RPA-CRISPR spatial isolation microfluidic chip based on thermal response hydrogel valve and detection method thereof

Abstract

The application discloses an RPA-CRISPR spatial isolation microfluidic chip based on a thermal response hydrogel valve and a detection method thereof, and belongs to the technical field of molecular biology. The RPA-CRISPR spatial isolation microfluidic chip comprises a fluid channel layer and a pressure-sensitive adhesive layer; a plurality of groups of detection units are symmetrically arranged in the center of the fluid channel layer; each group of detection units at least comprises a sample chamber, an RPA amplification cavity connected to the sample chamber and a thermal response hydrogel valve; the thermal response hydrogel valve is arranged between the RPA amplification cavity and a downstream cavity, and the thermal response hydrogel valve is prepared from gelatin hydrogel crosslinked by genipin. Compared with a traditional wax-based thermal sensitive valve, the genipin crosslinked gelatin hydrogel thermal response rate of the application is faster, and only about 120 seconds are needed to completely melt and open the passage; short-time heating at 45 DEG C does not need long-time high-temperature treatment, can avoid continuous high-temperature damage to the Cas12a protein which is sensitive to heat, and guarantees the biological activity of the CRISPR detection system and the fluorescence signal intensity.

Description

Technical Field

[0001] This invention belongs to the technical field of molecular biology, and specifically relates to an RPA-CRISPR spatial isolation microfluidic chip based on a thermally responsive hydrogel valve and its detection method. Background Technology

[0002] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-related protein (Cas) systems have become revolutionary tools in the field of molecular diagnostics due to their high specificity and programmability. The basic principle of nucleic acid detection methods based on the CRISPR / Cas system is to utilize CRISPR RNA (crRNA) to recognize the target nucleic acid sequence, activate the trans-cleavage activity of the Cas nuclease, and then cleave the non-specific single-stranded DNA fluorescent reporter probe, releasing a detectable fluorescent signal. However, CRISPR detection itself has inherent limitations in sensitivity, making it difficult to directly apply to the detection of low-concentration targets in clinical samples. Therefore, it usually requires a nucleic acid pre-amplification step to enrich the target sequence. This introduces a fundamental incompatibility problem: the CRISPR detection system initiates premature cleavage of the amplification products and primers before RPA amplification is complete, leading to a significant decrease in the final detection sensitivity.

[0003] To resolve this contradiction, researchers explored solutions primarily from two dimensions: Temporal separation strategy: This approach avoids compatibility issues by controlling the reaction sequence, for example, by chemically modifying crRNA to temporarily inhibit its activity, which is then restored under illumination of a specific wavelength of light. While this method achieves temporal control, its reliance on complex chemical synthesis and specialized photoactivation equipment limits its widespread application in practical scenarios.

[0004] Spatial separation strategy: This involves creating physically separated reaction chambers on a microfluidic chip, storing amplification and detection reagents separately, and triggering mixing through a specific mechanism. However, the spatial separation strategy of microfluidic passive valves still faces significant limitations. The physical pathways formed before and after valve opening can cause aerosol cross-contamination of amplification products, making complete isolation of the reaction chambers impossible and affecting the stability and reliability of detection results. While microfluidic active valve strategies offer better control precision, the high manufacturing cost of solenoid and pneumatic valves limits their large-scale application.

[0005] Active valves based on thermoresponsive materials have shown application potential in the RPA-CRISPR spatial separation field due to their controllable triggering methods and low manufacturing costs. Wax, as a common thermosensitive material, has been used for time-sequential release control of reagents. However, wax-based valves inherently suffer from slow response speeds, while CRISPR / Cas proteins are extremely sensitive to heat, and prolonged heating can easily lead to loss of activity. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an RPA-CRISPR spatial isolation microfluidic chip based on a thermally responsive hydrogel valve and its detection method.

[0007] This invention employs the following technical features: an RPA-CRISPR spatially isolated microfluidic chip based on a thermally responsive hydrogel valve, wherein the chip has a two-layer bonded structure, comprising: a fluid channel layer and a pressure-sensitive adhesive layer; wherein, the fluid channel layer is provided with multiple sets of detection units distributed in a centrally symmetrical manner; Each detection unit includes at least: a sample chamber, an RPA amplification chamber connected to the sample chamber, and a thermally responsive hydrogel valve; the thermally responsive hydrogel valve is disposed between the RPA amplification chamber and the downstream chamber, and the thermally responsive valve is made of gelatin hydrogel chemically crosslinked with genipin.

[0008] In a further embodiment, the downstream chamber includes: The waste liquid chamber is connected to the RPA amplification chamber via a thermal response valve; The transition chamber is connected to the waste liquid chamber; A CRISPR / Cas12a detection chamber is connected to the transition chamber; a passive valve is provided between the CRISPR / Cas12a detection chamber and the transition chamber.

[0009] In a further embodiment, the transition cavity is provided with an exhaust port, and a polyethylene porous filter membrane is disposed in the exhaust port.

[0010] In a further embodiment, the waste liquid chamber is a funnel-shaped cavity, and the waste liquid chamber and the transition cavity form a flow-diverting structure.

[0011] In a further embodiment, the fabrication process of the thermally responsive hydrogel valve is as follows: Genipin and gelatin were mixed and crosslinked at a predetermined mass ratio to obtain a gelatin hydrogel chemically crosslinked with genipin; by adjusting the mass ratio of genipin to gelatin, the gel-sol phase transition temperature of the gelatin hydrogel was increased to 42℃~47℃. The gelatin hydrogel, once formed, remains solid at the RPA amplification reaction temperature, achieving physical isolation between the RPA amplification chamber and the downstream chamber. When heated to 42℃~47℃, it rapidly melts into a liquid state, thus opening the valve passage.

[0012] A detection method, based on the RPA-CRISPR spatially isolated microfluidic chip with a thermally responsive hydrogel valve as described above, includes the following steps: DNA samples, RPA amplification mixture, genipin cross-linked gelatin hydrogel solution, and CRISPR / Cas12a detection premix were respectively added into the corresponding chambers of the chip. Allow the genipin crosslinked gelatin hydrogel to stand until it is completely cured, then close the thermal response valve and perform low-speed centrifugation to drive the DNA sample into the RPA amplification chamber and mix it thoroughly with the RPA amplification reaction solution in the chamber. The chip is placed in a constant temperature environment at the temperature required for the amplification reaction to carry out the RPA isothermal amplification reaction. The solid thermal response valve is kept closed to isolate the RPA amplification chamber from the downstream chamber. After the RPA amplification reaction is completed, the environment where the chip is located is heated to 42℃~47℃ and kept at a constant temperature for a predetermined time. The thermal response valve melts rapidly when heated, opening the fluid passage between the RPA amplification chamber and the waste liquid chamber. During high-speed centrifugation, the hydrogel solution preferentially enters the waste liquid chamber, followed by the amplification product filling the waste liquid chamber. Excess amplification product enters the CRISPR / Cas12a detection chamber after passing through the transition chamber and the passive valve. Maintain the temperature required for the CRISPR reaction, activate the CRISPR / Cas12a trans-cleavage fluorescent probe, collect the fluorescence signal, and interpret the target nucleic acid detection results.

[0013] In a further embodiment, the specific method for acquiring fluorescence signals and interpreting target nucleic acid detection results is as follows: The fluorescence signal in the detection chamber is excited by an LED light source, and the fluorescence image is acquired by a camera. After the image is automatically analyzed by an image recognition algorithm, the positive or negative result is output.

[0014] In a further embodiment, high-speed centrifugation further includes: the molten hydrogel and amplification products preferentially filling the waste liquid cavity under the action of centrifugal force.

[0015] In a further embodiment, the CRISPR reaction requires a temperature of 37°C and a predetermined duration of 10 minutes.

[0016] In a further embodiment, the low-speed centrifugation speed is 1500 rpm and the high-speed centrifugation speed is 3000 rpm.

[0017] The beneficial effects of this invention are: This invention achieves spatial physical isolation between the RPA amplification system and the CRISPR detection system through a genipin cross-linked gelatin thermal response valve, completely avoiding the problem of Cas enzyme in the CRISPR system prematurely cutting amplification primers and target amplification products during the amplification stage. Compared with the one-step method without valve synchronous reaction, the detection sensitivity is improved by 54 times, and the lowest detection limit can reach 2.9 copies / μL, which can stably detect low abundance trace amounts of target nucleic acid.

[0018] Compared to traditional wax-based thermosensitive valves, the genipin crosslinked gelatin hydrogel used in this invention has a faster thermal response rate, requiring only about 120 seconds to completely melt and open the pathway; short-term heating at 45°C eliminates the need for prolonged high-temperature treatment, thus avoiding damage to the heat-sensitive Cas12a protein from continuous high temperatures and ensuring the bioactivity and fluorescence signal intensity of the CRISPR detection system.

[0019] The chip relies on centrifugal force to achieve sequential delivery of reagents and fluid transfer between chambers. The entire reaction process is completed inside the chip, eliminating the need for manual pipetting or opening the cap. This blocks the diffusion of amplified product aerosols from the source, effectively avoiding cross-contamination between samples and improving the stability and repeatability of test results.

[0020] The chip is equipped with a funnel-shaped waste liquid chamber and a transition chamber for flow separation. The molten hydrogel can preferentially flow into the waste liquid chamber for storage under centrifugal force, preventing the molten hydrogel from entering the CRISPR detection chamber and avoiding the hydrogel matrix from inhibiting the CRISPR / Cas12a enzymatic reaction, thus ensuring the normal progress of the detection reaction.

[0021] The preferred method for exciting fluorescence using an LED light source is handheld, which is paired with a smartphone camera to capture fluorescence images. The image recognition algorithm automatically outputs the positive or negative result, eliminating the need for large-scale precision fluorescence detection equipment. This method is suitable for on-site nucleic acid testing in grassroots and field settings where instrument resources are limited.

[0022] The entire nucleic acid testing process takes less than 30 minutes; the chip is equipped with multiple independent detection units arranged in a central symmetric manner, which can simultaneously complete the testing of multiple samples in a single test, greatly improving the testing efficiency of batch samples. Attached Figure Description

[0023] Figure 1 This is a structural diagram of the RPA-CRISPR centrifugal microfluidic chip in Example 1.

[0024] Figure 2 The principle of generating the thermal response valve in Example 1.

[0025] Figure 3 The thermodynamic properties of gelatin hydrogelatin that has been chemically cross-linked with genipin are shown in the graph.

[0026] Figure 4 Photographs of gelatin hydrogels chemically cross-linked with genipin at different temperatures.

[0027] Figure 5 This is a comparison diagram of the melting process of genipin crosslinked gelatin hydrogel and paraffin at 45°C.

[0028] Figure 6 This is a flowchart of the detection method in Example 2.

[0029] Figure 7 The effect of different RPA reaction times on the final fluorescence intensity.

[0030] Figure 8 This is a schematic diagram of the complete nucleic acid detection process of the RPA-CRISPR centrifugal microfluidic chip in Example 2.

[0031] Figure 9 This is a schematic diagram of the automatic recognition and detection process of fluorescent images on a smartphone, which is the companion to Example 2.

[0032] Figure 10 The image shows the validation results of the RPA-CRISPR centrifugal microfluidic chip for clinical HER2 gene samples.

[0033] Figure 11 The graph shows the batch repeatability and detection limit performance characterization of the RPA-CRISPR centrifugal microfluidic chip.

[0034] Figure 12 This is the result of a one-step RPA-CRISPR detection.

[0035] Figure 1 The labels in the diagram are as follows: 1. Fluid channel layer; 2. Sample chamber; 3. RPA amplification chamber; 4. Thermal response valve; 5. Waste liquid chamber; 6. Transition chamber; 7. CRISPR / Cas12a detection chamber; 8. Passive valve; 9. Exhaust port. Detailed Implementation

[0036] The present invention will now be further described in conjunction with the embodiments and accompanying drawings.

[0037] Example 1 This embodiment addresses the issues of reduced sensitivity in existing RPA-CRISPR one-step detection methods due to incompatibility between amplification and detection reaction conditions, as well as the problems of aerosol cross-contamination and slow valve material response in existing spatial separation strategies. It provides a centrifugal microfluidic chip based on a genipin cross-linked gelatin thermal response valve 4.

[0038] like Figure 1 As shown, an RPA-CRISPR spatially isolated microfluidic chip based on a thermally responsive hydrogel valve is disclosed. The chip has a two-layer bonded structure, including a fluid channel layer 1 and a pressure-sensitive adhesive layer. The fluid channel layer 1 contains multiple sets of detection units distributed in a centrally symmetrical manner. Each detection unit includes at least: a sample chamber 2 for receiving the sample to be tested, an RPA amplification chamber 3 for containing the RPA amplification mixture, and a downstream chamber. The RPA amplification chamber 3 is connected to the sample chamber 2.

[0039] It also includes: a thermally responsive hydrogel valve 4, disposed between the RPA amplification chamber 3 and the downstream chamber, the thermally responsive valve 4 being made of gelatin hydrogel chemically crosslinked with genipin.

[0040] In a further embodiment, the downstream chamber includes: Waste liquid chamber 5 is connected to RPA amplification chamber 3 via thermally responsive hydrogel valve 4; waste liquid chamber 5 is used to receive molten hydrogel material and part of amplification products.

[0041] Transition chamber 6 is connected to the waste liquid chamber 5; A CRISPR / Cas12a detection chamber 7 is connected to the transition chamber 6; the CRISPR / Cas12a detection chamber 7 is used to contain CRISPR / Cas12a detection reagents. A passive valve 8 is provided between the CRISPR / Cas12a detection chamber 7 and the transition chamber 6.

[0042] In this embodiment, the transition cavity 6 is provided with an exhaust port 9, and a polyethylene porous filter membrane is provided in the exhaust port. The exhaust port 9 is used to discharge the air trapped inside the transition cavity 6 during the fluid transport of the amplification product through the transition cavity 6, eliminate the obstruction of fluid flow by air bubbles in the cavity, ensure that the amplification product can smoothly and completely push open the passive valve 8 and flow into the downstream CRISPR / Cas12a detection cavity, and avoid the residual air bubbles occupying the reagent holding space and interfering with the subsequent CRISPR fluorescence detection signal reading.

[0043] Considering that molten hydrogel residue can easily enter downstream along with amplification products, causing channel blockage and interfering with fluorescence detection, the waste liquid chamber in this embodiment is a funnel-shaped chamber, which forms a flow-diverting structure with the transition chamber. This flow-diverting structure allows for the layered retention of high-viscosity gel solution, separating and purifying the nucleic acid supernatant, effectively preventing microfluidic channel blockage and reducing signal interference from impurities in subsequent CRISPR / Cas12a detection.

[0044] In a further embodiment, the thermoresponsive hydrogel valve is composed of gelatin hydrogel chemically cross-linked with genipin, and its preparation process is as follows: Genipin and gelatin are mixed and crosslinked at a predetermined mass ratio to obtain a gelatin hydrogel chemically crosslinked with genipin. By adjusting the mass ratio of genipin to gelatin (the mass ratio of genipin to gelatin ranges from 0.5:1000 to 4:1000), as the proportion of genipin added increases, the crosslinking density of the hydrogel increases and the phase transition temperature gradually rises. A mass ratio of 3:1000 is preferred as the optimized crosslinking formulation, which increases the gel-sol phase transition temperature of the gelatin hydrogel to 42°C to 47°C. Preferably, the gel-sol phase transition temperature is increased from 30°C to 43 to 45°C, and most preferably to 43°C.

[0045] The gelatin hydrogel, once formed, remains solid at the RPA amplification reaction temperature, achieving physical isolation between the RPA amplification chamber and the downstream chamber. When heated to 42℃~47℃, it rapidly melts into a liquid state, thus opening the valve passage.

[0046] The working mechanism of the thermal response valve described in this embodiment is as follows: Figure 2 As shown, genipin undergoes a covalent cross-linking reaction with the free amino groups on the gelatin molecular chain, while the carbonyl group of the gelatin molecule can interact with the hydroxyl groups on the genipin residues to construct a novel hydrogen bond network; the synergistic effect of the covalent bond and hydrogen bond network significantly improves the thermal stability of the modified gelatin hydrogel.

[0047] At low temperature (4℃), gelatin molecular chains form a large number of triple helix structures, supplemented by covalent cross-linking and hydrogen bond networks, and the hydrogel maintains a dense solid state. When the system is heated to the phase transition temperature range of about 45℃, the gelatin triple helix structure dissociates, the hydrogen bond network is destroyed, and the hydrogel quickly changes from a solid gel to a liquid sol.

[0048] Based on the aforementioned temperature-induced reversible phase transition characteristics, the modified gelatin hydrogel after molding can remain stable in a solid state at the conventional RPA amplification reaction temperature. The solid gel achieves physical isolation between the RPA amplification chamber and the downstream chamber, blocking fluid communication between the chambers. When the system is heated to the phase transition range of 42℃~47℃, the hydrogel rapidly melts into a liquid sol, opening the passage between the chambers and completing the valve opening action.

[0049] To investigate the effect of genipin crosslinking modification on the temperature-induced phase transition properties of gelatin hydrogels, this embodiment conducted viscosity-temperature tests and rheological modulus temperature scanning characterization on gelatin with different ratios of modification. The test results are as follows: Figure 3 As shown.

[0050] like Figure 3 The viscosity-temperature curve of (A): The gel-sol phase transition temperature of unmodified pure gelatin is about 30℃. At the conventional RPA amplification temperature of 37℃, the viscosity decreases significantly and cannot stably maintain the solid isolation cavity. After chemical crosslinking with genipin, the viscosity curve of gelatin hydrogel shifts to the high temperature range as a whole, and the gel-sol phase transition temperature increases to 43~45℃. The phase transition temperature gradually increases with the increase of the genipin crosslinking ratio. Among them, the modified formulation with a genipin to gelatin mass ratio of 3:1000 has both the optimal phase transition temperature and the fastest reversible phase transition response rate suitable for RPA system.

[0051] like Figure 3Temperature-scanning rheological curves of storage modulus G' and loss modulus G'' in (B): Taking the optimized modified formulation with a mass ratio of 3:1000 as an example, the gel-sol transition temperature corresponding to the intersection of the storage modulus and loss modulus curves is 43℃. This temperature is higher than the RPA isothermal amplification temperature of 37℃, which can ensure that the hydrogel valve remains solid and sealed during the amplification stage. When the system temperature rises to above 43℃, the storage modulus quickly falls below the loss modulus, and the gel quickly transforms into a liquid sol, realizing the controllable opening of the valve passage.

[0052] To visually verify the effect of different crosslinking ratios on the macroscopic morphology of the temperature-induced phase transition of gelatin hydrogels, the static inverted morphology of each group of samples was observed at three characteristic temperatures: 25℃, 37℃, and 45℃. The actual results are as follows: Figure 4 As shown.

[0053] Group A consists of unmodified pure gelatin, while groups B through F correspond to cross-linked samples with a genipin:gelatin mass ratio of 0.5:1000, 1:1000, 2:1000, 3:1000, and 4:1000, respectively.

[0054] At room temperature (25°C), all gelatin samples were solid gels that did not flow when inverted and could be stably molded. When the temperature was raised to the RPA amplification operating temperature of 37℃, the uncrosslinked gelatin (Group A) and the low crosslinking ratios of 0.5:1000 / 1:1000 / 2:1000 (Groups B / C / D) all melted into liquid. After being inverted, the fluid flowed significantly, making it impossible to achieve cavity isolation and sealing. The optimized ratio of 3:1000 (Group E) sample remained intact and solid, and was able to stably isolate the RPA amplification cavity from the downstream chamber during the 37℃ amplification stage. Although the high crosslinking ratio of 4:1000 (Group F) also remained solid, its phase transition temperature was too high.

[0055] When the temperature is raised to 45°C, the cross-linked gelatin in the 3:1000 ratio (Group E) completely melts into a liquid state, and the pipeline is open. If the proportion of genipin is further increased, the melting point of the hydrogel phase change will rise to above 45°C, which will prolong the heating time for valve melting and opening, and reduce the efficiency of the detection process.

[0056] Based on the results of rheological testing and physical morphology observation, it can be seen that the optimal crosslinking formulation is a genipin to gelatin mass ratio of 3:1000. The phase change temperature is suitable for the RPA isothermal amplification system, which can achieve the controllable thermal response valve effect of solid sealing at 37℃ and rapid melting of the passage at 45℃.

[0057] To compare the melting response rate of the modified gelatin hydrogel valve in this embodiment with that of a traditional paraffin thermal valve, a comparative experiment was conducted simultaneously at a phase transition temperature of 45°C to observe the melting process of both valves. Real-time morphological changes were recorded as follows: Figure 5 As shown.

[0058] As can be seen from the time-series photographs, the genipin crosslinked modified gelatin melts rapidly under heating conditions of 45℃, with a complete melting response time of only about 120 s; at the same temperature, the paraffin-based valve melts very slowly, taking up to 17 minutes to completely melt. Quantitative comparison shows that the melting rate of the modified gelatin is approximately 8.5 times that of the paraffin valve.

[0059] Compared to traditional paraffin thermal response valves, the genipin cross-linked gelatin thermal valve of this invention has an extremely fast melting and conduction speed, which greatly shortens the waiting time for stepwise nucleic acid detection of microfluidic chips, improves the overall detection throughput and operating efficiency, and is more suitable for the needs of rapid on-site nucleic acid diagnosis.

[0060] Example 2 Based on the RPA-CRISPR spatially isolated microfluidic chip based on a thermally responsive hydrogel valve disclosed in the embodiments, this embodiment provides a detection method, such as... Figure 6 As shown, it includes the following steps: The DNA sample, RPA amplification mixture, genipin crosslinked gelatin hydrogel solution, and CRISPR / Cas12a detection premix were respectively added to the corresponding chambers of the chip; among which... Allow the genipin crosslinked gelatin hydrogel to stand until it is completely cured, then close the thermal response valve. Then perform the first low-speed centrifugation to drive the DNA sample into the RPA amplification chamber and mix it thoroughly with the RPA amplification reaction solution in the chamber. The chip is placed in a constant temperature environment at the temperature required for the amplification reaction to carry out the RPA isothermal amplification reaction. The solid thermal response valve is kept closed to isolate the RPA amplification chamber from the downstream chamber. After the RPA amplification reaction is completed, the environment where the chip is located is heated to 42℃~47℃ and kept at a constant temperature for a predetermined time. The thermal response valve melts rapidly when heated, opening the fluid passage between the RPA amplification chamber and the waste liquid chamber. After a second high-speed centrifugation, the hydrogel solution preferentially enters the waste liquid chamber, followed by the amplification product filling the waste liquid chamber. Excess amplification product enters the CRISPR / Cas12a detection chamber after passing through the transition chamber and the passive valve. Maintain the temperature required for the CRISPR reaction, activate the CRISPR / Cas12a trans-cleavage fluorescent probe, collect the fluorescence signal, and interpret the target nucleic acid detection results.

[0061] The DNA sample extracted and purified using a commercial kit, the RPA amplification mixture, the genipin cross-linked gelatin hydrogel solution, and the CRISPR / Cas12a detection premix were added to the corresponding chambers of the chip. After addition, all sample inlets were sealed with a pressure-sensitive membrane, leaving only the vent hole in the transition chamber to balance the internal gas pressure of the chip. The chip was then allowed to stand at room temperature or 4°C until the genipin cross-linked gelatin hydrogel with a mass ratio of 3:50 was completely solidified. The solid gel formed a thermally responsive valve to achieve chamber isolation. After the valve is solidified and closed, perform low-speed centrifugation: set the centrifugation speed to 1500 rpm and the centrifugation time to 15 s, drive the DNA sample into the RPA amplification chamber, and then oscillate back and forth at ±1000 rpm and 60° amplitude for 120 s to fully mix the DNA sample with the RPA amplification reaction solution in the chamber.

[0062] The chip was placed in a constant temperature environment of 37℃ for 5-30 minutes to carry out the RPA isothermal amplification reaction, such as... Figure 7 As shown, 37℃ is lower than the 43℃ phase transition temperature of modified gelatin. The thermal response valve in the cured state remains closed, isolating the RPA amplification chamber from the downstream chamber and preventing the detection reagent from reacting prematurely with the amplification product.

[0063] After the RPA amplification reaction is completed, the environment where the chip is located is heated to 42℃~47℃, preferably 45℃, and kept at a constant temperature for a predetermined time, such as 120 s. The melting rate of this modified gelatin is 8.5 times that of the paraffin valve. The thermal response valve melts rapidly when heated, opening the fluid passage between the RPA amplification chamber and the waste liquid chamber.

[0064] High-speed centrifugation was performed, with the centrifugation speed set at 3000 rpm and the centrifugation time at 30 s. The molten gel residue was retained by the diversion structure formed by the funnel-shaped waste liquid chamber and the transition chamber. After the supernatant amplification product containing only the target nucleic acid filled the transition chamber, it entered the CRISPR / Cas12a detection chamber through the passive valve. The air bubbles in the transition chamber were discharged simultaneously through the vent hole, ensuring smooth fluid transport.

[0065] The CRISPR / Cas12a nucleic acid detection reaction was initiated by maintaining a constant temperature of 37℃. After Cas12a specifically recognizes the target amplification product, it activates the cleavage activity, lyses the fluorescent probe to generate a fluorescent signal, and collects the fluorescent signal to interpret the target nucleic acid detection results.

[0066] The detection method in this embodiment describes the physical state of the chip at each stage throughout the entire process, as follows: Figure 8As shown in the figure, four process nodes are sequentially displayed: empty chip, reagent addition completion, amplification product centrifugation and splitting, and CRISPR fluorescence detection. The magnified view of the red dashed line clearly shows the fluid filling and stratified splitting effect of a single set of downstream chambers (waste liquid chamber, transition chamber, passive valve, and CRISPR detection chamber), intuitively verifying that the funnel splitting structure, vent, passive valve, and thermal response valve work together to achieve the complete functions of step-by-step isolation, solid-liquid separation, and nucleic acid fluorescence detection.

[0067] In a further embodiment, the specific method for acquiring fluorescence signals and interpreting target nucleic acid detection results is as follows: The fluorescence signal in the detection chamber is excited by an LED light source, and the fluorescence image is acquired by a camera. After the image is automatically analyzed by an image recognition algorithm, the positive or negative result is output.

[0068] Preferably, a handheld blue LED light is used as the fluorescence excitation source, along with an optical filter to simultaneously filter out ambient stray light and scattered light from the chip substrate, effectively improving the signal-to-noise ratio of the fluorescence image; the chip fluorescence image is acquired using a smartphone's built-in camera, and the image recognition algorithm is integrated into a smartphone detection app. The complete processing flow of the app is as follows: Figure 9 Step-by-step demonstration: Open the dedicated detection APP and import the original fluorescence image captured. First, the color fluorescence image is converted to grayscale to remove color interference. Then, pixel intensity normalization correction is performed to eliminate signal deviation caused by shooting distance and uneven lighting. Finally, based on the preset fluorescence grayscale threshold, the positive / negative (P / N) determination is automatically completed and the visual detection result is directly output.

[0069] This preferred solution does not rely on large, precision fluorescence detection instruments; the entire device is portable and low-cost, making it suitable for rapid on-site diagnostic scenarios. Simultaneously, the standardized image algorithm avoids subjective errors from human visual interpretation, ensuring objective and consistent detection results. Furthermore, it is worth noting that the image recognition algorithm described in this embodiment is an existing recognition technology and is not considered an improvement in this embodiment, therefore it will not be elaborated upon.

[0070] Finally, the RPA-CRISPR spatially isolated microfluidic chip based on the thermoresponsive hydrogel valve of this invention was used to detect 60 clinical HER2 gene samples, and the pathological test results were used as the gold standard. The complete detection process and validation results are as follows: Figure 9 As shown.

[0071] like Figure 10 As shown in (A), after nucleic acid extraction, the clinical blood sample was simultaneously divided into two parts: one part was used to complete RPA amplification-CRISPR fluorescence detection using the centrifugal microfluidic chip of Example 1, and the fluorescence signal was automatically interpreted by a mobile APP; the other part was used to perform quantitative detection using a commercial qPCR instrument to obtain the Ct value.

[0072] like Figure 10 As shown in (B), a single chip can complete the detection of multiple samples in parallel. Different copy numbers of HER2 targets correspond to different green fluorescence intensities, which can intuitively distinguish the strength of positive results. Figure 10 The heatmap (C) in the image shows the detection results of 60 samples using two methods, with the brightness of the color blocks corresponding to the fluorescence signal intensity.

[0073] like Figure 10 As can be seen in (D) of Example 2, the normalized fluorescence intensity output by the detection method is significantly correlated with the Ct value measured by qPCR. The smaller the Ct value (the higher the target concentration), the higher the fluorescence intensity of the chip, proving that the fluorescence signal can accurately quantify the target load.

[0074] like Figure 10 Statistical results of diagnostic parameters (E) in the study: Among 60 clinical samples, 34 were positive and 26 were negative by qPCR; the chip of this invention detected 36 positive and 24 negative cases; the overall sensitivity of the method was 95.7%, the specificity was 100%, the positive predictive value (PPV) was 100%, the negative predictive value (NPV) was 86.7%, the clinical diagnostic accuracy was excellent, and there were no false positive samples.

[0075] The parallel repeatability and lowest detection limit of the chip in Example 1 were further characterized, and the test results are as follows: Figure 11 As shown.

[0076] like Figure 11 As shown in Figure (A), HER2 plasmid standards of equal concentration were added to the nine independent detection units of the chip, and the fluorescence intensity of each channel was collected and extracted to calculate the intra-batch coefficient of variation. The low degree of variation indicates that the multi-channel parallel detection of this microfluidic chip has good repeatability and stable signals between batches.

[0077] like Figure 11 As shown in the standard curve of gradient concentration in (B), the concentration range of HER2 plasmid was set. A normalized fluorescence intensity of 0.15 was used as the threshold for determining positive or negative results. As the target concentration increased, the fluorescence intensity exhibited a typical S-shaped increase. Based on this threshold, the detection limit of the chip in Example 1 for the HER2 gene was determined to be 2.9. Low concentrations of the target can still be detected stably, and the detection sensitivity is high.

[0078] like Figure 12 As shown in the gradient concentration standard curve of the one-step RPA-CRISPR method, the detection limit for the HER2 gene can reach 156. Therefore, the method proposed in this invention has a sensitivity increased by 54 times.

Claims

1. An RPA-CRISPR spatially isolated microfluidic chip based on a thermally responsive hydrogel valve, characterized in that, The chip has a two-layer bonding structure, including a fluid channel layer and a pressure-sensitive adhesive layer; wherein, the fluid channel layer is provided with multiple sets of detection units distributed in a centrally symmetrical manner; Each detection unit includes at least: a sample chamber, an RPA amplification chamber connected to the sample chamber, and a thermally responsive hydrogel valve; the thermally responsive hydrogel valve is disposed between the RPA amplification chamber and the downstream chamber, and the thermally responsive valve is made of gelatin hydrogel chemically crosslinked with genipin.

2. The RPA-CRISPR spatial isolation microfluidic chip based on a thermally responsive hydrogel valve according to claim 1, characterized in that, The downstream chamber includes: The waste liquid chamber is connected to the RPA amplification chamber via a thermal response valve; The transition chamber is connected to the waste liquid chamber; A CRISPR / Cas12a detection chamber is connected to the transition chamber; a passive valve is provided between the CRISPR / Cas12a detection chamber and the transition chamber.

3. The RPA-CRISPR spatial isolation microfluidic chip based on a thermally responsive hydrogel valve according to claim 2, characterized in that, The transition cavity is provided with an exhaust port, and a polyethylene porous filter membrane is provided in the exhaust port.

4. The RPA-CRISPR spatial isolation microfluidic chip based on a thermally responsive hydrogel valve according to claim 2, characterized in that, The waste liquid chamber is a funnel-shaped cavity, and the waste liquid chamber and the transition cavity form a flow-diverting structure.

5. The RPA-CRISPR spatial isolation microfluidic chip based on a thermally responsive hydrogel valve according to claim 1, characterized in that, The fabrication process of the thermally responsive hydrogel valve is as follows: Genipin and gelatin were mixed and crosslinked at a predetermined mass ratio to obtain a gelatin hydrogel chemically crosslinked with genipin; by adjusting the mass ratio of genipin to gelatin, the gel-sol phase transition temperature of the gelatin hydrogel was increased to 42℃~47℃. The gelatin hydrogel, once formed, remains solid at the RPA amplification reaction temperature, achieving physical isolation between the RPA amplification chamber and the downstream chamber. When heated to 42℃~47℃, it rapidly melts into a liquid state, thus opening the valve passage.

6. A detection method, based on the RPA-CRISPR spatially isolated microfluidic chip based on a thermally responsive hydrogel valve as described in any one of claims 2 to 5, characterized in that, Includes the following steps: DNA samples, RPA amplification mixture, genipin cross-linked gelatin hydrogel solution, and CRISPR / Cas12a detection premix were respectively added into the corresponding chambers of the chip. Allow the genipin crosslinked gelatin hydrogel to stand until it is completely cured, then close the thermal response valve and perform low-speed centrifugation to drive the DNA sample into the RPA amplification chamber and mix it thoroughly with the RPA amplification reaction solution in the chamber. The chip is placed in a constant temperature environment at the temperature required for the amplification reaction to carry out the RPA isothermal amplification reaction. The solid thermal response valve is kept closed to isolate the RPA amplification chamber from the downstream chamber. After the RPA amplification reaction is completed, the environment where the chip is located is heated to 42℃~47℃ and kept at a constant temperature for a predetermined time. The thermal response valve melts rapidly when heated, opening the fluid passage between the RPA amplification chamber and the waste liquid chamber. During high-speed centrifugation, the hydrogel solution preferentially enters the waste liquid chamber, followed by the amplification product filling the waste liquid chamber. Excess amplification product enters the CRISPR / Cas12a detection chamber after passing through the transition chamber and the passive valve. Maintain the temperature required for the CRISPR reaction, activate the CRISPR / Cas12a trans-cleavage fluorescent probe, collect fluorescence signals, and interpret the target nucleic acid detection results.

7. The detection method according to claim 6, characterized in that, The specific method for collecting fluorescence signals and interpreting target nucleic acid detection results is as follows: The fluorescence signal in the detection chamber is excited by an LED light source, and the fluorescence image is acquired by a camera. After the image is automatically analyzed by an image recognition algorithm, the positive or negative result is output.

8. The detection method according to claim 6, characterized in that, High-speed centrifugation also includes the following: the molten hydrogel and amplification products preferentially fill the waste liquid cavity under the action of centrifugal force.

9. The detection method according to claim 6, characterized in that, The CRISPR reaction requires a temperature of 37°C and a predetermined duration of 10 minutes.

10. The detection method according to claim 6, characterized in that, The low-speed centrifugation speed is 1500 rpm, and the high-speed centrifugation speed is 3000 rpm.

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