A chip for detecting ultra-micro nucleic acid based on cascade reaction and a detection method thereof

Abstract

The application provides a kind of ultra-micro nucleic acid detection chip based on cascade reaction and its detection method, including identification area, reaction area, marking area and signal output area connected in turn;The recognition area is loaded with Cas13a-crRNA ribonucleoprotein complex, trigger-RNA and enzyme reaction buffer;The reaction area is loaded with silver-loaded DNA probe and urease.The application uses the side cutting activity of CRISPR-Cas13a system, combined with Ag metal chelation toehold mediated strand displacement reaction and urease-silver ion inhibition system, to design a microfluidic on-chip detection chip for visual detection of ultra-micro target nucleic acid without nucleic acid amplification.The ultra-micro nucleic acid detection chip does not need amplification, has high sensitivity, is visualized by naked eye, and converts extremely small nucleic acid recognition signal into significant color change signal through cascade signal amplification mechanism.

Description

Technical Field

[0001] This invention belongs to the field of micro-volume nucleic acid detection technology, and relates to a nucleic acid detection chip based on cascade reaction and its detection method and application, especially to an ultra-micro-volume nucleic acid detection chip based on cascade reaction and its detection method. Background Technology

[0002] Current nucleic acid detection technologies primarily rely on polymerase chain reaction (PCR) and its derivatives. While PCR offers high sensitivity, it requires sophisticated thermal cycling equipment and has a long detection time. Isothermal amplification techniques (such as RPA and LAMP), although not requiring thermal cycling, often carry the risks of nonspecific amplification and aerosol contamination.

[0003] CRISPR-Cas systems (especially Cas12 and Cas13) are widely used in next-generation molecular diagnostics due to their high specificity. However, most CRISPR-based detection methods (such as Sherlock) still typically require a pre-amplification step (such as RPA) to reach the detection limit, increasing operational complexity and the risk of contamination. Existing amplification-free CRISPR detection methods often have weak signals, rely on expensive fluorescence detection equipment, and are difficult to implement for point-of-care testing (POCT) with visual visualization.

[0004] Therefore, designing a more suitable nucleic acid detection method to solve the aforementioned technical problems of existing nucleic acid detection has become one of the urgent issues for many front-line researchers in the industry. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a nucleic acid detection chip based on cascade reaction and its detection method and application, especially an ultra-micro nucleic acid detection chip based on cascade reaction. The nucleic acid detection chip provided by the present invention has the characteristics of no amplification required, high sensitivity, and visual visualization. Through the cascade signal amplification mechanism, the extremely small amount of nucleic acid recognition signal is converted into a significant color change signal, thereby realizing the detection of ultra-micro nucleic acid.

[0006] This invention provides a nucleic acid detection chip based on a cascade reaction, comprising a recognition area, a reaction area, a labeling area, and a signal output area connected in sequence;

[0007] The recognition region is loaded with the Cas13a-crRNA ribonucleoprotein complex, trigger RNA, and enzyme reaction buffer.

[0008] The reaction zone is loaded with silver-loaded DNA probes and urease.

[0009] Preferably, the marked area is loaded with urea and a pH-sensitive indicator;

[0010] The signal output area is a visualization window used to present the color change results of the system;

[0011] The chip also includes a chip substrate;

[0012] The identification area, reaction area, marking area, and signal output area are disposed on the chip substrate.

[0013] Preferably, the connection specifically refers to connecting four functional liquid storage areas through channels;

[0014] The identification area, reaction area, and marking area are each independently selected from the closed cavity;

[0015] The identification area is equipped with a sample inlet;

[0016] The exit of the identification area is connected to the entrance of the reaction area through a microchannel.

[0017] Preferably, the outlet of the reaction zone is connected to the inlet of the marking zone via a microchannel;

[0018] The outlet of the marking area is connected to the inlet of the reaction area via a microchannel;

[0019] The channel includes a serpentine channel;

[0020] An expansion structure is provided at the connection points between one and more of the four functional liquid storage zones and the channel.

[0021] Preferably, the chip is also provided with a pneumatic drive interface for connecting an external pressure drive device to control the fluid to flow sequentially through each functional liquid storage area to complete the cascade reaction;

[0022] The Cas13a is the LwaCas13a protein derived from Leptotrichia wadei;

[0023] The crRNA consists of a constant region that binds to Cas13a and a guide region that is complementary to the target RNA;

[0024] The trigger RNA is a single-stranded RNA molecule whose sequence contains a recognition site for Cas13a trans-cleavage.

[0025] Preferably, the trigger-RNA is completely complementary to the strand substitution region of the toehold region of the silver-loaded DNA probe;

[0026] The silver-loaded DNA probe is a partially double-stranded DNA structure with a single-stranded toehold protrusion.

[0027] The double-stranded region contains at least one CC mismatch site;

[0028] The CC mismatch site specifically binds to Ag(I) to form a C-Ag(I)-C artificial base pair, realizing Ag + Stable chelation and isolation.

[0029] This invention also provides a nucleic acid detection method based on a cascade reaction, comprising the following steps:

[0030] 1) The sample to be tested is mixed with Cas13a-crRNA ribonucleoprotein complex, trigger-RNA and enzyme reaction buffer to obtain the primary product;

[0031] 2) After mixing the primary product obtained in the above steps, the silver-loaded DNA probe, and urease again, a secondary product is obtained;

[0032] 3) After further mixing the secondary product obtained in the above steps, urea and pH-sensitive indicator, a colorimetric product is obtained. The detection result is determined by the color change of the colorimetric product.

[0033] Preferably, the nucleic acid detection method is an ultra-micro nucleic acid detection method;

[0034] The sample to be tested includes single-stranded RNA;

[0035] Step 1) specifically involves molecular recognition and primary signal amplification.

[0036] In step 1), during the mixed incubation process, if the target RNA is present in the sample to be tested, the Cas13a-crRNA complex specifically binds to the target RNA and activates the trans-cleavage activity of Cas13a, cleaving the trigger RNA in the system.

[0037] If there is no target RNA in the sample to be tested, Cas13a will not be activated and the trigger RNA will remain intact.

[0038] Preferably, step 2) is specifically a secondary signal transduction step;

[0039] The silver-loaded DNA probe is a double-stranded nucleic acid probe containing a CC mismatch site;

[0040] The CC mismatch site chelates Ag via the C-Ag(I)-C artificial base pair. + ;

[0041] If the trigger RNA in the primary product is degraded during the remixing process in step 2), the strand displacement reaction cannot be triggered, and Ag... + Maintain the chelated state;

[0042] If the trigger RNA in the primary product is intact, it acts as the initiator strand to trigger the strand displacement reaction of the silver-loaded DNA probe, releasing chelated Ag. + Free Ag + It binds to the active site of urease and inhibits its catalytic activity.

[0043] Preferably, step 3) specifically involves signal output and result interpretation.

[0044] In step 3), if the catalytic activity of urease in the secondary product is inhibited, urea cannot be hydrolyzed, the pH value of the system does not change, and the colorimetric product does not change color.

[0045] If the urease in the secondary product catalyzes the hydrolysis of urea to generate ammonia and increases the pH value of the system, the color of the pH-sensitive indicator will change with the change of the pH value of the system. The qualitative interpretation of the detection result can be completed by observing the color change of the colorimetric product with the naked eye.

[0046] The logic for the determination includes:

[0047] Positive result: Target RNA was present in the test sample, trigger RNA was degraded, and no free Ag was found. + Upon release, urease retains its catalytic activity, catalyzing the hydrolysis of urea and raising the pH of the system; phenol red indicator turns deep red.

[0048] Negative result: No target RNA was found in the test sample; the trigger RNA remained intact, and the trigger strand displacement reaction released Ag. + The urease activity was inhibited, the pH of the system did not change significantly, and the phenol red indicator remained golden yellow.

[0049] This invention provides a cascade reaction-based nucleic acid detection chip, comprising a recognition region, a reaction region, a labeling region, and a signal output region connected sequentially. The recognition region contains a Cas13a-crRNA ribonucleoprotein complex, trigger RNA, and an enzyme reaction buffer. The reaction region contains a silver-loaded DNA probe and urease. Compared to existing technologies, this invention addresses the problems of current nucleic acid detection methods, such as reliance on nucleic acid amplification, expensive equipment, and complex operation. It specifically designs a cascade reaction-based nucleic acid detection chip with a specific structure and composition. This is a microfluidic on-chip detection chip that utilizes the incidental cleavage activity of the CRISPR-Cas13a system combined with Ag metal chelate toehold-mediated strand displacement reaction and a urease-silver ion inhibition system to achieve visualized detection of ultra-micro target nucleic acids without nucleic acid amplification. This ultra-micro nucleic acid detection chip requires no amplification, has high sensitivity, and is visually visible. Through a cascade signal amplification mechanism, it converts extremely small nucleic acid recognition signals into significant color change signals.

[0050] This invention presents a microfluidic nucleic acid detection chip based on the CRISPR-Cas13a cascade enzyme reaction system. Through spatial partitioning and fluid control, it integrates recognition, signal conversion, enzymatic amplification, and color readout processes into a single microfluidic platform, achieving integrated detection from target RNA recognition to visualized signal output. It is suitable for rapid multi-channel streaming detection and POCT (Point-of-Care Testing) applications. The chip requires no amplification reaction: utilizing the high signal turnover rate of Cas13a (cutting multiple trigger RNAs) and the high catalytic efficiency of urease (hydrolyzing large amounts of urea) to achieve dual cascade amplification, it can detect ultra-small amounts of nucleic acid without PCR or RPA amplification; it has high specificity: combining the single-base resolution of the CRISPR system with the high specificity of Toehold chain substitution, it can achieve single-base mutation detection, effectively addressing the rapid detection of highly variable pathogens such as viruses; it is low-cost and portable: eliminating the need for expensive thermal cyclers or fluorescence microscopes, and results can be interpreted visually, making it suitable for on-site testing; its modular design allows for the detection of different viruses or gene targets simply by changing the crRNA, making it highly versatile. Attached Figure Description

[0051] Figure 1 A simplified schematic diagram illustrating the principle of the cascade reaction provided by this invention;

[0052] Figure 2 This is a simplified schematic diagram of the microfluidic chip design provided by the present invention;

[0053] Figure 3 These are the feasibility verification data provided in the embodiments of the present invention;

[0054] Figure 4 These are the sensitivity test results in the embodiments of the present invention. Detailed Implementation

[0055] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention and not for limiting the claims of the present invention.

[0056] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0057] There are no particular restrictions on the purity of any raw materials used in this invention. However, it is preferred to use analytical grade or conventional purity in the field of nucleic acid detection reagents.

[0058] All terms and abbreviations used in this invention are conventional terms and abbreviations in the field. Each term and abbreviation is clear and distinct in its relevant application area, and those skilled in the art can understand it clearly, accurately, and uniquely based on the terms and abbreviations.

[0059] This invention provides a nucleic acid detection chip based on a cascade reaction, comprising a recognition area, a reaction area, a labeling area, and a signal output area connected in sequence;

[0060] The recognition region is loaded with the Cas13a-crRNA ribonucleoprotein complex, trigger RNA, and enzyme reaction buffer.

[0061] The reaction zone is loaded with silver-loaded DNA probes and urease.

[0062] In this invention, the marked area is preferably loaded with urea and a pH-sensitive indicator.

[0063] In this invention, the signal output area is preferably a visualization observation window used to present the color change results of the system.

[0064] In this invention, the chip preferably also includes a chip substrate.

[0065] In this invention, the identification area, reaction area, marking area and signal output area are preferably disposed on the chip substrate.

[0066] In this invention, the connection is preferably a channel connecting four functional liquid storage areas.

[0067] In this invention, the identification area, reaction area, and marking area are each preferably selected independently from a closed cavity.

[0068] In this invention, the identification area is preferably provided with a sample inlet.

[0069] In this invention, the outlet of the identification area is preferably connected to the inlet of the reaction area via a microchannel.

[0070] In this invention, the outlet of the reaction zone is preferably connected to the inlet of the marking zone via a microchannel.

[0071] In this invention, the outlet of the marking area is preferably connected to the inlet of the reaction area via a microchannel.

[0072] In this invention, the channel preferably includes a serpentine channel.

[0073] In this invention, one or more of the four functional liquid storage zones are preferably provided with an expansion structure at their connection points with the channel.

[0074] In this invention, the chip is preferably also provided with a pneumatic drive interface for connecting an external pressure drive device to control the fluid to flow sequentially through each functional liquid storage area to complete the cascade reaction.

[0075] In this invention, the Cas13a is preferably the LwaCas13a protein derived from Leptotrichia wadei.

[0076] In this invention, the crRNA preferably consists of a constant region that binds to Cas13a and a guide region that is complementary to the target RNA.

[0077] In this invention, the trigger RNA is preferably a single-stranded RNA molecule whose sequence contains a Cas13a trans-cleavage recognition site.

[0078] In this invention, the trigger-RNA is preferably completely complementary to the strand substitution region of the toehold region of the silver-loaded DNA probe.

[0079] In this invention, the silver-loaded DNA probe is preferably a partially double-stranded DNA structure with a single-stranded toehold protrusion.

[0080] In this invention, the double-stranded region preferably contains at least one CC mismatch site.

[0081] In this invention, the CC mismatch site preferably binds specifically to Ag(I) to form a C-Ag(I)-C artificial base pair, thereby realizing Ag + Stable chelation and isolation.

[0082] This invention provides a nucleic acid detection method based on a cascade reaction, comprising the following steps:

[0083] 1) The sample to be tested is mixed with Cas13a-crRNA ribonucleoprotein complex, trigger-RNA and enzyme reaction buffer to obtain the primary product;

[0084] 2) After mixing the primary product obtained in the above steps, the silver-loaded DNA probe, and urease again, a secondary product is obtained;

[0085] 3) After further mixing the secondary product obtained in the above steps, urea and pH-sensitive indicator, a colorimetric product is obtained. The detection result is determined by the color change of the colorimetric product.

[0086] In this invention, the sample to be tested is first mixed with Cas13a-crRNA ribonucleoprotein complex, trigger-RNA and enzyme reaction buffer to obtain the primary product.

[0087] In this invention, the nucleic acid detection method is preferably an ultra-micro nucleic acid detection method.

[0088] In this invention, the sample to be tested preferably includes single-stranded RNA.

[0089] In this invention, step 1) is preferably the step of molecular recognition and first-level signal amplification.

[0090] In this invention, during step 1), if target RNA is present in the sample to be tested during the mixed incubation process, the Cas13a-crRNA complex preferably specifically binds to the target RNA and activates the trans-cleavage activity of Cas13a to cleave the trigger RNA in the system.

[0091] If there is no target RNA in the sample to be tested, it is preferable that Cas13a is not activated and the trigger RNA remains intact.

[0092] In this invention, the primary product obtained in the above steps, the silver-loaded DNA probe, and urease are mixed again to obtain the secondary product.

[0093] In this invention, step 2) is preferably a secondary signal transduction step.

[0094] In this invention, the silver-loaded DNA probe is preferably a double-stranded nucleic acid probe containing a CC mismatch site.

[0095] In this invention, the CC mismatch site preferably chelates Ag via a C-Ag(I)-C artificial base pair. + .

[0096] In this invention, during the remixing process in step 2), if the trigger RNA in the primary product is degraded, then preferably the strand displacement reaction cannot be triggered, and Ag... + Maintain the chelated state;

[0097] If the trigger RNA in the primary product is intact, it is preferred to act as the initiator strand to trigger the strand displacement reaction of the silver-loaded DNA probe, releasing the chelated Ag. + Free Ag + It binds to the active site of urease and inhibits its catalytic activity.

[0098] Finally, the secondary product obtained in the above steps, urea, and pH-sensitive indicator are mixed to obtain a colorimetric product. The detection result is determined by the color change of the colorimetric product.

[0099] In this invention, step 3) is specifically preferred to be the step of signal output and result interpretation.

[0100] In this invention, in step 3), if the catalytic activity of urease in the secondary product is inhibited, preferably urea cannot be hydrolyzed, the pH value of the system does not change, and the colorimetric product does not change color.

[0101] If the urease in the secondary product catalyzes the hydrolysis of urea to generate ammonia and increases the pH value of the system, the color of the preferred pH-sensitive indicator changes with the pH value of the system. The qualitative interpretation of the detection result is completed by observing the color change of the chromogenic product with the naked eye.

[0102] In this invention, the logic for the determination preferably includes:

[0103] Positive result: Target RNA was present in the test sample, trigger RNA was degraded, and no free Ag was found. + Upon release, urease retains its catalytic activity, catalyzing the hydrolysis of urea and raising the pH of the system; phenol red indicator turns deep red.

[0104] Negative result: No target RNA was found in the test sample; the trigger RNA remained intact, and the trigger strand displacement reaction released Ag. + The urease activity was inhibited, the pH of the system did not change significantly, and the phenol red indicator remained golden yellow.

[0105] The present invention provides the application of the nucleic acid detection chip or the nucleic acid detection method described in any one of the above technical solutions in nucleic acid detection.

[0106] This invention aims to complete and refine the overall technical solution, better ensure the composition and structure of the nucleic acid detection chip, and further improve the detection effect of the nucleic acid detection chip. Specifically, the ultra-micro nucleic acid detection chip based on cascade reactions, its detection method, and applications may include the following:

[0107] A method for detecting ultra-micro nucleic acids based on a cascade reaction includes the following steps:

[0108] S1 Molecular Recognition and Primary Signal Amplification: The test sample is mixed with the Cas13a-crRNA complex and trigger RNA and incubated. If the target RNA is present in the test sample, the Cas13a-crRNA complex specifically binds to the target RNA and activates the trans-cleavage activity of Cas13a, cleaving the trigger RNA in the system. If the test sample does not contain target RNA, Cas13a is not activated, and the trigger RNA remains intact.

[0109] S2 secondary signal transduction: The reaction product of S1 is mixed with a silver-loaded DNA probe and urease. The silver-loaded DNA probe is a double-stranded nucleic acid probe containing a CC mismatch site, which chelates Ag through a C-Ag(I)-C artificial base pair.+ If the trigger RNA in S1 is degraded, the strand displacement reaction cannot be triggered, and Ag... + Maintaining the chelated state preserves the urease catalytic activity completely. If the trigger RNA in S1 is intact, it acts as the initiator chain to trigger the strand displacement reaction of the silver-loaded DNA probe, releasing the chelated Ag+ into the system and free Ag. + It binds to the active site of urease, inhibiting the catalytic activity of urease;

[0110] S3 signal output and result interpretation: Mix the reaction product of S2 with urea and pH-sensitive indicator, and determine the detection result by the color change of the system;

[0111] Urease can catalyze the hydrolysis of urea to generate ammonia and increase the pH value of the system. The color of the pH-sensitive indicator changes reversibly with the change of the pH value of the system.

[0112] Specifically, the Cas13a is the LwaCas13a protein derived from Leptotrichia wadei.

[0113] Specifically, the crRNA consists of a constant region that binds to Cas13a and a guide region that is complementary to the target RNA. The sequence complementarity of the guide region and the target RNA is ≥95%, and single base mismatches can significantly reduce or block the activation of Cas13a.

[0114] Specifically, the trigger-RNA is a single-stranded RNA molecule whose sequence contains a preferential recognition site for Cas13a trans-cleavage and is completely complementary to the toehold region and strand substitution region of the silver-loaded DNA probe.

[0115] Specifically, the silver-loaded DNA probe is a partially double-stranded DNA structure with a single-stranded toehold protrusion. The double-stranded region contains at least one CC mismatch site. The CC mismatch site specifically binds to Ag(I) to form a C-Ag(I)-C artificial base pair, thereby achieving stable chelation and isolation of Ag+.

[0116] Specifically, the pH-sensitive indicator is phenol red, which is golden yellow at pH < 6.8 and dark red at pH > 8.4.

[0117] Specifically, the result determination logic is as follows:

[0118] Positive result: Target RNA was present in the test sample, trigger RNA was degraded, and no free Ag was found. + The urease is released and retains its catalytic activity. The addition of urea can catalyze its hydrolysis and increase the pH of the system. The phenol red indicator turns dark red.

[0119] Negative result: No target RNA was found in the test sample; the trigger RNA remained intact, and the trigger strand displacement reaction released Ag. + It inhibits urease activity, and there is no significant hydrolysis reaction after the addition of urea. The pH of the system does not change significantly, and the phenol red indicator remains golden yellow.

[0120] The present invention also provides a microfluidic chip for ultra-micro nucleic acid detection based on cascade reaction, used to implement the detection method described in any of the above technical solutions. The chip includes a chip substrate, and the chip substrate is provided with a recognition area, a reaction area, a labeling area, a signal output area, and microchannels connecting each functional area in sequence.

[0121] The recognition area is a closed cavity preloaded with Cas13a-crRNA complex, trigger-RNA and enzyme reaction buffer for performing step S1. The recognition area is provided with a sample inlet, and its outlet is connected to the inlet of the reaction area through a microchannel.

[0122] The reaction zone is a closed cavity, preloaded with silver-loaded DNA probes and urease, for performing step S2.

[0123] The marking area is a closed cavity preloaded with urea and a pH-sensitive indicator for performing step S3. The outlet of the marking area is connected to the signal output area through a microchannel.

[0124] The signal output area is a visualization window used to present the color change results of the system;

[0125] The chip is also equipped with a pneumatic drive interface for connecting an external pressure drive device to control the fluid to flow sequentially through each functional area to complete the cascade reaction.

[0126] Specifically, the microchannels connecting the functional areas have a width of 200 μm and a height of 100 μm. The microchannels regulate the fluid flow rate and reaction time through their length, width, and curved structure, ensuring stable laminar transport of the fluid at low Reynolds numbers.

[0127] Specifically, the cavity volume of the recognition area is 10 μL, and the microchannel between the recognition area and the reaction area is equipped with a narrow-channel passive valve to restrict the spontaneous flow of fluid through capillary pressure, thereby preventing the reagents from mixing and reacting prematurely.

[0128] Specifically, the inlet of the channel connecting the reaction zone and the recognition zone is configured as an expansion structure, and the outlet is configured with a serpentine mixing channel to reduce the flow rate, enhance molecular diffusion and reagent mixing efficiency, and improve the chain displacement reaction efficiency.

[0129] Specifically, the cavity volume of the marking area is 20 μL, and the inner wall of the cavity is hydrophilically treated; the cavity volume of the signal output area is 40 μL, which is a circular visualization window and has a standard colorimetric control area inside.

[0130] No pre-amplification of nucleic acid is required, completely eliminating the risk of amplification contamination: This invention constructs a dual-cascade amplification system by amplifying the primary signal from Cas13a trans-cleavage and combining it with the secondary enzymatic signal amplification catalyzed by urease. Under the premise of completely eliminating the need for nucleic acid pre-amplification steps such as PCR and RPA, it achieves the detection of ultra-micro target nucleic acids as low as 100aM, which greatly simplifies the operation process, shortens the detection time, and fundamentally avoids the risks of aerosol contamination and false positives caused by amplification.

[0131] High specificity with single-base resolution capability: This invention combines the single-base recognition capability of the CRISPR-Cas13a system with the high sequence specificity of the toehold chain substitution reaction. The reaction can be blocked by a single base mismatch in the key region, which can accurately distinguish between single nucleotide polymorphisms (SNPs) and viral mutation sites, and significantly reduce the probability of false positives.

[0132] Visually interpretable results without the need for expensive equipment, suitable for POCT scenarios: This invention regulates pH changes through a urease-silver ion inhibition system and combines it with phenol red indicator to achieve high-contrast color change output. The results can be directly interpreted by the naked eye, without the need for expensive optical equipment such as fluorescence microscopes and ELISA readers, which greatly reduces the detection cost and is suitable for on-site real-time detection scenarios such as grassroots, field, and bedside.

[0133] Modular design and high versatility: The detection system of this invention is modularly designed. Only the guide region sequence of crRNA needs to be changed to adapt to different target RNA detection needs. It can quickly develop detection products for different targets such as viruses, pathogens, and tumor markers, and has extremely high versatility.

[0134] Integrated microfluidic chip for automated detection: This invention integrates a complete detection system into a microfluidic chip, and achieves sequential control of fluid through pneumatic drive. The entire detection process can be completed simply by adding the sample to be tested. It is easy to operate, requires no professional operators, and has excellent potential for commercial application.

[0135] Furthermore,

[0136] Nucleic acid molecular detection principle based on cascade enzyme reaction

[0137] Molecular recognition mechanism

[0138] This invention utilizes the CRISPR-Cas13a system to specifically recognize and initially amplify the signal of target RNA. Cas13a (CRISPR-associated protein 13a) is an RNA-targeting endonuclease derived from bacteria and archaea, belonging to type VI effector proteins in class II CRISPR systems. Unlike classic DNA-targeting CRISPR-Cas systems (such as Cas9 and Cas12), Cas13a specifically recognizes and cleaves single-stranded RNA. Its unique feature is that it not only possesses cis cleavage activity but also significant trans cleavage capability.

[0139] The ribonucleoprotein (RNP) complex formed by the assembly of Cas13a and crRNA is the fundamental structure for achieving specific recognition and catalytic functions. crRNA typically consists of two parts: a constant region (direct repeat), responsible for binding to the Cas13a protein and maintaining the stability of the complex structure; and a variable guide region (spacer), whose sequence is completely or nearly completely complementary to the target RNA, thus endowing the system with a high degree of sequence-specific recognition capability. When Cas13a (derived from the Cas13a protein of *Leptotrichia wadei*) binds to the designed crRNA, a complex in a state of quiescent activation is formed. In this state, the catalytic center of Cas13a (usually composed of two HEPN domains) is spatially constrained and has not yet exhibited significant nucleic acid cleavage activity. At this point, the system possesses high specific recognition potential, but signal output has not yet been initiated.

[0140] When the target RNA molecule enters the solution system, its sequence undergoes base pairing with the spacer region of the crRNA. In the critical recognition region, a single base mismatch can significantly reduce or completely block Cas13a activation. This property gives Cas13a single-base resolution capability, enabling it to distinguish point mutations, single nucleotide polymorphisms (SNPs), and viral mutations.

[0141] Once the target RNA successfully pairs with the crRNA, the Cas13a protein undergoes a significant conformational change. This conformational shift rearranges the previously separated HEPN domains to form a complete catalytic active site, thereby activating its endonuclease function. At this point, Cas13a first performs cis-cleavage on the target RNA paired with the crRNA; this step completes molecular recognition and initial signal triggering. Once activated by the target RNA, Cas13a enters a continuously active state, and its catalytic center is no longer limited to recognizing specific sequences, but instead performs non-specific cleavage on any single-stranded RNA molecules in the surrounding environment.

[0142] First-order signal transduction and amplification

[0143] Cas13a exhibits high catalytic efficiency in its trans-cleavage activity, rapidly degrading large amounts of RNA substrates and thus achieving exponential signal amplification. A specific type of RNA molecule, trigger RNA, is further introduced as a key medium for first-stage signal amplification. The trigger RNA sequence does not participate in initial recognition but is rapidly cleaved after Cas13a activation. By pre-adding a large amount of trigger RNA to the reaction system, the recognition event of a single target molecule can be transformed into a cleavage event of numerous trigger RNA molecules, significantly enhancing the detection signal. Specifically, when the LwaCas13a-crRNA complex recognizes and binds to the target RNA, its conformation changes, activating trans-cleavage activity. At this point, the trigger RNA in the system becomes the primary cleavage substrate. Each activated Cas13a molecule can sequentially cleave 10⁴–10⁵ trigger RNA molecules, thereby achieving the first layer of signal amplification.

[0144] Because a single target RNA molecule can activate one Cas13a molecule, and that Cas13a molecule can cleave dozens or even hundreds of trigger RNA molecules, the system can detect extremely low concentrations of the target molecule. Secondly, signal amplification depends on the correct activation of Cas13a, which in turn depends on the precise pairing between the crRNA and the target RNA. Therefore, with proper crRNA design, the system can effectively avoid nonspecific background signals. Thirdly, the trans-cleavage kinetics of Cas13a are rapid, producing significant signal changes within minutes under suitable conditions, enabling rapid detection.

[0145] Signal acquisition mechanism of primary signal and secondary transduction

[0146] After the sample enters the first-stage signal channel where it is recognized by Cas13a and undergoes signal transduction of the trigger RNA, it enters the second-stage signal conversion and amplification module, namely the toehold nucleic acid molecular probe system based on metal ion chelation. This probe consists of a partially double-stranded structure containing a single-stranded overhang (toehold region) for initial binding to the trigger RNA. The probe structure is precisely designed to be thermodynamically metastable; that is, the double-stranded structure is regulated by free energy difference, remaining stable in the absence of trigger RNA while undergoing strand displacement reactions in its presence.

[0147] In toehold, this invention utilizes a cytosine-cytosine (C:C) mismatch to selectively introduce the metal ion Ag(I), and forms a metal-mediated C–Ag(I)–C artificial base pair through base-metal cation interactions, thereby affecting Ag. + A stable chelation is achieved, preventing it from participating in subsequent reactions in the untriggered state. Upon the appearance of the trigger RNA, it first rapidly binds to the probe via the toehold region, then gradually replaces the original strand through branching migration, thus completing the strand replacement reaction. During this process, silver ions (Ag) in the C-Ag(I)-C chelated state... + As the probe structure dissociates and is released from the chelated state into the solution, it becomes a free metal ion. This process realizes the signal transduction from nucleic acid signal to chemical signal.

[0148] Secondary signal output mechanism

[0149] Released Ag + It functions as an inhibitor of urease in the system. Urease is an enzyme that catalyzes the hydrolysis of urea, and its catalytic reaction is as follows:

[0150]

[0151] This reaction produces ammonia (NH3), thus raising the pH of the solution. Under normal circumstances, urease has high catalytic activity and can rapidly decompose urea, leading to alkalization of the solution. However, when Ag... + When present, it can bind to the sulfhydryl group or other key coordination sites in the urease molecule, thereby inhibiting the enzyme's catalytic activity. Ag + The higher the concentration, the stronger the inhibitory effect and the lower the urea decomposition rate. Based on this mechanism, by regulating Ag... + The amount of urease released can indirectly control urease activity, thereby regulating the degree of pH change in the solution.

[0152] For detection, this study typically introduces a pH-sensitive phenol red dye into the detection system. Its color changes with pH, ​​appearing golden yellow under acidic conditions and deep red under alkaline conditions. Therefore, in the absence of target RNA, Cas13a is not activated, the trigger RNA is not cleaved, the toehold probe does not undergo strand displacement, and Ag... + When chelated, urease activity remains normal, urea is fully decomposed, the solution pH increases, and the solution turns deep red. Conversely, in the presence of target RNA, the first-stage amplification by Cas13a generates a large number of trigger RNA fragments, triggering the toehold strand displacement reaction and releasing a large amount of Ag. +This significantly inhibits urease activity. At this point, urea decomposition is hindered, pH changes are weakened, and the color of the colorimetric system remains unchanged at a golden yellow. Qualitative detection can be achieved by visually observing the color difference. A complete schematic diagram is shown below. Figure 1 As shown, Figure 1 A simplified schematic diagram illustrating the principle of the cascade reaction provided by this invention.

[0153] In terms of detection logic, this invention designs two competing reaction paths, depending on the presence of target nucleic acid in the sample:

[0154] Path 1: The sample contains the target nucleic acid (positive result)

[0155] Recognition and activation: The Cas13a-crRNA complex specifically binds to the target RNA, and the Cas13a enzyme is activated.

[0156] Cutting (gate): Activated Cas13a degrades single-stranded trigger RNA in the system.

[0157] Strand displacement reaction blockade: The trigger RNA is degraded and cannot trigger the toehold strand displacement reaction.

[0158] Ag + Confinement: The chain displacement reaction does not occur, the pre-designed silver-loaded probe remains stable, and silver ions (Ag) remain trapped. + (It) has not been released.

[0159] Enzyme activity maintenance: Free Ag in the system + At extremely low concentrations, urease activity cannot be inhibited. Urease remains highly active.

[0160] Color development: Active urease hydrolyzes urea, changing the pH of the solution.

[0161] Result: The solution containing phenol red indicator turned red.

[0162] Pathway 2: The sample does not contain the target nucleic acid (negative result)

[0163] Inactive: Cas13a cannot bind to the target and is in an inactive state.

[0164] No cleavage: The trigger RNA in the system remains intact.

[0165] SDR initiation: The intact trigger RNA serves as the initiation strand, undergoing a strand displacement reaction with the silver-loaded probe.

[0166] Ag + Release: Strand displacement reactions cause allosteric changes or dissociation of DNA, releasing high concentrations of silver ions (Ag). + ).

[0167] Enzyme activity inhibition: Released Ag + It binds to the sulfhydryl group at the active site of urease, strongly inhibiting urease activity.

[0168] No reaction: The urea was not hydrolyzed and the solution environment remained unchanged.

[0169] Result: The solution containing phenol red indicator turned yellow.

[0170] System Composition

[0171] Detection modules: Cas13a protein, specific crRNA.

[0172] Signal transmission module:

[0173] ssRNA ligation probe: A specific RNA sequence designed as the toehold trigger for the SDR reaction and containing a Cas13a cleavage site (such as a U-rich sequence).

[0174] Silver-loaded DNA complex: A double-stranded or hairpin structure pre-formed by DNA strand hybridization, containing specific C-Ag-C mismatches for loading Ag. + Or Ag + It is physically isolated from the DNA structure in other ways.

[0175] Signal output module: urease, urea, phenol red indicator.

[0176] Microfluidic chip design

[0177] To achieve simplified operation, intuitive result interpretation, and strong commercial application potential in nucleic acid detection, this invention designs a microfluidic nucleic acid detection chip based on the CRISPR-Cas13a cascade enzyme reaction system. This chip integrates recognition, signal conversion, enzymatic amplification, and color readout processes into a single microfluidic platform through spatial partitioning and fluid control, realizing integrated detection from target RNA recognition to visualized signal output. It is suitable for rapid multi-channel streaming detection and POCT (Point-of-Care Testing) applications.

[0178] The chip consists of four main functional liquid reservoirs and connecting microchannels: a recognition region, a reaction region, a labeling region, and a signal output region. These reservoirs are arranged in layers on the chip plane and connected by microchannels 200 μm wide and 100 μm high. This ensures stable laminar transport under low Reynolds number (Re << 1) conditions, avoiding uneven mixing and runaway reactions caused by turbulence. See also Figure 2 , Figure 2 A simplified schematic diagram of the microfluidic chip design provided by this invention. Figure 2 In the middle, from left to right, the liquid storage areas are: identification area, reaction area, marking area, and signal output area.

[0179] The recognition region is located at the beginning of the fluid path and is pre-loaded with LwaCas13a, crRNA, trigger RNA, and enzyme reaction auxiliary buffer. This region is a relatively independent, closed circular cavity with a volume of approximately 10 μL, which reduces reagent consumption, dead zones, and air bubble retention. To avoid premature contact with downstream reactions, the structure generates significant capillary pressure through narrow channels. A passive valve is implemented through hydrodynamic impedance, interfacial stability, and diffusion restriction. Without external pressure, the liquid will hardly spontaneously flow downstream, thus preventing premature reagent reaction and realizing the microfluidic logic of sequentially initiating the cascade enzyme reaction.

[0180] Secondly, the reaction zone, connecting to the recognition zone, is the core module for signal conversion. This region contains components such as toehold structures, Ag(I), and urease. Its structure is similar to that of the recognition zone, but the channel connection is designed with an expanded structure to reduce the flow rate and enhance molecular diffusion, thereby improving the efficiency of the chain displacement reaction. Simultaneously, a serpentine channel is introduced at the outlet to enhance passive mixing, balancing isolation and mixing.

[0181] The labeling region stores urea and phenol red indicators, and its structural design emphasizes stability and uniformity. Since this region primarily participates in the color development process following the enzymatic reaction, it is designed as a 20-microliter reservoir to ensure sufficient visual contrast for color changes. The material in this region is hydrophilically treated to prevent uneven droplet adhesion and localized concentration deviations.

[0182] The signal output area, located at the end of the chip, is the final readout region. This area is a 40-microliter circular observation window, ensuring uniform color distribution and reducing visual errors caused by boundary effects, facilitating the detection of color changes by the naked eye or optical equipment. In future designs, a scale or reference color area will be added to this area to improve quantitative analysis capabilities.

[0183] In terms of the connection structure, the microchannels between the liquid storage zones not only serve the functions of transport and mixing, but also provide fluid resistance. The flow rate and volume are jointly regulated by the channel length, width, and tortuous structure, thereby achieving control over the reaction sequence and time intervals.

[0184] Finally, the chip structure includes a pneumatic interface for connecting an external air cylinder or miniature air pump. Gas is pressurized to drive liquid sequentially into each functional area, enabling an automated flow process. The chip is compatible with both manual and automatic operation modes, making it suitable for both laboratory environments and rapid on-site testing.

[0185] The present invention provides a cascade reaction-based ultra-micro nucleic acid detection chip, its detection method, and its applications. The nucleic acid detection chip designed in this invention, with a specific structure and composition, is a microfluidic on-chip detection chip that utilizes the incidental cleavage activity of the CRISPR-Cas13a system combined with Ag metal chelate toehold-mediated strand displacement reaction and a urease-silver ion inhibition system to achieve visualized detection of ultra-micro target nucleic acids without nucleic acid amplification. This ultra-micro nucleic acid detection chip requires no amplification, has high sensitivity, and is visible to the naked eye. Through a cascade signal amplification mechanism, it converts extremely small nucleic acid recognition signals into significant color change signals.

[0186] This invention presents a microfluidic nucleic acid detection chip based on the CRISPR-Cas13a cascade enzyme reaction system. Through spatial partitioning and fluid control, it integrates recognition, signal conversion, enzymatic amplification, and color readout processes into a single microfluidic platform, achieving integrated detection from target RNA recognition to visualized signal output. It is suitable for rapid multi-channel streaming detection and POCT (Point-of-Care Testing) applications. The chip requires no amplification reaction: utilizing the high signal turnover rate of Cas13a (cutting multiple trigger RNAs) and the high catalytic efficiency of urease (hydrolyzing large amounts of urea) to achieve dual cascade amplification, it can detect ultra-small amounts of nucleic acid without PCR or RPA amplification; it has high specificity: combining the single-base resolution of the CRISPR system with the high specificity of Toehold chain substitution, it can achieve single-base mutation detection, effectively addressing the rapid detection of highly variable pathogens such as viruses; it is low-cost and portable: eliminating the need for expensive thermal cyclers or fluorescence microscopes, and results can be interpreted visually, making it suitable for on-site testing; its modular design allows for the detection of different viruses or gene targets simply by changing the crRNA, making it highly versatile.

[0187] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a nucleic acid detection chip based on cascade reaction and its detection method and application provided by the present invention. However, it should be understood that these embodiments are implemented under the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operation processes, only to further illustrate the features and advantages of the present invention, and not to limit the claims of the present invention. The scope of protection of the present invention is not limited to the following embodiments.

[0188] Example 1

[0189] Construction of detection system

[0190] Nucleic acid sequence design: All sequences are 5'-3' in length. The trigger-RNA sequence is complementary to the toehold and substitution region of the silver-loaded DNA probe. See Table 1, which shows the specific nucleic acid sequences in Example 1 of this invention.

[0191] Table 1

[0192]

[0193] Preparation of silver-loaded DNA probes: Double-stranded DNA containing CC mismatch sites was prepared and annealed in the presence of AgNO3 to form a C-Ag-C structure. The Ag... + Chelated within the DNA double helix. Specific procedure: 5 µL of 300 nM Rec strand, 5 µL of 300 nM Block strand, and 5 µL of 300 M AgNO3. Incubate at room temperature for 20 minutes in the presence of 0.6 M NaNO3. Remove free Ag by dialysis or column chromatography. + Store at 4°C. Ag ion loading was verified by measuring melting curves using PAGE electrophoresis or real-time quantitative PCR. Ag ion concentration after column chromatography was measured using iodometric titration.

[0194] Example 2

[0195] Testing process

[0196] Cas13a Reaction Stage: At the start of the detection, the sample to be tested (RNA extraction solution) is added to a microinjection pump, and a low-pressure pneumatic drive of 1 kPa is applied to propel the sample through the molecular recognition cell. Simultaneously, a low-pressure pneumatic drive of 1 kPa is applied to the molecular recognition cell to ensure thorough 1:1 mixing of the enzyme reaction system and the sample. During this stage, the flow rate should be controlled at a low level to avoid bubble formation and liquid surface instability. This ensures sufficient contact between the nucleic acid sample and the pre-loaded system. If a target is present, the trigger RNA is cleaved; if no target is present, the trigger RNA remains intact.

[0197] Signal cascading stage: Preloading Ag in the chain replacement pool + Chelated toehold nucleic acid probes and urease. A low-pressure pneumatic drive of 1 kPa is applied simultaneously as the sample liquid flows through the reaction chamber. The liquids are mixed 1:1, and a displacement reaction occurs. Initially, in the positive pathway (containing the target RNA), the trigger RNA has been cleaved and cannot trigger the toehold strand displacement reaction; Ag... + Still stably chelated by the nucleic acid structure, free Ag in solution + At extremely low concentrations, urease remains active. In the negative pathway (no target RNA), the trigger RNA remains intact, initiating a toehold chain displacement reaction, causing the nucleic acid structure to dissociate and releasing Ag. + Released Ag + It binds to urease, alters the urease conformation, inhibits its catalytic activity, and results in the inactivation of urease.

[0198] Colorimetric Readout Stage: The reaction solution is advanced into the colorimetric cell, which contains urea and a pH indicator (phenol red). The colorimetric cell allows for naked-eye observation of the colorimetric reaction to obtain the test results. Figure 3 See also Figure 3 , Figure 3 These are the feasibility verification data provided in the embodiments of the present invention.

[0199] Urease activity is maintained in positive samples, catalyzing the hydrolysis of urea: (NH2)2CO + H2O → 2NH3 + CO2, where NH3 dissolves in water to generate OH-. - This leads to an increase in pH. In negative samples, urease is acted upon by Ag. + Inhibition prevents urea from being broken down, resulting in a constant pH. The corresponding pH change is represented by a color change using phenol red. In positive samples, a change from yellow to red is observed, while in negative samples, the developing solution remains yellow.

[0200] Optimal reaction conditions and detection limit determination: The system exhibited optimal performance when the trigger RNA concentration was 100 fM and the toehold probe concentration was 1 pM. Absorbance was measured at 560 nm using a microplate reader; significant absorbance changes were observed even at sample concentrations as low as 100 aM. Figure 4 This has become an effective high-sensitivity detection method.

[0201] See Figure 4 , Figure 4 These are the sensitivity test results in the embodiments of the present invention.

[0202] The foregoing has provided a detailed description of the cascade reaction-based ultra-micro nucleic acid detection chip, its detection method, and its applications. Specific examples have been used to illustrate the principles and implementation methods of the invention. The descriptions of these embodiments are merely for the purpose of helping to understand the method and core ideas of the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including manufacturing and using any device or system, and implementing any combined method. It should be noted that for those skilled in the art, several improvements and modifications can be made to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims. The scope of protection of this patent is defined by the claims and may include other embodiments that can be conceived by those skilled in the art. If these other embodiments have structural elements that are not different from the wording of the claims, or if they include equivalent structural elements that are not substantially different from the wording of the claims, then these other embodiments should also be included within the scope of the claims.

Claims

1. A nucleic acid detection chip based on a cascade reaction, characterized in that, It includes a recognition area, a reaction area, a marking area, and a signal output area that are connected in sequence; The recognition region is loaded with the Cas13a-crRNA ribonucleoprotein complex, trigger RNA, and enzyme reaction buffer. The reaction zone is loaded with silver-loaded DNA probes and urease.

2. The nucleic acid detection chip according to claim 1, characterized in that, The marked area is loaded with urea and a pH-sensitive indicator; The signal output area is a visualization window used to present the color change results of the system; The chip also includes a chip substrate; The identification area, reaction area, marking area, and signal output area are disposed on the chip substrate.

3. The nucleic acid detection chip according to claim 1, characterized in that, Specifically, the connection refers to the connection of four functional liquid storage areas through channels; The identification area, reaction area, and marking area are each independently selected from the closed cavity; The identification area is equipped with a sample inlet; The exit of the identification area is connected to the entrance of the reaction area through a microchannel.

4. The nucleic acid detection chip according to claim 3, characterized in that, The outlet of the reaction zone is connected to the inlet of the marking zone via a microchannel; The outlet of the marking area is connected to the inlet of the reaction area via a microchannel; The channel includes a serpentine channel; An expansion structure is provided at the connection points between one and more of the four functional liquid storage zones and the channel.

5. The nucleic acid detection chip according to claim 1, characterized in that, The chip is also equipped with a pneumatic drive interface for connecting an external pressure drive device to control the fluid to flow sequentially through each functional liquid storage area to complete the cascade reaction. The Cas13a is the LwaCas13a protein derived from Leptotrichia wadei; The crRNA consists of a constant region that binds to Cas13a and a guide region that is complementary to the target RNA; The trigger RNA is a single-stranded RNA molecule whose sequence contains a recognition site for Cas13a trans-cleavage.

6. The nucleic acid detection chip according to claim 1, characterized in that, The trigger-RNA is completely complementary to the strand substitution region of the toehold region of the silver-loaded DNA probe; The silver-loaded DNA probe is a partially double-stranded DNA structure with a single-stranded toehold protrusion. The double-stranded region contains at least one CC mismatch site; The CC mismatch site specifically binds to Ag(I) to form a C-Ag(I)-C artificial base pair, realizing Ag + Stable chelation and isolation.

7. A nucleic acid detection method based on a cascade reaction, characterized in that, Includes the following steps: 1) The sample to be tested is mixed with Cas13a-crRNA ribonucleoprotein complex, trigger-RNA and enzyme reaction buffer to obtain the primary product; 2) After mixing the primary product obtained in the above steps, the silver-loaded DNA probe, and urease again, a secondary product is obtained; 3) After further mixing the secondary product obtained in the above steps, urea and pH-sensitive indicator, a colorimetric product is obtained. The detection result is determined by the color change of the colorimetric product.

8. The nucleic acid detection method according to claim 7, characterized in that, The nucleic acid detection method is an ultra-micro nucleic acid detection method; The sample to be tested includes single-stranded RNA; Step 1) specifically involves molecular recognition and primary signal amplification. In step 1), during the mixed incubation process, if the target RNA is present in the sample to be tested, the Cas13a-crRNA complex specifically binds to the target RNA and activates the trans-cleavage activity of Cas13a, cleaving the trigger RNA in the system. If there is no target RNA in the sample to be tested, Cas13a will not be activated and the trigger RNA will remain intact.

9. The nucleic acid detection method according to claim 7, characterized in that, Step 2) is specifically a secondary signal transduction step; The silver-loaded DNA probe is a double-stranded nucleic acid probe containing a CC mismatch site; The CC mismatch site chelates Ag via the C-Ag(I)-C artificial base pair. + ; If the trigger RNA in the primary product is degraded during the remixing process in step 2), the strand displacement reaction cannot be triggered, and Ag... + Maintain the chelated state; If the trigger RNA in the primary product is intact, it acts as the initiator strand to trigger the strand displacement reaction of the silver-loaded DNA probe, releasing chelated Ag. + Free Ag + It binds to the active site of urease and inhibits its catalytic activity.

10. The nucleic acid detection method according to claim 7, characterized in that, Step 3) specifically refers to the steps of signal output and result interpretation. In step 3), if the catalytic activity of urease in the secondary product is inhibited, urea cannot be hydrolyzed, the pH value of the system does not change, and the colorimetric product does not change color. If the urease in the secondary product catalyzes the hydrolysis of urea to generate ammonia and increases the pH value of the system, the color of the pH-sensitive indicator will change with the change of the pH value of the system. The qualitative interpretation of the detection result can be completed by observing the color change of the colorimetric product with the naked eye. The logic for the determination includes: Positive result: Target RNA was present in the test sample, trigger RNA was degraded, and no free Ag was found. + Upon release, urease retains its catalytic activity, catalyzing the hydrolysis of urea and raising the pH of the system; phenol red indicator turns deep red. Negative result: No target RNA was found in the test sample; the trigger RNA remained intact, and the trigger strand displacement reaction released Ag. + The urease activity was inhibited, the pH of the system did not change significantly, and the phenol red indicator remained golden yellow.

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