Fluorescence detection kit for detecting small biological molecules using G-quadruplex as quenching group in nucleic acid aptamers, usage method and application

Abstract

The present invention relates to a fluorescence detection kit, a method of use and an application for detecting biomolecules using G-quadruplex as a quenching group in nucleic acid aptamers, and belongs to the field of biochemical analysis technology. The present invention is based on the property that G-quadruplex has a strong quenching effect on fluorescent dyes, and utilizes target molecules to induce G-rich nucleic acid aptamers to form secondary structures such as G-quadruplexes, and the amount of the secondary structures such as the formed G-quadruplexes is related to the concentration of the target molecules, thereby using G-quadruplexes as label-free quenching groups for detecting biomolecules. At the same time, the two ends of the nucleic acid aptamers are extended to form tail chains that can form complementary base pairs, which are used to embed non-labeled fluorescent dyes, and the increase of fluorescent signal is achieved by increasing the logarithm of complementary bases. The present invention does not need to label nucleic acid aptamers, has low production cost, low detection cost, and simple detection principle, easy to operate, reduces the requirements for operators, and is more conducive to practical application.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemical analysis, and in particular to a fluorescence detection kit for detecting biological small molecules by using a G-quadruplex as a quenching group in a nucleic acid aptamer. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Nucleic acids play a vital role in biological systems as storage media for cellular genetic information. Over the past few decades, scientists have exploited the unique structure and recognition capabilities of nucleic acids to achieve a variety of scientific research and technological applications. One emerging application of nucleic acids is the development of aptamers, or aptamers. Aptamers were first discovered in the 1990s through the systematic evolution of ligands by exponential enrichment (SELEX) process. Aptamers are single-stranded DNA or RNA molecules that can bind to various targets with high affinity and specificity. Their excellent selectivity and high affinity for their targets offer advantages over traditional recognition units in detection applications, even surpassing protein antibodies. Aptamers are generally more stable under various environmental conditions, reusable, and cost-effective to synthesize, with simplified synthesis and modification. Therefore, aptamers offer broad application prospects as an alternative to traditional antibodies in basic research, drug testing, clinical diagnosis, and the treatment of various diseases.

[0004] G-quadruplexes are among the most important non-classical nucleic acid secondary structures due to their high stability and widespread distribution. G-quadruplexes are specialized nucleic acid secondary structures formed by guanine-rich oligonucleotides. The core unit of a G-quadruplex is the G-quadruplex, composed of four guanine bases linked by Hoogsteen hydrogen bonds. Stacking of two or more guanine quartets forms a G-quadruplex. Unlike DNA, which typically exists as a double helix, G-quadruplexes can form diverse topologies depending on the sequence of the oligonucleotides that comprise the structure and the orientation of the nucleic acid strands. Due to its flexible sequence design and diverse structure, G-quadruplexes have become an important unit for high-sensitivity analytical detection.

[0005] Fluorescence detection methods offer significant advantages in rapid and sensitive analysis due to their high sensitivity, simple operation, and short detection time. Existing technologies facilitate the quantitative detection of small biomolecules using nucleic acid aptamers, but these methods require labeling the aptamers, specifically by modifying the aptamer nucleotides with fluorescent or quenching groups. While modified fluorescent groups offer more stable stoichiometric fluorescence responses and quenching groups offer more efficient quenching, labeling nucleic acids significantly increases synthesis and detection costs. Therefore, there remains a need for simple, low-cost methods for detecting small biomolecules. Summary of the Invention

[0006] In response to the problems existing in the above-mentioned prior art, the present invention provides a fluorescent detection kit, method of use and application for detecting biological small molecules using G-quadruplex as a quenching group in nucleic acid aptamers, which is efficient, simple, sensitive, selective and has low detection cost and does not require labeling of fluorescent groups and quenching groups.

[0007] In order to achieve the above object, the technical solution provided by the present invention is:

[0008] A fluorescence detection kit for detecting small biological molecules using a G-quadruplex as a quenching group in a nucleic acid aptamer, comprising the following solutions:

[0009] The detection solution includes 100-2000nM detection nucleic acid, a buffer solution with a concentration of 100-1000mmol / L and a pH of 4-10, a surfactant with a mass fraction of 0.1%-10.0%, and a preservative. The detection nucleic acid includes two parts: 1. a nucleic acid aptamer that can recognize the biological small molecule to be detected and form a G-quadruplex or a G-rich secondary structure; 2. a stem structure formed by complementary base sequences connected to the two ends of the nucleic acid aptamer, which can be embedded in a fluorescent dye to produce fluorescence.

[0010] Fluorescent color developing solution: including a label-free fluorescent dye with a concentration of 0.1×-10.00× (the original solution concentration is 10000×); a buffer solution with a concentration of 100-1000mM and a pH of 4-10.

[0011] Furthermore, a preservative may be added to the detection liquid to prevent the detection liquid from becoming moldy due to long-term storage.

[0012] Furthermore, the nucleic acid aptamer is a nucleotide fragment obtained from a nucleic acid molecule library that can bind to a target substance with high specificity and selectivity.

[0013] Furthermore, the nucleic acid aptamer can be induced by the small molecule to form a G-quadruplex or other guanine-rich structures.

[0014] Furthermore, the complementary base sequence in the stem-like structure is Am-aptamer-Tn (m=1-20, n=1-20), and the label-free fluorescent dye can be embedded in the structure and generate fluorescence.

[0015] Furthermore, the surfactant includes Tween 20, Tween 60, Tween 80 or Triton X100.

[0016] Furthermore, the label-free fluorescent dye includes SYBR Gold, SYBR Green I, SYBR Green II, SYBR Safe, ethidium bromide (EB), Genefinder or Geneview.

[0017] Furthermore, the preservative includes gentamicin or cinnamaldehyde.

[0018] Furthermore, the buffer solution in the detection solution includes Tris-HCl buffer solution, PBS buffer solution or HEPES buffer solution.

[0019] A method for using a fluorescence detection kit for detecting small biological molecules using a G-quadruplex as a quenching group in a nucleic acid aptamer comprises the following steps:

[0020] Step 1: Take 80 μL of the sample to be tested and add it to the wells of a 96-well all-black microplate.

[0021] In step 2, 10 μL of the test solution is added to the sample in a 96-well black microplate and allowed to react for 3-20 minutes. The nucleic acids in the test solution, induced by the biomolecule to be tested, form specific three-dimensional structures. The aptamers partially form G-quadruplexes or other guanine-rich structures, and the proportion of these structures formed is proportional to the concentration of the biomolecule to be tested. The complementary base sequences at both ends of the aptamers are drawn together and hybridized to form a stem-like structure due to the specific three-dimensional structure formed by the aptamers.

[0022] In step 3, add 10 μL of fluorescent colorimetric solution to the sample to be tested in a 96-well black microplate and allow to react for 1-10 minutes. The label-free fluorescent dye embeds into the stem structure formed by the detection nucleic acid in step 2, generating strong fluorescence. Because the G-quadruplex or other guanine-rich structures formed by the detection nucleic acid in step 2 have the ability to quench fluorescence, different concentrations of the biomolecule to be tested result in different degrees of fluorescence quenching.

[0023] Step 4: Measure the fluorescence intensity.

[0024] Furthermore, the fluorescence intensity is obtained by using a fluorescence microplate reader, a fluorescence spectrophotometer or a simple fluorescence detector.

[0025] A fluorescent detection kit for detecting small biological molecules using a G-quadruplex as a quenching group in a nucleic acid aptamer is used for detecting small biological molecules; for detecting tumor markers, etc.

[0026] Furthermore, the biological small molecules include metal ions and mycotoxins.

[0027] Furthermore, the tumor markers include serum carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), prostate-specific antigen (PSA), human chorionic gonadotropin (HCG), etc.

[0028] The present invention uses G-quadruplex as a quenching group. Due to the low oxidation potential of guanine (G), the low oxidation potential and high electron cloud density of G-quadruplex rich in G have a strong quenching effect on fluorescent dyes. Therefore, G-quadruplex can be used as a label-free quenching group to achieve quenching of nearby fluorescence. Due to the presence of target molecules, more G-rich nucleic acid aptamers can induce G-rich nucleic acid aptamers to form secondary structures such as G-quadruplexes. The amount of the formed G-quadruplexes and G-rich secondary structures is related to the concentration of the target molecules. Therefore, G-quadruplexes and G-rich secondary structures can be used as label-free quenching groups for detecting small biomolecules. The two ends of the nucleic acid aptamer are extended to form tail chains that can form complementary base pairs for embedding non-labeled fluorescent dyes to achieve the acquisition of fluorescent signals. By increasing the logarithm of the complementary bases, the increase of the fluorescent signal can be achieved.

[0029] The present invention uses potassium ions and ochratoxin A (OTA) as examples to demonstrate that both parallel G-quadruplex structures and antiparallel G-quadruplex structures can be used as quenching groups to construct fluorescence sensors for detecting small biological molecules.

[0030] Compared with the prior art, the present invention has the following beneficial effects: for the first time, the present invention utilizes the G-quadruplex or G-rich nucleic acid secondary structure formed by the nucleic acid aptamer itself as a quenching group; by increasing the number of pairs of complementary bases, it is used to embed a non-labeled fluorescent dye to achieve the acquisition of a fluorescent signal. A scheme for sensitive detection of small biological molecules without labeling quenchers, labeling fluorophores, and competitive reactions has been constructed, and based on this, a low-cost, high-throughput, ultra-sensitive detection kit for small biological molecules has been constructed. The present invention does not require labeling of nucleic acid aptamers, so the production cost of the kit is low and the detection cost is low. The present invention does not use a competitive reaction, so the detection limit is low. Since the detection principle is simple and the operation is easy, the requirements for operators are reduced, which is more conducive to practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 a is a schematic diagram of the principle of the method for fluorescent detection of potassium ions of the present invention.

[0032] Figure 1 b is a schematic diagram of the principle of the method for fluorescent detection of ochratoxin A of the present invention.

[0033] Figure 2a This is a fluorescence intensity curve diagram of the method used to detect potassium ions (potassium ion concentration 0-16.0 μM).

[0034] Figure 2b This is a fluorescence intensity curve diagram of the method used to detect potassium ions (potassium ion concentration 0-500.0μM).

[0035] Figure 3a This is a fluorescence intensity curve diagram of the method used to detect ochratoxin A (ochratoxin A concentration 0-16.0 nM).

[0036] Figure 3b This is a fluorescence intensity curve diagram of the method used to detect ochratoxin A (ochratoxin A concentration 0-500.0 nM).

[0037] Figure 4 The fluorescence quenching intensities of different metal ions measured based on the method of the present invention.

[0038] Figure 5 The fluorescence quenching intensities of different mycotoxins measured based on the method of the present invention. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solutions and advantages of the present invention clearer, the above scheme is further described below in conjunction with the accompanying drawings and specific examples. It should be understood that the specific examples are used to illustrate the present invention and are not limited to limiting the scope of the present invention. The implementation conditions used in the examples can be further adjusted according to the differences between the candidate nucleic acid aptamers and the targets. The implementation conditions not specified are generally the conditions in conventional experiments.

[0040] Example 1

[0041] A fluorescence detection kit for detecting potassium ions based on nucleic acid aptamers using parallel G-quadruplexes as quenchers. In this example, the base sequence of the DNA molecule forming the parallel G-quadruplex structure is AmGGGTGGGTGGGTGGGTn (m=6, n=6). The solutions used in the kit are as follows:

[0042] Detection solution: includes 200nM detection nucleic acid; Tris-HCl buffer solution with a concentration of 100mM and a pH of 8.4; and Tween 20 surfactant with a mass fraction of 2.0%.

[0043] Fluorescence color development solution: including 3.00× concentration of label-free fluorescent dye SYBR Green I (the original solution concentration is 10000×) used for fluorescence detection; and 100 mM Tris-HCl buffer solution with a pH of 8.4.

[0044] Testing steps:

[0045] Potassium ion standard solutions of 0.00 μM, 0.24 μM, 0.49 μM, 0.98 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.50 μM, 125.0 μM, 250.0 μM, and 500.0 μM and goat serum samples diluted 1000 times were prepared respectively.

[0046] Take 80 μL of standard sample or test sample and add it to the wells of 96-well all-black microplate.

[0047] Take 10 μL of the test solution and add it to the sample to be tested in a 96-well black microplate and react for 3 minutes.

[0048] Take 10 μL of fluorescent color development solution and add it to the sample to be tested in a 96-well black microplate and react for 1 minute.

[0049] After the reaction is completed, the fluorescence intensity is obtained by a fluorescence microplate reader, and the content of the sample to be tested is obtained according to the pre-made standard curve.

[0050] 1. Test results of potassium ion standard solution:

[0051] The results showed that when potassium ions were absent, the reaction system had a high fluorescence intensity. As the potassium ion concentration increased, the fluorescence intensity gradually decreased. Figure 2b The relationship between the fluorescence intensity of the emitted fluorescence at an emission wavelength of 525 nm and the concentration of potassium ions is given. Figure 2a As shown in Figure 2, the prepared potassium ion fluorescence sensor has the characteristics of high accuracy, wide linear range (0.24 to 15.63 μM), and low detection limit (0.24 μmol / L) for the detection of potassium ions. The fluorescence intensity of the system and the potassium ion concentration show a linear relationship, and the regression equation obtained by fitting is F K + =8130.76-214.47C K + (μM)(R 2 =0.996).

[0052] To test the anti-interference performance of the kit, potassium ions were replaced with other metal cations such as NH4 + ,Na + ,Ca 2+ ,Mg 2+ ,Zn 2+ ,Cd 2+ , where the concentration of metal ions is 62.5 μM, the above detection steps are used for detection, and the results are as follows Figure 4 As shown, the horizontal axis is different similar metal cations, and the vertical axis is the fluorescence quenching intensity measured after adding other metal cations. Compared with the fluorescence signal caused by other metal ions, the same concentration of K + This significantly quenched the fluorescence response of the detection kit, proving that the kit has strong anti-interference performance.

[0053] 2. Goat serum sample dilution test results:

[0054] Goat serum samples were diluted 1000-fold with Tris-HCl buffer and tested using the above test steps. K was calculated according to the linear calibration equation. + The results showed that the K + The concentration was 6.56mmol / L. The goat serum sample was determined by atomic absorption spectrometry, and the K + The concentration was 6.41±0.54mmol / L (RSD=2.8%, n=6). Comparing the two sets of data, the measured value was in good agreement with the standard value, indicating that this method can be used to determine the concentration of K in serum. + Concentration detection.

[0055] like Figure 1 As shown in Figure a, potassium ions can induce potassium ion-specific aptamers to form parallel G-quadruplex structures. In the absence of potassium ions, the nucleic acid chains exhibit a random coiled state, allowing the fluorescent dye to embed into the complementary double strands at the nucleic acid tails. As the potassium ion concentration increases, the aptamers form parallel G-quadruplex structures, generating a strong photoinduced electron transfer effect between the G-quadruplex and the fluorescent group, thereby quenching the fluorescence of the fluorescent dye embedded in the complementary double strands at the nucleic acid tails, thereby enabling the detection of potassium ions. This kit can be used to measure the potassium ion concentration in human serum. The results demonstrate that the prepared fluorescent kit for potassium ion detection based on parallel G-quadruplex quenching of the fluorophore has excellent practical application value.

[0056] Example 2

[0057] A fluorescence detection kit for detecting potassium ions based on nucleic acid aptamers using parallel G-quadruplexes as quenchers. In this example, the base sequence of the DNA molecule forming the parallel G-quadruplex structure is AmGGGTGGGTGGGTGGGTn (m=3, n=20). The solutions used in the kit are as follows:

[0058] Detection solution: including 2000nM detection nucleic acid; PBS buffer solution with a concentration of 500mM and a pH of 4.0; a surfactant of Tween 60 with a mass fraction of 10.0%; and a preservative of gentamicin 1g / L.

[0059] Fluorescence color development solution: including 0.10× concentration of label-free fluorescent dye SYBR Green II (the original solution concentration is 10000×) used for fluorescence detection; and 500 mM Tris-HCl buffer solution with a pH of 4.0.

[0060] Testing steps:

[0061] Potassium ion standard solutions of 0.00 μM, 0.24 μM, 0.49 μM, 0.98 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.50 μM, 125.0 μM, 250.0 μM, and 500.0 μM and goat serum samples diluted 1000 times were prepared respectively.

[0062] Take 80 μL of standard sample or test sample and add it to the wells of 96-well all-black microplate.

[0063] Take 10 μL of the test solution and add it to the sample to be tested in a 96-well black microplate and react for 10 minutes.

[0064] Take 10 μL of fluorescent color development solution and add it to the sample to be tested in a 96-well black microplate and react for 5 minutes.

[0065] After the reaction is completed, the fluorescence intensity is obtained by a fluorescence microplate reader, and the content of the sample to be tested is obtained according to the pre-made standard curve.

[0066] 1. Test results of potassium ion standard solution:

[0067] The results showed that when potassium ions were absent, the reaction system had a high fluorescence intensity. As the potassium ion concentration increased, the fluorescence intensity gradually decreased. The prepared potassium ion fluorescence sensor had the characteristics of high accuracy, wide linear range (0.98 to 62.5 μM), and low detection limit (0.98 μmol / L) for the detection of potassium ions. The fluorescence intensity of the system showed a linear relationship with the potassium ion concentration. The regression equation obtained by fitting was F K + =7130.76-615.57C K + (μM)(R 2 =0.996).

[0068] To test the anti-interference performance of the kit, potassium ions were replaced with other metal cations such as NH4 + ,Na+ ,Ca 2+ ,Mg 2 + ,Zn 2+ ,Cd 2+ The concentration of metal ions was 62.5 μM. The above detection steps were used for detection. Compared with the fluorescence signals caused by other metal ions, the fluorescence signals of K + This significantly quenched the fluorescence response of the detection kit, proving that the kit has strong anti-interference performance.

[0069] 2. Goat serum sample dilution test results:

[0070] Goat serum samples were diluted 1000-fold with Tris-HCl buffer and tested using the above test steps. K was calculated according to the linear calibration equation. + The results showed that the K + The concentration was 6.56mmol / L. The goat serum sample was determined by atomic absorption spectrometry, and the K + The concentration was 6.41±0.54mmol / L (RSD=2.8%, n=6). Comparing the two sets of data, the measured value was in good agreement with the standard value, indicating that this method can be used to determine the concentration of K in serum. + Concentration detection.

[0071] like Figure 1 As shown in Figure a, potassium ions can induce potassium ion-specific aptamers to form parallel G-quadruplex structures. In the absence of potassium ions, the nucleic acid chains exhibit a random coiled state, allowing the fluorescent dye to embed into the complementary double strands at the nucleic acid tails. As the potassium ion concentration increases, the aptamers form parallel G-quadruplex structures, generating a strong photoinduced electron transfer effect between the G-quadruplex and the fluorescent group, thereby quenching the fluorescence of the fluorescent dye embedded in the complementary double strands at the nucleic acid tails, thereby enabling the detection of potassium ions. This kit can be used to measure the potassium ion concentration in human serum. The results demonstrate that the prepared fluorescent kit for potassium ion detection based on parallel G-quadruplex quenching of the fluorophore has excellent practical application value.

[0072] Example 3

[0073] A fluorescence detection kit for detecting potassium ions based on nucleic acid aptamers using parallel G-quadruplexes as quenchers. In this example, the base sequence of the DNA molecule forming the parallel G-quadruplex structure is AmGGGTGGGTGGGTGGGTn (m=20, n=3). The solutions used in the kit are as follows:

[0074] Detection solution: including 1000 nM detection nucleic acid; HEPES buffer solution with a concentration of 1000 mM and a pH of 8.4; and Tween 80 as a surfactant with a mass fraction of 0.1%.

[0075] Fluorescence color development solution: including 10.00× concentration of label-free fluorescent dye SYBR Safe (original solution concentration is 10000×) used for fluorescence detection; and 1000 mM Tris-HCl buffer solution with a pH of 10.0.

[0076] Testing steps:

[0077] Potassium ion standard solutions of 0.00 μM, 0.24 μM, 0.49 μM, 0.98 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.50 μM, 125.0 μM, 250.0 μM, and 500.0 μM and goat serum samples diluted 1000 times were prepared respectively.

[0078] Take 80 μL of standard sample or test sample and add it to the wells of 96-well all-black microplate.

[0079] Take 10 μL of the test solution and add it to the sample to be tested in a 96-well black microplate and react for 20 minutes.

[0080] Take 10 μL of fluorescent color development solution and add it to the sample to be tested in a 96-well black microplate and react for 10 minutes.

[0081] After the reaction is completed, the fluorescence intensity is obtained by a fluorescence microplate reader, and the content of the sample to be tested is obtained according to the pre-made standard curve.

[0082] 1. Test results of potassium ion standard solution:

[0083] The results showed that when potassium ions were absent, the reaction system had a high fluorescence intensity. As the potassium ion concentration increased, the fluorescence intensity gradually decreased. The prepared potassium ion fluorescence sensor had the characteristics of high accuracy, wide linear range (1.95 to 125.0 μM), and low detection limit (1.95 μmol / L) for the detection of potassium ions. The fluorescence intensity of the system showed a linear relationship with the potassium ion concentration. The regression equation obtained by fitting was F K + =4132.65-314.47C K + (μM)(R 2 =0.996).

[0084] To test the anti-interference performance of the kit, potassium ions were replaced with other metal cations such as NH4 + ,Na + ,Ca2+ ,Mg 2 + ,Zn 2+ ,Cd 2+ The concentration of metal ions was 62.5 μM. The above detection steps were used for detection. Compared with the fluorescence signals caused by other metal ions, the fluorescence signals of K + This significantly quenched the fluorescence response of the detection kit, proving that the kit has strong anti-interference performance.

[0085] 2. Goat serum sample dilution test results:

[0086] Goat serum samples were diluted 1000-fold with Tris-HCl buffer and tested using the above test steps. K was calculated according to the linear calibration equation. + The results showed that the K + The concentration was 6.56mmol / L. The goat serum sample was determined by atomic absorption spectrometry, and the K + The concentration was 6.41±0.54mmol / L (RSD=2.8%, n=6). Comparing the two sets of data, the measured value was in good agreement with the standard value, indicating that this method can be used to determine the concentration of K in serum. + Concentration detection.

[0087] like Figure 1 As shown in Figure a, potassium ions can induce potassium ion-specific aptamers to form parallel G-quadruplex structures. In the absence of potassium ions, the nucleic acid chains exhibit a random coiled state, allowing the fluorescent dye to embed into the complementary double strands at the nucleic acid tails. As the potassium ion concentration increases, the aptamers form parallel G-quadruplex structures, generating a strong photoinduced electron transfer effect between the G-quadruplex and the fluorescent group, thereby quenching the fluorescence of the fluorescent dye embedded in the complementary double strands at the nucleic acid tails, thereby enabling the detection of potassium ions. This kit can be used to measure the potassium ion concentration in human serum. The results demonstrate that the prepared fluorescent kit for potassium ion detection based on parallel G-quadruplex quenching of the fluorophore has excellent practical application value.

[0088] Example 4

[0089] A fluorescence detection kit for detecting OTA based on nucleic acid aptamers using antiparallel G-quadruplex as the quencher. In this example, the base sequence of the DNA molecule forming the antiparallel G-quadruplex structure is AmGATCGGGTGTGGGTGGCGTAAAGGGAGCATCTn (m=4, n=4). The solutions used in the kit are as follows:

[0090] The detection solution includes 200 nM detection nucleic acid; a Tris-HCl buffer solution with a concentration of 100 mM and a pH of 8.4; and a surfactant, Tween 20, with a mass fraction of 2.0%.

[0091] Fluorescence color development solution: including the label-free fluorescent dye SYBR Green I used for fluorescence detection, with a concentration of 3.00×; Tris-HCl buffer solution with a concentration of 100 mM and a pH of 8.4.

[0092] A fluorescence detection kit for detecting OTA based on nucleic acid aptamers using antiparallel G-quadruplex as a quencher. Detection steps:

[0093] OTA standard solutions of 0.00nM, 0.24nM, 0.49nM, 0.98nM, 1.95nM, 3.91nM, 7.81nM, 15.63nM, 31.25nM, 62.50nM, 125.0nM, 250.0nM, and 500.0nM and actual OTA samples after sample pretreatment were prepared respectively.

[0094] Take 80 μL of the sample to be tested and add it to the wells of a 96-well all-black microplate.

[0095] Take 10 μL of the test solution and add it to the sample to be tested in a 96-well black microplate and react for 20 minutes.

[0096] Take 10 μL of fluorescent color development solution and add it to the sample to be tested in a 96-well black microplate and react for 10 minutes.

[0097] After the reaction is completed, the fluorescence intensity is obtained by a fluorescence spectrophotometer, and the content of the sample to be tested is obtained according to a pre-made standard curve.

[0098] 1. OTA standard solution test results:

[0099] The results showed that when OTA was absent, the reaction system had a high fluorescence intensity, and as the OTA concentration increased, the fluorescence intensity gradually decreased. Figure 3b The relationship between the fluorescence intensity of the emitted fluorescence at an emission wavelength of 525 nm and the concentration of OTA is given. The prepared OTA fluorescence sensor has high detection accuracy for OTA, such as Figure 3a As shown in the figure, the prepared OTA fluorescence sensor has a wide linear detection range for OTA (0.24 to 15.63 nM) and a low detection limit (0.24 nmol / L). The fluorescence intensity of the system and the OTA concentration show a linear relationship, and the regression equation obtained by fitting is F OTA =7174.24-65.42C OTA (nM)(R 2 =0.987).

[0100] In order to test the anti-interference performance of the kit, OTA was replaced with other mycotoxins such as ochratoxin B (OTB), aflatoxin B1 (AFB1), aflatoxin B1 (AFG1), fumonisin B1 (FB1), patulin (Patulin), zearalenone (ZEA), deoxycholesterol (DON), where the concentration of the mycotoxins was 62.5nM. The above detection steps were used for detection, and the results were as follows: Figure 5 As shown, the horizontal axis represents different similar mycotoxins, and the vertical axis represents the fluorescence quenching intensity measured after adding other mycotoxins. Compared with the fluorescence signals caused by other mycotoxins, the same concentration of OTA significantly quenches the fluorescence response of the detection kit, proving that the kit has strong anti-interference performance.

[0101] 2. OTA actual sample test results:

[0102] Based on the physicochemical properties of OTA, a two-liquid extraction method was used to pre-treat a real-world OTA sample (red wine). As shown in the table below, the recoveries of four different OTA concentrations ranged from 97.75% to 107.58%, demonstrating that this method is feasible and reliable for detecting OTA in real-world samples.

[0103]

[0104] like Figure 1 As shown in Figure b, ochratoxin A (OTA) can induce OTA-specific aptamers to form antiparallel G-quadruplex structures. In the absence of OTA, the nucleic acid chains exhibit a random coiled state, allowing the fluorescent dye to intercalate into the complementary double strands at the nucleic acid tails. As the OTA concentration increases, the aptamers form antiparallel G-quadruplex structures, generating a strong photoinduced electron transfer effect between the G-quadruplex and the fluorophore, thereby quenching the fluorescence of the fluorescent dye embedded in the complementary double strands at the nucleic acid tails, enabling the detection of OTA. This kit can be used to determine the concentration of OTA in foods and feeds such as wine, grape juice, beer, wheat, and corn. The results demonstrate that the prepared fluorescence kit for detecting aflatoxin A based on antiparallel G-quadruplex quenching of the fluorophore has excellent practical application value.

[0105] The descriptions presented in the above exemplary embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to be exhaustive or to limit the present invention to the precise forms described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described in order to explain the specific principles of the present invention and its practical applications, so that other persons skilled in the art can easily understand, implement and utilize the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be defined by the appended claims and their equivalents.

[0106] Example 5

[0107] A fluorescence detection kit for detecting OTA based on nucleic acid aptamers using antiparallel G-quadruplex as the quencher. In this example, the base sequence of the DNA molecule forming the antiparallel G-quadruplex structure is AmGATCGGGTGTGGGTGGCGTAAAGGGAGCATCTn (m=5, n=20). The solutions used in the kit are as follows:

[0108] The detection solution includes 500 nM detection nucleic acid; a Tris-HCl buffer solution with a concentration of 1000 mM and a pH of 10; and a surfactant of Tween 80 with a mass fraction of 5.0%.

[0109] Fluorescence colorimetric solution: including the label-free fluorescent dye ethidium bromide (EB) used for fluorescence detection at a concentration of 7.00×; and a Tris-HCl buffer solution at a concentration of 100 mM and a pH of 10.0.

[0110] A fluorescence detection kit for detecting OTA based on nucleic acid aptamers using antiparallel G-quadruplex as a quencher. Detection steps:

[0111] OTA standard solutions of 0.00nM, 0.24nM, 0.49nM, 0.98nM, 1.95nM, 3.91nM, 7.81nM, 15.63nM, 31.25nM, 62.50nM, 125.0nM, 250.0nM, and 500.0nM and actual OTA samples after sample pretreatment were prepared respectively.

[0112] Take 80 μL of the sample to be tested and add it to the wells of a 96-well all-black microplate.

[0113] Take 10 μL of the test solution and add it to the sample to be tested in a 96-well black microplate and react for 20 minutes.

[0114] Take 10 μL of fluorescent color development solution and add it to the sample to be tested in a 96-well black microplate and react for 10 minutes.

[0115] After the reaction is completed, the fluorescence intensity is obtained by a fluorescence spectrophotometer, and the content of the sample to be tested is obtained according to a pre-made standard curve.

[0116] OTA standard solution test results:

[0117] The results show that when OTA is absent, the reaction system has a high fluorescence intensity. As the OTA concentration increases, the fluorescence intensity gradually decreases. The relationship between the fluorescence intensity of the emitted fluorescence corresponding to the emission wavelength of 366nm and the OTA concentration is shown in Figure 2. The prepared OTA fluorescence sensor has high detection accuracy for OTA, such as Figure 3a As shown in the figure, the prepared OTA fluorescence sensor has a wide linear detection range for OTA (0.98 to 125.0 nM) and a low detection limit (0.98 nmol / L). The fluorescence intensity of the system and the OTA concentration show a linear relationship, and the regression equation obtained by fitting is F OTA =8280.42-423.54C OTA (nM)(R 2 =0.987).

[0118] like Figure 1 As shown in Figure b, ochratoxin A (OTA) can induce OTA-specific aptamers to form antiparallel G-quadruplex structures. In the absence of OTA, the nucleic acid chains exhibit a random coiled state, allowing the fluorescent dye to intercalate into the complementary double strands at the nucleic acid tails. As the OTA concentration increases, the aptamers form antiparallel G-quadruplex structures, generating a strong photoinduced electron transfer effect between the G-quadruplex and the fluorophore, thereby quenching the fluorescence of the fluorescent dye embedded in the complementary double strands at the nucleic acid tails, enabling the detection of OTA. This kit can be used to determine the concentration of OTA in foods and feeds such as wine, grape juice, beer, wheat, and corn. The results demonstrate that the prepared fluorescence kit for detecting aflatoxin A based on antiparallel G-quadruplex quenching of the fluorophore has excellent practical application value.

[0119] The descriptions presented in the above exemplary embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to be exhaustive or to limit the present invention to the precise forms described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described in order to explain the specific principles of the present invention and its practical applications, so that other persons skilled in the art can easily understand, implement and utilize the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be defined by the appended claims and their equivalents.

[0120] Example 6

[0121] A fluorescence detection kit for detecting OTA based on nucleic acid aptamers using antiparallel G-quadruplex as the quencher. In this example, the base sequence of the DNA molecule forming the antiparallel G-quadruplex structure is AmGATCGGGTGTGGGTGGCGTAAAGGGAGCATCTn (m=14, n=14). The solutions used in the kit are as follows:

[0122] The detection solution includes 1200 nM detection nucleic acid; a Tris-HCl buffer solution with a concentration of 100 mM and a pH of 8.4; and a surfactant with a mass fraction of Triton X100 of 10.0%.

[0123] Fluorescent colorimetric solution: including the label-free fluorescent dye Geneview used for fluorescence detection at a concentration of 3.00×; and a Tris-HCl buffer solution at a concentration of 100 mM and a pH of 10.0.

[0124] A fluorescence detection kit for detecting OTA based on nucleic acid aptamers using antiparallel G-quadruplex as a quencher. Detection steps:

[0125] OTA standard solutions of 0.00nM, 0.24nM, 0.49nM, 0.98nM, 1.95nM, 3.91nM, 7.81nM, 15.63nM, 31.25nM, 62.50nM, 125.0nM, 250.0nM, and 500.0nM and actual OTA samples after sample pretreatment were prepared respectively.

[0126] Take 80 μL of the sample to be tested and add it to the wells of a 96-well all-black microplate.

[0127] Take 10 μL of detection solution and add it to the sample to be tested in a 96-well black microplate and react for 5 minutes.

[0128] Take 10 μL of fluorescent color development solution and add it to the sample to be tested in a 96-well black microplate and react for 10 minutes.

[0129] After the reaction is completed, the fluorescence intensity is obtained by a simple fluorescence detector, and the content of the sample to be tested is obtained according to the pre-made standard curve.

[0130] OTA standard solution test results:

[0131] The results show that when OTA is absent, the reaction system has a high fluorescence intensity. As the OTA concentration increases, the fluorescence intensity gradually decreases. The relationship between the fluorescence intensity of the emitted fluorescence corresponding to the emission wavelength of 525nm and the OTA concentration is shown in Figure 2. The prepared OTA fluorescence sensor has high detection accuracy for OTA, such as Figure 3aAs shown in the figure, the prepared OTA fluorescence sensor has a wide linear detection range for OTA (0.24 to 15.63 nM) and a low detection limit (0.24 nmol / L). The fluorescence intensity of the system and the OTA concentration show a linear relationship, and the regression equation obtained by fitting is F OTA =6217.24-56.42C OTA (nM)(R 2 =0.987).

[0132] like Figure 1 As shown in Figure b, ochratoxin A (OTA) can induce OTA-specific aptamers to form antiparallel G-quadruplex structures. In the absence of OTA, the nucleic acid chains exhibit a random coiled state, allowing the fluorescent dye to intercalate into the complementary double strands at the nucleic acid tails. As the OTA concentration increases, the aptamers form antiparallel G-quadruplex structures, generating a strong photoinduced electron transfer effect between the G-quadruplex and the fluorophore, thereby quenching the fluorescence of the fluorescent dye embedded in the complementary double strands at the nucleic acid tails, enabling the detection of OTA. This kit can be used to determine the concentration of OTA in foods and feeds such as wine, grape juice, beer, wheat, and corn. The results demonstrate that the prepared fluorescence kit for detecting aflatoxin A based on antiparallel G-quadruplex quenching of the fluorophore has excellent practical application value.

[0133] The descriptions presented in the above exemplary embodiments are only intended to illustrate the technical solutions of the present invention and are not intended to be exhaustive or to limit the present invention to the precise forms described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described in order to explain the specific principles of the present invention and its practical applications, so that other persons skilled in the art can easily understand, implement and utilize the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be defined by the appended claims and their equivalents.

Claims

1. A fluorescence detection kit for detecting small biological molecules using G-quadruplex as a quenching group in nucleic acid aptamers, characterized in that: Includes the following solutions: The detection solution comprises 100-2000 nM detection nucleic acid, a buffer solution with a concentration of 100-1000 mmol / L and a pH of 4-10, and a surfactant with a mass fraction of 0.1%-10.0%. The detection nucleic acid comprises two parts: (1) a nucleic acid aptamer that can recognize the biological small molecule to be detected and form a G-quadruplex or a G-rich secondary structure, and (2) a stem structure formed by complementary base sequences connected to both ends of the nucleic acid aptamer, and the stem structure can be embedded in a fluorescent dye to generate fluorescence; the complementary base sequence in the stem structure is Am-nucleic acid aptamer-Tn, wherein m=1-20, n=1-20, and the label-free fluorescent dye can be embedded in the structure to generate fluorescence; Fluorescent colorimetric solution: including a label-free fluorescent dye with a concentration of 0.1~10.00×; a buffer solution with a concentration of 100-1000mM and a pH of 4-10.

2. A fluorescence detection kit for detecting small biological molecules using a G-quadruplex as a quenching group in a nucleic acid aptamer according to claim 1, characterized in that: A preservative is added to the detection solution.

3. The fluorescence detection kit for detecting small biological molecules using G-quadruplex as a quenching group in nucleic acid aptamers according to claim 1, characterized in that: The nucleic acid aptamer is a nucleotide fragment obtained from a nucleic acid molecule library and capable of combining with a target substance with high specificity and selectivity.

4. The fluorescence detection kit for detecting small biomolecules using a G-quadruplex as a quenching group in a nucleic acid aptamer according to claim 1, characterized in that: The surfactant includes Tween 20, Tween 60, Tween 80 or Triton X100.

5. The fluorescence detection kit for detecting small biomolecules using G-quadruplex as a quenching group in nucleic acid aptamers according to claim 1, characterized in that: The buffer solution in the detection solution includes Tris-HCl buffer solution, PBS buffer solution or HEPES buffer solution.

6. The fluorescence detection kit for detecting small biological molecules using G-quadruplex as a quenching group in nucleic acid aptamers according to claim 2, characterized in that: The preservatives include gentamicin or cinnamaldehyde.

7. A method for using a fluorescence detection kit for detecting small biomolecules using a G-quadruplex as a quenching group in a nucleic acid aptamer according to any one of claims 1 to 6, characterized in that: The steps include: Step 1: Take 80 mL of the sample to be tested and add it to the wells of a 96-well all-black microplate; Step 2: Add 10 mL of the test solution to the sample in a 96-well black microplate and allow to react for 3-20 minutes. Step 3: Add 10 mL of fluorescent colorimetric solution to the sample to be tested in a 96-well black microplate and allow to react for 1-10 minutes. Step 4: Measure the fluorescence intensity using a fluorescence microplate reader, a fluorescence spectrophotometer or a simple fluorescence detector.

8. Use of a fluorescence detection kit for detecting small biological molecules using a G-quadruplex as a quenching group in nucleic acid aptamers according to any one of claims 1 to 6, characterized in that: Used for the detection of small biological molecules.

9. Use of a fluorescence detection kit for detecting biological small molecules using a G-quadruplex as a quenching group in nucleic acid aptamers according to claim 8, characterized in that: Used to detect tumor markers.

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