Nanopore sensor based on crisper-cas12a and its application in igf-1 protein detection

Abstract

This invention provides a CRISPR-Cas12a-based nanopore sensor and its application in IGF-1 protein detection, belonging to the fields of biosensing and molecular diagnostics. The nanopore sensor includes: a solid-state nanopore unit, which is a glass capillary nanoparticle with an opening at the tip and a conductive layer covering the inner wall of the nanoparticle; a reporter molecule chemically bonded to the conductive layer; a protein recognition and signal conversion unit, including a DNA aptamer capable of specifically binding to Atlantic salmon IGF-1 protein and a single-stranded DNA complementary strand partially complementary to the DNA aptamer; and a signal amplification unit containing the Cas12a protein and crRNA complementary to the target DNA strand. This sensor achieves efficient, sensitive, and specific conversion from protein to electrical signal through a cleverly designed molecular competition system.

Description

Technical Field

[0001] This invention relates to the fields of biosensing and molecular diagnostics, and in particular to a CRISPR-Cas12a-based nanopore sensor and its application in the detection of IGF-1 protein. Background Technology

[0002] In the intensive salmon farming industry, real-time and precise monitoring of the concentration of insulin-like growth factor-1 (IGF-1) protein, a core regulator of fish growth, is crucial for achieving scientific feeding, health early warning, and improved economic benefits. Currently, direct detection of IGF-1 in fish mainly relies on enzyme-linked immunosorbent assay (ELISA). Although this method has acceptable specificity, it has inherent drawbacks that cannot be overcome: (1) the preparation of high-quality, highly specific fish IGF-1 monoclonal antibodies is time-consuming and extremely expensive; (2) the detection process is cumbersome, involving multiple incubation and washing steps, which is time-consuming; and (3) it relies on large-scale ELISA readers and other optical equipment, making it difficult to deploy and achieve automated continuous monitoring at the farming site. These shortcomings severely restrict the application of ELISA in precision aquaculture scenarios.

[0003] Nucleic acid aptamers are a class of single-stranded DNA or RNA molecules that can bind to target molecules (such as proteins and small molecules) with high specificity and high affinity; they are known as "chemical antibodies." Compared with protein antibodies, aptamers have many advantages, including simple synthesis, low cost, small batch-to-batch variability, good stability, and ease of chemical modification. However, traditional detection methods based on aptamers (such as colorimetric and fluorescence methods) generally suffer from insufficient sensitivity, especially when detecting low-abundance protein targets in complex biological matrices, often failing to reach the detection limits required for practical applications.

[0004] Clustered regularly spaced short palindromic repeats and their associated protein Cas12a (CRISPR-Cas12a) system have become a powerful tool for nucleic acid detection and signal amplification due to their "trans-cleavage" activity. When the Cas12a / crRNA complex recognizes and binds to its target double-stranded DNA, Cas12a is activated and can non-specifically cleave single-stranded DNA in the surrounding environment. This property has been used to construct highly sensitive nucleic acid detection platforms. However, the CRISPR-Cas12a system itself cannot directly recognize protein targets. Solid-state nanopores, especially nanopipettes drawn from glass capillaries, have become ideal substrates for constructing next-generation biosensors due to their high mechanical strength, controllable size, and ease of surface chemical modification. Their label-free, real-time monitoring of single-molecule events via electrical readout offers unique advantages for miniaturizing, integrating, and deploying detection devices in real-time. In the current technology, there is no attempt to combine aptamer recognition, CRISPR signal amplification, and nanopore sensing. Traditional detection strategies are mostly step-by-step and offline, which suffer from drawbacks such as low signal conversion efficiency, complex operation steps, and difficulty in integration. Therefore, there is an urgent need in this field for an innovative integrated solution that can cleverly transform protein detection into nucleic acid activation events and couple the powerful signal amplification capability of CRISPR with the convenient electrical readout of nanopores, thereby achieving ultrasensitive, rapid, and field-friendly detection of key protein biomarkers such as IGF-1.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a CRISPR-Cas12a-based nanopore sensor and its application in IGF-1 protein detection. It offers a novel biosensor based on a three-tiered signal transduction mechanism of "competitive binding-CRISPR activation-nanopore readout." This sensor, through a cleverly designed molecular competition system, transforms the IGF-1 protein binding event into a specific nucleic acid signal that activates the CRISPR-Cas12a system. Ultimately, it generates an easily detectable electrical signal through changes in the nanopore interface properties, thus achieving efficient, sensitive, and specific conversion from protein to electrical signal.

[0007] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides a CRISPR-Cas12a-based nanopore sensor, comprising: The solid-state nanopore unit is a nanostraw made of glass capillary, the tip of which has an opening and the inner wall of which is covered with a conductive layer. A reporter molecule, wherein the reporter molecule is disposed on the conductive layer by chemical bonds; The protein recognition and signal conversion unit includes a DNA aptamer capable of specifically binding to Atlantic salmon IGF-1 protein, and a single-stranded DNA complementary strand that is partially complementary to the DNA aptamer sequence. The signal amplification unit contains the Cas12a protein and crRNA complementary to the target DNA strand.

[0008] Furthermore, the tip of the nanostraw has an opening diameter of 70nm-90nm.

[0009] Furthermore, the nucleotide sequence of the reporter molecule is shown in SEQ ID NO.1, and the concentration is 50 nM-100 nM.

[0010] Furthermore, the 5' end of the reporter molecule is modified with a thiol group.

[0011] SEQ ID NO.1 5'-HS-(CH2)6-CATATTAATTTGATGGATATTTT-3'.

[0012] Furthermore, the nucleotide sequence of the DNA aptamer is shown in SEQ ID NO.2; the nucleotide sequence of the complementary strand of the single-stranded DNA is shown in SEQ ID NO.3; and the nucleotide sequence of the crRNA is shown in SEQ ID NO.4.

[0013] SEQ ID NO.2 5'-ATTGGCACTCCACGCATAGGGGGAGGCCCCCCTCGAAAATGTTACTTTGCCGACTGCCTATGCGTGCTACCGTGAA-3'; SEQ ID NO.3 5'-CAGTCGGCAAAGTAACATTT-3'; SEQ ID NO.4 5'-UAAUUUCUACUAAGUGUAGAUAAAUGUUACUUUGCCGACUG-3'.

[0014] Furthermore, the conductive layer is a gold film with a thickness of 10 nm.

[0015] Furthermore, the molar ratio of Cas12a protein to crRNA is 1:1.

[0016] This invention also provides a method for fabricating a CRISPR-Cas12a-based nanopore sensor, comprising the following steps: S1. Drawing glass capillaries into nano-straws with open tips; S2. A conductive layer is deposited on the inner wall of the nanotube; S3. The reporter molecule is introduced into the nanoparticle straw and fixed to the surface of the conductive layer by chemical bonds; S4. Treat the fixed interface with a blocking agent to block non-specific binding sites.

[0017] Furthermore, the blocking agent is a bovine serum albumin solution with a concentration of 150 μg / mL to 250 μg / mL.

[0018] Furthermore, the concentration of the sealing agent is 200 μg / mL.

[0019] Furthermore, the concentration of the DNA aptamer was 1 μM.

[0020] The present invention also provides the application of the above-mentioned CRISPR-Cas12a-based nanopore sensor in the detection of insulin-like growth factor-1 in fish.

[0021] Furthermore, the fish in question is Atlantic salmon.

[0022] Furthermore, the aforementioned CRISPR-Cas12a nanopore sensor is applied to the detection of insulin-like growth factor-1 in fish, and the specific steps include: Sa, mix the DNA aptamer with a single-stranded DNA complementary strand whose partial sequence is complementary to the DNA aptamer at a certain molar ratio, and anneal in a buffer to form an aptamer-complementary strand complex. Sb, Cas12a protein and crRNA were mixed and incubated to synthesize the Cas12a / crRNA ribonucleoprotein complex; Sc. Apply voltage to the ligand-complementary strand complex prepared by Sa to enrich the target DNA on the conductive layer; Sd, the sample to be tested is mixed with the aptamer-complementary chain complex and incubated; Se, Inject the Cas12a / crRNA ribonucleoprotein complex into a solid nanopore unit; Sf: Apply a negative voltage to the tip of the nanoparticle to incubate the reaction; After the reaction is complete, the solution is replaced with electrode buffer solution for signal detection, and the concentration of insulin-like growth factor-1 is calculated.

[0023] Furthermore, in the Sa, the molar ratio of the DNA aptamer to the complementary single-stranded DNA strand whose partial sequence is complementary to the DNA aptamer is 1:1.

[0024] Furthermore, in the Sa, the mixed incubation conditions are 35℃-38℃ for 25min-35min.

[0025] Furthermore, in the Sc, the specific conditions for the negative voltage are: -1V, with a duration of 30s; the incubation conditions in the Sc are 35℃-38℃, 5min-15min.

[0026] Furthermore, the Sd specifically includes: calculating the insulin-like growth factor-1 concentration based on a pre-established standard curve by measuring the current-voltage curve within a scan range of -1.0V to +1.0V as a response signal.

[0027] Furthermore, the electrode buffer contains 30 mM KCl and 20 mM Tris-HCl.

[0028] The present invention has the following technical effects: (1) By designing an aptamer-complementary strand competitive binding system, the recognition event of the protein target (IGF-1) was cleverly converted into a specific nucleic acid signal (free complementary strand) that can activate the CRISPR-Cas12a system. This conversion mechanism is the core innovation of this invention, enabling the powerful signal amplification capability of the CRISPR system to be applied to the field of protein detection.

[0029] (2) It achieves efficient in-situ integration of CRISPR signal amplification and nanopore readout. The activated Cas12a directly cuts the fixed reporter molecule at the nanopore interface, causing changes in the interface properties, and is sensitively detected by macroscopic electrical signals such as the ion current rectification effect. This in-situ serial mode of "recognition-activation-cutting-detection" avoids the signal loss and operational complexity of traditional stepwise detection.

[0030] (3) High detection sensitivity and strong specificity. On the one hand, the trans-cleavage activity of CRISPR-Cas12a is used to amplify the enzymatic signal, which significantly improves the detection sensitivity to the fM level. On the other hand, the detection specificity is guaranteed by two factors: the primary specific recognition of the protein by the aptamer and the sequence specific recognition of the complementary strand by the crRNA, which together ensure an extremely low false positive rate.

[0031] (4) Simple sample processing and fast detection process. This method directly targets proteins, completely bypassing the cumbersome and unstable RNA extraction, reverse transcription and nucleic acid amplification steps. Samples only need simple pretreatment before loading, and combined with the rapid CRISPR reaction (usually 10-30 minutes), it is expected to complete the entire detection process from sample to result within 1 hour, which is much faster than traditional ELISA. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This is a schematic diagram illustrating the detection principle of the sensor of the present invention; Figure 2 The stepwise current-voltage response diagram for the functionalization process of nanostraws; Figure 3This diagram illustrates the optimization of reaction conditions. A represents the results of surface modification of the sensor with different concentrations of reDNA; B represents the optimal reaction temperature of the CRISPR system; C represents the optimized crRNA / Cas12a ratio in the CRISPR cleavage reaction; and D represents the optimized Cas12a concentration. Figure 4 For the standard curve established in the examples and for verifying the analytical performance of the sensor, A is a linear relationship between the sensor's response signal and the logarithm of the IGF-1 protein concentration; B is a graph showing the linear relationship between the sensor's response signal and the logarithm of the IGF-1 protein concentration. -1V Plot a standard curve with lgC (the logarithm of IGF-1 concentration) on the ordinate and lgC (the logarithm of IGF-1 concentration) on the abscissa. C represents the effect of the protein-free negative control on the sensor; D represents the repeatability test results. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] In a first aspect, the present invention provides a CRISPR-Cas12a-based nanopore sensor, comprising: The solid-state nanopore unit is a nanostraw made of glass capillary, the tip of which has an opening and the inner wall of which is covered with a conductive layer. A reporter molecule, wherein the reporter molecule is disposed on the conductive layer by chemical bonds; The protein recognition and signal conversion unit includes a DNA aptamer capable of specifically binding to Atlantic salmon IGF-1 protein, and a single-stranded DNA complementary strand that is partially complementary to the DNA aptamer sequence. The signal amplification unit contains the Cas12a protein and crRNA complementary to the target DNA strand.

[0036] Glass surfaces possess a certain degree of chemical activity, facilitating the deposition of a uniform and well-adhered conductive metallic layer on their inner walls (especially the inner walls of tips) through methods such as thermal evaporation, sputtering, or chemical plating. Even without relying on gold-sulfur bonds, the abundant silanol groups (Si-OH) on the glass surface provide a foundation for subsequent silanization chemical modifications. Various functional groups (such as amino, carboxyl, and biotin) can be introduced into the glass surface using silane coupling agents to immobilize reporter molecules or other biometric elements. This allows for greater flexibility in sensor construction.

[0037] Because the glass itself is transparent, researchers can directly observe the position and morphology of the nanoparticle tip under a microscope during experiments. This is extremely convenient for precisely manipulating the nanoparticle to the detection area (e.g., a specific location near a cell or microfluidic channel).

[0038] Meanwhile, the glass capillary has good mechanical strength and is not easily deformed. In complex detection environments (such as solution flow, multiple rinsing, and long-term measurement), the nanopore structure at its tip can remain stable, ensuring the reliability and lifespan of the sensor readout.

[0039] In some embodiments, the tip of the nanostraw has an opening diameter of 70nm-90nm.

[0040] This size range ensures that the nanopores have a suitable ion current rectification effect, allowing macromolecules (such as the Cas12a complex) to pass through or approach while maintaining sensitivity to changes in surface charge, which is the structural basis for subsequent high-sensitivity electrical detection.

[0041] In some embodiments, the nucleotide sequence of the reporter molecule is shown in SEQ ID NO.1, and the concentration is 50 nM-100 nM.

[0042] This sequence is a random ssDNA sequence without secondary structure, ensuring that it is easily and efficiently trans-cleaved by Cas12a. At this concentration, it ensures that there are enough reporter molecules at the interface to generate a detectable initial signal, but does not cause steric hindrance due to excessive density, which would affect the cleavage efficiency of Cas12a. It is a balance point between signal strength and cleavage efficiency.

[0043] In some embodiments, the 5' end of the reporter molecule is modified with a thiol group.

[0044] In some embodiments, the nucleotide sequence of the DNA aptamer is shown in SEQ ID NO.2; the nucleotide sequence of the complementary strand of the single-stranded DNA is shown in SEQ ID NO.3; and the nucleotide sequence of the crRNA is shown in SEQ ID NO.4.

[0045] The aptamer provides primary specificity for recognizing the IGF-1 protein. The complementary strand is both a competitive binding target of the aptamer and a key to activating the CRISPR system. Its sequence is designed to be part of the aptamer and a target of crRNA, achieving a molecular link between recognition and activation. The crRNA provides secondary specificity for recognizing the complementary strand, which is a second guarantee of detection specificity, ensuring that Cas12a is activated only when the target complementary strand is present, greatly reducing false positives.

[0046] In some embodiments, the conductive layer is a gold film with a thickness of 10 nm.

[0047] The gold film not only provides good conductivity for electrical signal measurement, but more importantly, its surface is easily modified with thiolized reporter molecules through gold-sulfur bonds (Au-S), which achieves stable and directional fixation of reporter molecules.

[0048] In some embodiments, the molar ratio of the Cas12a protein to crRNA is 1:1.

[0049] The ratio of Cas12a protein to crRNA was optimized to ensure that the two could fully form a ribonucleoprotein complex with target recognition and cleavage activity, avoiding waste or non-specific binding due to excess of one component, thus ensuring the efficiency and stability of signal amplification.

[0050] Secondly, the present invention also provides a method for fabricating a CRISPR-Cas12a-based nanopore sensor, comprising the following steps: S1. Drawing glass capillaries into nano-straws with open tips; S2. A conductive layer is deposited on the inner wall of the nanotube; S3. The reporter molecule is introduced into the nanoparticle straw and fixed to the surface of the conductive layer by chemical bonds; S4. Treat the fixed interface with a blocking agent to block non-specific binding sites.

[0051] The standardized pulling and deposition process of this preparation method ensures the consistency of the sensor substrate and successfully constructs a sensing interface to be cut. The reporter molecule is stably fixed in the signal readout region, and the sealing step effectively prevents the non-specific adsorption of other proteins or nucleic acids in subsequent reactions, thereby reducing the background signal of detection and improving the signal-to-noise ratio.

[0052] In some embodiments, the blocking agent is a bovine serum albumin solution with a concentration of 150 μg / mL to 250 μg / mL.

[0053] Bovine serum albumin (BSA) is a commonly used inert protein blocking agent. BSA at this concentration range is sufficient to form a dense monolayer on the gold membrane surface, effectively blocking non-specific binding sites, while not being too thick to hinder ion transport or affect Cas12a activity. This is a crucial auxiliary step in ensuring detection specificity and sensitivity.

[0054] In some embodiments, the concentration of the sealing agent is 200 μg / mL.

[0055] Thirdly, the present invention also provides the application of the above-mentioned CRISPR-Cas12a-based nanopore sensor in the detection of insulin-like growth factor-1 in fish.

[0056] In some embodiments, the fish is Atlantic salmon.

[0057] In some embodiments, the steps of detecting insulin-like growth factor-1 in fish using the CRISPR-Cas12a nanopore sensor described above include: Sa, mix the DNA aptamer with a single-stranded DNA complementary strand whose partial sequence is complementary to the DNA aptamer at a certain molar ratio, and anneal in a buffer to form an aptamer-complementary strand complex. Sb, Cas12a protein and crRNA were mixed and incubated to synthesize the Cas12a / crRNA ribonucleoprotein complex; Sc. Apply voltage to the ligand-complementary strand complex prepared by Sa to enrich the target DNA on the conductive layer; Sd, the sample to be tested is mixed with the aptamer-complementary chain complex and incubated; Se, Inject the Cas12a / crRNA ribonucleoprotein complex into a solid nanopore unit; Sf: Apply a negative voltage to the tip of the nanoparticle to incubate the reaction; After the reaction is complete, the solution is replaced with electrode buffer solution for signal detection, and the concentration of insulin-like growth factor-1 is calculated.

[0058] In some embodiments, the concentration of the DNA aptamer is 1 μM.

[0059] In some embodiments, in Sa, the molar ratio of the DNA aptamer to the complementary single-stranded DNA strand whose partial sequence is complementary to the DNA aptamer is 1:1.

[0060] In some embodiments, the conditions for mixed incubation in Sa are 35°C-38°C for 25-35 minutes.

[0061] In some embodiments, the specific conditions for the negative voltage in the Sc are: -1V for a duration of 30s; and the incubation conditions in the Sc are 35℃-38℃ for 5min-15min.

[0062] In some embodiments, the Sd specifically includes: calculating the insulin-like growth factor-1 concentration based on a pre-established standard curve by measuring a current-voltage curve within a scan range of -1.0V to +1.0V as a response signal.

[0063] In some embodiments, the electrode buffer contains 30 mM KCl and 20 mM Tris-HCl.

[0064] The following is a detailed explanation using specific embodiments: Example 1: Design and fabrication of key nucleic acid elements All primers were synthesized and purified by Sangon Biotech (Shanghai) Co., Ltd.

[0065] (1) Reporter gene (reDNA) design The reDNA design is shown in SEQ ID NO.1: 5'-HS-(CH2)6-CATATTAATTTGATGGATATTTT-3'.

[0066] (2) Aptamer search and complementary chain design An aptamer (Apt) specifically binding to IGF-1 was designed as shown in SEQ ID NO.2: 5'-ATTGGCACTCCACGCATAGGGGGAGGCCCCCCTCGAAAATGTTACTTTGCCGACTGCCTATGCGTGCTACCGTGAA-3'. A partially complementary single-stranded cDNA was designed as shown in SEQ ID NO.3: 5'-CAGTCGGCAAAGTAACATTT-3'. The aptamer (SEQ ID NO.2) and the complementary strand (SEQ ID NO.3) were mixed in annealing buffer at a molar ratio of 1:1. The mixture was heated at 95°C for 5 minutes and then slowly cooled to room temperature to form a stable double-stranded complex.

[0067] (3) Guide RNA design Guide RNA (crRNA) was designed based on cDNA as shown in SEQ ID NO.4: 5'-UAAUUUCUACUAAGUGUAGAUAAAUGUUACUUUGCCGACUG-3'. Fabrication of the nanosensor.

[0068] Example 2: Establishment of a method for the fabrication and detection of a CRISPR-nanopore sensor (1) Fabrication of nanosensors Nanoparticles were prepared using a Sutter P-97 laser needle puller. A borosilicate glass capillary (Sutter Instrument, BF100-50-10) was used to obtain nanoparticles with a tip diameter of approximately 80±10 nm.

[0069] The nanoparticle pipette was fixed on the rotating sample stage of the coating instrument, and DC magnetron sputtering was performed in an Ar atmosphere using a high-purity gold target as the sputtering source for 120 seconds to coat its inner wall with a conductive layer.

[0070] (2) Immobilization and surface sealing of reporter molecules Prepare a 100 nM solution of thiolized DNA (reporter molecule) and inject it into the tip of a nanopipette, ensuring the gold-modified region is completely filled. Place the nanopipette in a 4°C refrigerator and allow it to react overnight (12-16 hours) in the dark. Subsequently, inject blocking buffer containing 200 μg / mL bovine serum albumin (BSA), incubate at room temperature for 1 hour, and rinse three times with pure water.

[0071] (3) Stepwise modification of nanosensors and their electrochemical characterization Two Ag / AgCl electrodes were inserted into a functionalized nanopipette and electrode buffer solution, serving as the working and reference electrodes, respectively. Cyclic voltammetry curves were recorded at a scan rate of 100 mV / s within a voltage range of -1.0 V to +1.0 V using a CHI660E electrochemical workstation (Shanghai Chenhua). During the development of the nanosensor, current was gradually recorded. Potential (I) Changes in P). For example Figure 2 As shown, due to the dissociation of surface silanol, the negatively charged bare nanoparticles exhibit negative rectification (black curve). After gold plating, an increase in ion current at -1V can be observed (red curve), which is due to Cl... - Ions adsorbed onto the gold layer, thus enhancing the negative charge. When the reporter molecule was further immobilized to the inner surface via Au-S bonds, a significant nonlinear IP curve (blue curve) was observed due to the large amount of negative charge carried by the DNA molecule. Further addition of the apt / cDNA double strand to the nanopipette did not cause a change in current (green curve). However, in the presence of IGF-1, a significant decrease in current was observed in the negative voltage region (purple curve), indicating that cas12a was activated, cleaving the reporter molecule from the inner wall, resulting in a significant reduction in the negative charge on the inner surface. These results validate the feasibility of the proposed CRISPR / Cas nanosensor for the electrochemical detection of IGF-1.

[0072] Example 3: Optimization of Reaction Conditions (1) Optimization of molecular modification concentration in reports To obtain optimal reaction conditions, the concentration of reporter molecules (reDNA) modified on the inner wall of the nanosensor was first optimized. Different concentrations of reDNA were prepared to modify the surface of the sensor, such as... Figure 3 As shown in Figure A, saturation is achieved when the reDNA concentration reaches 100 nM.

[0073] (2) Optimization of the crRNA / Cas12a ratio in the CRISPR cleavage reaction To obtain optimal reaction conditions, the crRNA:Cas12a ratio in the CRISPR system was optimized. The crRNA:Cas12a ratios were set to 0.5:1, 0.75:1, 1:1, 1:1.5, and 1:2. The CRISPR Cas12a detection system was prepared, and after incubation at 37°C for 30 min, the fluorescence intensity was measured using a microplate reader with an excitation wavelength of 484 nm and an emission wavelength of 530 nm. Figure 3 As shown in C, the relative fluorescence intensity increases with the increase of the crRNA:Cas12a ratio. When the crRNA:Cas12a ratio is 1:1, the relative fluorescence intensity is the highest, and the Cas12a protein cleavage activity is the highest.

[0074] (3) Optimization of Cas12a concentration The concentrations of Cas12a protein and crRNA were optimized at a 1:1 ratio of crRNA to Cas12a. Figure 3 As shown in D, the relative fluorescence intensity increases with increasing concentration. When the protein concentration is 20 nM, a strong fluorescence signal can be generated. Therefore, a concentration of 20 nM for Cas12a protein and crRNA was selected for subsequent experiments.

[0075] (4) Optimization of reaction temperature in CRISPR system The reaction temperatures were set to 27℃, 32℃, 37℃, 42℃, and 47℃, respectively. After incubating in a water bath at the set temperatures for 30 minutes, the samples were transferred to an ELISA plate, and the fluorescence intensity was measured using an ELISA reader. Figure 3 As shown in B, the relative fluorescence intensity of Cas12a protein reaches its highest point at 37℃ within the temperature range of 27℃-47℃, indicating the greatest cleavage activity. The optimal reaction temperature for Cas12a protein is 37℃, and excessively high temperatures will affect its protein activity.

[0076] Example 4: Performance Verification Analysis (1) Linear range test Concentration gradient standards were prepared using recombinant Atlantic salmon IGF-1 protein: 1 fM, 10 fM, 1 pM, 10 pM, and 100 pM. Each concentration was measured three times, following the method established above. Within the concentration range of 1 fM to 100 pM, the sensor response signal showed a good linear relationship with the logarithm of the IGF-1 protein concentration. Figure 4 As shown in Figure A, the ion current rectification enhances with increasing IGF-1 protein concentration, indicating that the sensor possesses stable quantitative capability within this concentration range. -1V Plot a standard curve with the vertical axis representing the IGF-1 concentration and the logarithm of IGF-1 concentration (lgC) on the horizontal axis, as shown below. Figure 4As shown in B. Within the concentration range of 1 fM–100 pM, I… -1V It shows a good linear relationship with lgC, and the linear regression equation is: y = 0.99863x - 1.40977 (R²). 2 =0.99673), the detection limit is approximately 0.72 fM.

[0077] (2) Selective verification The effects of Atlantic salmon IGF-1 protein, IGF-2 protein, salmon growth hormone GH, insulin, and a negative control without protein on the sensor were tested separately. Each sample was tested three times. The results are as follows: Figure 4 As shown in Figure C, the nanosensor only generates a strong signal response to IGF-1, and shows no significant cross-reaction to other non-target molecules, demonstrating the good specificity of the method.

[0078] (3) Repeatability test The reproducibility of 10 functionalized nanosensors was assessed using current-voltage (IV) curves, such as... Figure 4 As shown in D, the nanosensor exhibits high repeatability.

[0079] Example 5: Actual Sample Detection To evaluate the analytical performance of the CRISPR-nanopore sensor described in this invention in real, complex biological samples, we conducted testing and verification using actual salmon serum samples.

[0080] (1) Actual sample collection and preparation of spiked samples Healthy adult Atlantic salmon (n=5) were obtained from a fish farm. Blood was collected via the tail vein, allowed to stand at room temperature for 30 minutes, and then centrifuged for 15 minutes to separate the serum. Each serum sample was divided into two aliquots: one for detection using the method of this invention, and the other for parallel validation using a commercial Atlantic salmon IGF-1 ELISA kit. Known serum samples with undetectable or extremely low IGF-1 levels were used as blank matrix. Recombinant Atlantic salmon IGF-1 protein standards were quantitatively added to these aliquots to prepare spiked serum samples with final concentrations of 1 pM, 10 pM, and 100 pM. All serum samples were diluted 10-fold to reduce matrix effects and then used directly for subsequent detection. (2) Preparation of detection solution Take 10 μL of diluted serum sample to be tested, mix it with 90 μL of detection buffer containing 1 nM aptamer-complementary strand complex (Apt-cDNA), and incubate at 37°C for 30 minutes to form the test reaction solution.

[0081] (3) Assembly and sample loading of CRISPR reaction system 20 μL of the pre-assembled CRISPR-Cas12a / crRNA complex (with a final concentration of 20 nM for both crRNA and Cas12a protein) was injected into the tip region of the nanopore using a microsyringe.

[0082] (4) Electroosmotic enrichment and reaction Immediately apply a -1V bias voltage across the nanopore for 30 seconds to enrich the reaction molecules from step (1) at the nanopore interface using electroosmosis. Then, incubate at 37°C for 10 minutes to allow the competitively released cDNA to activate Cas12a, which in turn cleaves the reporter molecules on the inner wall of the nanopore.

[0083] (5) Signal reading After the reaction, the nanopores were washed three times with ultrapure water and then replaced with electrode buffer (30 mM KCl, 20 mM Tris-HCl, pH=7.5). Using an electrochemical workstation, IV curves were recorded at a scan rate of 100 mV / s within the range of -1.0 V to +1.0 V. The ion current (Ik) at -1 V was also recorded. -1v ).

[0084] The measured I -1v Substitute into Example 4 ( Figure 4 A) Establish the standard curve (y = 0.99863x - 1.40977, where y = I -1v , x=lg[IGF-1]), calculate the concentration of IGF-1 in the sample.

[0085] The experimental results are shown in Tables 1 and 2.

[0086] Table 1: Recovery rate of IGF-1 spiked in serum samples (n=3) As shown in Table 1, within the spiked concentration range of 1 pM to 100 pM, the average recovery rate ranged from 94.5% to 112.0%, and the relative standard deviation (RSD) was less than 10%. This indicates that the sensor of the present invention has good accuracy and precision in complex serum matrices, with minimal matrix interference and reliable quantitative results.

[0087] Five actual Atlantic salmon serum samples were tested in parallel using the sensor of this invention and a commercially available ELISA kit. The results are shown in Table 2.

[0088] Table 2: Comparison of test results for actual serum samples The two methods showed good consistency, with relative deviations within ±6%. Paired t-test analysis indicated no statistically significant difference between the two methods (p>0.05). This confirms the effectiveness and reliability of the CRISPR-nanopore sensor of this invention for the quantitative detection of IGF-1 in actual fish serum samples.

[0089] This embodiment verifies the application potential of the competitive activation CRISPR-nanopore sensor described in this invention in real-world complex biological samples through spiked recovery experiments and comparison experiments with actual samples and standard methods. The sensor exhibits high accuracy, sensitivity, and resistance to matrix interference, enabling rapid, sensitive, and specific detection of Atlantic salmon IGF-1 protein. The results are consistent with those of commercial ELISA methods, while avoiding the drawbacks of ELISA, such as reliance on expensive antibodies, cumbersome operation, and the need for large instruments. This provides a powerful new tool for rapid detection in aquaculture fields or laboratories.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A nanopore sensor based on CRISPR-Cas12a, characterized in that, include: The solid-state nanopore unit is a nanostraw made of glass capillary, the tip of which has an opening and the inner wall of which is covered with a conductive layer. A reporter molecule, wherein the reporter molecule is disposed on the conductive layer by chemical bonds; The protein recognition and signal conversion unit includes a DNA aptamer capable of specifically binding to Atlantic salmon IGF-1 protein, and a single-stranded DNA complementary strand that is partially complementary to the DNA aptamer sequence. The signal amplification unit contains the Cas12a protein and crRNA complementary to the target DNA strand.

2. The CRISPR-Cas12a-based nanopore sensor according to claim 1, characterized in that, The tip of the nanostraw has an opening diameter of 70nm-90nm.

3. The CRISPR-Cas12a-based nanopore sensor according to claim 1, characterized in that, The nucleotide sequence of the reporter molecule is shown in SEQ ID NO.1, and the concentration is 50 nM-100 nM.

4. The CRISPR-Cas12a-based nanopore sensor according to claim 1, characterized in that, The nucleotide sequence of the DNA aptamer is shown in SEQ ID NO.2; the nucleotide sequence of the complementary strand of the single-stranded DNA is shown in SEQ ID NO.3; and the nucleotide sequence of the crRNA is shown in SEQ ID NO.

4.

5. The CRISPR-Cas12a-based nanopore sensor according to claim 2, characterized in that, The conductive layer is a gold film with a thickness of 10 nm.

6. The CRISPR-Cas12a-based nanopore sensor according to claim 1, characterized in that, The molar ratio of Cas12a protein to crRNA is 1:

1.

7. A method for fabricating a CRISPR-Cas12a-based nanoporous sensor as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Drawing glass capillaries into nano-straws with open tips; S2. A conductive layer is deposited on the inner wall of the nanotube; S3. Introduce the reporter molecule into the nanoparticle straw and fix it to the surface of the conductive layer by chemical bonds; S4. Treat the fixed interface with a blocking agent to block non-specific binding sites.

8. The method for fabricating a CRISPR-Cas12a-based nanoporous sensor according to claim 7, characterized in that, The blocking agent is bovine serum albumin solution with a concentration of 150 μg / mL to 250 μg / mL.

9. The application of a CRISPR-Cas12a-based nanopore sensor as described in any one of claims 1-6 in the detection of insulin-like growth factor-1 in fish.

10. The application according to claim 9, characterized in that, The fish in question is an Atlantic salmon.

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