Biosensor and method for detecting NF-kB p50

Abstract

The invention discloses a biosensor and a method for detecting NF-kB p50, the biosensor comprises: an electrode, the surface of which is bound with a DNA tetrahedral structure; the DNA tetrahedral structure is used for being combined with a detection probe DNA; the detection probe DNA comprises a detection probe DNA nucleic acid sequence and an electrochemical luminescence group connected with the detection probe DNA nucleic acid sequence. The biosensor can realize reversible dissociation of detection probe DNA under an alkaline condition, so that at least 10 times of repeated detection cycles are supported, and the detection sensitivity and specificity are not influenced. According to the detection method, a differential pulse voltammetry is adopted, a collateral cleavage signal amplification strategy of a CRISPR-Cas12a system is combined, high-sensitivity detection of NF-kappa B p50 is achieved, the method is suitable for complex biological samples and has high sensitivity, high specificity and good reuse performance, the lowest detection limit can reach the 100 fly mole level, and the linear detection range covers 100 aM to 80000 aM.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly to a biosensor and a method for detecting NF-κB p50. Background Art

[0002] As a key transcription factor, NF-κB p50 is involved in regulating immune responses, inflammatory reactions, and cell survival, and is closely related to various diseases such as cancer, autoimmune diseases, and chronic inflammation. Due to its central role in cellular processes, accurate detection of NF-κB p50 is crucial for early diagnosis, disease monitoring, and treatment evaluation. However, existing detection technologies still face challenges in achieving highly sensitive and specific detection of low-abundance biomarkers such as NF-κB p50.

[0003] In recent years, electrochemical biosensors have attracted much attention as a promising alternative to traditional detection methods such as enzyme-linked immunosorbent assay (ELISA) and Western blotting. These sensors have significant advantages in terms of sensitivity, rapid response, and integration with portable devices for point-of-care testing. However, in the case of detecting trace amounts of NF-κB p50, existing biosensors have the defects of low sensitivity, being single-use only, and relatively high detection costs. Summary of the Invention

[0004] The present invention provides a biosensor and a method for detecting NF-κB p50 to solve the problems that existing biosensors can only be used once and have relatively high detection costs. The present invention is achieved through the following technical solutions:

[0005] A biosensor, comprising:

[0006] An electrode, on the surface of which a DNA tetrahedron structure is bound; the DNA tetrahedron structure is used for binding to a detection probe DNA; the detection probe DNA contains a detection probe DNA nucleic acid sequence and an electrochemiluminescent group linked to the detection probe DNA nucleic acid sequence;

[0007] The DNA tetrahedron structure is assembled from DNA strand T1, DNA strand T2, DNA strand T3, and DNA strand T4; the nucleic acid sequence of DNA strand T1 is as shown in SEQ ID No.1; the nucleic acid sequence of DNA strand T2 is as shown in SEQ ID No.2; the nucleic acid sequence of DNA strand T3 is as shown in SEQ ID No.3; the nucleic acid sequence of DNA strand T4 is as shown in SEQ ID No.4;

[0008] The detection probe DNA nucleic acid sequence is as shown in SEQ ID No.5.

[0009] Optionally, the electrode is a gold electrode; the 5' ends of the DNA strands T2, T3, and T4 are all connected with sulfhydryl groups;

[0010] Preferably, the electrochemiluminescent group is a ferrocene group; the ferrocene group is connected to the 3' end of the nucleic acid sequence of the detection probe DNA.

[0011] The preparation method of the above biosensor comprises the following steps:

[0012] Mix the DNA strands T1, T2, T3, and T4, incubate at pH 8.0 - 9.0 and a temperature of 94.8 - 95.2 °C for 10 - 12 minutes, and then cool to 3.8 - 4.2 °C to assemble and form a DNA tetrahedron structure;

[0013] Coat the DNA tetrahedron structure on the electrode so that the DNA tetrahedron structure binds to the electrode surface.

[0014] A method for detecting NF-κB p50 comprises the following steps:

[0015] Step 1) Assemble the probe S1 and the probe S2 to form an S1 / S2 double strand, and then mix and react the S1 / S2 double strand with the sample containing NF-κB p50 to be detected, so that the S1 / S2 double strand binds to NF-κB p50 to form a probe-NF-κB p50 conjugate; then add ExoIII enzyme to decompose the S1 / S2 double strand that has not bound to NF-κB p50 to obtain a mixture; the nucleic acid sequence of the probe S1 is as shown in SEQ ID No.6, and the nucleic acid sequence of the probe S2 is as shown in SEQ ID No.7;

[0016] Step 2) Add a hairpin probe to the mixture, and the probe-NF-κB p50 conjugate reacts with the hairpin probe and the Exo III enzyme to obtain a solution containing intermediate DNA; the nucleic acid sequence of the hairpin probe is as shown in SEQ ID No.8;

[0017] Step 3) Mix and react equal amounts of the solution containing intermediate DNA with the H1 single strand and the H2 single strand respectively to obtain a solution containing the H1a single strand and a solution containing the H2a single strand; then mix and react the solution containing the H1a single strand and the solution containing the H2a single strand to obtain a solution containing the H1a / H2a double strand; the nucleic acid sequence of the H1 single strand is as shown in SEQ ID No.9, the nucleic acid sequence of the H2 single strand is as shown in SEQ ID No.10, the nucleic acid sequence of the H1a single strand is as shown in SEQ ID No.11; the nucleic acid sequence of the H2a single strand is as shown in SEQ ID No.12;

[0018] Step 4) React the solution containing the H1a / H2a double strand with the CRISPR-Cas12a / crRNA complex and the detection probe DNA to obtain a reaction product. The nucleic acid sequence of the crRNA is shown as SEQ ID No. 13. Use the above-mentioned biosensor to perform chemiluminescence detection on the detection reaction product, and then obtain the NF-κB p50 content of the sample containing NF-κB p50 to be measured according to the chemiluminescence detection result.

[0019] Optionally, in Step 4), the NF-κB p50 content of the sample containing NF-κB p50 to be measured is obtained by the standard curve method, that is, a standard curve (the relationship curve between the NF-κB p50 content and the DPV signal) established by pre-detecting a series of standard samples with known concentrations using the same detection method as above. By comparing the chemiluminescence signal with the standard curve and calculating, quantitative analysis of NF-κB p50 can be performed, so that the concentration of this protein in the sample can be accurately obtained.

[0020] Optionally, the method further includes:

[0021] Step 5) Regeneration treatment of the biosensor: Treat the biosensor after detection in Step 4) in an alkaline solution to dissociate the DNA tetrahedron structure from the detection probe DNA.

[0022] Optionally, the pH of the alkaline solution is 9.8 - 10.2;

[0023] Preferably, the alkaline solution is a TAE buffer solution with a pH of 9.8 - 10.2.

[0024] Optionally, in Step 1), probe S1 and probe S2 are mixed in a hybridization buffer, heated at 90 - 95 °C for 5 minutes, and then slowly cooled to 25 °C at a rate of 1 °C per minute to form the S1 / S2 double strand. Add the S1 / S2 double strand to the sample containing NF-κB p50 to be measured, incubate at 36 - 38 °C for 30 - 32 minutes to form a probe-NF-κB p50 conjugate, and then add Exo III enzyme to continue the reaction for 30 - 32 minutes to decompose the S1 / S2 double strand that is not bound to NF-κB p50.

[0025] Preferably, the content of the S1 / S2 double strand added to the sample containing NF-κB p50 to be measured is 100 nmol / ; the content of Exo III enzyme added to the sample containing NF-κB p50 to be measured is 40 U - 60 U;

[0026] Preferably, the hybridization buffer is a TAE buffer solution with a pH of 7.4.

[0027] Optionally, in step (ii), 480-520 nmol / L hairpin probe is added to the mixed solution, and incubated at 36-38 °C for 60-100 minutes.

[0028] Preferably, the nucleic acid sequence of the intermediate DNA is shown in SEQ ID No. 14.

[0029] Optionally, in step (iii), the single-stranded H1 and H2 are first heated at 94-96 °C for 4-6 minutes and then cooled to room temperature to form a stable hairpin structure;

[0030] The single-stranded H1 and H2 of the hairpin structure and the intermediate DNA are incubated at 36-38 °C for 115-125 min respectively; then Exo III enzyme is added for reaction for 58-62 min to obtain a solution containing the H1a fragment and a solution containing the H2a fragment respectively;

[0031] The solution containing the H1a fragment and the solution containing the H2a fragment are mixed, heated at 60-70 °C for 8-12 minutes and then naturally cooled to room temperature to form the H1a / H2a double strand.

[0032] Optionally, in step (iv), the solution containing the H1a / H2a double-strand is reacted with the CRISPR-Cas12a / crRNA complex and the detection probe DNA, specifically including:

[0033] A reaction solution containing 20-40 nmol / L CRISPR-Cas12a / crRNA complex and 80-120 nmol / L detection probe DNA is added to the solution containing the H1a / H2a double-strand, and mixed and reacted for 30-60 minutes;

[0034] Preferably, in step (iv), a reaction solution containing 30 nmol / L CRISPR-Cas12a / crRNA complex and 100 nmol / L detection probe DNA is added to the solution containing the H1a / H2a double-strand, and mixed and reacted for 30 minutes;

[0035] Preferably, the reaction solution in step (iv) also contains Tris-HCl, KCl, MgCl, glycerol, and DTT;

[0036] Preferably, differential pulse voltammetry is used to perform chemiluminescence detection on the detection reaction product. Compared with the prior art, the present invention has the following beneficial effects:

[0037] The electrode is modified with a DNA tetrahedron structure to improve the stability and signal response efficiency of the sensor; the exonuclease III (Exo III)-mediated cyclic amplification and CRISPR / Cas12a collateral cleavage amplification are utilized to achieve ultrasensitive detection of NF-κB p50; differential pulse voltammetry (DPV) can be combined for electrochemical signal readout to improve the detection sensitivity and specificity. In addition, the present invention innovatively introduces a pH-responsive probe regeneration mechanism. By dissociating the DNA1 probe from the electrode surface under alkaline conditions, the non-destructive regeneration and repeated use of the biosensor are realized, ensuring that it still maintains high sensitivity and stability after multiple detection cycles, breaking through the key limitations of current biosensing technologies. It provides a new idea for the regeneration of DNA nanoprobes. By integrating the advantages of high sensitivity, specificity and repeatability, this sensor provides a powerful tool for accurately monitoring biomolecular interactions and promoting the development of biosensing technologies. This sensor can be further applied to the detection of other transcription factors or protein biomarkers, with strong versatility and expandability.

[0038] The method of the present invention proposes a highly sensitive and reusable electrochemical biosensor based on the exonuclease III (Exo III), CRISPR / Cas12a system and DNA tetrahedron nanostructure multi-level amplification mechanism. The sensor can generate sensitive and monitorable electrochemical signals under the condition of pH = 7.0 to achieve accurate detection of NF-κB p50. The enzyme cycle amplification of Exo III and the collateral cleavage effect of CRISPR / Cas12a act synergistically to jointly enhance the signal response. The DNA tetrahedron-modified electrode further improves the stability and specificity of the system, providing an efficient platform for sensitive signal transduction. The detection method of the present invention is applicable to complex biological samples (such as serum, cell lysate or tissue homogenate), and has high sensitivity, high specificity and good repeatability performance. The lowest detection limit can reach the level of 100 femtomoles (fM), and the linear detection range covers 100 aM to 80,000 aM. The biosensor can achieve reversible dissociation of the DNA1 probe under alkaline conditions of pH = 10.0, thus supporting at least 10 repeated detection cycles without affecting the detection sensitivity and specificity. The detection method uses differential pulse voltammetry (DPV), combined with the collateral cleavage signal amplification strategy of the CRISPR-Cas12a system, to achieve high-sensitivity detection of NF-κB p50. At the same time, this sensor can be further applied to the detection of other transcription factors or protein biomarkers, with strong versatility and expandability. Brief Description of the Drawings

[0039] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the specific embodiments or the prior art. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0040] Figure 1 Schematic diagram of the influence of reaction parameters on the detection result.

[0041] Figure 2 Detection performance of NF-κB p50 by a biosensor based on differential pulse voltammetry (DPV).

[0042] Figure 3 Specificity and pH-responsive regeneration result diagram of a CRISPR-Cas12a-based differential pulse voltammetry (DPV) biosensor.

[0043] Figure 4 Flowchart of Example 2. Specific embodiments

[0044] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and do not limit the present invention.

[0045] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0046] All nucleic acid probes, DNA tetrahedron structure-related nucleic acid sequences, crRNAs of the CRISPR / Cas12a system, and related nucleic acid molecules used in the present invention are synthesized by GenScript (China), in order to ensure the accuracy, stability, and repeatability of experimental data. The specific information is as follows:

[0047] All nucleic acid sequences, including T1 (SEQ ID No.1), T2 (SEQ ID No.2), T3 (SEQ ID No.3), T4 (SEQ ID No.4) of the DNA tetrahedron structure, detection probe DNA (SEQ ID No.5), probe S1 (SEQ ID No.6), probe S2 (SEQ ID No.7), hairpin probe HP (SEQ ID No.8), H1 single strand (SEQ ID No.9), H2 single strand (SEQ ID No.10), and crRNA (SEQ ID No.13), were synthesized by GenScript Biotech, dissolved in DEPC-treated water or 1×PBS buffer, and formulated at the concentration required for the experiment (20 - 100 μmol / L).

[0048] The crRNA (SEQ ID No.13) and related nucleic acid molecules required for the CRISPR / Cas12a system used in the present invention have undergone strict quality control to ensure their high efficiency and specificity. All nucleic acid probes and DNA structures are stored at -20°C, and are treated with RNase inhibitor before the experiment to ensure the integrity and stability of the samples. The nucleic acid sequence of the intermediate DNA obtained during the experiment is shown in SEQ ID No.14, the nucleic acid sequence of the H1a single strand is shown in SEQ ID No.11, and the nucleic acid sequence of the H2a single strand is shown in SEQ ID No.12.

[0049] Sequence Listing

[0050]

[0051]

[0052] Example 1

[0053] This example provides a biosensor, including:

[0054] An electrode, on the surface of which a DNA tetrahedron structure is bound; the DNA tetrahedron structure is used to bind to the detection probe DNA; the detection probe DNA contains a detection probe DNA nucleic acid sequence and a ruthenium compound linked to the detection probe DNA nucleic acid sequence;

[0055] The DNA tetrahedron structure is assembled from DNA strand T1, DNA strand T2, DNA strand T3, and DNA strand T4; the nucleic acid sequence of DNA strand T1 is as shown in SEQ ID No.1; the nucleic acid sequence of DNA strand T2 is as shown in SEQ ID No.2; the nucleic acid sequence of DNA strand T3 is as shown in SEQ ID No.3; the nucleic acid sequence of DNA strand T4 is as shown in SEQ ID No.4;

[0056] The nucleic acid sequence of the detection probe DNA is as shown in SEQ ID No. 5.

[0057] The preparation method of the electrode includes:

[0058] Assembly of DNA tetrahedron structure: Four DNA strands (T1, T2, T3, T4) are mixed at equal concentrations in Tris-MgCl buffer (pH 8.0) with a final concentration of 1.0 μmol / L. The mixture is heated at 95 °C for 10 minutes and then rapidly cooled to 4 °C within 30 seconds to promote the correct folding of the tetrahedron structure. The assembled DNA tetrahedron structure is used for subsequent construction of the biosensor.

[0059] Modification of the gold electrode: A gold electrode with a diameter of 2 mm is polished successively with alumina polishing paste (0.3 μm and 0.05 μm), and ultrasonically cleaned with ethanol and ultrapure water to remove surface impurities. Subsequently, 10 μL of the pre-assembled DNA tetrahedron solution (1.0 μmol / L) is dropped onto the electrode surface and fixed at room temperature for 12 hours using the gold-thiol interaction. After the electrode is rinsed with Tris-MgCl buffer to remove the unbound DNA, the surface active sites are blocked with 1 mmol / L hexanethiol for 30 minutes. Finally, the electrode is thoroughly rinsed with ethanol and ultrapure water, and the obtained biosensor is used for subsequent detection.

[0060] The above biosensor has a pH-responsive regeneration mechanism: When the sensor is immersed in an alkaline environment (pH ≥ 10.0), the high pH environment can cause the hydrogen bonds between the detection probe DNA and the complementary strand (or tetrahedron structure) to break, and the interaction between the detection probe DNA fixed on the DNA tetrahedron structure and the electrode surface is disrupted. And under alkaline conditions, the phosphate backbone of the DNA strand carries more negative charges, enhancing the electrostatic repulsion between strands and promoting the dissociation of the detection probe DNA from the electrode surface. The dissociation of the detection probe DNA restores the biosensor to its initial state, enabling it to be reused for subsequent detection cycles. Therefore, by immersing the sensor of the present invention in a buffer solution with pH ≥ 10.0, incubating briefly and then rinsing, the next round of detection can be carried out. The sensor of the present invention can reversibly dissociate the probe under alkaline conditions and maintain stable sensor performance (> 10 cycles), with high specificity, rapid response (completed within 2 hours) and potential for modular expansion. The synergistic effect of its multi-level signal amplification and intelligent regeneration design breaks through the limitations of traditional methods such as low sensitivity, high cost and single use, providing an efficient and economical solution for clinical diagnosis, dynamic monitoring of chronic diseases and the development of multi-target biosensing technologies.

[0061] Example 2

[0062] This embodiment provides a method for detecting NF-κB p50 based on the above biosensor, and there are still significant challenges in the required sensitivity. Therefore, a signal amplification strategy must be adopted.

[0063] Exonuclease III (Exo III) and the CRISPR / Cas12a system have shown unique advantages in biosensor applications. As a highly specific enzyme, Exo III can perform multi-level signal amplification by continuously degrading probes and recycling protein-DNA complexes. This enzymatic amplification effect enables the biosensor system to detect low-abundance targets such as NF-κB p50 more efficiently and reliably. The CRISPR / Cas12a system, with its signal amplification ability triggered by collateral cleavage activity, has been successfully applied to the biosensing field in recent years. When combined with Exo III-assisted amplification, CRISPR / Cas12a can further improve the detection sensitivity through a synergistic effect: Exo III is responsible for DNA signal amplification, while CRISPR / Cas12a generates collateral amplification signals that are easy to detect electrochemically.

[0064] The method of the present invention is as Figure 4 shown and includes the following steps:

[0065] Step 1) Preparation of the probe solution: Mix probe S1 and probe S2 in TAE buffer (Tris-acetate-ethylenediaminetetraacetic acid buffer), heat at 90 °C for 5 minutes, and then slowly cool to 25 °C at a rate of 1 °C / minute to ensure sufficient hybridization.

[0066] Protein binding and Exo III digestion: Mix the S1 / S2 double-stranded solution (100 nM) with the sample containing NF-κB p50 to be tested, incubate at 37 °C in PBS buffer for 30 minutes, and then incubate at 37 °C for another 30 minutes to catalyze the enzymatic reaction.

[0067] Step 2) Hairpin probe (HP) reaction: Add 500 nM hairpin probe (HP) to the mixture and incubate for 60 minutes to form an intermediate DNA solution.

[0068] Step 3) Preparation of the H1a / H2a double strand: To avoid the degradation of the H1a / H2a double strand by Exo III, the enzymatic digestion reactions of H1 with the intermediate DNA and H2 with the intermediate DNA are carried out separately:

[0069] First, the single strands of H1 and H2 were heated at 95°C for 5 minutes respectively and then slowly cooled (for 4 hours) to room temperature to form stable hairpin structures. Equal amounts of the above intermediate DNA solution were added to the hairpin probes of H1 and H2 (1 μM) respectively, and incubated at 37°C for 2 hours. Then, Exo III (0.5 U / μL) was added and digestion continued for 1 hour, and the reaction was terminated by heating at 65°C for 10 minutes. The solutions containing H1a and H2a fragments were mixed, heated at 65°C for 10 minutes and then naturally cooled to room temperature to form a solution containing H1a / H2a double strands.

[0070] Step (4) CRISPR-Cas12a cleavage reaction and differential pulse voltammetry (DPV) signal acquisition: 50 μL of reaction solution was added to the solution containing H1a / H2a double strands in step (3) and mixed well. The reaction solution contained 20 mM Tris-HCl, 100 mM KCl, 5 mM MgCl, 5% glycerol, 1 mM DTT, 20 nM CRISPR-Cas12a / crRNA complex and 100 nM detection probe DNA, and the reaction was carried out for 30 minutes to complete the cleavage of the detection probe DNA. Subsequently, the regenerable biosensor was immersed in the reaction solution for 30 minutes, and the DPV signal was recorded. By comparing and calculating the DPV signal with the standard curve (the relationship curve between the content of NF-κB p50 and the DPV signal) established in advance by the same detection method as above, the NF-κB p50 can be quantitatively analyzed, and thus the concentration of this protein in the sample can be accurately obtained.

[0071] Step (5) Sensor regeneration is achieved by dissociating the detection probe DNA under alkaline conditions (TEA solution with pH = 10.0), ensuring that it can be reused for subsequent detection cycles.

[0072] The method of the present invention uses a reusable CRISPR / Cas12a biosensor, and realizes ultrasensitive detection (femtomolar level) of NF-κB p50 by integrating Exo III enzyme cascade amplification, CRISPR / Cas12a collateral cleavage and DNA tetrahedron enhancement strategies.

[0073] Example 3

[0074] In this example, according to the detection method in Example 2, the addition amount of Exo III in step (1) was optimized. The addition amounts of Exo III in step (1) were modified to 0 U, 10 U, 20 U, 30 U, 50 U, and 60 U respectively. Other experimental conditions were the same as those in Example 2, and the DPV signals were as Figure 1 shown in A.

[0075] According to Figure 1 the influence of the Exo III concentration on the DPV signal in A, 40 U was determined as the optimal concentration because the enzyme would be saturated after this concentration.

[0076] Example 4

[0077] In this example, according to the detection method in Example 2, the Exo III reaction time in step (ii) was optimized. The reaction times in step (ii) were respectively modified to 0 min, 20 min, 40 min, 60 min, 80 min, 1000 U, and samples containing 0.1 fM and 50 fM NF-κB p50 were respectively detected. Other experimental conditions were the same as those in Example 2, and the DPV signals were as Figure 1 shown in B.

[0078] According to Figure 1 shown in B, the influence of the Exo III reaction time on the DPV signal was that the signal was stable at 60 minutes, which was the optimal reaction time.

[0079] Example 5

[0080] In this example, according to the detection method in Example 2, the addition amount of CRISPR-Cas12a / crRNA in step (iv) was optimized. The addition amounts of CRISPR-Cas12a / crRNA in step (iv) were respectively modified to 0 nM, 5 nM, 10 nM, 20 nM, 40 nM, 60 nM. Other experimental conditions were the same as those in Example 2, and the DPV signals were as Figure 1 shown in C.

[0081] According to Figure 1 shown in C, through the optimization of the CRISPR-Cas12a / crRNA concentration, 20 nM was determined as the optimal concentration to balance sensitivity and specificity.

[0082] Example 6

[0083] In this example, according to the detection method in Example 2, the reaction time for Cas12a-mediated cleavage of the detection probe DNA in step (iv) was optimized. The reaction times in step (iv) were respectively modified to 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min. Other experimental conditions were the same as those in Example 2, and the DPV signals were as Figure 1 shown in D.

[0084] According to Figure 1 shown in D, regarding the influence of the Cas12a-mediated cleavage time on the DPV signal, the signal reached a plateau at 30 minutes, which was the optimal signal amplification time. These optimizations together ensured the high sensitivity and high specificity of this biosensor in the detection of NF-κB p50.

[0085] Test Example 1

[0086] In this test example, a series of NF-κB p50 with different concentrations were detected according to the detection method of Example 2 to clarify the detection performance of the method of the present invention.

[0087] Figure 2 is the detection performance of NF-κB p50 by a biosensor based on differential pulse voltammetry (DPV). Figure 2 A shows the relationship between the concentration of NF-κB p50 and the DPV signal intensity. Figure 2 B shows the linear correlation between the logarithm of the NF-κB p50 concentration and the DPV signal. The results show that as the concentration increases, the signal decreases significantly, and the lowest signal is observed at a concentration of 100 femtomolar (fM). This result reflects the efficient triple signal amplification mechanism composed of DNA cleavage mediated by exonuclease III (Exo III), hairpin probe hybridization, and CRISPR-Cas12a activation.

[0088] Test Example 2

[0089] In this test example, the method in Example 2 was used to detect various different proteins to clarify the specificity of the method of the present invention. And the pH response mechanism of the biosensor was tested.

[0090] Figure 3 is the result diagram of the specificity and pH-responsive regeneration of a CRISPR-Cas12a-based differential pulse voltammetry (DPV) biosensor. First, based on the test method of Example 2, the above biosensor was used to test samples containing bovine serum albumin (BSA), sialic acid-binding immunoglobulin-like lectin-5 (Siglec-5), and prostate-specific antigen (PSA) respectively. The results are as follows. Figure 3 As shown in A, the biosensor showed high specificity. For nuclear factor κB p50 (100,000 aM), the DPV signal decreased significantly, while the signals generated by non-specific proteins bovine serum albumin (BSA), sialic acid-binding immunoglobulin-like lectin-5 (Siglec-5), and prostate-specific antigen (PSA) were similar to the blank control.

[0091] The biosensor was alternately placed in a solution containing the detection probe DNA and a TAE buffer with pH = 10 to test its reusability. The results are as follows. Figure 3 As shown in B, the pH-induced regeneration process restored the performance of the biosensor under alkaline conditions (pH 10.0), with a 99.2% signal reduction, indicating that the biosensor had been effectively restored. And in five cycles, the consistent regeneration rate exceeded 94.7%, confirming its stability and reusability.

[0092] Test Example 3

[0093] To evaluate the practical applicability of the developed pH-induced regenerative biosensor for detecting nuclear factor κB p50 (NF-κB p50), the protein was added to human serum samples diluted tenfold. Different concentrations of NF-κB p50 (100 aM, 1000 aM, 5000 aM, 10000 aM, 50000 aM, and 80000 aM) were added to these diluted sample matrices, and the corresponding differential pulse voltammetry (DPV) signals were recorded. The NF-κB p50 was quantitatively analyzed by comparing the DPV response with a pre-established calibration curve, enabling the precise determination of the concentration of this protein in the samples.

[0094] By analyzing the recovery rate and relative standard deviation (RSD), the performance of the biosensor in this biologically relevant environment was evaluated to ensure accurate detection even in the presence of potential interferents. As shown in Table 1, the biosensor exhibited excellent reproducibility and precision, with all RSD values being lower than 6%. The recovery rate ranged from 99.61% to 106.23%, confirming the system's ability to reliably detect and quantify NF-κB p50 in diluted human serum samples.

[0095] These results highlight the robustness and reliability of the biosensor in clinical and diagnostic applications, especially in complex biological matrices. The stable recovery rate and low RSD values indicate that the biosensor is minimally affected by potential matrix effects, such as interfering proteins or biomolecules in serum. This ability underscores its practical value in detecting trace amounts of NF-κB p50, an important biomarker, in complex biological fluids with high sensitivity and specificity.

[0096] Table 1. Determination of NF-κB p50 in tenfold diluted human serum using the proposed biosensor (n = 3)

[0097]

[0098] The practical application of the biosensor in serum samples demonstrated its suitability for real diagnostic scenarios where accurate and precise detection of low-abundance proteins is crucial. Even in challenging environments, the DPV biosensor maintained high recovery rates and reproducibility, showing promise as a reliable tool for clinical diagnosis and disease monitoring, especially for detecting NF-κB p50 in patient samples.

[0099] Obviously, the above embodiments are merely examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A biosensor, characterized in that: include: An electrode, wherein a DNA tetrahedron structure is bound to the surface of the electrode; the DNA tetrahedron structure is used to bind to a detection probe DNA; the detection probe DNA comprises a detection probe DNA nucleic acid sequence and an electrochemical luminescent group connected to the detection probe DNA nucleic acid sequence; The DNA tetrahedron structure is formed by assembling DNA chain T1, DNA chain T2, DNA chain T3 and DNA chain T4; the nucleic acid sequence of DNA chain T1 is shown in SEQ ID No.1; the nucleic acid sequence of DNA chain T2 is shown in SEQ ID No.2; the nucleic acid sequence of DNA chain T3 is shown in SEQ ID No.3; the nucleic acid sequence of DNA chain T4 is shown in SEQ ID No.4; The detection probe DNA nucleic acid sequence is shown as SEQ ID No.

5.

2. The biosensor according to claim 1, characterized in that The electrode is a gold electrode; the 5' ends of the DNA strands T2, T3 and T4 are all connected with thiol groups; Preferably, the electrochemiluminescent group is a ferrocene group; the ferrocene group is connected to the 3' end of the detection probe DNA nucleic acid sequence.

3. The method for preparing the biosensor according to claim 1 or 2, characterized in that: The steps include: DNA strands T1, T2, T3 and T4 are mixed, kept warm at pH 8.0-9.0 and 94.8-95.2°C for 10-12 minutes, and then cooled to 3.8-4.2°C to assemble into a DNA tetrahedral structure; The DNA tetrahedron structure is coated on the electrode so that the DNA tetrahedron structure is combined on the surface of the electrode.

4. A method for detecting NF-κB p50, characterized in that: The steps include: Step 1) Assemble probe S1 and probe S2 to form S1 / S2 double strands, then mix the S1 / S2 double strands with the sample to be tested containing NF-κB p50 to react, so that the S1 / S2 double strands bind to NF-κB p50 to form a probe-NF-κB p50 complex; then add Exo III enzyme to decompose the S1 / S2 double strands that are not bound to NF-κB p50 to obtain a mixed solution; the nucleic acid sequence of the probe S1 is shown in SEQ ID No.6, and the nucleic acid sequence of the probe S2 is shown in SEQ ID No.7; Step 2) adding a hairpin probe to the mixed solution, and reacting the probe-NF-κB p50 complex with the hairpin probe and the Exo III enzyme to obtain a solution containing intermediate DNA; the nucleic acid sequence of the hairpin probe is shown in SEQ ID No. 8; Step 3) Mixing equal amounts of the solution containing the intermediate DNA with the H1 single strand and the H2 single strand respectively to obtain a solution containing the H1a single strand and a solution containing the H2a single strand, respectively; then mixing the solution containing the H1a single strand and the H2a single strand to obtain a solution containing the H1a / H2a double strand; the nucleic acid sequence of the H1 single strand is shown in SEQ ID No.9, the nucleic acid sequence of the H2 single strand is shown in SEQ ID No.10, the nucleic acid sequence of the H1a single strand is shown in SEQ ID No.11; the nucleic acid sequence of the H2a single strand is shown in SEQ ID No.12; Step 4) reacting the H1a / H2a double-stranded solution with the CRISPR-Cas12a / crRNA complex and the detection probe DNA to obtain a reaction product, wherein the nucleic acid sequence of the crRNA is shown in SEQ ID No. 13; performing chemiluminescence detection on the detection reaction product using the biosensor according to claim 1 or 2, and then obtaining the NF-κB p50 content of the NF-κB p50 sample to be tested according to the chemiluminescence detection result.

5. The method according to claim 4, characterized in that The method further comprises: Step 5) Biosensor regeneration treatment: The biosensor after detection in step 4) is treated in an alkaline solution to dissociate the DNA tetrahedral structure from the detection probe DNA.

6. The method according to claim 5, characterized in that The pH of the alkaline solution is 9.8 to 10.2; Preferably, the alkaline solution is a TAE buffer solution with a pH of 9.8 to 10.

2.

7. The method according to claim 4, characterized in that In the step 1), the probe S1 and the probe S2 are mixed in a hybridization buffer, heated at 90-95° C. for 5 minutes, and then slowly cooled to 25° C. at a rate of 1° C. / min to form an S1 / S2 double chain; the S1 / S2 double chain is added to a sample containing NF-κB p50 to be tested, incubated at 36-38° C. for 30-32 minutes to form a probe-NF-κBp50 complex, and then Exo III enzyme is added to continue the reaction for 30-32 minutes to decompose the S1 / S2 double chain that is not bound to NF-κBp50; Preferably, the content of S1 / S2 double chain added to the sample containing NF-κB p50 to be tested is 100 nmol / ; the content of Exo III enzyme added to the sample containing NF-κB p50 to be tested is 40U-60U; Preferably, the hybridization buffer is TAE buffer with a pH of 7.

4.

8. The method according to claim 4, characterized in that In the step 2), 480-520 nmol / L of the hairpin probe is added to the mixed solution, and the mixture is incubated at 36-38° C. for 60-100 minutes; Preferably, the nucleic acid sequence of the intermediate DNA is shown as SEQ ID No.

14.

9. The method according to claim 4, characterized in that In the step 3), the H1 single strand and the H2 single strand are heated at 94-96° C. for 4-6 minutes and then cooled to room temperature to form a stable hairpin structure; The H1 single strand and the H2 single strand of the hairpin structure were incubated with the intermediate DNA at 36-38° C. for 115-125 minutes respectively; then Exo III enzyme was added to react for 58-62 minutes to obtain a solution containing the H1a fragment and a solution containing the H2a fragment respectively; The solution containing the H1a fragment is mixed with the solution containing the H2a fragment, heated at 60-70°C for 8-12 minutes, and then naturally cooled to room temperature to form an H1a / H2a double chain.

10. The method according to claim 4, characterized in that In the step 4), the H1a / H2a double-stranded solution is reacted with the CRISPR-Cas12a / crRNA complex and the detection probe DNA, specifically comprising: Add the reaction solution containing 20-40 nmol / L CRISPR-Cas12a / crRNA complex and 80-120 nmol / L detection probe DNA to the H1a / H2a double-stranded solution, and mix and react for 30-60 minutes; Preferably, in the step 4), a reaction solution containing 30 nmol / L CRISPR-Cas12a / crRNA complex and 100 nmol / L detection probe DNA is added to the H1a / H2a double-stranded solution, and the mixture is reacted for 30 minutes; Preferably, the reaction solution in step 4) further comprises Tris-HCl, KCl, MgCl, glycerol, and DTT; Preferably, in step 4), differential pulse voltammetry is used to perform chemiluminescence detection on the detection reaction product.

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