Cascade positive feedback ultrasensitive rapid detection biosensor and application thereof

Abstract

The invention belongs to the technical field of biological detection, and relates to a cascade positive feedback ultrasensitive rapid detection biosensor and application thereof. The biosensor comprises a nucleic acid amplification reaction network unit and a signal conversion unit, the nucleic acid amplification reaction network unit comprises at least three cyclic amplification reaction modules, and each cyclic amplification reaction module comprises an FB (at) (LP (at) HRP-Track + WLnDn) compound and an Fn chain; the signal conversion unit comprises TMB (Tetramethylbenzidine) and H2O2. The biosensor provided by the invention has the advantages of strong specificity, high sensitivity, high detection speed, simplicity and convenience in operation, lower cost and the like; meanwhile, in combination with selection of detection markers, on-site early diagnosis of the prawn WSSV is expected to be realized, and a new direction is provided for design of biosensors.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a cascaded positive feedback ultrasensitive rapid detection biosensor and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] White spot syndrome virus (WSSV) in shrimp is a highly lethal, stress-dependent virus with a wide range of hosts, a long incubation period, and strong infectivity. It has been reported that WSSV can cause 100% mortality of infected species in an entire aquaculture farm within 3-10 days of the initial disease outbreak, resulting in billions of dollars in economic losses to the aquaculture industry annually. Historically, every WSSV outbreak has been a devastating blow to the aquaculture industry; therefore, WSSV detection is urgently needed.

[0004] Currently, detection techniques for WSSV are mainly divided into three categories: histopathological detection methods, immunological detection methods, and molecular biological detection methods. Although each existing detection technique has its advantages, they still have significant limitations in practical applications. Clinically, highly variable coloration, primarily reddish or pinkish discoloration, is observed in infected shrimp. Gross pathology reveals delayed hemolymph coagulation, excessive decontamination of the gills and exoskeleton, and electron microscopy reveals that the histological characteristics of WSSV infection include enlarged cell nuclei in ectodermal and mesodermal tissues. Because WSSV has an incubation period ranging from several days to several months, histopathological detection methods are only suitable for late-stage infections; early-stage infection cannot be confirmed by microscopic examination alone. Immunological methods, such as enzyme-linked immunosorbent assay (ELISA), are simple to operate and suitable for on-site detection, but their sensitivity is limited, making it difficult to meet the needs of early infection detection. Molecular biological detection methods have become the mainstream technique for WSSV detection. Techniques: Polymerase chain reaction (PCR) has high detection sensitivity, but it cannot quantify the target sequence, and PCR has a high false negative and false positive rate and requires electrophoresis of the products, which easily leads to cross-contamination; qPCR does not require additional product processing and can target sequences, achieving absolute quantification of viral load; Nested PCR improves detection sensitivity through two rounds of amplification; however, nested PCR2 has cumbersome operation steps, requiring two pairs of primers and two rounds of amplification, increasing the risk of contamination, and like PCR and qPCR, it relies on expensive equipment and professional laboratory personnel, making it unsuitable for use in aquaculture fields; Loop-mediated isothermal amplification (LCM)... LAMP (Low-Ambient Propagation), an emerging nucleic acid amplification technology, does not require a dedicated PCR instrument and can complete the reaction in 30-60 minutes at a constant temperature of 63-65℃. Recently, Kumar et al. proposed a visualization method for detecting WSSV using LAMP, but its quantitative ability and sensitivity have been somewhat reduced. Recombinase isothermal amplification (RPA) is another promising detection method, specifically detecting WSSV at a constant temperature of 39℃, making it ideal for rapid on-site screening. RPA can be combined with other technologies, such as LFA and fluorescence detection, to suit different detection needs. While LAMP and RPA simplify operation, primer design is complex, specificity requirements are high, and amplification efficiency is affected by various factors. All nucleic acid amplification-based technologies are susceptible to interference from inhibitors in the sample and are difficult to implement in farms. Emerging methods combine the CRISPR-Cas system with other methods for higher specificity and sensitivity. CRISPR is an emerging technology for rapid disease detection and various applications. This system uses related endonucleases to identify and eliminate foreign sequences based on similarity.The combination of CRISPR-Cas and RPA has led to the development of two methods: Specific High-sensitivity Enzymatic Reporter unlocking (SHERLOCK) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR). These methods utilize different Cas enzymes (SHERLOCK uses Cas13 to target RNA, while DETECTR uses Cas12 to target DNA) to achieve highly specific, sensitive, rapid, and on-site detection of pathogen nucleic acids. This has revolutionized the early diagnosis and control of diseases in aquaculture. However, due to the limitations of proteases, these methods have certain requirements regarding the environment and detection conditions, thus limiting their popularity in aquaculture due to their qualitative nature. Therefore, developing a WSSV detection technology that combines ultra-high sensitivity, rapid response, low environmental requirements, ease of operation, and controllable cost has become an urgent need in the aquaculture field. Furthermore, for viruses like WSSV, which have long incubation periods, high infectivity, and significant harm, early and accurate detection of WSSV infection is a crucial step in controlling the spread of epidemics and reducing economic losses. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a cascaded positive feedback ultrasensitive rapid detection biosensor and its applications. The biosensor provided by this invention has advantages such as high specificity, high sensitivity, fast detection speed, simple operation, and low cost. Furthermore, by combining this with the selection of detection biomarkers, it is expected to achieve early on-site diagnosis of WSSV in shrimp and also provides a new direction for biosensor design.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In the first aspect, a cascaded positive feedback ultrasensitive rapid detection biosensor includes a nucleic acid amplification reaction network unit and a signal conversion unit; The signal conversion unit includes 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2; The nucleic acid amplification reaction network unit includes at least three cyclic amplification reaction modules, each cyclic amplification reaction module including a detection complex (FB@(LP@HRP-Track+WL)). n D n (complex) and F n The detection complex comprises magnetic beads with both Track and W chains simultaneously attached to their surface. Liposomes containing horseradish peroxidase (HRP) are attached to the Track chains.n Chains hybridized with D via complementary hybridization. n Chain and W chain are connected to form W / L n / D n Three-chain system: Track chain, W chain, L chain n Chain, D n Chain and F n All strands are single-stranded DNA, F n The strand has a hairpin structure, n is 1 or m, where m is any integer from 2 to the number of cyclic amplification reaction modules. The W strand contains 8-17 DNAzymes, the Track strand contains sites that can be recognized and cleaved by the DNAzymes, and the target sequence can replace the D1 strand in the W / L1 / D1 triplet, and D... m-1 Capable of replacing W / L m / D m D in the triple chain m Chain, and F n The chain can identify the replaced W / L n / D n Trichain and L n Chain complementarity allows the W chain to change from the replaced W / L chain. n / D n The DNAzyme in the W strand is then freed in the triple strand, and then recognizes and cleaves the corresponding site of the Track strand, thereby releasing the liposome containing horseradish peroxidase from the surface of the magnetic bead.

[0007] When the target sequence is present, in the first-stage cyclic amplification reaction module, the target sequence can replace the D1 chain in the W / L1 / D1 triplet, after which W / L n / D n Trichain can be F n The chain can be identified, and is consistent with the replaced W / L. n / D n L in the triple chain n Chain complementarity, thus enabling the W chain to transform from the replaced W / L chain. n / D n Within the triplet, DNAzyme in the W strand recognizes and cleaves the corresponding site on the Track strand, thereby releasing liposomes containing horseradish peroxidase from the magnetic bead surface. The target sequence replaces the D1 strand released from the W / L1 / D1 triplet with the next W / L1 chain. n / D n Triple chain substitution, in the m-th stage of the cyclic amplification reaction module, the reaction is the same as in the first stage of the cyclic amplification reaction module, the difference being: D from the previous stage... m-1 Replacement W / L m / D m D in the triple chain mThe chain; thereby, through the reaction of at least three stages of cyclic amplification reaction modules, a large number of liposomes containing HRP are released; the liposomes are able to be penetrated by H2O2, react with HRP to produce ·OH, causing the membrane to rupture and release HRP. Finally, the free HRP catalyzes the colorimetric reaction of H2O2 and TMB, thereby converting the invisible nucleic acid reaction into a visible analytical signal.

[0008] Secondly, the application of the biosensor described in the first aspect of the present invention in detecting biomarkers for white spot syndrome virus in shrimp.

[0009] The application of the biomarkers for detecting white spot syndrome virus in shrimp described in this invention can be for the purpose of disease diagnosis and treatment, or for non-disease diagnosis and treatment.

[0010] Thirdly, a detection kit for white spot syndrome virus in shrimp includes the biosensor and buffer solution described in the first aspect of the present invention, wherein the biomarker detected by the detection kit is WSSV-miR-n24.

[0011] To facilitate early detection, the selection of biomarkers is crucial. Viral biomarkers include nucleic acids (including RNA and DNA) and proteins. Among them, microRNAs (miRNAs) play a vital role in regulating gene expression and their stability in various biological fluids, including blood, and are crucial in post-transcriptional gene regulation. Therefore, in the early stages of viral invasion, the expression levels of corresponding miRNAs may increase or decrease rapidly. This characteristic gives them a significant advantage as early diagnostic biomarkers. Studies have shown that the WSSV-encoded microRNA biomarker WSSV-miR-n24 can be detected in shrimp as early as 6 hours after infection. Furthermore, WSSV-miR-n24, as a key regulatory factor encoded by the virus, effectively inhibits apoptosis by specifically targeting the 3' untranslated region of the critical apoptosis gene Caspase 8 in shrimp, thereby promoting viral survival and proliferation in the host. This unique biological function makes it an ideal molecular biomarker for early WSSV infection. The detection kit provided by this invention offers advantages such as high sensitivity, rapid response, low requirements for the detection environment, ease of operation, and controllable cost in WSSV detection.

[0012] The beneficial effects of this invention are as follows: 1. The biosensor provided by this invention possesses the advantage of ultra-high sensitivity. This invention modularly cascades base-mediated strand displacement (TMSD) and DNAzyme-catalyzed cleavage, introducing a positive feedback loop. The designed nucleic acid amplification reaction network unit allows a single target sequence to trigger multiple, cyclic signal amplification processes, achieving a leap from "linear amplification" to "exponential amplification," thereby realizing ultra-high sensitivity detection. Furthermore, this invention uses liposome-encapsulated horseradish peroxidase (HRP@LP) as a signal conversion unit, releasing it in large quantities from magnetic beads via DNAzyme cleavage. H2O2 penetration of the liposome membrane triggers HRP release and catalyzes a colorimetric reaction, achieving an effective conversion from nucleic acid recognition events to high-magnification chemically amplified signals, further enhancing detection sensitivity. Experiments show that this biosensor has a detection limit as low as 7.9 aM (approximately 4800 copies / mL) for the target biomarker WSSV-miR-n24, meeting the detection requirements for extremely low viral loads during the viral incubation period or early infection, providing a possibility for early diagnosis.

[0013] 2. The biosensor provided by this invention has the advantage of a wide linear range. Experiments show that the detection signal of this biosensor exhibits a good linear relationship across a range of nine orders of magnitude (1 aM to 1 pM) of target concentration, enabling the same method to detect both early trace infections and assess viral load in the mid-to-late stages, thus having a wide range of applications.

[0014] 3. The biosensor provided by this invention has the advantage of rapid response. Kinetic studies show that the nucleic acid amplification reaction network unit of the biosensor of this invention, in an optimized three-stage cascade mode, can complete the entire detection process within 15 minutes, significantly faster than traditional methods such as PCR and LAMP (which typically require 1-2 hours or more), thus facilitating rapid on-site decision-making and emergency response to epidemics.

[0015] 4. The biosensor provided by this invention has excellent specificity and anti-interference ability. Experiments show that in a complex simulated shrimp tissue nucleic acid matrix, the biosensor can still maintain the detection capability at the aM level (39.8 aM), demonstrating its good robustness and potential for detection in practical samples.

[0016] 5. The biosensor provided by this invention is easy to operate during the detection process and is suitable for point-of-care testing (POCT). The entire detection is carried out at a constant temperature (30℃), eliminating the need for complex temperature cycling control equipment. The detection results can be read using an ELISA reader or a UV-Vis spectrophotometer, and even visual colorimetric detection can be achieved (see the color change of the well plate in the standard curve), with low instrument dependence. Combined with magnetic bead separation, the operation steps are simple, eliminating the need for post-processing such as electrophoresis, reducing the risk of cross-contamination. The core components are artificially synthesized DNA / RNA sequences and conventional biochemical reagents, eliminating the need for expensive proteases (such as Cas enzymes) or special instruments; in addition, the preparation processes of materials such as liposomes and magnetic beads are mature and the cost is relatively low, which is conducive to large-scale production and widespread application. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1 A schematic diagram of the biosensor provided by this invention.

[0019] Figure 2 This is a diagram showing the feasibility verification results in an embodiment of the present invention; (A) Polyacrylamide gel electrophoresis imaging after three cycles: Lane 1: W / L1 / D1; Lane 2: W / L2 / D2; Lane 3: W / L3 / D3; Lane 4: W; Lane 5: Track; Lane 6: W+Track; Lane 7: F1; Lane 8: F2; Lane 9: F3; Lane 10: L1F1; Lane 11: L2F2; Lane 12: L3F3; Lane 13: Target; Lane 14: W / L n / D n + Track + F n + Target; Lane 15: W / L n / D n + Track + F n Lane 16: W / L n / D n+ Track, the results showed that clearly cleaved Track chain bands could only be observed in the presence of T (lane 14), the concentration of F chain in the system was 2 μM, and the concentration of other chains was 1 μM; UV-vis absorption spectrum verified the feasibility (B) catalytic colorimetric development of free HRP and HRP coated in liposomes; (C) catalytic colorimetric development of HRP-GA-Track and HRP@LP-Track in the first cycle, with [W / L1 / D1] = 15 nM, [T] = 1 nM, [F1] = 30 nM, [Track] = 100 nM, [Mg 2+ [15 mM; (D) Whether the target object is present under different cycles, [W / L] n / D n [F] = 15 nM, [T] = 1 nM, [F] = 15 ... n ]=30 nM, [Track]=100 nM, [Mg 2+ =15 mM, the total reaction volume is 200 μL, and FB (10 mg / mL) is taken as 20 μL.

[0020] Figure 3 In the embodiments of the present invention, LP, LP@HRP, LP@HRP-Track, FB, and FB@(LP@HRP-Track+WL) are... n D n Characterization of DLS (A) and ZETA potential (B) of ).

[0021] Figure 4The following is a diagram showing the optimization results of different F-chain lengths in the embodiments of the present invention; (A) Polypropylene gel electrophoresis: Lane 1: W; Lane 2: L1; Lane 3: D1; Lane 4: W / L1 / D1; Lane 5: Track; Lane 6: W / L1 / D1+ Track; Lane 7: F6; Lane 8: W / L1 / D1+ Track + F6; Lane 9: W / L1 / D1+ Track + F6; Lane 10: F7; Lane 11: W / L1 / D1+ Track + F7; Lane 12: W / L1 / D1+ Track + F7; Lane 13: F8; Lane 14: W / L1 / D1+Track + F8; Lane 15: W / L1 / D1+ Track + F8 Lane 16: Target. The substitution ability of the F chain was determined by comparing the presence or absence of segmented Track chain bands and the presence of W / L1 / D1 when the target was present. The concentration of the F chain in the system was 2 μM, and the concentration of other chains was 1 μM. (B) UV-vis F / F0; Optimization of UV-vis absorption spectrum signal and signal-to-noise ratio conditions; (C) Track chain concentration; (D) WLD concentration; (E) Amount of FB-LP complex; (F) Mg 2+ Concentration. In the optimized system, after removing the concentration of the optimized substance, the overall system concentration remains: [W / L1 / D1] = 15 nM, [T] = 1 nM, [F] = 30 nM, [Track] = 100 nM, [Mg 2+ =15mM, the total reaction volume is 200 μL, and FB (10 mg / mL) is taken as 20 μL.

[0022] Figure 5 The following is a diagram showing the optimized reaction time structure in this embodiment of the invention: (A) is the UV-vis absorption spectrum signal of the first module; (B) is the signal-to-noise ratio (SNR) of the first module; (C) is the UV-vis absorption spectrum signal of the first + second module; (D) is the SNR of the first + second module; (E) is the UV-vis absorption spectrum signal of the first + second + third module; (F) is the SNR of the first + second + third module; the optimal reaction times are 60 min, 30 min, and 15 min, [W / L] n / D n [F] = 15nM, [T] = 1 nM, ...F] = 15nM, [T] = 1 nM, [F] = 15nM, [F] = 1 nM, [F] n ]=30 nM, [Track]=100 nM, [Mg 2+ =15 mM, the total reaction volume is 200 μL, and FB (10 mg / mL) is taken in 20 μL.

[0023] Figure 6(A) is the standard curve detected in this embodiment of the invention; (B) is the standard curve of the first module [10 nM, 1 nM, 500 pM, 100 pM, 10 pM, 1 pM, 500 fM, 100 fM, 10 fM, 1 fM, 0], R 2 =0.99636, detection limits are 501 fM; (B) is the standard curve of the first + second module [1 nM, 100 pM, 10 pM, 1 pM, 500 fM, 100 fM, 10 fM, 1 fM, 0], R 2 =0.99696, detection limits are 8.9 fM; (C) is the standard curve of the first + second + third modules [10 pM, 1 pM, 100 fM, 10 fM, 1 fM, 500 aM, 100 aM, 10 aM, 1 aM, 0], R 2 =0.998, detection limits were 7.9 aM; (D) is the standard curve of real shrimp tissue nucleic acid [10 pM, 1 pM, 100 fM, 10 fM, 1 fM, 500 aM, 100 aM, 10 aM, 1 aM, 0], R 2 =0.99561, detection limits are 39.8 aM; [W / L n / D n [F] = 15 nM, [T] = 1 nM, [F] = 15 ... n ]=30 nM, [Track]=100 nM, [Mg 2+ =15 mM, the total reaction volume is 200 μL, and FB (10 mg / mL) is taken as 20 μL. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0026] To achieve detection that combines ultra-high sensitivity, rapid response, low requirements for detection environment, simple operation and controllable cost, this invention proposes a cascaded positive feedback ultra-sensitive rapid detection biosensor and its application.

[0027] A typical embodiment of the present invention provides a cascaded positive feedback ultrasensitive rapid detection biosensor, comprising a nucleic acid amplification reaction network unit and a signal conversion unit; The signal conversion unit includes 3,3',5,5'-tetramethylbenzidine and H2O2; The nucleic acid amplification reaction network unit includes at least three cyclic amplification reaction modules, each cyclic amplification reaction module including a detection complex and F. n The detection complex comprises magnetic beads with both Track and W chains simultaneously attached to their surface. Liposomes containing horseradish peroxidase are attached to the Track chains. n Chains hybridized with D via complementary hybridization. n Chain and W chain are connected to form W / L n / D n Three-chain system: Track chain, W chain, L chain n Chain, D n Chain and F n All strands are single-stranded DNA, F n The strand has a hairpin structure, n is 1 or m, where m is any integer from 2 to the number of cyclic amplification reaction modules. The W strand contains 8-17 DNAzymes, the Track strand contains sites that can be recognized and cleaved by the DNAzymes, and the target sequence can replace the D1 strand in the W / L1 / D1 triplet, and D... m-1 Capable of replacing W / L m / D m D in the triple chain m Chain, and F n The chain can identify the replaced W / L n / D n Trichain and L n Chain complementarity allows the W chain to change from the replaced W / L chain. n / D n The DNAzyme in the W strand is then freed in the triple strand, and then recognizes and cleaves the corresponding site of the Track strand, thereby releasing the liposome containing horseradish peroxidase from the surface of the magnetic bead.

[0028] In some embodiments, the number of cyclic amplification reaction modules is 3 to 5. Studies have shown that having 3 to 5 cyclic amplification reaction modules, especially 3, can further reduce costs while maintaining extremely high sensitivity.

[0029] F n The chain has a hairpin structure, which avoids the defect of severe leakage and reduced specificity caused by straight chains. In some embodiments, F n The hairpin structure of the chain has 7 or 8 bases at the stem. F nThe number of stem bases in the hairpin structure of the chain has a significant impact on the reaction rate and efficiency. When the number of stem bases is 7 or 8, especially 7, it has higher reaction efficiency and specificity.

[0030] In some embodiments, the target sequence is as shown in SEQ ID NO:3, i.e., the nucleic acid sequence of WSSV-miR-n24.

[0031] Specifically, n is 3, and the nucleic acid sequence of the W chain is shown in SEQ ID NO:1.

[0032] Specifically, n is 3, and the nucleic acid sequence of the Track chain is shown in SEQ ID NO:2.

[0033] Specifically, n is 3, L n The nucleic acid sequences of the strands are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. n The nucleic acid sequences of the strands are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively. n The nucleic acid sequences of the strands are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively.

[0034] In some embodiments, the liposomes are composed of DPPC, cholesterol, and DSPE-PEG-NHS in a molar ratio of 4.5~5.5:2.7~3.3:1.8~2.2. These liposomes can be configured to have NHS ester groups on their surface, which can react with the primary amine groups at the ends of the Track chain to form amide bonds, thereby linking the Track chain to the liposome.

[0035] In some embodiments, the surface of the magnetic beads is connected to the Track chain and W chain via biotin-streptavidin binding.

[0036] Another embodiment of the present invention provides an application of the above-mentioned biosensor in detecting biomarkers for white spot syndrome virus in shrimp.

[0037] Specifically, the sequence of the marker of white spot syndrome virus in shrimp is used as the target sequence of the biosensor. The marker of white spot syndrome virus in shrimp is incubated with the nucleic acid amplification reaction network unit for a set time, and then magnetic separation is performed to obtain the supernatant. Then, a signal conversion unit is added to the supernatant and the optical signal is detected.

[0038] In some embodiments, when the markers of shrimp white spot syndrome virus are incubated with nucleic acid amplification reaction network units, W / L n / D nThe total concentration was 14.5–15.5 nM, the T-chain concentration was 0.9–1.1 nM, and the F-chain concentration was... n The total concentration of the chain was 27–33 nM, the concentration of the track chain was 90–110 nM, and Mg... 2+ The concentration was 14–15 mM. Studies have shown that the detection effect is better under these conditions.

[0039] A third embodiment of the present invention provides a detection kit for white spot syndrome virus in shrimp, comprising the above-mentioned biosensor and a buffer solution, wherein the biomarker detected by the detection kit is WSSV-miR-n24.

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0041] Example Experimental materials and reagents Potassium chloride (analytical grade), potassium dihydrogen phosphate (analytical grade), disodium hydrogen phosphate dodecahydrate (analytical grade), hydrogen peroxide (30%), and chloroform (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. Horseradish peroxidase (analytical grade) was purchased from Sigma-Aldrich. 3,3',5,5'-Tetramethylbenzidine (analytical grade) was purchased from BBI Life Sciences Co., Ltd. DEPC water, 6× glycerol gel loading buffer (analytical grade), tetramethylethylenediamine (analytical grade), and tris(hydroxymethyl)aminomethane (analytical grade) were purchased from Sangon Biotech (Shanghai) Co., Ltd. Cholesterol (analytical grade) was purchased from Maclean's Biochemical Technology Co., Ltd. Ammonium persulfate (analytical grade), DSPE-PEG-NHS (analytical grade), and DPPC (analytical grade) were purchased from Aladdin (Shanghai) Biochemical Technology Co., Ltd. MD34-100000 dialysis bags were purchased from Hunan Yibo Biotechnology Co., Ltd. Streptomyces affinity magnetic beads, TB buffer, and 30% acrylamide solution were purchased from Beyotime Biotechnology Co., Ltd. A 20 bp DNA ladder marker was purchased from Takara Biotechnology Co., Ltd. The magnetic bead-based nucleic acid extraction kit was purchased from Qingdao Jianma Gene Technology Co., Ltd. All DNA and RNA sequences (Table 1) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Some analytical reagents did not require further purification. All solutions were prepared using 0.1% DEPC water.

[0042] Table 1 Oligonucleotide Sequences

[0043] Note: The underlined portion represents the catalytic sequence of the track strand and DNAzyme strand (8-17E DNAzyme). rA is located on the track strand and represents adenosine ribonucleotide.

[0044] (a) Methods 1. Fabrication of a cascaded positive feedback ultrasensitive rapid detection biosensor 1.1. Preparation of HRP@LP liposomes HRP-coated liposomes (HRP@LP) were prepared using a thin-film hydration-extrusion method: DPPC, cholesterol, and DSPE-PEG-NHS were weighed in a molar ratio of 5:3:2 and dissolved in 4 mL of chloroform. The mixture was sonicated for 10 min to thoroughly mix the components and transferred to a round-bottom flask. The flask was placed in a rotary evaporator and evaporated at 45 °C for 30 min to form a uniform lipid film. 10 mL of PBS buffer (0.01 M, pH=7.4) containing HRP (0.2 mg / mL) was added to the flask and hydrated at 37 °C for 2 h to obtain a crude liposome suspension. The suspension was sonicated on ice for 10 min to regularize the liposome diameter and extruded 5 times through a 0.22 μm polycarbonate membrane extruder to obtain liposomes with uniform particle size. The extruded liposomes were transferred to a dialysis bag and dialyzed with PBS buffer at 4 °C for 24 h (with buffer changed every 8 h) to remove free HRP, yielding HRP@LP, which was stored at 4 °C. Liposome particle size and zeta potential were determined by dynamic light scattering (DLS, Malvern Zetasizer Nano ZS90), and morphology was observed by scanning electron microscopy (SEM, Hitachi SU8010). Sample preparation: 10 μL of HRP@LP suspension diluted 10 times was dropped onto a silicon wafer, and after natural drying, it was sprayed with platinum for observation.

[0045] 1.2. Oligonucleotide chain preparation The W chain, L1 chain, L2 chain, L3 chain, D1 chain, D2 chain, D3 chain, F1 chain, F2 chain, F3 chain, Track chain, and target compound WSSV-miR-n24 used in the experiment were all synthesized by Sangon Biotech (Shanghai) Co., Ltd., purified by HPLC, and then dissolved in Tris-HCl buffer (10 mM Tris, 150 mM NaCl, pH 7.5) to 100 μM as the stock solution. The stock solution was stored at -20℃ protected from light and diluted to the required concentration according to experimental needs before use.

[0046] 1.3. W / L n / D n Preparation of triple chain complex Take 450 nM W chain and L respectively n Chains (L1 / L2 / L3), D n20 μL each of the chains (D1 / D2 / D3) were mixed thoroughly and then annealed: incubated at 90℃ for 10 min, followed by cooling to 25℃ to form W / L n / D n The triple-stranded complex was stored at 4°C for later use. Complex formation was verified by 15% non-denaturing polyacrylamide gel electrophoresis: 10 μL of the complex sample was mixed with 2 μL of 6× loading buffer and loaded into a gel. Electrophoresis was performed at 300 V for 60 min using 1× TBE buffer. After electrophoresis, the gel was stained with SYBR Red nucleic acid dye for 30 min, and the bands were observed using a gel imaging system (Bio-Rad).

[0047] 1.4. FB@(LP @ HRP-Track+WL n D n Preparation of the complex First, HRP@LP and Track chains were incubated at a volume ratio of 40:1 for 3 h, allowing the NHS on the surface of HRP@LP to react with the primary amino group at the 3' end of the Track chain to form an amide bond, thus synthesizing Track-HRP@LP. Then, unbound Track chains were removed by dialysis to obtain Track-HRP@LP. 20 μL of streptavidin-modified magnetic beads (10 mg / mL) were washed three times to remove the solution, and 100 μL W / L was added at a volume ratio of 1:9. n / D n Tristanine (15 nM) and Track-HRP@LP (final Track chain concentration 100 nM) were incubated at 30°C for 1.5 h to achieve a W / L ratio of [missing information]. n / D n The triplet and Track chains bind to the magnetic bead surface via biotin-streptavidin interaction. The supernatant is removed by magnetic separation, and the mixture is washed three times with Tris-HCl buffer and resuspended to 100 μL to form FB@(LP@HRP-Track), which is then stored at 4°C. Complex assembly is verified by DLS and Zeta potential: FB, FB@(LP@HRP-Track), and FB@(LP@HRP-Track+WL) are measured separately. n D n The particle size and potential changes.

[0048] 2. Optimization of testing procedures and conditions 2.1 Testing Process Reaction start-up: Add 100 μL FB@(LP@HRP-Track+WL) n D nTo the Tris-HCl buffer (10 mM Tris-HCl, 100 mM NaCl, 15 mM MgCl2, pH=7.5) of the complex, add 5 μL of the target compound WSSV-miR-n24 at different concentrations (final concentration 1 aM~10 nM) and 5 μL of 600 nM F n F n Before adding the components, they need to be annealed at 90℃ for 10 minutes, then cooled to 25℃ to maintain their hairpin structure. After adding all components and mixing them evenly, they are placed in a 30℃ constant temperature incubator for incubation (60 min for the first module, 30 min for the first module + second module, and 15 min for the first module + second module + third module). The detection scheme includes FB@(LP@HRP-Track+WL1D1, F1) in the first module; FB@(LP@HRP-Track+WL2D2, F2) in the second module; and FB@(LP@HRP-Track+WL3D3, F3) in the third module.

[0049] Magnetic separation: After incubation, place the reaction tube on a magnetic rack and let it stand for 2 minutes to allow the magnetic bead complex to precipitate. Collect the supernatant (containing the released HRP@LP).

[0050] Signal detection: Take 30 μL of supernatant, add 25 μL of TMB substrate solution, 25 μL of H2O2 and 120 μL of NaAc-Hac (0.2 M HAc and 0.2 M NaAc·3H2O, pH=3.6) buffer solution, and incubate at room temperature for 20 min. Measure the absorbance at 652 nm using a microplate reader (ThermoScientific Multiskan Go) or a UV-Vis spectrophotometer. Each sample was tested in triplicate. Results are expressed as mean ± standard deviation.

[0051] 2.2. Comparative Experiment Procedure Single HRPs are linked to the Track chain via glutaraldehyde, and the results are compared with the Track-HRP@LP signal. The Track-HRP synthesis method is as follows: Step 1: Track@GA@HRP Reaction: Take the Track chain (final concentration 100 nM) and 60 μL of HRP solution (0.2 mg / mL) into a centrifuge tube. Add 60 μL of 0.5% glutaraldehyde solution (dissolved in 0.1 M, pH 6.8 PBS) to the tube, and gently mix with a pipette. Incubate the mixture at room temperature (approximately 25°C) in the dark for 1.5 h, then place the mixture in a 4°C refrigerator and react overnight (approximately 12-16 h) with gentle shaking. Low temperature and slow reaction promote the formation of the correct heterocrosslinks and reduce protein polymerization. This step allows one aldehyde group of glutaraldehyde to bind to the amino group on the HRP surface, and the other aldehyde group to bind to the amino group on the Track chain.

[0052] Step 2: Removal of free glutaraldehyde: Unreacted glutaraldehyde was removed using a dialysis bag. The final HRP-Track chain conjugate solution was collected. The purified conjugate was aliquoted and stored at 4°C in the dark.

[0053] 2.3. Optimization of Reaction Conditions F-chain structure and length optimization: Hairpin-shaped F-chains (6, 7, and 8 bases in the stem) were designed respectively, and the chain substitution efficiency was verified by 15% non-denaturing polyacrylamide gel electrophoresis: W / L1 / D1 complex, F-chain, and Track chain were mixed and incubated at 37℃ for 30 min. The product bands were observed after electrophoresis. At the same time, the signal-to-noise ratio (F / F0, with target signal / without target signal) was measured by UV-vis to determine the optimal F-chain structure and length.

[0054] Key concentration optimization: A track chain concentration gradient was set (25, 50, 100, 200, 400 nM). By adding surfactant, the results of incubation with different track chain concentrations and magnetic beads were compared to obtain the optimal track chain concentration; W / L n D n Concentration gradients (5, 10, 15, 20, 25 nM), Mg 2+ Concentration gradients (5, 10, 15, 20, 25 mM) were used to measure the absorbance and signal-to-noise ratio at different concentrations. The concentration with the highest signal-to-noise ratio was selected as the optimal concentration.

[0055] Reaction time optimization: Set the reaction time gradient for the first module (5-150 min), the first + second module (5-70 min), and the first + second + third module (1-40 min). Measure the absorbance values ​​at different time points and select the time when the absorbance reaches a stable plateau and the background signal is at its lowest as the optimal reaction time.

[0056] 3. Evaluation of Detection Performance 3.1. Sensitivity and Linear Range Under optimized conditions, WSSV-miR-n24 standards at concentrations of 1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 1 nM, and 10 nM were added, and the corresponding absorbance values ​​were measured. A standard curve was plotted with the logarithm of the target analyte concentration on the x-axis and the absorbance values ​​on the y-axis. Calculate the detection limit, where σ is the standard deviation of the blank signal; determine the linear range of detection based on the linear fitting equation of the standard curve.

[0057] 3.2. Anti-interference capability Simulated real shrimp tissue nucleic acid background: Nucleic acid extraction method: Using a nucleic acid extraction kit, (1) Take 20 mg shrimp meat + 180 μL water and grind into a cell suspension. Centrifuge at 10,000 rpm for 1 min, remove the supernatant, add 250 μL lysis buffer A and 40 μL proteinase K to a centrifuge tube, vortex to mix, centrifuge briefly, and incubate in a metal bath at 55℃ for 30 min. (2) Take 300 μL binding buffer B to a centrifuge tube, let stand for 5 min, centrifuge to collect the supernatant, add 30 μL of fully resuspended magnetic beads, vortex to mix, and bind at room temperature for 10 min, mixing 3-5 times during this period. Then place the centrifuge tube on a magnetic rack and let stand until the solution is completely clear, and remove the supernatant. (3) Add 600 μL washing buffer C, vortex to mix, let stand for 4 min, place the centrifuge tube on a magnetic rack and let stand until the solution is completely clear, and remove the supernatant. (4) Add 600 μL of washing buffer D, vortex to mix, let stand for 4 min, place the centrifuge tube on a magnetic rack and let stand until the solution is completely clear, then remove the supernatant. (5) Add 600 μL of washing buffer E, vortex to mix, let stand for 4 min, place the centrifuge tube on a magnetic rack and let stand until the solution is completely clear, then remove the supernatant. Repeat the above operation once. (6) Briefly centrifuge to place the magnetic beads at the bottom of the tube, place it on a magnetic rack and discard the residual ethanol, then dry at 56°C with the cap open for 3 min. (7) Add 30 μL of elution buffer to resuspend, vortex for 1 min, then incubate the centrifuge tube in a 56°C metal bath for 10 min. (8) Place the centrifuge tube on a magnetic rack and let stand until the magnetic beads are completely adsorbed, carefully transfer the nucleic acid solution to a new centrifuge tube and store it under appropriate conditions. Dilute to 100 μL with Tris-HCl buffer to prepare the interference matrix. Ensure all solution components that could damage liposomes are thoroughly washed. This is the amount of nucleic acid extracted for one set of experiments. Add the target analyte WSSV-miR-n24 (1 aM, 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM) to the interference matrix, then add it to the detection system. Measure the absorbance values, plot a standard curve, and calculate the detection limit. Compare the detection limit with the standard curve of the buffer system.

[0058] (II) Results and Discussion 1. Principle of Cascaded Positive Feedback Ultrasensitive Rapid Detection Biosensors The principle of the biosensor constructed in this embodiment is as follows: Figure 1 As shown, the biosensor consists of three functionally defined and interconnected modules. The overall design concept draws inspiration from the cascaded signal transduction in living organisms, achieving effective detection of weak input signals through multi-stage amplification. It comprises a nucleic acid amplification reaction network containing target recognition and a signal conversion section. In the first module's cyclic amplification reaction, target T first hybridizes with the exposed anchorage of the L1 strand in W / L1 / D1 on FB and attaches to the end of L1. Compared to the L1 / D1 double strand, T has a stronger binding energy to L1, leading to a strand displacement reaction. D1 is gradually replaced by T to form W / L1 / T, while simultaneously exposing an anchorage region in the middle of L1 for the adhesion of the fuel chain F1. Subsequently, F1 undergoes a strand displacement reaction with W / L1 / T, releasing T and W. The release of W activates its DNAzyme function; an 8-17 DNAzyme with RNA cleavage activity was selected, with a recognition sequence of 5'-rAG-3'. 2+Under the influence of the magnetic bead, a tethered walking device is formed, acting on the cleavage recognition site on the Track chain. The Track chain is linked to the horseradish peroxidase (HRP)-encapsulated liposome (HRP@LP) via amide bonds to form HRP@LP-Track, cleaving the Track chain and continuing to act on other uncleaved Track chains on the magnetic bead, continuously detaching HRP@LP from the magnetic bead and into a free state. The target substance T, released along with W, will again undergo a chain displacement reaction with the unreacted W / L1 / D1, forming the inner loop in the first module's cyclic amplification reaction. The D1 chain displaced by T in the first module's cyclic amplification reaction can then act as a trigger chain, initiating the second module's cyclic amplification reaction in the same way; the D2 chain displaced by D1 in the second module's cyclic amplification reaction can then trigger the third module's cyclic amplification reaction... In this way, the system constructs a cascaded positive feedback network triggered by a single target substance, amplified step by step, and self-reinforcing, transforming weak input signals into powerful internal driving forces. Each module employs a similar but different sequence design, ensuring the specificity and orthogonality of the reaction and avoiding non-specific cross-reactions. After a certain reaction time, the sheared HRP@LP is transferred along with the supernatant to a new centrifuge tube via magnetic separation. 3,3',5,5'-Tetramethylbenzidine (TMB) and H2O2 are added. H2O2 penetrates the phospholipid bilayer membrane and reacts with HRP to generate ·OH, causing membrane rupture and releasing HRP. The free HRP catalyzes the colorimetric reaction between H2O2 and TMB, producing a quantitatively readable absorbance signal, thus converting the invisible nucleic acid reaction into a visible analytical signal. This signal transduction method has the advantages of strong amplification and low background interference, thereby achieving ultrasensitive detection of the target analyte T.

[0059] 2. Characterization and feasibility verification of biosensors First, the correctness and efficiency of each chain substitution reaction pathway were verified by non-denaturing polyacrylamide gel electrophoresis (PAGE). Experimental results showed ( Figure 2 In lane A), the reaction between W and the Track strand in lane 6 resulted in two bands, Tra1 and Tra2, migrating faster than the original Track strand. This is because W's DNAzyme cleavage function recognized the cleavage site on the Track strand and cleaved it, thus verifying the feasibility of W cleaving the Track strand in the system. Lane 16 represents the W / L ratio of all triplet strands. n D n The Track chain did not generate Tra1 and Tra2 chain bands, verifying the W / L. n D nThe stability of the entire detection complex FB@(LP@HRP-Track+WL) n D n The substance itself does not exhibit non-specific reactions; lanes 14 and 15 show the results of the entire detection system with and without the target analyte T, revealing that only in the presence of T is there a significant L reaction. n F n The appearance of Tra1 and Tra2 chain belts confirms the design's W / L. n D n L n F n The successful execution of the complex and the various displacement reactions, as well as the specificity of the entire system, were noteworthy. Particularly noteworthy was the observation of a distinct T band in lane 14, indicating the presence of the target compound T and its W / L ratio during cycling. n D n The disappearance of [something] is sufficient to prove the success of the reaction, thus preliminarily verifying its feasibility.

[0060] Further confirmation through UV-Vis spectroscopy revealed that HRP@LP can be penetrated by H2O2 and react with HRP within it to generate free radicals. This leads to the cleavage of the phospholipid bilayer and the release of HRP, which then catalyzes the colorimetric reaction of H2O2 and TMB to produce a signal. Furthermore, in HRP solutions of the same concentration, LP exhibits a certain degree of HRP encapsulation, and the signal generated by free HRP catalysis is 1.2 times stronger than that generated by HRP@LP catalysis. Figure 2 The reason for the signal difference (B in the original text) is that some HRP that was not coated in the membrane was dialyzed out during the synthesis of HRP@LP; in the first cycle, the separation effect of FB resulted in the HRP@LP catalytic colorimetric signal being 1.4 times that of HRP alone via the binding of glutaric dialdehyde (GA) to the Track chain. Figure 2 The C in the sample, along with the synthesized blank liposome LP, showed no signal generation when LP coexisted with TMB and H2O2, proving that the signal was not caused by the components in the liposome. Furthermore, TMB and H2O2 alone also produced no signal, comprehensively verifying that the colorimetric signal was generated only when HRP, TMB, and H2O2 coexisted and interacted. Finally, the signal difference between different cycles with and without the target analyte was verified. Figure 2 The feasibility of the entire strategy was verified for the first time using UV-vis (D). The signal output of the biosensor is strictly dependent on the presence of the target, and the HRP encapsulated in the liposomes retains high catalytic activity after release, which establishes the functional integrity of the entire signal transduction pathway.

[0061] To ensure the reliability and repeatability of the system, the morphology and size of key components were characterized. Dynamic light scattering (DLS) was used to analyze the particle size distribution of LP (D > 220 nm), HRP@LP (D > LP), HRP@LP-Track (HRP@LP + 27.2 nm ≥ D > HRP@LP), and the final composite (2LP + 254.4 nm ≥ D > 200 + LP nm). The results show that these nanoparticles have correspondingly reasonable size distributions. Figure 3 (A in the middle).

[0062] Zeta potential analysis revealed the variation pattern of surface charge on nanoparticles ( Figure 3 (B) The original liposomes are negatively charged, and HRP is also negatively charged. After loading HRP, the Zeta potential of HRP@LP shifts negatively by 1.98 mV and 3.69 mV compared to LP and HRP, respectively. Furthermore, because the DNA strand is negatively charged, the Zeta potential of HRP@LP-Track further shifts negatively by 3.09 mV compared to HRP@LP after linking to the Track strand. FB is negatively charged and ultimately assembles with magnetic beads and triplet to form a stable complex FB@(LP@HRP-Track+WL). n D n The potential shifted negatively by 4.6 mV compared to HRP@LP-Track. This charge change is consistent with the expectations of each assembly step, confirming the success of the assembly process. As for the electrostatic repulsion during assembly, it certainly exists; however, in the high-salt environment of the Tris-HCl buffer solution, this electrostatic repulsion was partially shielded, and compared to the forces of streptavidin-biotin and amide bonds, the electrostatic forces were relatively weak and not dominant.

[0063] 3. Condition Optimization To achieve optimal performance, the system underwent multi-parameter optimization. First, during electrophoresis feasibility verification experiments, it was found that the straight F chain consistently exhibited severe leakage, meaning that Tra1 and Tra2 chain bands existed even without T. Therefore, the F chain structure was modified to a hairpin structure with weaker binding to the L chain. The number of bases in the hairpin's stem significantly impacts the reaction rate and efficiency: an excessively short stem F chain may cause the hairpin itself to be unstable, reverting to a straight chain and resulting in non-specific binding of the LF chain, leading to low reaction efficiency; conversely, an excessively long stem F chain may cause the hairpin structure to remain stable after binding to the anchor points on the L chain, failing to form a stable LF chain to displace W and T, also resulting in low reaction efficiency. Therefore, an appropriate number of bases in the F chain stem is necessary to significantly reduce the leakage risk of the detection system while maintaining sufficient binding force with the L chain to displace W and the target chain. Finally, the signal-to-noise ratio F / F was determined by gel electrophoresis and UV-vis analysis. 0（ Target signal present / No target signal present Result ( Figure 4 A and B in the study determined that the optimal stem length of the F chain was 7 bases. At this length, the reaction maintained both high efficiency and good specificity. Secondly, the W / L ratio in the entire detection system was [not specified]. n D n Excessive W chains result in unused W chains in the detection system, leading to waste. Furthermore, a large number of W chains implies a small number of Track chains, directly impacting the amount of HRP released, resulting in a weaker signal and reduced amplification. Conversely, too few W chains mean a large number of Track chains, increasing their operating time. For dynamic chain displacement responses, a longer response time increases the probability of non-specific reactions, affecting the specificity of the detection strategy. Therefore, the W / L ratio of the system was optimized by comparing F / F0 and the final signal. n D n The concentrations and track chain concentrations were 15 nM and 100 nM, respectively. Figure 4 (C, D in the text); In addition, the synthesized complex FB@(LP@HRP-Track+WL was diluted. n D n It was found that the final F / F0 also increased with increasing complex concentration, proving that the complex synthesis method fully utilized the binding sites on FB (). Figure 3 The signal intensity increased with increasing complex concentration over the same reaction time, up to the maximum effective value of the UV-vis signal. This verified that the reaction system could fully react and shear the Track-LP prepared according to the experimental method within the experimental time, thereby generating a signal. Finally, the Mg... 2+ Concentration optimization is needed because Mg 2+ Mg is a key ion for DNA zyme cleavage. 2+ At low concentrations, the shear function is not activated, and Mg 2+ Excessive concentration can affect the structure of the DNA strand, causing unexpected changes in the entire strand displacement reaction, resulting in the formation of Mg from F / F0. 2+ The optimal concentration is 15 mM ( Figure 4 (F in the text). These optimized parameters ensure the efficient operation of the system.

[0064] As the number of cycle modules increases, the background in the signalless reaction system increases more rapidly. Since chain substitution is a dynamic and rapid process, even seemingly non-binding double strands will gradually develop gaps over time, allowing fuel chains to bind and subsequently undergo non-specific reactions to generate a signal. Therefore, optimizing the reaction time is crucial to minimizing background signal, maximizing signal, and achieving the highest sensitivity and specificity. Thus, the reaction time for different cycles was optimized in detail. Figure 5A significant reduction in the time required to reach the optimal signal was observed (60 min for the first module, 30 min for the first + second modules, and only 15 min for the first + second + third modules). This phenomenon initially reveals the powerful accelerating effect of the cascade structure on reaction kinetics, suggesting an efficient kinetic mechanism within it. This time reduction not only improves detection efficiency but also reduces the accumulation of non-specific background signals, contributing to an improved signal-to-noise ratio.

[0065] (III) Demonstration of the dynamic amplification capability of the detection strategy 1. Detection performance of the ultra-sensitive detection strategy Under optimized conditions, the system demonstrated superior detection performance. The signal response at different target analyte concentrations was compared. Figure 6 (A, B, and C in the diagram) It was found that the entire system, from the first module to the first + second + third modules, exhibited a good linear relationship within the concentration range of 1 aM to 1 nM, demonstrating its wide dynamic range detection capability. Furthermore, the standard curves all showed a linear relationship between signal and the logarithm of concentration. This is because in the entire reaction system, a single target analyte can always correspond to the release of multiple liposomes, and each liposome corresponds to a signal; therefore, the relationship between the target analyte and the signal is exponential. With the introduction of cascaded cycling, the detection sensitivity increased by orders of magnitude: the detection limits of the first module, the second module, and the first + second + third modules reached 501 fM, 8.9 fM, and 7.9 aM, respectively. This increase in sensitivity directly demonstrates the powerful effectiveness of the cascaded amplification strategy. Particularly noteworthy is that the first + second + third modules achieved a detection capability at the aM level, which is of great significance in the field of early diagnosis of shrimp viruses, as the viral load is usually extremely low in the early stages of viral infection.

[0066] The linear range of detection also expands significantly with the increase of the cascade stage. The linear range of the first module is 1 pM - 1 nM, the first + second module expands to 100 fM - 100 pM, and the first + second + third module further expands to 1 aM - 1 pM, spanning nearly 10 orders of magnitude. This wide dynamic range enables the system to adapt to the detection needs of different infection stages.

[0067] In practical applications, biosensors need to operate within complex biological matrices. To evaluate the practical application potential of biosensors, tests were conducted against a complex background simulating shrimp tissue nucleic acids. Figure 6 (D) The first + second + third module system maintained a detection capability of 39.8 aM in complex matrices, which is on the same order of magnitude as the performance in the buffer system (7.9 aM), with only a slight decrease. This demonstrates the good robustness and anti-interference ability of the biosensor, laying the foundation for its application in practical sample detection.

[0068] 2. Comparison with other detection methods To objectively evaluate the performance advantages of this system, it was systematically compared with several commonly used nucleic acid detection methods, including quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and enzyme-linked immunosorbent assay (ELISA).

[0069] Regarding sensitivity, recent studies have shown that nested PCR has a detection limit of approximately 700 copies / mL for WSSV, and requires two rounds of amplification and two pairs of primers, making the procedure cumbersome. LAMP technology, due to primer interactions during isothermal amplification, even with primer modification (such as locked nucleic acid modification), has a detection limit of approximately 2 × 10⁻⁶. 4 The combination of RPA and LFA bands was detected at 37 °C for 30 min. Here, the 5' end of the FITC-labeled probe binds to the WSSV RPA product, and the biotinylated 5' end of the reverse primer binds to streptavidin on the band. With appropriate optimization, this technique can achieve 2 × 10⁻⁶ copies / mL; 7 The detection limit is 1 copy / mL; based on Cas13, the diagnosis is called specific high-sensitivity enzyme reporter gene unlocking (SHERLOCK), which initially detects white spot syndrome virus (WSSV) with a sensitivity of up to 1×10⁻⁶. 4 copy / mL; the detection limit of ELSA for WSSV is 10. 4 While simple to operate and design, the copy / microgram protein assay is suitable for point-of-care testing (POCT), but its detection capability is significantly weaker than PCR and LAMP. The biosensor in this embodiment uses a three-module cascaded positive feedback amplification to reduce the detection limit to 7.9 aM, which translates to approximately 4800 copies / mL based on the extracted nucleic acid volume and the final reaction system volume. Its sensitivity is comparable to, or even better than, nested PCR, LAMP, and SHERLOCK, while its ease of operation is comparable to ELISA, making it suitable for POCT and meeting the detection needs in the early stages of WSSV infection.

[0070] In summary, the biosensor in this embodiment does not require stringent reaction conditions or large instruments. It has significant advantages in terms of sensitivity, speed, simplicity, and cost in resource-constrained aquaculture sites, providing a high-performance solution for shrimp virus detection and also contributing to the popularization of advanced detection technologies.

[0071] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A cascaded positive feedback ultrasensitive rapid detection biosensor, characterized in that, Includes nucleic acid amplification reaction network units and signal conversion units; The signal conversion unit includes 3,3',5,5'-tetramethylbenzidine and H2O2; The nucleic acid amplification reaction network unit includes at least three cyclic amplification reaction modules, each cyclic amplification reaction module including a detection complex and F. n The detection complex comprises magnetic beads with both Track and W chains simultaneously attached to their surface. Liposomes containing horseradish peroxidase are attached to the Track chains. n Chains hybridized with D via complementary hybridization. n Chain and W chain are connected to form W / L n / D n Three-chain system: Track chain, W chain, L chain n Chain, D n Chain and F n All strands are single-stranded DNA, F n The strand has a hairpin structure, n is 1 or m, where m is any integer from 2 to the number of cyclic amplification reaction modules. The W strand contains 8-17 DNAzymes, the Track strand contains sites that can be recognized and cleaved by the DNAzymes, and the target sequence can replace the D1 strand in the W / L1 / D1 triplet, and D... m-1 Capable of replacing W / L m / D m D in the triple chain m Chain, and F n The chain can identify the replaced W / L n / D n Trichain and L n Chain complementarity allows the W chain to change from the replaced W / L chain. n / D n The DNAzyme in the W strand is then freed in the triple strand, and then recognizes and cleaves the corresponding site of the Track strand, thereby releasing the liposome containing horseradish peroxidase from the surface of the magnetic bead.

2. The biosensor as described in claim 1, characterized in that, The number of the cyclic amplification reaction modules is 3 to 5.

3. The biosensor as described in claim 1, characterized in that, F n The hairpin structure of the chain has 7 or 8 bases in the stem.

4. The biosensor as described in claim 1, characterized in that, The target sequence is shown in SEQ ID NO:3, which is the nucleic acid sequence of WSSV-miR-n24.

5. The biosensor as described in claim 4, characterized in that, n is 3, and the nucleic acid sequence of the W chain is shown in SEQ ID NO:1; Alternatively, n is 3, and the nucleic acid sequence of the Track chain is shown in SEQ ID NO:2; Or, n is 3, L n The nucleic acid sequences of the strands are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. n The nucleic acid sequences of the strands are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively. n The nucleic acid sequences of the strands are shown in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively.

6. The biosensor as described in claim 1, characterized in that, The liposomes are composed of DPPC, cholesterol, and DSPE-PEG-NHS in a molar ratio of 4.5~5.5:2.7~3.3:1.8~2.

2.

7. The biosensor as described in claim 1, characterized in that, The surface of the magnetic beads is connected to the Track and W chains via biotin-streptavidin binding.

8. The application of the biosensor according to any one of claims 1 to 7 in detecting biomarkers for white spot syndrome virus in shrimp.

9. The application as described in claim 8, characterized in that, Using the sequence of the marker of white spot syndrome virus in shrimp as the target sequence of the biosensor, the marker of white spot syndrome virus in shrimp and the nucleic acid amplification reaction network unit are incubated for a set time, and then magnetic separation is performed to obtain the supernatant. Then, a signal conversion unit is added to the supernatant and the optical signal is detected.

10. A detection kit for white spot syndrome virus in shrimp, characterized in that, The kit includes the biosensor and buffer solution shown in any of claims 1 to 7, wherein the biomarker detected by the kit is WSSV-miR-n24.

Images