate the transfer of the waste liquid to the sample waste liquid chamber, while the second siphon channel is not opened and activated because the second burst valve connected in series with it is not burst. Nucleic acid extraction: The microfluidic chip is rotated at a first speed, causing the nucleic acid lysis buffer in the lysis buffer chamber to break through the first burst valve, stopping centrifugation. The first siphon channel is activated due to capillary force, transferring the lysis buffer to the sample chamber. Preferably, the microfluidic chip is rotated at a second speed to accelerate the transfer of the lysis buffer to the sample chamber. Then, the microfluidic chip is rotated clockwise and counterclockwise at a third speed less than the second speed to ensure that the target microorganisms bound to the immunomagnetic beads adsorbed on the inner wall of the sample chamber come into full contact with the nucleic acid lysis buffer, releasing the nucleic acid from the target microorganisms. Nucleic acid amplification and detection: The microfluidic chip is rotated at a first rotation speed so that the nucleic acid solution obtained in the sample chamber breaks through the second burst valve, stopping centrifugation. The second siphon channel is activated due to capillary force, allowing the nucleic acid solution to be transferred to the detection chamber. Preferably, the microfluidic chip is rotated at a second rotation speed to accelerate the transfer of the nucleic acid solution to the detection chamber. The nucleic acid solution reacts with the pre-embedded nucleic acid amplification reagent in the detection chamber, and then the nucleic acid concentration is detected. When the microfluidic chip rotates at a second or third speed, the pressure exerted by the liquid in the chamber on the burst valve is insufficient to break the first and second burst valves.

9. The method according to claim 8, characterized in that, The first rotational speed is 2500-3500 rpm, preferably 3000 rpm; And / or, the second rotational speed is 1000-2500 rpm, preferably 1250-2000 rpm; And / or, the third rotational speed is 500-1000 rpm, preferably 800 rpm.

Images