A self-driven hybridization chain reaction-based method for detecting sequence combination of biomolecules, a kit and application thereof

Abstract

The application provides a nucleic acid sequence and a method for detecting biomolecules based on a self-driven hybrid chain reaction (SHCR), which comprises a priming chain AI, a blocking chain B, a hairpin probe H1, a hairpin probe H2, a hairpin probe H3 and a hairpin probe H4, the priming chain AI comprises an aptamer sequence of a to-be-detected biomolecule and a starting sequence I, the sequence of the starting sequence I is a*‑b*, wherein a* and b* are gene fragments; the 5' end of the hairpin probe H1 is connected with b*, and the 3' end of the hairpin probe H3 is connected with a*. The application further provides a kit for detecting biomolecules based on the SHCR and a method for detecting biomolecules, in particular, a method for detecting a kanamycin residual amount in food. Through ingenious design, when the to-be-detected biomolecule exists, the sequence combination of the above biomolecule can initiate the self-driven hybrid chain reaction, the priming chain can be autonomously supplemented, the self-driven reaction process is reversely activated, thereby a more significant FRET signal is generated, and the detection sensitivity is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular detection technology, specifically relating to a sequence combination, reagent kit, and application of biomolecules based on a self-driven hybridization chain reaction. Background Technology

[0002] Kanamycin, an aminoglycoside antibiotic with potent antibacterial activity, is widely used to treat various serious infections by interfering with bacterial protein expression. However, inappropriate use or abuse of kanamycin can lead to many adverse effects in humans, such as ototoxicity, nephrotoxicity, and antibiotic resistance. To ensure consumer safety, many countries have set maximum residue limits (MRLs) for kanamycin in different foods. For example, the maximum MRL for kanamycin in animal-derived foods is 100 μg / kg in muscle, 150 μg / kg in cow and sheep milk, and 600 μg / kg in liver.

[0003] Conventional analytical methods for determining kanamycin mainly include high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), enzyme-linked immunosorbent assay (ELISA), and gas chromatography-mass spectrometry (GC-MS). However, these methods typically require cumbersome operations, are time-consuming, and involve expensive equipment. Recently, other methods such as spectrophotometry, electrochemical methods, and surface-enhanced Raman scattering (SERS) biosensors have been developed for kanamycin detection. However, they are still hampered by complex nanomaterial modification and insufficient signal gain. Due to the high specificity, programmability, and stability of DNA, some DNA-based signal amplification strategies have been used for highly sensitive kanamycin detection. However, these methods typically require expensive enzymes, are susceptible to external environmental interference, have limited stability, and involve cumbersome reactions. Therefore, researchers have developed isothermal non-enzymatic amplification techniques, such as cascade hybridization (HCR), catalytic hairpin self-assembly (CHA), and DNAzyme catalytic reactions. To improve signal amplification capabilities, most nucleic acid isothermal amplification techniques focus on integrating different nucleic acid amplification technologies to develop various cascaded DNA nanosystems, but these systems still suffer from low signal amplification capabilities and efficiency. Summary of the Invention

[0004] To address the aforementioned problems, the first aspect of this invention provides a method for detecting nucleic acid sequences of biomolecules based on a self-driven hybridization chain reaction (SHCR), comprising an initiating chain AI, a blocking chain B, hairpin probes H1, H2, H3, and H4. The initiating chain AI includes an aptamer sequence of the biomolecule to be tested and a starting sequence I, wherein the sequence of the starting sequence I is a*-b*, where a* and b* are gene fragments. The 5' end of the hairpin probe H1 is attached to b*, and the 3' end of the hairpin probe H3 is attached to a*. Each hybrid H1-H3 generated during the self-driven hybridization chain reaction forms a catalytically active assembly unit a*-b*, and each assembly unit a*-b* can serve as an initiating chain, thereby accelerating the reaction process.

[0005] Furthermore, hairpin probes H1-H4 are all hairpin structures, with the stem of each hairpin structure consisting of a double strand formed by the complementarity of b and b*; the closed strand B includes the complementary strand of the start sequence I; hairpin probe H1 includes the sequence b*-abcb*, where a and c are gene fragments, c is the loop of the hairpin structure, and a is the 5' single-stranded sticky end extending from the stem of the hairpin structure; hairpin probe H2 includes the sequence b*-dbc*, and is labeled with the fluorophore Cy3 at the 5' end, where d is a gene fragment, d is... The hairpin structure has a loop portion, and c* is a 3' single-stranded sticky end extending from the stem portion of the hairpin structure; hairpin probe H3 includes the sequence d*-beb*-a*, where e is a gene fragment, e is the loop portion of the hairpin structure, and d* is a 5' single-stranded sticky end extending from the stem portion of the hairpin structure; hairpin probe H4 includes the sequence b*-a*-be*, and is labeled with the fluorophore Cy5 at the 3' end, where a* is the loop portion of the hairpin structure, and e* is a 3' single-stranded sticky end extending from the stem portion of the hairpin structure.

[0006] Furthermore, the aptamer sequence is the aptamer sequence of kanamycin.

[0007] Furthermore, the sequences of the hairpin probes H1-H4 are shown in SEQ ID No. 1-4.

[0008] Furthermore, the sequence that triggers chain AI is shown in SEQ ID No. 5, and the sequence that closes chain B is any one of SEQ ID Nos. 6-9.

[0009] The second aspect of this application provides a kit for detecting biomolecules based on a self-driven hybridization chain reaction, comprising a sequence combination of any one of the first aspects.

[0010] The third aspect of this application provides a method for biomolecular detection using sequence combinations provided in any one of the first aspects or a kit provided in the second aspect.

[0011] Furthermore, biomolecular detection methods are used to detect kanamycin residues in food.

[0012] Furthermore, the specific steps of the biomolecular detection method include: mixing the initiating chain AI and the blocking chain B in hydroxyethylpiperazine thiosulfate buffer, incubating at room temperature to obtain the complex AI / B, then mixing the complex AI / B and hairpin probes H1-H4 with the analyte, incubating at room temperature, and measuring the fluorescence intensity of the reaction system.

[0013] Furthermore, the concentration ratio of closed chain B to initiating chain AI is 1.5.

[0014] This invention designs a sequence combination, kit, and method for detecting biomolecules, particularly kanamycin residues in food, based on a self-driven hybridization chain reaction (SHCR). Through ingenious design, the sequence combination of the biomolecules in the presence of the analyte can initiate a self-driven hybridization chain reaction, allowing the initiating chain to self-replenish and reverse-activate the self-driven reaction process, leading to accelerated continuous reactions and thus improving the signal amplification capability of the sensing system until all participating reactants are exhausted. Compared to traditional hybridization chain reactions (HCR), SHCR generates a more significant FRET signal, improving detection sensitivity. A highly sensitive and reliable method based on SHCR can be established to monitor kanamycin residues in food, which is of great significance for ensuring food safety and consumer health. Attached Figure Description

[0015] For ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0016] Figure 1 This is a schematic diagram illustrating the principle of biomolecule detection based on self-driven hybridization chain reaction in one embodiment of the present invention.

[0017] Figure 2 This is a comparative analysis diagram of reaction systems based on HCR and SHCR in another embodiment of the present invention;

[0018] Figure 3 This is a comparison of fluorescence analysis of reaction systems based on HCR and SHCR in another embodiment of the present invention;

[0019] Figure 4 This is a comparative diagram of the selectivity test of the SHCR-based reaction for Kanamycin detection in another embodiment of the present invention;

[0020] Figure 5 This is an optimization result of the closed chain in the SHCR system in another embodiment of the present invention. Detailed Implementation

[0021] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the embodiments and features described herein can be combined with each other.

[0022] Table 1 lists the sequence information for each of the following embodiments.

[0023] Table 1 DNA Sequence Information

[0024]

[0025]

Example 1

[0026] Figure 1 This is a schematic diagram illustrating the principle of self-driven hybridization chain reaction detection of the biomolecule Kanamycin in one embodiment of the present invention. Hairpin probes were designed using NUPACK software, and the relevant nucleic acid sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The structure of the hairpin probes is as follows: Figure 1 As shown in the dashed box on the right, where:

[0027] The initiation chain AI consists of the aptamer sequence of Kanamycin and the initiation sequence I. The sequence of the initiation sequence I is a*-b*, where a* and b* are gene fragments.

[0028] Closed chain B consists of the complementary chain of the starting sequence I;

[0029] The hairpin probes H1-H4 are all hairpin structures, with the stem of each hairpin structure consisting of a double strand formed by complementary b and b* bases. In the absence of target biomolecules, each hairpin structure has sufficient complementary base pairing in its stem to maintain its stability and fluorescence. However, in the presence of target biomolecules, it can initiate a self-driven hybridization chain reaction, forming a network of DNA nanostructures, which undergo fluorescence resonance energy transfer to achieve the detection of the target.

[0030] The hairpin probe H1 consists of five parts: b*, a, b, c, and b*, forming the sequence b*-abcb*. c is the loop of the hairpin structure, a is the 5' single-stranded sticky end extending from the stem of the hairpin structure, and the 5' end of H1 is connected to a split I(a*-b*) fragment (b*).

[0031] The hairpin probe H2 consists of four parts: b*, d, b, and c*, forming the sequence b*-dbc*. Its 5' end is labeled with the fluorophore Cy3, d is the loop part of the hairpin structure, and c* is the 3' single-stranded sticky end extending from the stem part of the hairpin structure.

[0032] The hairpin probe H3 consists of five parts: d*, b, e, b*, and a*, forming the sequence d*-beb*-a*. e is the loop of the hairpin structure, d* is the 5' single-stranded sticky end extending from the stem of the hairpin structure, and the 3' end of H3 is connected to a split I(a*-b*) fragment (a*).

[0033] The hairpin probe H4 consists of four parts: b*, a*, b, and e*, forming the sequence b*-a*-be*. Its 3' end is labeled with the fluorophore Cy5. a* is the loop part of the hairpin structure, and e* is the 3' single-stranded sticky end extending from the stem of the hairpin structure.

[0034] like Figure 1 As shown, in the absence of the target biomolecule Kanamycin, AI hybridizes with B to form the AI / B complex to prevent signal leakage caused by the release of the initiating sequence I. The AI / B complex, hairpin probes H1, H2, H3, and H4 maintain their stability and cannot undergo the HCR reaction. In the presence of the target biomolecule Kanamycin, the aptamer sequence in the AI / B complex immediately binds to the target biomolecule, leading to the exposure of the initiating sequence I and initiating the HCR reaction. The initiating sequence I opens the hairpin probe H1 based on the Toehold-mediated chain substitution reaction principle, generating the intermediate I-H1, thereby exposing the sequence cb*, which hybridizes with H2 to generate the intermediate I-H1-H2. After H2 is opened, the released sequence b*-d can hybridize with H3 to generate the intermediate I-H1-H2-H3, bringing the two split a*-b* unit fragments closer together to form a complete catalytically active assembly unit a*-b*. H3 exposes the eb* sequence, which can open H4 to generate the intermediate product I-H1-H2-H3-H4, bringing the fluorophores Cy3 and Cy5 closer together, thus inducing fluorescence resonance energy transfer and providing signal output. Once opened, H4 releases the same sequence a*-b* as the starting sequence I, which then opens the next H1. Therefore, the starting sequence I can initiate alternating hybridization between H1, H2, H3, and H4, generating double-stranded DNA nanowires I-(H1-H2-H3-H4). N This generates a large number of catalytically active assembly units a*-b* and FRET signals. Each assembly unit a*-b* can serve as a starting sequence to accelerate the cross-catalytic reaction, continuously activating the HCR reaction and initiating the SHCR reaction, thereby generating more assembly units a*-b* and significant FRET signals. Therefore, the SHCR amplification reaction proposed in this embodiment exhibits exponential amplification efficiency (1:N). N The amplification efficiency is far higher than that of traditional HCR (1:N), thus greatly improving the detection sensitivity of Kanamycin.

[0035] The above principle can also be applied to other biomolecules such as nucleic acids and proteins besides Kanamycin. As long as a suitable initiating chain AI and a blocking chain B are designed to expose the initiating sequence I, a self-driven hybridization chain reaction can be initiated.

[0036]

Example 2

[0037] This embodiment is based on a self-driven hybridization chain reaction for the detection of Kanamycin. The specific steps include: mixing the initiating chain AI and the blocking chain B in hydroxyethylpiperazine thiosulfate (HEPES) buffer and incubating at room temperature for 30 min to obtain the AI / B complex; then mixing the AI / B complex and hairpin probes H1-H4 with the analyte containing the target biomolecule Kanamycin and incubating at room temperature for 3 h; and measuring the fluorescence intensity of the reaction system. The concentration of the initiating chain AI is 100 nM, the concentration of the blocking chain B is 150 nM, and the concentration of the hairpin probes H1-H4 is 100 nM each.

[0038]

Example 3

[0039] This embodiment compares the conventional HCR and the SHCR reaction system proposed in this application. The conventional HCR uses a hairpin probe sequence of H... 1T +H2+H 3T +H4, the hairpin probe sequence of SHCR is H1+H2+H3+H4, H1-H4, H 1T H 3T All were 100 nM. All hairpin probe powders were first dissolved in phosphate buffer, and their absorbance was measured using a UV spectrophotometer to calculate the accurate concentration. Then, all hairpin probes were prepared to 4 μM using HEPES buffer and reacted in PCR at 95°C for 5 min and 25°C for 2 h to allow for stable hairpin formation. All reactions were carried out in HEPES buffer with a concentration of 10 mM, pH 7.2, containing 1 M NaCl and 50 mM MgCl2. Subsequently, the reaction was performed according to the method in Example 2.

[0040] Figure 2 (A) is a comparison chart of the results of fluorescence spectroscopy analysis of the HCR and SHCR reaction systems in this embodiment. Figure 2 (A) It can be seen that when the initiator Kanamycin is added to the SHCR system lacking H1 or H3 hairpins, the fluorescence of the system does not change. Figure 2 (A) curves b and d). The effect of self-assembled I(a*-b*) on the overall scale-up reaction was investigated by replacing the split I(a*-b*) fragments (a*) and (b*) in hairpin probes H1 and H3 with adenine deoxynucleotides. 1T and H 3TWhen the HCR system replacing H1 and H3 was incubated with the analyte for a typical conventional HCR amplification reaction, a relatively low fluorescence response was obtained. Figure 2 (A) curve f), while the complete SHCR system produced a significant fluorescence response when analyzing the target analyte Kanamycin. Figure 2 (A) curve h). These results demonstrate that the self-driven hybridization chain reaction proposed in this embodiment has significant signal amplification capability.

[0041] Figure 2 (B) is a schematic diagram showing the results of characterizing SHCR products using gel electrophoresis. The incubated reaction solution was mixed with loading buffer and then added to a 12% acrylamide gel. The electrophoresis apparatus voltage was set to 120V. After 3.5 hours, the gel was removed and stained with GelRed. Finally, the DNA was visualized under ultraviolet light using a chemiluminescence imaging system. The electrophoresis results are as follows: Figure 2 As shown in (B), where a and b are the results of conventional HCR electrophoresis, and c and d are the results of SHCR electrophoresis, specifically as follows: (a) Background of the HCR system, without Kanamycin; (b) HCR system, with 100 nM Kanamycin added; (c) Background of the SHCR system, without Kanamycin; (d) SHCR system, with 100 nM Kanamycin added. Figure 2 (B) It can be seen that, due to the continuous accumulation of the initiation chain used to activate the HCR reaction in the SHCR system, the SHCR system activated by the target analyte produces more high molecular weight products compared to the traditional HCR system.

[0042] Figure 2 (C) and (D) are schematic diagrams showing the results of characterizing the SHCR products using atomic force microscopy. Freshly peeled mica sheets were pretreated with 90 μL of (3-aminopropyl)trimethoxysilane (APTES) and 30 μL of N,N-diisopropylethylamine (DIPEA) vapor for 2 h to impart a positive charge; this process was performed in a desiccator. The incubated reaction solution sample was diluted to 20 nM and dropped onto the mica sheets. After 15 min, it was washed three times with ultrapure water, dried under nitrogen, and scanned using an atomic force microscope. The results are shown below. Figure 2 As shown in (C) and (D), the Kanamycin-induced SHCR system yielded a large number of micrometer-long linear DNA nanostructures with a height of approximately 2 nm. Figure 2 C), while in the SHCR system without Kanamycin, only tiny spots of individual hairpins were observed. Figure 2 D).

[0043] Figure 2This demonstrates the successful construction of the cross-catalyzed SHCR system and the significant signal amplification capability of the SHCR system.

[0044] Figure 3 This is a graph showing the fluorescence intensity of the reaction system measured by a fluorescence spectrometer. Figure 3 (A) It can be seen that when Kanamycin is not added to the SHCR system, each DNA probe can maintain its own stability, and the ratio fluorescence of the system only undergoes a slight change. Figure 3 Curve a) in (A) shows that when different concentrations of Kanamycin are added, the change in ratio fluorescence intensity is positively correlated with the concentration of Kanamycin, which can be used to detect Kanamycin. Figure 3 (B) As can be seen, with the increase of Kanamycin concentration, the fluorescence intensity of the system gradually decreases at (λ = 565 nm) and gradually increases at (λ = 670 nm). This change in fluorescence intensity shows a good linear relationship with the Kanamycin concentration in the range of 50 pM-10 nM, and the detection limit is 40 pM. Figure 3 (C) demonstrates that this embodiment can achieve rapid and highly sensitive detection of Kanamycin. The SHCR system and the traditional HCR system were simultaneously used to detect different concentrations of target DNA molecules, and the results are as follows: Figure 3 As shown in (D), it can be seen that SHCR amplifies the reaction further compared to the traditional HCR amplification reaction, and the SHCR system has higher sensitivity and better detection effect.

[0045]

Example 4

[0046] Enrofloxacin (ENR), chloramphenicol (CAP), tetracycline (TET), sulfadiazine (SMT), and ampicillin (AMP) were selected as interfering components to investigate the selectivity of this method for the detection of kanamycin. The results are as follows: Figure 4 As shown. By Figure 4 (A, B) It can be seen that the fluorescence of the system only changes significantly when it reacts with Kanamycin. The fluorescence changes caused by ENR, CAP, TET, SMT and AMP are very small, indicating that this method has good selectivity for the detection of Kanamycin.

[0047]

Example 5

[0048] A self-driven hybridization chain reaction-based method for the analysis of Kanamycin was applied to the detection of Kanamycin in milk powder samples. Milk powder samples were diluted with ultrapure water to obtain the desired concentration. Spiked samples were prepared by adding the required amount of Kanamycin to the diluted samples. The final amounts of Kanamycin added to the milk powder were 100, 500, and 1000 pM. Before detection, the pH of the samples was adjusted to 4.6 by adding 20% ​​glacial acetic acid and incubated in water at 45°C for 10 minutes. Subsequently, the samples were centrifuged at 5000 rpm for 20 minutes to remove protein and fat. Finally, the filtrate was filtered through a 0.22 μm filter, and the pH was adjusted to 7.4 to obtain the milk powder sample to be tested. After the reaction was carried out according to the method in Example 2, the fluorescence intensity of the system was measured using a fluorescence spectrometer, and the Kanamycin content was determined by the strength of the FRET signal. Using a sensor based on the SHCR aptamer, the initial Kanamycin value of the milk powder sample used in the spiked recovery experiment was determined to be 406.2 pM. Meanwhile, the samples were also tested using a commercial ELISA kit as a control.

[0049] The application potential of the SHCR detection system in real-world samples was evaluated by analyzing milk powder samples contaminated with Kanamycin and supplemented with different concentrations of Kanamycin standards. As shown in Table 2, the recoveries from milk powder ranged from 97.8% to 102.3%, with an RSD of less than 5%. Furthermore, the reliability of the SHCR method proposed in this invention was compared with that of a commercial Kanamycin-ELISA kit; no significant differences were observed between the two methods.

[0050] Table 2 Comparison of SHCR and ELISA kit results in detecting kanamycin in milk powder samples.

[0051]

[0052] In summary, the above embodiments demonstrate that SHCR-based sensors can reliably determine antibiotics in real samples. The flexibility and programmability of SHCR sensing systems facilitate the extensive exploration of various biomolecules or proteins in real samples, and they have great potential in food analysis.

[0053]

Example 6

[0054] In this embodiment, closed chains B1-B4 were designed and optimized experimentally. Figure 5 The optimization results for the closed chain are as follows, Figure 5 (A) Design of DNA probes for closed strands B1, B2, B3, and B4; Figure 5(B) FRET signals of the SHCR sensing platform in the presence of different closed chains; (C) FRET signals of the SHCR sensing platform with different B / AI ratios; (D) fluorescence kinetic curves of the SHCR sensing platform in (a) without kanamycin and (b) with 100 nM kanamycin. It can be seen that among the closed chains B1-B4, closed chain B3 is optimal; the optimal B / AI ratio is 1.5.

[0055] Although the contents of this application have been specifically shown and described in conjunction with preferred embodiments, those skilled in the art should understand that any changes in form and detail made to this application without departing from the spirit and scope of this application as defined by the appended claims and without inventive effort are within the scope of protection of this application.

Claims

1. A kit for detecting a biomolecule based on a self-driven hybridization chain reaction, characterized by comprising: comprises an adaptor sequence of a biological molecule to be detected and a starting sequence I, the sequence of the starting sequence I is wherein are all gene fragments; the 5' end of the hairpin probe H1 is connected with , the 3' end of the hairpin probe H3 is connected with ; The hairpin probes H1-H4 are all hairpin structures, the stems of which are all b and complementary to form a double strand; The hairpin probe H1 comprises a sequence wherein a and c are both gene fragments, c is a loop of the hairpin structure, and a is a 5' single-stranded sticky end extending from the stem of the hairpin structure. The hairpin probe H2 comprises a sequence and is labeled with a fluorophore Cy3 at the 5' end, wherein d is a gene fragment, d is a loop of the hairpin structure, a 3' single-stranded sticky end extending from the stem of the hairpin structure; The hairpin probe H3 comprises the sequence wherein e is a gene fragment, e is a loop of the hairpin structure, a 5' single-stranded sticky end extending from the stem of the hairpin structure; The hairpin probe H4 comprises the sequence and is labeled at the 3' end with the fluorophore Cy5, wherein is the loop part of the hairpin structure, is the 3' single-stranded sticky end extending from the stem part of the hairpin structure; The sequences of the hairpin probes H1-H4 are shown as SEQ ID No. 1-4.

2. The kit for detecting a biomolecule based on a self-driven hybridization chain reaction according to claim 1, wherein The blocking chain B comprises a complementary strand of the initiation sequence I.

3. The kit for detecting biomolecules based on self-driven hybridization chain reaction according to claim 1, wherein, The aptamer sequence is an aptamer sequence of kanamycin.

4. The kit for detecting biomolecules based on self-driven hybridization chain reaction according to claim 1, wherein, The sequence of the initiation chain AI is shown as SEQ ID No. 5, and the sequence of the blocking chain B is any one of SEQ ID No. 6-9.

5. A method for detecting biomolecules in food using the kit of any one of claims 1-4.

6. The method of claim 5, wherein, The method is used for detecting kanamycin residues in food.

7. The method of claim 6, wherein, The specific steps include: mixing the initiation chain AI and the blocking chain B in hydroxyethylpiperazine ethanesulfonic acid buffer, obtaining the complex AI / B after incubation at room temperature, mixing the complex AI / B and the hairpin probes H1-H4 with the test substance, and measuring the fluorescence intensity of the reaction system after incubation at room temperature.

8. The method of claim 7, wherein, The concentration ratio of the blocking chain B to the initiation chain AI is 1.5.

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