A biosensor and a method for detecting nf-kb p50

Abstract

The application discloses a biosensor and a method for detecting NF-kappa B p50, comprising: an electrode, wherein a DNA tetrahedron structure is combined on the surface of the electrode; the DNA tetrahedron 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 electrochemiluminescence group connected with the detection probe DNA nucleic acid sequence. The biosensor can realize reversible dissociation of the detection probe DNA under alkaline conditions, thereby supporting at least 10 repeated detection cycles without affecting detection sensitivity and specificity. The detection method adopts a differential pulse voltammetry method, combines with a side branch cutting signal amplification strategy of a CRISPR-Cas12a system, and realizes high-sensitivity detection of the NF-kappa B p50. The method is suitable for complex biological samples, has high sensitivity, high specificity and good repeated use performance, and the minimum detection limit can reach 100 femtomolar level, and the linear detection range covers 100 aM to 80000 aM.

Description

Technical Field

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

[0002] NF-κB p50, a key transcription factor, participates in the regulation of immune responses, inflammatory responses, 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, current detection technologies still face challenges in achieving high sensitivity and specificity for 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 assays (ELISA) and Western blotting. These sensors offer significant advantages in sensitivity, rapid response, and integration with portable devices for point-of-care detection. However, for the detection of trace NF-κB p50, existing biosensors suffer from drawbacks such as low sensitivity, single-use capability, and high detection costs. Summary of the Invention

[0004] This invention provides a biosensor and a method for detecting NF-κB p50, addressing the problems of existing biosensors being single-use and having high detection costs. This invention is achieved through the following technical solution:

[0005] A biosensor, comprising:

[0006] An electrode, the surface of which is bound with a DNA tetrahedral structure; the DNA tetrahedral structure is used to bind 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 tetrahedral structure is formed by assembling DNA strands T1, T2, T3, and T4; the nucleic acid sequence of DNA strand T1 is shown in SEQ ID No. 1; the nucleic acid sequence of DNA strand T2 is shown in SEQ ID No. 2; the nucleic acid sequence of DNA strand T3 is shown in SEQ ID No. 3; and the nucleic acid sequence of DNA strand T4 is shown in SEQ ID No. 4.

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

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

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

[0011] The above-mentioned method for preparing the biosensor includes the following steps:

[0012] DNA strands T1, T2, T3, and T4 were mixed and incubated at pH 8.0-9.0 and a temperature of 94.8-95.2℃ for 10-12 minutes, then cooled to 3.8-4.2℃ to assemble into a DNA tetrahedral structure.

[0013] The DNA tetrahedral structure is coated onto the electrode, thereby binding the DNA tetrahedral structure to the electrode surface.

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

[0015] Step 1) Assemble probe S1 and probe S2 to form S1 / S2 double strands, then mix the S1 / S2 double strands with the NF-κBp50-containing sample to be tested, allowing the S1 / S2 double strands to bind to NF-κB p50 and form a probe-NF-κB p50 conjugate; then add ExoIII enzyme to decompose the S1 / S2 double strands that are not bound to NF-κB p50, obtaining a mixture; the nucleic acid sequence of probe S1 is shown in SEQ ID No. 6, and the nucleic acid sequence of probe S2 is shown in SEQ ID No. 7;

[0016] Step 2) Add the hairpin probe to the mixture. The probe-NF-κB p50 ligand 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 shown in SEQ ID No. 8.

[0017] (Step 3) Equal volumes of the solution containing intermediate DNA were mixed and reacted with H1 single-stranded DNA and H2 single-stranded DNA, respectively, to obtain solutions containing H1a single-stranded DNA and H2a single-stranded DNA, respectively; then, the solutions containing H1a single-stranded DNA and H2a single-stranded DNA were mixed and reacted to obtain a double-stranded solution containing H1a / H2a; the nucleic acid sequence of the H1 single-stranded DNA is shown in SEQ ID No. 9, the nucleic acid sequence of the H2 single-stranded DNA is shown in SEQ ID No. 10, the nucleic acid sequence of the H1a single-stranded DNA is shown in SEQ ID No. 11, and the nucleic acid sequence of the H2a single-stranded DNA is shown in SEQ ID No. 12;

[0018] Step 4) The H1a / H2a double-stranded solution is reacted with the CRISPR-Cas12a / crRNA complex and the detection probe DNA to obtain the reaction product. The nucleic acid sequence of the crRNA is shown in SEQ ID No. 13. The above-mentioned biosensor is used to detect the reaction product by chemiluminescence, and the NF-κB p50 content of the sample containing NF-κB p50 is obtained according to the chemiluminescence detection result.

[0019] Optionally, in step four), the NF-κB p50 content of the sample to be tested containing NF-κB p50 is obtained by the standard curve method. That is, the same detection method is used to pre-detect a series of standard samples with known concentrations to establish a standard curve (NF-κB p50 content versus DPV signal relationship curve). By comparing the chemiluminescence signal with the standard curve, the NF-κB p50 can be quantitatively analyzed, thereby accurately obtaining the concentration of the protein in the sample.

[0020] Optionally, the method further includes:

[0021] 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.

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

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

[0024] Optionally, in step one), probe S1 and probe S2 are mixed in 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 S1 / S2 double strands; the S1 / S2 double strands are added to the sample containing NF-κBp50 to be tested, and incubated at 36–38°C for 30–32 minutes to form probe-NF-κB p50 conjugates; then Exo III enzyme is added and the reaction is continued for 30–32 minutes to decompose the S1 / S2 double strands that are not bound to NF-κB p50;

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

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

[0027] Optionally, in step two, 480-520 nmol / L hairpin probes are added to the mixture 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 three), the H1 single chain and the H2 single chain 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 H1 and H2 single strands of the hairpin structure were incubated with the intermediate DNA at 36–38 °C for 115–125 min; then Exo III enzyme was added and the reaction was carried out for 58–62 min to obtain solutions containing the H1a fragment and the H2a fragment, respectively.

[0031] A solution containing the H1a fragment is mixed with a 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.

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

[0033] 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 for 30–60 minutes.

[0034] Preferably, in step four), the 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 mixed for 30 minutes.

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

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

[0037] The electrode is modified with a DNA tetrahedral structure to improve the sensor's stability and signal response efficiency. Ultrasensitive detection of NF-κB p50 is achieved through Exo III-mediated cyclic amplification and CRISPR / Cas12a side-branch cleavage amplification. Differential pulse voltammetry (DPV) can be combined for electrochemical signal readout, further enhancing sensitivity and specificity. Furthermore, this invention innovatively introduces a pH-responsive probe regeneration mechanism. By dissociating the DNA1 probe from the electrode surface under alkaline conditions, the biosensor can be regenerated non-destructively and reused, ensuring high sensitivity and stability even after multiple detection cycles, overcoming key limitations of current biosensing technology. This provides a new approach to DNA nanoprobe regeneration. By integrating high sensitivity, specificity, and reproducibility, this sensor offers a powerful tool for accurately monitoring biomolecular interactions and advancing biosensing technology. The sensor can be further applied to the detection of other transcription factors or protein biomarkers, demonstrating strong versatility and scalability.

[0038] This invention presents a highly sensitive, reusable electrochemical biosensor based on a multi-stage amplification mechanism of exonuclease III (Exo III), a CRISPR / Cas12a system, and a DNA tetrahedral nanostructure. The sensor generates a sensitive and measurable electrochemical signal at pH 7.0, enabling precise detection of NF-κB p50. The cyclic amplification of Exo III and the side-branch cleavage effect of CRISPR / Cas12a synergistically enhance the signal response. The DNA tetrahedral modified electrode further improves the system's stability and specificity, providing an efficient platform for sensitive signal transduction. The detection method of this invention is applicable to complex biological samples (such as serum, cell lysates, or tissue homogenates) and exhibits high sensitivity, high specificity, and good reusability, with a detection limit reaching the 100 femtomolar (fM) level and a linear detection range covering 100 aM to 80,000 aM. The biosensor can achieve reversible dissociation of the DNA1 probe under alkaline conditions at pH 10.0, thus supporting at least 10 repeated detection cycles without affecting detection sensitivity and specificity. The detection method employs differential pulse voltammetry (DPV) combined with the side branch cleavage signal amplification strategy of the CRISPR-Cas12a system to achieve high-sensitivity detection of NF-κB p50. Furthermore, this sensor can be further applied to the detection of other transcription factors or protein biomarkers, demonstrating strong versatility and scalability. Attached Figure Description

[0039] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required for the specific embodiments or the prior art are 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 based on these drawings without creative effort.

[0040] Figure 1 This is a schematic diagram illustrating the influence of reaction parameters on the detection results.

[0041] Figure 2 To assess the detection performance of a biosensor based on differential pulse voltammetry (DPV) for NF-κB p50.

[0042] Figure 3 This is a graph showing the specificity and pH-responsive regeneration results of a CRISPR-Cas12a-based differential pulse voltammetry (DPV) biosensor.

[0043] Figure 4 This is a flowchart of Example 2. Detailed Implementation

[0044] Various exemplary embodiments of the present invention will now 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, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is only for describing particular embodiments and is not intended to limit the present invention.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] All nucleic acid probes, DNA tetrahedral structure-related nucleic acid sequences, CRISPR / Cas12a system crRNA, and related nucleic acid molecules used in this invention were synthesized by GenScript Biotech (GenScript, China) to ensure the accuracy, stability, and reproducibility of experimental data. Specific information is as follows:

[0047] All nucleic acid sequences, including DNA tetrahedral structures T1 (SEQ ID No. 1), T2 (SEQ ID No. 2), T3 (SEQ ID No. 3), T4 (SEQ ID No. 4), 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 prepared according to the required experimental concentration (20-100 μmol / L).

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

[0049] sequence list

[0050]

[0051]

[0052] Example 1

[0053] This embodiment provides a biosensor, including:

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

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

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

[0057] The preparation method of this electrode includes:

[0058] Assembly of DNA tetrahedral structures: Four DNA strands (T1, T2, T3, T4) were mixed at equal concentrations in Tris-MgCl buffer (pH 8.0) to a final concentration of 1.0 μmol / L. The mixture was heated at 95°C for 10 minutes, followed by rapid cooling to 4°C over 30 seconds to promote proper folding of the tetrahedral structure. The assembled DNA tetrahedral structures were used for subsequent biosensor construction.

[0059] Gold electrode modification: A 2 mm diameter gold electrode was sequentially polished with alumina polishing paste (0.3 μm and 0.05 μm), followed by ultrasonic cleaning with ethanol and ultrapure water to remove surface impurities. Then, 10 μL of pre-assembled DNA tetrahedral solution (1.0 μmol / L) was drop-coated onto the electrode surface, and fixation was performed at room temperature for 12 hours using gold-thiol interactions. After rinsing with Tris-MgCl buffer to remove unbound DNA, the surface active sites were blocked with 1 mmol / L hexathiol for 30 minutes. Finally, the electrode was thoroughly rinsed with ethanol and ultrapure water, and the resulting biosensor was used for subsequent detection.

[0060] The aforementioned biosensor possesses a pH-responsive regeneration mechanism: when the sensor is immersed in an alkaline environment (pH≥10.0), the high pH environment causes the hydrogen bonds between the detection probe DNA and its complementary strand (or tetrahedral structure) to break, disrupting the interaction between the detection probe DNA fixed on the DNA tetrahedral structure and the electrode surface. Furthermore, under alkaline conditions, the phosphate backbone of the DNA strand carries more negative charge, enhancing the electrostatic repulsion between the strands and causing the detection probe DNA to dissociate from the electrode surface. This dissociation of the detection probe DNA restores the biosensor to its initial state, allowing it to be reused in subsequent detection cycles. Therefore, immersing the sensor of this invention in a buffer solution with pH≥10.0, followed by a brief incubation and rinsing, allows for the next round of detection. The sensor of this invention reversibly dissociates the probe under alkaline conditions and maintains stable sensor performance (>10 cycles), exhibiting high specificity, rapid response (completed within 2 hours), and modular expansion potential. Its synergistic effect of multi-stage signal amplification and intelligent regeneration design breaks through the limitations of traditional methods such as low sensitivity, high cost and single use, and provides an efficient and economical solution for clinical diagnosis, dynamic monitoring of chronic diseases and development of multi-target biosensing technology.

[0061] Example 2

[0062] This embodiment provides a method for detecting NF-κB p50 based on the above-mentioned biosensor. However, the required sensitivity still presents a significant challenge, necessitating the use of signal amplification strategies.

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

[0064] The method of the present invention is as follows Figure 4 As shown, it includes the following steps:

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

[0066] Protein binding and Exo III digestion: The S1 / S2 double-stranded solution (100 nM) was mixed with the sample containing NF-κB p50 to be tested, and incubated in PBS buffer at 37°C for 30 minutes, and then incubated 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 a solution containing intermediate DNA.

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

[0069] First, the H1 and H2 single strands were heated at 95°C for 5 minutes and then slowly cooled (4 hours) to room temperature to form a stable hairpin structure. Equal volumes of the above intermediate DNA solution were added to the H1 and H2 hairpin probes (1 μM) and incubated at 37°C for 2 hours. Then, Exo III (0.5 U / μL) was added for further digestion 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: Add 50 μL of reaction solution to the solution containing H1a / H2a double strands from Step 3 and mix thoroughly. The reaction solution contains 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. React for 30 minutes to complete the cleavage of the detection probe DNA. Then, immerse the regenerative biosensor in the reaction solution for 30 minutes and record the DPV signal. By comparing the DPV signal with a pre-established standard curve (NF-κB p50 content vs. DPV signal relationship curve) using the same detection method, NF-κB p50 can be quantitatively analyzed, thus accurately determining the concentration of this protein in the sample.

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

[0072] The method of this invention employs a reusable CRISPR / Cas12a biosensor, which integrates Exo III enzyme cascade amplification, CRISPR / Cas12a side branch cleavage, and DNA tetrahedral enhancement strategies to achieve ultrasensitive detection (femtomolar level) of NF-κB p50.

[0073] Example 3

[0074] In this embodiment, the Exo III addition amount in step one) is optimized according to the detection method in Example 2. The Exo III addition amount in step one) is modified to 0U, 10U, 20U, 30U, 50U, and 60U, respectively. All other experimental conditions are the same as in Example 2, and the DPV signal is as follows: Figure 1 As shown in Figure A.

[0075] according to Figure 1 The effect of Exo III concentration on DPV signal in A: 40 U was determined to be the optimal concentration because the enzyme would saturate at this concentration.

[0076] Example 4

[0077] In this embodiment, the Exo III reaction time in step two) was optimized according to the detection method in Example 2. The reaction time in step two) was modified to 0 min, 20 min, 40 min, 60 min, 80 min, and 1000 U, respectively, and samples containing 0.1 fM and 50 fM NF-κB p50 were detected. All other experimental conditions were the same as in Example 2, and the DPV signal was as follows. Figure 1 As shown in B.

[0078] according to Figure 1 As shown in Figure B, the effect of Exo III reaction time on the DPV signal is shown. The reaction time stabilizes at 60 minutes, which is the optimal reaction time.

[0079] Example 5

[0080] In this embodiment, the detection method in Example 2 was followed, and the amount of CRISPR-Cas12a / crRNA added in step four) was optimized. The amount of CRISPR-Cas12a / crRNA added in step four) was modified to 0 nM, 5 nM, 10 nM, 20 nM, 40 nM, and 60 nM, respectively. All other experimental conditions were the same as in Example 2, and the DPV signal was as follows: Figure 1 As shown in C.

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

[0082] Example 6

[0083] In this embodiment, the reaction time of the Cas12a-mediated lysis detection probe DNA in step four) was optimized according to the detection method in Example 2. The reaction time in step four) was modified to 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively. All other experimental conditions were the same as in Example 2, and the DPV signal was as follows: Figure 1 As shown in D.

[0084] according to Figure 1 As shown in Figure D, the effect of Cas12a-mediated lysis time on the DPV signal is analyzed. The signal reaches a plateau at 30 minutes, which is the optimal signal amplification time. These optimizations collectively ensure the high sensitivity and high specificity of this biosensor in NF-κB p50 detection.

[0085] Experimental Example 1

[0086] This experimental example uses the detection method of Example 2 to detect a series of different concentrations of NF-κB p50 to clarify the detection performance of the method of the present invention.

[0087] Figure 2 This describes the detection performance of a biosensor based on differential pulse voltammetry (DPV) for NF-κB p50. Figure 2 A represents the relationship between NF-κB p50 concentration and DPV signal intensity. Figure 2 B represents the linear correlation between the logarithm of NF-κB p50 concentration and the DPV signal. The results showed that the signal significantly decreased with increasing concentration, with the lowest signal observed at a concentration of 100 femtomolars (fM). This result reflects a highly efficient triple signal amplification mechanism comprised of DNA cleavage mediated by exonuclease III (Exo III), hairpin probe hybridization, and CRISPR-Cas12a activation.

[0088] Experimental Example 2

[0089] This experimental example uses the method described in Example 2 to detect various proteins to clarify the specificity of the method of the present invention. Furthermore, the pH response mechanism of the biosensor was tested.

[0090] Figure 3 This is a graph showing the specificity and pH-responsive regeneration results of the CRISPR-Cas12a-based differential pulse voltammetry (DPV) biosensor. First, based on the testing method of Example 2, the above-mentioned 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 Figure A, the biosensor exhibits high specificity. For nuclear factor κB p50 (100,000 aM), the DPV signal is significantly reduced, 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) are similar to those of the blank control.

[0091] The biosensor was alternately placed in a solution containing the detection probe DNA and in TAE buffer at pH 10 to test its reusability. Results are as follows: Figure 3 As shown in Figure B, the pH-induced regeneration process restored the biosensor's performance under alkaline conditions (pH 10.0), with a 99.2% signal reduction, indicating that the biosensor was effectively recovered. Furthermore, the consistent regeneration rate exceeding 94.7% across five cycles confirms its stability and reusability.

[0092] Experimental Example 3

[0093] To evaluate the practical applicability of the developed pH-induced regeneration 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. NF-κB p50 was quantified by comparing the DPV response with a pre-established calibration curve, enabling precise determination of the protein concentration in the sample.

[0094] The performance of this biosensor in this biologically relevant environment was evaluated by analyzing recoveries and relative standard deviations (RSDs), ensuring accurate detection even in the presence of potential interfering factors. As shown in Table 1, the biosensor exhibited excellent reproducibility and precision, with all RSD values ​​below 6%. Recovery rates ranged from 99.61% to 106.23%, confirming that the system can reliably detect and quantify NF-κBp50 in diluted human serum samples.

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

[0096] Table 1. Measurement of NF-κB p50 (n=3) in human serum diluted 10-fold using the proposed biosensor

[0097]

[0098] The practical application of this biosensor in serum samples demonstrates its suitability for real-world diagnostic scenarios where accurate and precise detection of low-abundance proteins is crucial. Even in challenging environments, the DPV biosensor maintains high recovery and reproducibility, making it a promising tool for clinical diagnostics and disease monitoring, particularly for detecting NF-κB p50 in patient samples.

[0099] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting NF-κB p50, characterized in that, Includes the following steps: Step 1) Assemble probe S1 and probe S2 to form S1 / S2 double strands, then mix the S1 / S2 double strands with the NF-κB p50-containing sample to be tested, allowing the S1 / S2 double strands to bind to NF-κB p50 and form a probe-NF-κB p50 conjugate; then add ExoIII enzyme to decompose the S1 / S2 double strands that are not bound to NF-κB p50, obtaining a mixture; the nucleic acid sequence of probe S1 is shown in SEQ ID No. 6, and the nucleic acid sequence of probe S2 is shown in SEQ ID No. 7; Step 2) Add the hairpin probe to the mixture. The probe-NF-κB p50 ligand 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 shown in SEQ ID No.

8. (Step 3) Equal volumes of the solution containing intermediate DNA are mixed and reacted with H1 single-stranded DNA and H2 single-stranded DNA, respectively, to obtain solutions containing H1a single-stranded DNA and H2a single-stranded DNA, respectively; the solutions containing H1a single-stranded DNA and H2a single-stranded DNA are then mixed and reacted to obtain a double-stranded solution containing H1a / H2a; the nucleic acid sequence of the H1 single-stranded DNA is shown in SEQ ID No. 9, the nucleic acid sequence of the H2 single-stranded DNA is shown in SEQ ID No. 10, the nucleic acid sequence of the H1a single-stranded DNA is shown in SEQ ID No. 11, and the nucleic acid sequence of the H2a single-stranded DNA is shown in SEQ ID No.

12. Step 4) The H1a / H2a double-stranded solution is reacted with the CRISPR-Cas12a / crRNA complex and the detection probe DNA to obtain the reaction product. The nucleic acid sequence of the crRNA is shown in SEQ ID No.

13. The reaction product is electrochemically detected using a biosensor, and the NF-κB p50 content of the sample containing NF-κB p50 is obtained based on the electrochemical detection results. The biosensor includes: An electrode, the surface of which is bound with a DNA tetrahedral structure; the DNA tetrahedral structure is used to bind to a detection probe DNA; the detection probe DNA includes a detection probe DNA nucleic acid sequence and an electrochemical group connected to the detection probe DNA nucleic acid sequence; the electrochemical group is a ferrocene group; the ferrocene group is connected to the 3' end of the detection probe DNA nucleic acid sequence; The DNA tetrahedral structure is formed by assembling DNA strands T1, T2, T3, and T4; the nucleic acid sequence of DNA strand T1 is shown in SEQ ID No. 1; the nucleic acid sequence of DNA strand T2 is shown in SEQ ID No. 2; the nucleic acid sequence of DNA strand T3 is shown in SEQ ID No. 3; and the nucleic acid sequence of DNA strand T4 is shown in SEQ ID No.

4. The DNA nucleic acid sequence of the detection probe is shown in SEQ ID No.

5.

2. The method according to claim 1, characterized in that, The electrode is a gold electrode; the 5' ends of DNA strands T2, T3, and T4 are all connected to thiol groups.

3. The method according to claim 1, characterized in that, The preparation method of biosensors includes the following steps: DNA strands T1, T2, T3, and T4 were mixed and incubated at pH 8.0-9.0 and a temperature of 94.8-95.2°C for 10-12 minutes, then cooled to 3.8-4.2°C to assemble a DNA tetrahedral structure. The DNA tetrahedral structure is coated onto the electrode, thereby binding the DNA tetrahedral structure to the electrode surface.

4. The method according to claim 1, characterized in that, The method further includes: 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.

5. The method according to claim 4, characterized in that, The pH of the alkaline solution is 9.8 to 10.

2.

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

2.

7. The method according to claim 1, characterized in that, In step one), probe S1 and probe S2 are mixed in 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 S1 / S2 double strands. The S1 / S2 double strands are added to the sample containing NF-κB p50 to be tested, and incubated at 36-38°C for 30-32 minutes to form probe-NF-κB p50 binding complexes. Then, Exo III enzyme is added and the reaction is continued for 30-32 minutes to decompose the S1 / S2 double strands that are not bound to NF-κB p50. The concentration of S1 / S2 double strands in the NF-κB p50 sample to be tested was 100 nmol / L; the concentration of Exo III enzyme in the NF-κB p50 sample to be tested was 40 U~60 U. The hybridization buffer is a TAE buffer with a pH of 7.

4.

8. The method according to claim 1, characterized in that, In step two), 480-520 nmol / L hairpin probes are added to the mixture and incubated at 36-38°C for 60-100 minutes. The nucleic acid sequence of the intermediate DNA is shown in SEQ ID No.

14.

9. The method according to claim 1, characterized in that, In step three), the H1 single chain and the H2 single chain are first heated at 94~96°C for 4~6 minutes and then cooled to room temperature to form a stable hairpin structure. The H1 and H2 single strands of the hairpin structure were incubated with the intermediate DNA at 36-38°C for 115-125 min; then ExoIII enzyme was added and the reaction was carried out for 58-62 min to obtain solutions containing the H1a fragment and 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 the H1a / H2a double strand.

10. The method according to claim 1, characterized in that, In step four), the H1a / H2a double-stranded solution is reacted with the CRISPR-Cas12a / crRNA complex and the detection probe DNA, specifically including: 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 for 30-60 minutes. The reaction solution in step four) contains Tris-HCl, KCl, MgCl2, glycerol, and DTT; In step four), differential pulse voltammetry is used to electrochemically detect the reaction products.

11. The method according to claim 10, characterized in that, In step four), the 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 mixed for 30 minutes.

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