G-quadruplex structure switch-type aptamer sensor for detecting enrofloxacin and application thereof

Abstract

The application relates to the technical field of biochemistry, in particular to a G-quadruplex structure switch type aptamer sensor for detecting enrofloxacin and an application thereof. The nucleic acid sequence of the G-quadruplex structure switch type aptamer comprises a target aptamer and a G-rich DNA sequence, the G-rich DNA sequence comprises two fragments assembled at two ends of the target aptamer, which can not only provide a high-sensitivity signal reporting function, but also maintain the ability of the aptamer to specifically recognize a target. The G-quadruplex structure switch type aptamer is mixed with a fluorescent probe to construct a G-quadruplex structure switch type aptamer sensor, which is applied to the detection of enrofloxacin, can effectively improve the weak change problem of the binding structure of the enrofloxacin aptamer and the target, has a wide linear range and a low detection limit, can realize quantitative analysis of enrofloxacin in various food and water samples, and has great application potential in actual sample detection.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically, to a G-quadruplex switch-type nucleic acid aptamer sensor for detecting enrofloxacin and its application. Background Technology

[0002] Enrofloxacin (ENR), also known as ethylciprofloxacin, is a synthetic third-generation quinolone broad-spectrum antibiotic. Due to its low cost and good antibacterial activity, it is widely used as an animal-specific antibacterial agent in livestock and aquaculture. However, the misuse of enrofloxacin leads to its residues entering the food chain and environment, posing potential threats to ecosystems and human health, such as allergic reactions, teratogenic / carcinogenic mutations, and antibiotic resistance. Therefore, sensitive and accurate analysis of enrofloxacin residues in various foods and the environment is crucial for protecting public health. Existing methods for enrofloxacin detection mainly include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis, and enzyme-linked immunosorbent assay (ELISA). Although the accuracy of these methods is widely recognized, the need for expensive instruments, specialized operators, and longer processing times limits their practical application.

[0003] Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotides obtained through in vitro exponential enrichment ligand system evolution techniques. Compared to antibodies, nucleic acid aptamers offer advantages in stability, synthesis, and modification, and possess unique properties such as high affinity and strong specificity for their targets. Combined with various signal transduction methods (colorimetric, fluorescence, and electrochemical), nucleic acid aptamer-based sensors have become promising analytical tools in environmental analysis, food testing, and clinical diagnostics.

[0004] Sensitive label-free fluorescent aptamer sensors rely on significant structural changes in the nucleic acid aptamer before and after target binding, and the environmentally sensitive G-quadruplex (G4) can meet this requirement. Aptamer sequences rich in guanine (G) bases are found in K... + Na + Enrofloxacin aptamers can fold into a G4 structure in the presence of monovalent cations. To date, this G4-mediated antibiotic detection strategy, which provides significant structural changes through target binding, has become a research hotspot. However, currently reported enrofloxacin aptamer sequences cannot form a G4 structure, and their structural changes after target binding are weak. This makes the construction of enrofloxacin detection systems complex and expensive, hindering direct detection using fluorescent probes. Therefore, developing a simple, sensitive, cost-effective method based on G4 technology for the rapid detection of enrofloxacin is of great significance. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in related technologies. To this end, this invention provides a G-quadruplex-structured switchable nucleic acid aptamer sensor for detecting enrofloxacin and its application. The G-quadruplex-structured switchable nucleic acid aptamer sensor includes a G-quadruplex-structured switchable nucleic acid aptamer and a fluorescent probe. The nucleic acid sequence of the G-quadruplex-structured switchable nucleic acid aptamer includes a target aptamer and a G-rich DNA sequence. The G-rich DNA sequence splits into two fragments, which are respectively assembled at both ends of the target aptamer. The middle target aptamer is responsible for target recognition, while the two G-rich DNA fragments at both ends are responsible for signal reporting. This not only provides a highly sensitive signal reporting function but also maintains the aptamer's ability to specifically recognize the target. Applying this sensor to the detection of enrofloxacin can effectively improve the problem of weak structural changes in the binding of the enrofloxacin aptamer to the target, enabling quantitative analysis of enrofloxacin in various food and water samples, and has great application potential in practical sample detection.

[0006] To this end, the first aspect of the present invention provides a G-quadruplex structured switch-type nucleic acid aptamer, wherein the nucleic acid sequence of the G-quadruplex structured switch-type nucleic acid aptamer includes a target aptamer and a G-rich DNA sequence, wherein the G-rich DNA sequence includes two fragments, which are respectively assembled at both ends of the target aptamer;

[0007] The G-rich DNA sequence is 12–32 bp in length and includes at least four guanine sequence groups, with each fragment including at least two guanine sequence groups, and each guanine sequence group including at least three consecutive guanines.

[0008] This invention addresses the challenge of developing detection probes for enrofloxacin aptamers due to their inability to form G-quadruplex structures. It constructs a G-quadruplex-structured on / off nucleic acid aptamer sensor by engineering G-rich DNA sequences at both ends of the aptamer, thereby altering the original aptamer's conformation to a G4 structure. The constructed on / off nucleic acid aptamer comprises two regions: G4 regions at both ends responsible for signal reporting and a central aptamer region responsible for target identification. When mixed with the sample, the aptamer regions specifically bind to the sample, causing the G-quadruplex structure formed by the G-rich DNA sequences at both ends to open, thus enabling rapid detection of the sample.

[0009] According to an embodiment of the present invention, the nucleic acid sequence of the target aptamer is SEQ ID NO.16.

[0010] According to embodiments of the present invention, the G-rich DNA sequence includes one selected from SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.12, and SEQ ID NO.13.

[0011] According to an embodiment of the present invention, the sequence of the G-rich DNA is SEQ ID NO.1.

[0012] According to an embodiment of the present invention, the two segments are both AGGGACGGGA or TGAGGGAGGGG, or respectively AGGGAGGGA and GACGGGGGGG, or GGGTGGGTATGTCTT and TTTGACATGGGTAGGG, or GGGCAGGGA and GGGAACTGGG, or GGGCTTTTGGGC and GGGCTTTTGGGC.

[0013] According to an embodiment of the present invention, both segments are AGGGACGGGA.

[0014] According to an embodiment of the present invention, the nucleic acid sequence of the G-quadruplex switch aptamer includes one selected from SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22.

[0015] According to an embodiment of the present invention, the nucleic acid sequence of the G-quadruplex switch aptamer is SEQ ID NO. 17.

[0016] A second aspect of the present invention provides a G-quadruplex structured switchable nucleic acid aptamer sensor, the G-quadruplex structured switchable nucleic acid aptamer sensor comprising a G-quadruplex structured switchable nucleic acid aptamer and a fluorescent probe;

[0017] The G-quadruplex switch-type nucleic acid aptamer described therein is the G-quadruplex switch-type nucleic acid aptamer described in the first aspect.

[0018] The G-quadruplex structure switch-type nucleic acid aptamer sensor provided by this invention is based on a G-quadruplex structure switch-type nucleic acid aptamer that can provide a highly sensitive signal reporting function while maintaining the aptamer's ability to specifically recognize targets. When it is mixed with a sample containing enrofloxacin, the target aptamer specifically binds to enrofloxacin, causing the G4 structure to open and displacing the fluorescent probe, thereby achieving highly sensitive detection of enrofloxacin.

[0019] According to an embodiment of the present invention, the fluorescent probe includes at least one selected from thioflavone T and its derivatives, thiazole orange and its derivatives.

[0020] A third aspect of this invention provides the application of the G-quadruplex switching nucleic acid aptamer described in the first aspect or the G-quadruplex switching nucleic acid aptamer sensor described in the second aspect in the detection of enrofloxacin. The G-quadruplex switching nucleic acid aptamer described in the first aspect or the G-quadruplex switching nucleic acid aptamer sensor described in the second aspect can be used for the detection of enrofloxacin, effectively improving the problem of weak structural changes in the binding of the enrofloxacin aptamer to the target.

[0021] A fourth aspect of the present invention provides a method for detecting enrofloxacin, the method comprising:

[0022] (1) Constructing G-quadruplex switch-type nucleic acid aptamers;

[0023] (2) Mix the fluorescent probe and the G-quadruplex switch-type nucleic acid aptamer to construct a G-quadruplex switch-type nucleic acid aptamer sensor, and add the sample to be tested for detection;

[0024] in,

[0025] Step (1) includes assembling the G-rich DNA sequence with the target aptamer to obtain the G-quadruplex switch-type nucleic acid aptamer;

[0026] The G-quadruplex switch-type nucleic acid aptamer is the G-quadruplex switch-type nucleic acid aptamer described in the first aspect.

[0027] The detection method provided by this invention is based on the G-quadruplex switch-type nucleic acid aptamer described in the first aspect, which can achieve highly sensitive detection of enrofloxacin. This method is simple, has good specificity, requires no labeling or modification, and can realize quantitative analysis of enrofloxacin in various food and environmental samples, showing good application prospects.

[0028] According to an embodiment of the present invention, the concentration ratio of the fluorescent probe to the G-quadruplex switch-type nucleic acid aptamer is (100-1):1.

[0029] According to an embodiment of the present invention, the concentration ratio of the fluorescent probe to the G-quadruplex switch-type nucleic acid aptamer is 25:1.

[0030] According to an embodiment of the present invention, the reaction time between the fluorescent probe and the G-quadruplex switch-type nucleic acid aptamer in step (2) is 30-80 min.

[0031] According to an embodiment of the present invention, the reaction time between the fluorescent probe and the G-quadruplex switch-type nucleic acid aptamer in step (2) is 40 min.

[0032] According to an embodiment of the present invention, the fluorescent probe includes at least one selected from thioflavone T and its derivatives, thiazole orange and its derivatives.

[0033] The fifth aspect of this invention provides the application of the detection method described in the fourth aspect in the detection of enrofloxacin in livestock products and / or water bodies. The enrofloxacin detection method provided in the fourth aspect of this invention can achieve quantitative analysis of enrofloxacin in various livestock products and water bodies, and has great application potential in practical sample detection.

[0034] The present invention has the following advantages over the prior art:

[0035] (1) The G-quadruplex structure switch-type nucleic acid aptamer sensor provided by the present invention effectively improves the problem of weak structural change in the binding of enrofloxacin aptamer to the target. It is simple and easy to operate and has low detection cost when used for the detection of enrofloxacin. It has a wide linear detection range (0.05-20μM) and a low detection limit (26.7nM), and at the same time has good selectivity for enrofloxacin.

[0036] (2) This invention is the first to construct an enrofloxacin detection method based on G-quadruplex fluorescent probe, which greatly expands the application range of fluorescent sensors based on G-quadruplex technology.

[0037] (3) The method for detecting enrofloxacin provided by the present invention can realize the quantitative analysis of enrofloxacin in a variety of livestock products and water bodies, with good recovery rate (95.87%-104.36%), and has great application potential in actual sample detection.

[0038] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0039] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0040] Figure 1 The mechanism diagram of the detection of enrofloxacin based on the G-quadruplex structure switch-type nucleic acid aptamer sensor of the present invention is shown;

[0041] Figure 2 The fluorescence enhancement ratios of ThT after interacting with different G-rich DNA sequences are shown in Example 2 of this invention;

[0042] Figure 3 The following are fluorescence titration fitting curves of enrofloxacin with ENRA and G4-ENRA and ThT with G4-ENRA in Example 2 of the present invention.

[0043] Figure 4 The circular dichroism chromatograms of ENRA and G4-ENRA before and after binding with enrofloxacin in Example 2 of the present invention are shown.

[0044] Figure 5 The fluorescence spectra of G4-ENRA and ENRA before and after binding with enrofloxacin in Example 2 of the present invention are shown, along with a comparison of the changes in fluorescence values ​​corresponding to a wavelength of 497 nm.

[0045] Figure 6 The diagram shows the optimized concentration ratio of ThT to G4-ENRA (A) and the optimized reaction time (B) in Example 3 of the present invention.

[0046] Figure 7 The fluorescence spectra (A) of the system after adding different concentrations of enrofloxacin in Example 4 of the present invention and the standard curve (B) between the fluorescence change (1-F / F0)% and the concentration of enrofloxacin are shown.

[0047] Figure 8 The results of the specificity evaluation of enrofloxacin by the G-quadruplex structure-based switchable nucleic acid aptamer sensor in Example 5 of the present invention are shown. Detailed Implementation

[0048] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0049] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0050] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0051] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0053] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0054] According to an embodiment of the present invention, a first aspect of the present invention provides a G-quadruplex structured switch-type nucleic acid aptamer, wherein the nucleic acid sequence of the G-quadruplex structured switch-type nucleic acid aptamer includes a target aptamer and a G-rich DNA sequence, wherein the G-rich DNA sequence includes two fragments, which are respectively assembled at both ends of the target aptamer;

[0055] The G-rich DNA sequence is 12–32 bp in length and includes at least four guanine sequence groups, with each fragment including at least two guanine sequence groups, and each guanine sequence group including at least three consecutive guanines.

[0056] The target aptamer described herein is an aptamer capable of targeting enrofloxacin. Therefore, the G-quadruplex switching nucleic acid aptamer provides highly sensitive signal reporting while maintaining the aptamer's ability to specifically recognize the target. By engineering G-rich DNA sequences at both ends of the target aptamer, the conformation of the original target aptamer is altered to a G4 structure. When mixed with a sample containing enrofloxacin, the target aptamer specifically binds to enrofloxacin, opening the G4 structure and thus improving the problem of weak structural changes in the binding of the enrofloxacin aptamer to the target.

[0057] According to an embodiment of the present invention, the nucleic acid sequence of the target aptamer is SEQ ID NO.16. Using this target aptamer enables highly specific and strong-affinity recognition of the target, while reducing the number of bases by approximately half compared to conventional target aptamers, thus lowering the fabrication cost of the G-quadruplex structure switch-type nucleic acid aptamer sensor.

[0058] According to embodiments of the present invention, the G-rich DNA sequence includes one selected from SEQ ID NO.1, SEQ ID NO.4, SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.12, and SEQ ID NO.13. Using the G-rich DNA sequence can significantly enhance the fluorescence emission of fluorescent probes, such as thioflavin T (ThT), thereby promoting signal amplification and ensuring that the constructed G-quadruplex switch-type nucleic acid aptamer can achieve high sensitivity when used for detection.

[0059] According to an embodiment of the present invention, the two segments are both AGGGACGGGA or TGAGGGAGGGG, or respectively AGGGAGGGA and GACGGGGGGG, or GGGTGGGTATGTCTT and TTTGACATGGGTAGGG, or GGGCAGGGA and GGGAACTGGG, or GGGCTTTTGGGC and GGGCTTTTGGGC.

[0060] According to an embodiment of the present invention, both fragments are AGGGACGGGA. This maximizes the activation of the potential fluorescent properties of the fluorescent probe, promotes signal amplification, and improves the sensitivity of the constructed G-quadruplex switch-type nucleic acid aptamer for detection.

[0061] According to embodiments of the present invention, the nucleic acid sequence of the G-quadruplex switch-type nucleic acid aptamer includes one selected from SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22. The G-quadruplex switch-type nucleic acid aptamer having the aforementioned nucleic acid sequence exhibits an affinity for enrofloxacin comparable to that of the original aptamer, but significantly higher than its affinity for the fluorescent probe. This indicates that when enrofloxacin is present in the system, the constructed G-quadruplex switch-type nucleic acid aptamer can preferentially bind to it, thereby displacing the fluorescent probe.

[0062] According to an embodiment of the present invention, the nucleic acid sequence of the G-quadruplex switch-type nucleic acid aptamer is SEQ ID NO. 17. This ensures that the constructed G-quadruplex switch-type nucleic acid aptamer has an affinity for enrofloxacin comparable to that of the original aptamer, while being significantly higher than its affinity for fluorescent probes.

[0063] According to an embodiment of the present invention, a second aspect of the present invention provides a G-quadruplex structured switchable nucleic acid aptamer sensor, the G-quadruplex structured switchable nucleic acid aptamer sensor comprising a G-quadruplex structured switchable nucleic acid aptamer and a fluorescent probe;

[0064] The G-quadruplex switch-type nucleic acid aptamer described therein is the G-quadruplex switch-type nucleic acid aptamer described in the first aspect.

[0065] According to an embodiment of the present invention, the fluorescent probe includes at least one selected from thioflavone T and its derivatives, thiazole orange and its derivatives.

[0066] According to embodiments of the present invention, a third aspect of the present invention provides the application of the G-quadruplex switching nucleic acid aptamer described in the first aspect or the G-quadruplex switching nucleic acid aptamer sensor described in the second aspect in the detection of enrofloxacin.

[0067] According to an embodiment of the present invention, a fourth aspect provides a method for detecting enrofloxacin, the method comprising:

[0068] (1) Constructing G-quadruplex switch-type nucleic acid aptamers;

[0069] (2) Mix the fluorescent probe and the G-quadruplex switch-type nucleic acid aptamer to construct a G-quadruplex switch-type nucleic acid aptamer sensor, and add the sample to be tested for detection;

[0070] Step (1) includes assembling a G-rich DNA sequence with a target aptamer to obtain the G-quadruplex switch-type nucleic acid aptamer.

[0071] The G-quadruplex structure switch-type nucleic acid aptamer sensor is the G-quadruplex structure switch-type nucleic acid aptamer described in the first aspect.

[0072] This detection method is based on the G-quadruplex switch-type nucleic acid aptamer described in the first aspect, which can achieve highly sensitive detection of enrofloxacin. The method is simple, specific, and requires no labeling or modification. It can realize the quantitative analysis of enrofloxacin in various food and environmental samples and has good application prospects.

[0073] According to an embodiment of the present invention, the concentration ratio of the fluorescent probe to the G-quadruplex switch-type nucleic acid aptamer is (100-1):1. This significantly improves the detection sensitivity of enrofloxacin.

[0074] According to an embodiment of the present invention, the concentration ratio of the fluorescent probe to the G-quadruplex switch-type nucleic acid aptamer is 25:1. This significantly improves the detection sensitivity of enrofloxacin.

[0075] According to embodiments of the present invention, the fluorescent probe comprises at least one selected from thioflavone T and its derivatives, thiazole orange and its derivatives. The fluorescent probes used in this invention include, but are not limited to, thioflavone T and its derivatives, thiazole orange and its derivatives, and may also use other fluorescent probes currently reported in the literature that can recognize G-quadruplex structures.

[0076] According to an embodiment of the present invention, the reaction time between the fluorescent probe and the G-quadruplex switch-type nucleic acid aptamer in step (2) is 30-80 min. This significantly improves the detection sensitivity of enrofloxacin.

[0077] According to an embodiment of the present invention, the reaction time between the fluorescent probe and the G-quadruplex switch-type nucleic acid aptamer in step (2) is 40 min. This significantly improves the detection sensitivity of enrofloxacin.

[0078] According to embodiments of the present invention, a fifth aspect provides the application of the detection method described in the fourth aspect in the detection of enrofloxacin in livestock products and / or water bodies. The enrofloxacin detection method provided in the fourth aspect of the present invention can achieve quantitative analysis of enrofloxacin in various livestock products (e.g., chicken, fish, milk) and water bodies (e.g., lake water), and has great application potential in practical sample detection.

[0079] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0080] Table 1 shows the DNA sequence information used in the following embodiments of the present invention. Figure 1 This is a schematic diagram of the mechanism for detecting enrofloxacin using a G-quadruplex structure switch-type nucleic acid aptamer sensor provided by the present invention.

[0081] Table 1 DNA Sequence

[0082]

[0083]

[0084]

[0085] Example 1: Preparation of buffer solution

[0086] Accurately weigh a certain mass of HEPES buffer, KCl and NaCl, and dissolve them in ultrapure water (containing 0.025% Triton X-100) to obtain a buffer solution with a concentration of 25 mM HEPES, 20 mM KCl, 200 mM NaCl and 0.025% Triton X-100.

[0087] Example 2: Construction of a G-quadruplex switch-type nucleic acid aptamer sensor

[0088] The specific steps are as follows:

[0089] (1) Screening of G-rich DNA sequences: In order to ensure that the designed G-quadruplex switch-type nucleic acid aptamer can achieve high detection sensitivity, the G-rich DNA sequence used in this invention should be able to have a strong interaction with the fluorescent probe, and can maximize the activation of its potential fluorescent properties, thereby promoting signal amplification.

[0090] Take 10 μL of 10 μM of 15 G-rich DNA sequences (corresponding to the 15 DNA sequences in Table 1) and mix them with 25 μL of 100 μM of fluorescent probe ThT (thioflavin T). Add the buffer solution prepared in Example 1 to a total of 500 μL. After reacting in the dark for 40 min, perform fluorescence measurement and record the fluorescence value at 497 nm.

[0091] Figure 2 It is the fluorescence enhancement ratio after ThT interacts with different G-rich DNA sequences, where F is the fluorescence value after ThT interacts with different G-rich DNA sequences. ThT The value is the fluorescence value of ThT in the blank sample. By comparing F / F... ThT The fluorescence values ​​showed that the AA sequence could significantly enhance the fluorescence emission of ThT;

[0092] (2) Construction and affinity determination of G-quadruplex switch-type nucleic acid aptamers: The AA sequence screened in step (1) was taken and split into two DNA fragments (both AGGGACGGGA) and assembled at both ends of ENRA (target aptamer) to form G4-ENRA. The concentration of enrofloxacin was fixed at 2 μM, and different concentrations of G4-ENRA (0-8 μM) were added. The fluorescence spectrum was recorded at an excitation wavelength of 350 nm. The F / F0 value at 419 nm was fitted by the Hill function curve to obtain the affinity value K between G4-ENRA and enrofloxacin. d The affinity values ​​of ENRA and enrofloxacin were obtained using the same procedure, and their K values ​​were compared. d value.

[0093] Figure 3 This is a fluorescence titration fitting curve of enrofloxacin with ENRA, G4-ENRA, and ThT and G4-ENRA, respectively. It can be seen that the affinity (K0.05) of the engineered aptamer G4-ENRA for enrofloxacin is... d =161.6 nM) and the affinity of the original aptamer ENRA for enrofloxacin (K) d =352.9 nM) is equivalent, indicating that after assembling G-rich sequences at both ends of the enrofloxacin aptamer, the new aptamer still maintains a high affinity for the target; G4-ENRA's affinity for ThT K d=1.99μM, which is much lower than the affinity of G4-ENRA for enrofloxacin, indicating that when enrofloxacin is present in the system, the aptamer can preferentially bind to it, thereby displacing ThT;

[0094] (3) Circular dichroism characterization of the obtained G-quadruplex switch-type nucleic acid aptamers: ENRA and G4-ENRA were diluted to 3 μM with buffer solution and mixed with 0 and 500 μM enrofloxacin solutions, respectively. Circular dichroism was measured at 200-350 nm.

[0095] Figure 4 These are circular dichroism chromatograms of ENRA and G4-ENRA before and after binding with enrofloxacin. It can be seen that ENRA originally had a B-type aptamer structure, and its conformation did not change significantly after the addition of enrofloxacin. The engineered G4-ENRA exhibits a typical parallel G-quadruplex signal, indicating that the B-type aptamer was successfully converted into a G-quadruplex structure after assembling G-rich sequences at both ends of the aptamer. When enrofloxacin was added to the G4-ENRA system, the G-quadruplex structure formed by the G-rich short DNA sequences at both ends was opened due to the specific binding of the aptamer region to it. At this point, the characteristic peaks of the G-quadruplex structure disappeared, indicating that the G-quadruplex structure was destroyed.

[0096] (4) Fluorescence feasibility verification of the G-quadruplex structure switch-type nucleic acid aptamer sensor: G4-ENRA and ENRA were used as controls to verify that the present invention can improve the problem of weak binding between enrofloxacin aptamer and target, and significantly improve detection sensitivity. 10 μL of 10 μM G4-ENRA was diluted to 0.8 μM with buffer solution, and 25 μL of 100 μM ThT was added to construct the G-quadruplex structure switch-type nucleic acid aptamer sensor. After incubation at room temperature in the dark for 40 min, 125 μL of 200 μM enrofloxacin solution was added. After mixing, it was placed in the dark for another 30 min and fluorescence was measured at an excitation wavelength of 425 nm and an emission wavelength of 450-600 nm. The fluorescence value corresponding to 497 nm was recorded as F. For the blank group, 125 μL of enrofloxacin solution was replaced with ultrapure water, and the obtained fluorescence value was recorded as F0. The F0-F values ​​corresponding to G4-ENRA and ENRA were calculated respectively.

[0097] Figure 5Figure A shows the fluorescence spectra of G4-ENRA and ENRA before and after binding with enrofloxacin. It can be seen that when ThT binds to G4-ENRA, which has a G-quadruplex structure, the fluorescence intensity is significantly enhanced (curve f), while the enhancement is weak when binding to the original nucleic acid aptamer ENRA (curve d). After the addition of enrofloxacin, the aptamer can specifically bind to it, leading to the disruption of the G-quadruplex structure and causing structural changes in the aptamer. ThT is then released from the ThT / G4-ENRA complex, resulting in a decrease in the fluorescence intensity of the system (curve e). While adding ENR to the ENRA system also reduces the fluorescence intensity, the change is much smaller than that in the G4-ENRA system. Figure 5 As shown in Figure B, the fluorescence change of the sensor mediated by G4-ENRA is approximately 3.6 times that of the original nucleic acid aptamer ENRA, indicating that the G-quadruplex engineering of the nucleic acid aptamer can significantly improve the detection sensitivity.

[0098] Example 3: Exploration and Optimization of Experimental Parameters

[0099] Key parameters in the detection method were explored and optimized, including the concentration ratio of the fluorescent probe ThT to G4-ENRA and its reaction time, to improve the analytical performance of the method.

[0100] (1) Optimization of the concentration ratio of ThT to G4-ENRA: The concentration of ThT was fixed at 5 μM, and the concentration of G4-ENRA was set to 0.1-0.7 μM. The two were reacted at room temperature in the dark for 40 min, and then 125 μL of 200 μM enrofloxacin was added. After 30 min, the fluorescence value F at 497 nm was measured and recorded. In the blank group, 125 μL of enrofloxacin solution was replaced with ultrapure water, and the fluorescence value F0 was recorded. The optimal concentration ratio of [ThT] / [G4-ENRA] was selected based on the fluorescence change F0 / F.

[0101] Figure 6 Figure A shows the optimized concentration ratio of ThT to G4-ENRA. When the concentration of G4-ENRA is 0.2 μM, the fluorescence change F0 / F value of the system is the largest. As the concentration of G4-ENRA increases, the fluorescence change F0 / F value gradually decreases. Therefore, the optimal concentration ratio of [ThT] / [G4-ENRA] is 5:0.2, or 25:1.

[0102] (2) Optimization of the [ThT] / [G4-ENRA] reaction time: Take 10 μL of 10 μM G4-ENRA, dilute it to 0.8 μM with buffer solution, add 25 μL of 100 μM ThT, and incubate at room temperature in the dark for 1-80 min. Then add 125 μL of 200 μM enrofloxacin solution. After 30 min, measure the fluorescence and record the fluorescence value F at 497 nm. For the blank group, replace 125 μL of enrofloxacin solution with ultrapure water and record the fluorescence value F0. Select the optimal reaction time of [ThT] / [G4-ENRA] based on the fluorescence change F0 / F.

[0103] Figure 6 Figure B in the middle plot shows the optimal reaction time for ThT and G4-ENRA. As the reaction time increases, the fluorescence change F0 / F value gradually increases, reaching a stable state at 40 min. Therefore, 40 min is the optimal reaction time for ThT and G4-ENRA.

[0104] Example 4: Sensitivity detection of enrofloxacin by a G-quadruplex structure switch-type nucleic acid aptamer sensor

[0105] The specific steps are as follows:

[0106] 10 μL of 10 μM G4-ENRA was diluted to 0.8 μM with buffer solution, and 25 μL of 100 μM ThT solution was added. The mixture was reacted at room temperature in the dark for 40 min. Subsequently, 125 μL of enrofloxacin at different concentrations was added. After 30 min, fluorescence was measured, and the fluorescence value F at 497 nm was recorded. In the blank group, 125 μL of enrofloxacin solution was replaced with ultrapure water, and the fluorescence value F0 was recorded. The change in fluorescence value (1-F / F0)% was analyzed by linear regression with the enrofloxacin concentration, yielding y = 1.575x + 1.994(R²). 2 =0.986), detection limit is 26.7 nM, see Figure 7 The detection procedure for enrofloxacin using SEQ ID NO.18-22 containing nucleic acid aptamers is consistent with that of G4-ENRA.

[0107] Example 5: Specific detection of enrofloxacin by a G-quadruplex structure switch-type nucleic acid aptamer sensor

[0108] The specific steps are as follows:

[0109] 10 μL of 10 μM G4-ENRA was diluted to 0.8 μM with buffer solution, and 25 μL of 100 μM ThT was added. The mixture was reacted at room temperature in the dark for 40 min. Subsequently, it was mixed with 125 μL of 80 μM tetracycline (TET), ampicillin (AMP), amoxicillin (AMC), chloramphenicol (CPL), roxithromycin (ROX), erythromycin (EM), enrofloxacin (ENR), and a mixed antibiotic (MIX, a mixture of the aforementioned antibiotics). After 30 min, fluorescence was measured, and the fluorescence value at 497 nm was recorded. The results showed that the sensor has good specificity for enrofloxacin. Figure 8 .

[0110] Example 6: The specific steps for the quantitative analysis of enrofloxacin in real samples using a G-quadruplex structure switch-type nucleic acid aptamer sensor are as follows:

[0111] The above method was used to detect enrofloxacin in food and lake water, and the sample determination was carried out by spiked recovery.

[0112] (1) Food sample pretreatment:

[0113] Chicken and fish were chopped, and 0.5 g of each meat sample was accurately weighed and mixed with 5 mL of acetonitrile. Protein was removed by adding an appropriate amount of ethanol. The samples were then sonicated at room temperature for 10 min and centrifuged at 10,000 rpm for 10 min. The supernatant was collected, filtered through a 0.22 μm organic filter membrane, and the final solution was collected for subsequent measurements.

[0114] Dissolve 1 mL of milk in 5 mL of acetonitrile, vortex for 1 min, then centrifuge at 10000 rpm for 10 min, collect the supernatant, filter through a 0.22 μm organic filter membrane, and dilute 50 times for later use.

[0115] (2) Lake water sample: Centrifuge the lake water at 10,000 rpm for 6 minutes, and then filter the supernatant through a 0.22 μm microporous membrane.

[0116] The collected final filtrate was spiked with different concentrations of enrofloxacin for fluorescence measurement. The results were then substituted into the linear regression equation in Example 4 to calculate the detection limit of enrofloxacin. The results are shown in Table 2. The spiked recovery rate was 95.87%-104.36%, and the relative standard deviation was less than 4%, indicating good recovery.

[0117] Table 2. Detection results of enrofloxacin in real samples.

[0118]

[0119]

[0120] Note: * The average of three repetitions.

[0121] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0122] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A G-quadruplex switch-type nucleic acid aptamer, characterized in that, The nucleic acid sequence of the G-quadruplex switch aptamer includes a target aptamer and a G-rich DNA sequence. The G-rich DNA sequence includes two fragments, which are respectively assembled at both ends of the target aptamer. The G-rich DNA sequence is 12–32 bp in length and includes at least four guanine sequence groups, each fragment including at least two guanine sequence groups, and each guanine sequence group including at least three consecutive guanines. The nucleic acid sequence of the target aptamer is SEQ ID NO.

16.

2. The G-quadruplex switch-type nucleic acid aptamer according to claim 1, characterized in that, The two segments are both AGGGACGGGA or TGAGGGAGGGG, or AGGGAGGGA and GACGGGGGGG respectively, or GGGTGGGTATGTCTT and TTTGACATGGGTAGGG, or GGGCAGGGA and GGGAACTGGG, or GGGCTTTTGGGC and GGGCTTTTGGGC; Optionally, both segments are AGGGACGGGA.

3. The G-quadruplex switch-type nucleic acid aptamer according to claim 1, characterized in that, The nucleic acid sequence of the G-quadruplex switch aptamer includes one selected from SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22; Optionally, the nucleic acid sequence of the G-quadruplex switch aptamer is SEQ ID NO.

17.

4. A G-quadruplex structure switch-type nucleic acid aptamer sensor, characterized in that, The G-quadruplex structure switchable nucleic acid aptamer sensor includes a G-quadruplex structure switchable nucleic acid aptamer and a fluorescent probe; The G-quadruplex switching aptamer described therein is the G-quadruplex switching aptamer according to any one of claims 1-3; Optionally, the fluorescent probe includes at least one selected from thioflavone T and its derivatives, thiazole orange and its derivatives.

5. The application of the G-quadruplex switching nucleic acid aptamer according to any one of claims 1-3 or the G-quadruplex switching nucleic acid aptamer sensor according to claim 4 in enrofloxacin detection.

6. A method for detecting enrofloxacin, characterized in that, The detection method includes: (1) Constructing G-quadruplex switch-type nucleic acid aptamers; (2) Mix the fluorescent probe and the G-quadruplex switch-type nucleic acid aptamer to construct a G-quadruplex switch-type nucleic acid aptamer sensor, and add the sample to be tested for detection; in, Step (1) includes assembling the G-rich DNA sequence with the target aptamer to obtain the G-quadruplex switch-type nucleic acid aptamer; The G-quadruplex switch-type nucleic acid aptamer is the G-quadruplex switch-type nucleic acid aptamer as described in any one of claims 1-3.

7. The detection method according to claim 6, characterized in that, The concentration ratio of the fluorescent probe to the G-quadruplex switch-type nucleic acid aptamer is (100-1):

1.

8. The detection method according to claim 6, characterized in that, The concentration ratio of the fluorescent probe to the G-quadruplex switch-type nucleic acid aptamer is 25:

1.

9. The detection method according to claim 6, characterized in that, The fluorescent probe includes at least one selected from thioflavone T and its derivatives, thiazole orange and its derivatives.

10. The detection method according to claim 6, characterized in that, The reaction time between the fluorescent probe and the G-quadruplex switch aptamer in step (2) is 30-80 min.

11. The detection method according to claim 6, characterized in that, The reaction time between the fluorescent probe and the G-quadruplex switch aptamer in step (2) is 40 min.

12. The application of the detection method according to any one of claims 6 to 11 in the detection of enrofloxacin in livestock products and / or water bodies.

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