Theanine nucleic acid aptamer and application thereof in immunological detection

Abstract

The invention belongs to the field of theanine detection, and particularly discloses a theanine nucleic acid aptamer and application of the theanine nucleic acid aptamer in immunological detection. According to the method, an exponential enrichment ligand systematic evolution technology (SELEX) is utilized, carboxyl magnetic beads serve as a target fixing medium, theanine serves as a target, a nucleotide sequence specifically bound with the target is obtained through 14 rounds of screening, the dissociation constant reaches mu M after truncation optimization, and due to the advantages of being high in affinity, capable of being artificially synthesized, low in cost, easy to chemically modify and the like, the target can be specifically bound to the carboxyl magnetic beads. And the antibody can replace the theanine antibody to be used for immunological detection. The invention provides application of a theanine nucleic acid aptamer in immunohistochemical and ELISA (enzyme-linked immunosorbent assay) detection of theanine.

Description

Technical Field

[0001] The invention belongs to the field of theanine detection, and in particular relates to a theanine nucleic acid aptamer and its application in immunological detection. Background Art

[0002] Theanine (L-Theanine) is the main part of free amino acids in tea leaves, accounting for 1%-2% of the dry weight of tea leaves. It has many unique physiological functions. It can act as an analog of glutamine and an antagonist of caffeine, affecting the secretion and function of neurotransmitters in the central nervous system of humans and animals, and has a calming and anti-stress effect. In addition, theanine can also resist obesity, lower blood pressure, fight tumors, protect nerves, etc. At the same time, theanine has the fresh taste of monosodium glutamate and the sweetness of caramel. It is the main component of the fresh taste of tea leaves, which can relieve the bitterness of tea and enhance the sweetness. The content of theanine is a key factor affecting the taste of tea leaves, and largely determines the quality and economic value of tea leaves. Theanine can not only improve the flavor and quality of tea leaves, but also affect the physiological functions of tea trees. Theanine is a key substance that regulates the nitrogen metabolism of tea trees and contributes to the nitrogen cycle. The ammonium tolerance of tea trees is closely related to theanine metabolism. Tea trees can consume excess ammonium nitrogen in the body by synthesizing a large amount of theanine, thereby enhancing the ability to remove ammonia poisoning.

[0003] At present, various analytical techniques have been developed for the detection of theanine content, such as high performance liquid chromatography, amino acid analyzer, capillary electrophoresis, ninhydrin colorimetry, etc. Although these detection technologies meet the analytical requirements and can accurately detect the content of theanine, most of the technologies have inherent limitations and challenges. The instrument-based analysis methods have large instruments and equipment, expensive prices, complex experimental operations, and long detection times; while the simple instrument ninhydrin colorimetry has the disadvantages of low sensitivity and susceptibility to the influence of other amino acids. In addition, the theanine detection methods currently on the market do not involve in situ detection. Therefore, the development of a rapid, simple, reliable, sensitive, and in situ detection method for theanine detection is of great significance for theanine detection and spatial metabolomics in tea plants.

[0004] In recent years, biosensors based on nucleic acid aptamers have attracted great attention in the field of detection science due to their low cost, high selectivity and high sensitivity. Nucleic acid aptamers refer to small fragments of DNA or RNA that can bind to target substances with high specificity and affinity, which are screened from oligonucleotide synthetic libraries using the systematic evolution of ligands by exponential enrichment (SELEX). The binding of aptamers to targets is achieved through a specific three-dimensional conformation. It is based on secondary structures such as hairpins, stem loops, and G-quadruplexes, and achieves high-affinity binding with targets under specific conditions through hydrogen bonds, van der Waals forces and other interaction forces. The target range of nucleic acid aptamers is wide and is constantly increasing, including proteins, living cells, small biological molecules, heavy metal ions, viruses, etc. Compared with antibodies, aptamers are widely used in the field of analysis and detection due to their low cost, easy synthesis and modification, wider target range, better thermal stability, smaller molecular weight, and small batch-to-batch differences. They have been widely used in various fields such as medical diagnosis, environmental pollutant detection, and food hazard detection. Currently, there are very few research reports on theanine aptamers. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to provide a theanine nucleic acid aptamer and its application in immunological detection. The present invention utilizes the systematic evolution of ligands by exponential enrichment (SELEX) technology, takes carboxyl magnetic beads as the target fixation medium, and theanine as the target, and obtains an aptamer that specifically binds to the target through 14 rounds of screening. After truncation optimization, the dissociation constant reaches μM, and it specifically binds to theanine molecules; the nucleic acid aptamer screened and obtained by the present invention has good affinity and specificity, can be artificially synthesized, has low cost, short production cycle, and is easy to chemically modify. The aptamer can be prepared into sensors, molecular probes, detection reagents, etc. for the detection of theanine, and can replace the antibodies of theanine for immunological detection.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A theanine nucleic acid aptamer, the nucleotide sequence of which is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0008] SEQ ID NO:1 Sequence:

[0009] 5'-ATAGGAGTCACGACGACCAGAAGCGGCAGCAGCAGCCCGCCCGT GACCCAGCAGTCTGCCCGTATGTGCGTCTACCTCTTGACTAAT-3';

[0010] SEQ ID NO:2 Sequence:

[0011] 5'-GTCACGACGACCAGAAGCGGCAGCAGCAGCCCGCCCGTGACCCA GCAGTCTGC-3';

[0012] SEQ ID NO: 2 is a truncated and optimized sequence of SEQ ID NO: 1, and has a higher binding affinity with theanine, with a dissociation constant reaching μM.

[0013] The nucleotide sequence of the nucleic acid aptamer is modified, and the modification is selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium, and assimilation.

[0014] The nucleotide sequence of the nucleic acid aptamer is connected with a substance for labeling or detection, and the substance is selected from at least one of a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxin, a nanoluminescent material and an enzyme.

[0015] A detection probe or detection kit for theanine comprises the above-mentioned nucleic acid aptamer.

[0016] Application of the nucleic acid aptamer in preparing a detection probe or a detection kit for theanine.

[0017] Application of the above-mentioned nucleic acid aptamer, detection probe or detection kit in in situ detection of theanine.

[0018] The invention discloses an application of an immunohistochemical method based on nucleic acid aptamers in detecting theanine. The nucleic acid aptamers are labeled with digoxigenin and the signal molecule is alkaline phosphatase.

[0019] Application of aptamer-based enzyme-linked immunosorbent assay in the detection of theanine.

[0020] Application of the above-mentioned nucleic acid aptamers in the separation and enrichment of theanine.

[0021] Application of the above nucleic acid aptamer in the preparation of theanine targeting reagent.

[0022] The advantages of the present invention are that it provides a nucleic acid aptamer with high affinity for theanine, provides a new tea plant spatial metabolomics research strategy - an aptamer-based immunohistochemistry method, and provides an aptamer-based ELISA detection method for theanine.

[0023] The theanine aptamer obtained by the present invention can distinguish the sites of theanine in a tissue section environment with a thickness of 12 μm, accurately capture theanine, is not disturbed by complex environments, and has strong specific capture ability. Compared with previous metabolomics methods, the immunohistochemical method based on theanine aptamer proposed by the present invention can obtain the more microscopic spatial distribution characteristics of theanine in tea plants through the aptamer, and determine its sites in tissues, which will be a very effective new method for studying the spatial distribution of metabolites.

[0024] Compared with antibodies commonly used in traditional ELISA methods, the aptamer-based ELISA theanine detection method provided by the present invention has advantages over aptamers, including low cost, large-scale production, higher stability to high temperature and extreme pH values, long half-life and reversible denaturation, and easy modification of various functional group molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Agarose gel image of SELEX screening PCR results.

[0026] Figure 2 Affinity verification results of the aptamer library in the screening round.

[0027] Figure 3 This is the secondary structure of theanine nucleic acid aptamer.

[0028] Figure 4 Figure 2 shows the ITC data of affinity detection of theanine nucleic acid aptamer (SEQ ID NO: 1) obtained by the present invention and K d value.

[0029] Figure 5 The figure shows the docking result and binding site of theanine nucleic acid aptamer (SEQ ID NO: 1) and theanine molecule.

[0030] Figure 6 ITC data and K of theanine aptamer (SEQ ID NO: 2) and theanine affinity test d value.

[0031] Figure 7 This is a qualitative result diagram of the nanogold colorimetric experiment for specific verification of theanine nucleic acid aptamer (SEQ ID NO: 2).

[0032] Figure 8 This is a quantitative result diagram of the nanogold colorimetric experiment for specific verification of theanine nucleic acid aptamer (SEQ ID NO: 2).

[0033] Fig. 9 It is a control for incubation without theanine aptamer in tea stem, root, main vein and mesophyll sections in immunohistochemistry experiments.

[0034] Fig.10 This is a diagram showing the incubation results of adding theanine aptamer (SEQ ID NO: 2) to the sections of the mature zone, elongation zone and meristem zone of tea stem and root in immunohistochemistry experiment.

[0035] Fig.11 This is a diagram showing the incubation results of adding theanine aptamer (SEQ ID NO: 2) to the main veins and mesophyll sections of tea leaves in an immunohistochemical experiment. Three locations were selected: the leaf tip (LT), leaf middle (LC), and leaf base (LB).

[0036] Fig.12 ELISA detection of theanine nucleic acid aptamer (SEQ ID NO: 2). DETAILED DESCRIPTION

[0037] The present invention will be further illustrated and described below in conjunction with the embodiments.

[0038] Experimental materials and instruments:

[0039] Carboxyl magnetic beads (SMART Lifesciences), DNA urea denaturation-PAGE electrophoresis kit (Coolaber), UNIQ-10 column PAGE gel DNA recovery kit (Shanghai Sangon Biotechnology Co., Ltd., catalog number: B511135-0100), 1×PBS, 10×TBE, 2×DNA urea loading buffer (Coolaber), LATaq enzyme, 10×LATaqBuffer II (Mg 2+ Plus), dNTP Mixture (2.5 mM) (TaKaRa), FAM fluorescently labeled forward primer SEQ ID NO: 3 and Spacer18 spacer-labeled reverse primer SEQ ID NO: 4

[0040] (SEQ ID NO: 3: 5'-ATAGGAGTCACGACGACCAGAA-3'; SEQ ID NO: 4: 5'-dA20-Spacer18-ATTAGTCAAGAGGTAGACGCACATA-3') and an initial ssDNA library with unlabeled primers, a random sequence of 40 bases in the middle, and fixed primer sequences at both ends, SEQ ID NO: 5 (SEQ ID NO: 5: 5'

[0041] -ATAGGAGTCACGACGCCCAGAA-N40-TATGTGCGTCTACCTTGTACTAAT-3') were synthesized by Qingke.

[0042] Electrophoresis apparatus (Bio-Rad), electrophoresis apparatus (Liuyi), pH meter (Mettler Toledo), gel imaging analyzer (Bio-Rad), silent mixer (Qilin Bell WH-986), analytical balance (METTLER TOLEDO), constant temperature shaker, water bath (Jinghong), vortex mixer (SCILOGEX), ultra-micro spectrophotometer (Nanodrop)

[0043] Example 1 SELEX screening of theanine aptamers

[0044] 1.1 Carboxyl magnetic beads coupled to tea aminocarboxylate

[0045] (1) Solution preparation

[0046] Activation solution: 50 mM MES, pH 5.0;

[0047] EDC solution: 10 mg / ml, prepared with 50 mM MES (pH 5.0) solution;

[0048] Theanine solution: 1 mg / ml, prepared with 50 mM MES (pH 5.0) solution;

[0049] Blocking solution: 50 mM Tris, pH 7.4;

[0050] Washing solution: 0.1% Tween-20 in 1× PBS, pH 7.4;

[0051] (2) Magnetic bead activation

[0052] After mixing the magnetic beads, take 4 portions of 50μL 10mg / ml carboxyl magnetic beads in an EP tube, numbered as A, B, C, and D groups respectively. Place the EP tube on the magnetic separator for about 1 minute. When the solution becomes clear, use a pipette to discard the clear liquid. Remove the EP tube from the magnetic separator, add an activation solution equal to the volume of the suspension, use a gun tip to blow repeatedly 5-10 times, place the EP tube on the magnetic separator for about 1 minute, wait for the solution to become clear, use a pipette to discard the clear liquid, and repeat the wash once. Add 50μL EDC solution and mix well. Incubate at room temperature for 30 minutes. Make sure the magnetic beads are fully mixed, otherwise it will affect the activation efficiency. Place the EP tube on the magnetic separator for about 1 minute. When the solution becomes clear, use a pipette to discard the clear liquid. Wash twice with pre-cooled deionized water and activation solution as quickly as possible to avoid hydrolysis of the activation group.

[0053] (3) Magnetic beads coupled to theanine

[0054] In the four EP tubes treated with the above steps, add 50μl 1mg / ml theanine solution to groups A and B, and add 50μL activation solution to groups C and D. Mix thoroughly and incubate overnight at room temperature on a silent mixer (not less than 14h) to ensure that the magnetic beads are fully mixed. Place the EP tube containing the sample on a magnetic separator for about 1min after overnight incubation. After the solution becomes clear, collect the supernatant and magnetic beads from groups B and D respectively, and discard the supernatant and keep the magnetic beads from groups A and C. Wash the magnetic beads in groups B and D twice with cleaning solution to remove non-specific adsorption, then wash twice with 1×PBS buffer (pH7.4), add 20μL 1×PBS buffer (pH7.4), and water bath at 95℃ for 8min. After the EP tube after the water bath is ice-bathed for 8min, place it on a magnetic separator for about 1min, and collect the supernatant after the solution becomes clear. The absorbance of the supernatants of groups B and D after incubation, the supernatants after 95°C water bath, and theanine solution was measured using a Nanodrop micro-spectrophotometer to test the magnetic bead coupling effect, as shown in Table 1.

[0055] Table 1 Absorbance results of magnetic beads coupled with theanine

[0056]

[0057] (4) Block the remaining sites on the magnetic beads

[0058] Add 125 μl of blocking solution to the magnetic beads of groups A and C and incubate at room temperature for 30 min. Place the EP tube on the magnetic separator for about 1 min. When the solution becomes clear, discard the clear solution with a pipette, wash twice with the washing solution to remove non-specific adsorption, and wash twice with 1× PBS buffer (pH 7.4).

[0059] 1.2 Incubation of coupled carboxyl magnetic beads with library

[0060] (1) The 10 μM initial ssDNA library was first denatured in a 95°C water bath for 8 minutes and then immediately renatured in an ice bath for 8 minutes to form a specific structure.

[0061] (2) Add 400uLBB buffer to the EP tube in group C.

[0062] (3) Add 200uL of the initial ssDNA library to the above EP tube and incubate at room temperature for 30min.

[0063] (4) After incubation, all the supernatant was aspirated and added to the carboxyl magnetic beads coupled with theanine and incubated for 1 h.

[0064] (5) Aspirate and discard the supernatant, add 100 μL ddH2O, and incubate in a 95°C water bath for 8 minutes. After cooling, collect the supernatant, which is the first round of aptamer library obtained by carboxyl magnetic beads screening and used as a template for PCR amplification.

[0065] 1.3 PCR amplification

[0066] The aptamer library obtained by screening carboxyl magnetic beads was used as a PCR template, and the LAtaq enzyme system was selected to perform PCR amplification. The specific dosage is shown in Table 2 below;

[0067] Table 2

[0068] name Dosage LATaq enzyme 0.5μL <![CDATA[10×LATaqBufferII(Mg 2+ Plus)]]> 5μL dNTP Mixture (2.5 mM) 8μL Template 3μL FAM fluorescently labeled forward primer 2.5μL Spacer18 reverse primer 2.5μL dd sterile water 28.5μL

[0069] (1) Take 20 200 μL EP tubes and divide them into two groups. Add the ingredients in the above table into them accordingly.

[0070] (2) Vortex mix, centrifuge at low speed, and then place the EP tube into the PCR instrument.

[0071] (3) Edit the PCR program: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 95°C for 45 s, annealing at 60°C for 15 s, extension at 72°C for 15 s; final extension at 72°C for 1 min; incubation at 20°C for 15 min.

[0072] 1.4 Agarose gel electrophoresis to verify PCR results:

[0073] (1) Prepare 2% agarose gel: weigh 0.5 g agarose and add it to a conical flask. Then add 25 mL of 0.5% TBE solution to the conical flask and heat it in a microwave oven until the agarose is dissolved. Add 2.5 μL of nucleic acid dye, shake well, and pour it onto the gelatin plate. Insert a comb and let it stand for 30 min.

[0074] (2) After the gel has solidified, place the gel plate into the electrophoresis tank, add 0.5× TBE solution, remove the comb, and load 5 μL of sample.

[0075] (3) Perform electrophoresis at 120 V on a Bio-Rad electrophoresis apparatus for 30 min.

[0076] (4) After removing the gel, use a Bio-Rad gel imaging analyzer to image and determine the PCR effect.

[0077] 1.5 DNA-PAGE electrophoresis:

[0078] (1) Preparation of urea-PAGE gel

[0079] Prepare 30mL of 8% PAGE gel and shake it evenly. Pour the prepared gel into the gel plate (leakage test is required in advance), stop pouring the gel when the gel surface reaches the top, and insert the comb. Polymerize at room temperature for 60 minutes. After the gel solidifies, pull out the comb and rinse the gel holes with 1×TBE solution.

[0080] (2) Sample processing

[0081] The PCR amplified product sample was mixed with 2× DNA urea loading buffer in a volume ratio of 1:1, denatured at 95°C for three minutes, and placed on ice before use.

[0082] (3) Electrophoresis

[0083] Fix the gel plate on the 6-1 electrophoresis instrument and add enough 1×TBE to the upper and lower tanks. After 30 minutes of pre-electrophoresis, load the samples placed on ice, with one well of each gel being marked with a marker and the remaining wells being marked with PCR samples. Connect the power cord, turn on the switch, and select a power of 5-6w. Stop the electrophoresis and take out the gel. Use the Bio-Rad gel imaging analyzer to image the gel, find the position corresponding to the fluorescent marker, and cut the entire row horizontally.

[0084] 1.6 PAGE gel DNA recovery:

[0085] The desired bands were recovered using the UNIQ-10 column PAGE gel DNA recovery kit (Cat. No.: B511135-0100). The aptamer library obtained in this step was used for the next round of screening.

[0086] 1.7 Repeat the second to fourteenth rounds of screening

[0087] 1.8 Affinity verification of aptamer libraries in each round

[0088] Fluorescence spectrophotometry was used to verify the affinity changes between aptamer screening rounds. For the 14 rounds of aptamer libraries obtained by carboxyl magnetic bead screening, 20 nmol / L of aptamer library was incubated with magnetic beads coupled with theanine, the supernatant was discarded after incubation, the ssDNA bound to the magnetic beads was eluted, and the fluorescence intensity of the eluate was measured using a micro-fluorescence spectrophotometer.

[0089] 1.9 High-throughput sequencing and secondary structure prediction

[0090] Several rounds of aptamer libraries with higher affinity were selected and PCR amplified using unlabeled primers. The PCR products were purified and sent to Novogene for library construction and High-throughput sequencing (HTS) sequencing was completed using the Illumina sequencing platform. The sequencing data was analyzed using a self-written program to obtain candidate theanine nucleic acid aptamers to be tested. Finally, the secondary structure and free energy of the aptamers were predicted using the NUPACK website (http: / / www.nupack.org / ).

[0091] Test results:

[0092] As can be seen from Table 1, the absorbance value of the supernatant incubated with theanine and magnetic beads is less than that of the solution without theanine incubation, and the absorbance value of the eluate after theanine and magnetic beads incubation is greater than that of the eluate without theanine incubation, indicating that theanine is successfully coupled to the magnetic beads.

[0093] From the gel image after PCR ( Figure 1 ) can be seen from the figure that the band size is between 50-100 bp, which is consistent with the 87 nt size of the initial library, indicating that the screening process is reasonable and can be purified and recovered to continue the next round of screening.

[0094] The aptamer library of the same concentration was incubated with the target, and the fluorescence intensity of the ssDNA eluted after incubation was measured. The more ssDNA theanine bound, the greater the fluorescence intensity of the eluted solution, and the higher the affinity of the aptamer library in this round. Figure 2 The results showed that the affinity of the 5th / 7th / 13th rounds screened was high, and the affinity of the 7th round was the highest, so these three rounds were selected for high-throughput sequencing. The candidate theanine nucleic acid aptamer Apt-1 was obtained through high-throughput analysis, and its sequence is shown in SEQ ID NO: 1, and its secondary structure is shown in Figure 3 As shown in the left picture.

[0095] Example 2 Affinity verification and specificity verification and truncation optimization of theanine nucleic acid aptamer

[0096] 2.1 Aptamer affinity verification

[0097] The Kd value of theanine aptamer Apt-1 was determined by ITC. When the vacuum degree was higher than 400 mmHg, the sample was degassed for 10 minutes. The aptamer and theanine were dissolved in 1× binding buffer (binding buffer: 50mMTris, 5mMKCl, 100mMNaCl, 1mMMgCl2, pH=7.4). 350μL of 25μM aptamer was added to the titration needle. 50μL of 50mM theanine was added to the sample pool. The total number of titrations was set to 20, the volume of each titration was set to 2.5μL, the time was set to 200s, the reaction temperature was set to 25℃, and the stirring speed was set to 250rpm. The molar concentration of the sample and the name of the experimental data file were set in the titration syringe and sample pool. After running, the titration results were analyzed by Launch NanoAnalyze software to obtain the Kd value of the theanine aptamer sequence.

[0098] 2.2 Molecular docking and sequence truncation

[0099] The secondary structure of the aptamer Apt-1 was predicted using NUPACK, and the tertiary structure of the aptamer was generated using XiaoLab. The 3D structure of theanine was obtained from the PubChem website. The molecular docking software AutoDock was used to perform molecular docking simulation between the aptamer and the target to determine the optimal binding conformation. Based on the results of molecular docking and the secondary structure of the aptamer, the aptamer was truncated and optimized, and the non-binding sites were truncated to obtain the truncated aptamer Apt-2. The sequence is shown in SEQ ID NO: 2, and the secondary structure is shown in Figure 3 As shown in the figure on the right, after truncation, the Kd value of the truncated aptamer Apt-2 was determined using the ITC method.

[0100] 2.3 Verification of aptamer specificity

[0101] The specificity of the aptamer Apt-2 was verified by the nanogold colorimetric method. 1.25 μM aptamer (5 μL) was added to the EP tube, and incubated with 25 μL of 0.25 μM theanine, arginine, histidine, glutamic acid, serine and aspartic acid for 10 min, and then 135 μL of 13 nM nanogold particles was added and incubated for 5 min. After quickly adding 295 μL of 83.3 mM NaCl to the solution and incubating for 5 min, 200 μL of the solution was collected and placed on an ELISA plate for spectral scanning from 200 nm to 800 nm. At the same time, 200 μL of the solution was collected and photographed in an ELISA strip to determine the color change.

[0102] Test results:

[0103] In this study, the aptamer Apt-1 that can specifically recognize theanine was screened using the systematic evolution of ligands by exponential enrichment (SELEX). The dissociation constant between the aptamer and theanine was determined by ITC, and its Kd value was 2.772×10 -2 M( Figure 4 ). The binding site of the aptamer and theanine was simulated by molecular docking ( Figure 5 ), the aptamer was truncated and optimized, which greatly improved the binding affinity between the aptamer and theanine. The Kd value of the truncated aptamer Apt-2 was 2.033μM ( Figure 6 ).

[0104] In order to verify the specificity of theanine aptamer, this study used nanogold colorimetry to verify the binding specificity of theanine to the aptamer Apt-2, whose affinity was greatly improved after truncation. This study selected five amino acids with similar structures to theanine for aptamer specificity verification, namely arginine, histidine, glutamic acid, serine and aspartic acid. Nanogold colorimetric experiments are often used to characterize the binding affinity between small molecule targets and aptamers. AuNPs are stable colloidal solutions that are red in color. In a high-salt environment, AuNPs will aggregate, the solution will be bluish-purple, and the ultraviolet absorption spectrum will change. If an aptamer is added to the system, the aptamer will bind to the surface of AuNPs through electrostatic adsorption, protecting the AuNPs from dispersion in the solution and preventing aggregation. The color of the system will not change. When theanine is added, the high affinity of the aptamer to theanine causes the aptamer to preferentially bind to theanine. AuNPs lose the protection of the aptamer and will aggregate under high salt concentrations. The color of the solution gradually changes from red to bluish-purple. The degree of aggregation of AuNPs can be evaluated by measuring the absorbance ratio at wavelengths of 650nm and 520nm. After adding the NaCl solution, the color of the nanogold solution containing theanine changes most significantly ( Figure 7 ), the other five amino acids had no obvious color change in the nanogold solutions. The quantitative results also showed that the aptamer Apt-2 had the strongest binding ability with theanine ( Figure 8 ), and had no obvious binding ability with the other five amino acids. The binding ability of theanine aptamer to theanine was much higher than that of the other five amino acids.

[0105] Example 3 Detection of theanine in tea plant tissue sections by aptamer-based immunohistochemistry

[0106] 3.1 Collection and embedding of tissue specimens

[0107] The roots, stems and leaves of the tea tree were taken, cut into appropriate sizes, embedded in OCT, quickly frozen in liquid nitrogen and isopentane, and stored at -80°C.

[0108] 3.2 Slicing

[0109] The embedded blocks were sectioned using a freezing microtome with a section thickness of 12 μm.

[0110] 3.3 OCT removal

[0111] The slices were spread on a wet box, first soaked in distilled water for 5 min to wash off the OCT, then washed three times with PBS for 5 min, and wiped dry.

[0112] 3.4 Circle and closure

[0113] After drying the slices, use a tissue pen to draw a circle around the tissue. Add 5% BSA in the tissue circle to evenly cover the tissue and seal at room temperature for 30 minutes.

[0114] 3.5 Primary antibody (aptamer) incubation

[0115] The blocking solution was aspirated and 50 μL of 500 nmol / L digoxigenin-labeled theanine aptamer Apt-2 was added to the sliced ​​tissue and incubated at 37°C for 30 min.

[0116] 3.6 Secondary Antibody Incubation

[0117] The tissue sections were washed with PBS for 5 min three times and incubated with 50 μL of alkaline phosphatase (AP)-labeled digoxigenin antibody (1:100) at 37°C for 15 min.

[0118] 3.7 Staining

[0119] The tissue sections were washed with PBS for 5 min 3 times, then stained with 50 μL BCIP / NBT, incubated at room temperature for 90 min, the reaction was terminated with dd water, and finally the images were analyzed using an optical microscope.

[0120] Test results:

[0121] Fig. 9 The following are the controls without theanine aptamer in the stem, root, main vein and mesophyll sections. There are fewer purple signals in the tissues. The samples after incubation with theanine aptamer Apt-2 ( Fig.10 , Fig.11 ) showed that the signal of the stem was mainly in the cortex and phloem, and that of the root was mainly in the vascular tube. The spongy tissue in the mesophyll and the vascular tube in the main vein of the leaf slice showed purple signal, and the signal of the main vein was stronger than that of the mesophyll tissue. The experimental results show that the theanine aptamer obtained by the present invention can distinguish the sites of theanine in the environment of tissue slices with a thickness of 12 μm, and obtain the characteristics of the more microscopic spatial distribution of theanine in tea plants.

[0122] Example 4 Detection of theanine by aptamer-based enzyme-linked immunosorbent assay

[0123] 4.1 Coating and sealing

[0124] A 96-well plate was coated with theanine, and after washing, the wells were blocked with PBS buffer containing 1% BSA for 1 h and then washed 5 times with PBS buffer.

[0125] 4.2 Immune recognition

[0126] 20ug / ml of theanine aptamer Apt-2 labeled with biotin was added to each well after denaturation and renaturation at 95℃ for 8min and ice bath for 8min, and then incubated for 1 hour. The unbound molecules were washed 5 times with PBS buffer.

[0127] 4.3 Immune competition

[0128] 1g of fresh tea leaf sample was ground and added to 100mL PBS buffer, then placed in a constant temperature water bath at 100℃ for 30min, filtered and tested. The sample to be tested was added to each well, incubated for 1 hour, washed 5 times with PBS to wash away the biotin-labeled aptamer that was competitively bound, and the buffer without theanine was used as the control.

[0129] 4.4 Color development and termination

[0130] Streptavidin-HRP was added to each well for 30 minutes. TMB (tetramethylbenzidine) colorimetric solution was then added and incubated for 20 minutes, and terminated with H2SO4. The absorbance of each well was measured at 450nm by a microplate reader.

[0131] Test results:

[0132] The competitive ELISA method based on nucleic acid aptamers detects theanine. The control group does not contain theanine and will not compete for binding to the biotin-labeled theanine aptamer. Therefore, the OD value of the color development result in the control group well is greater than the OD value of the sample to be tested. The final color development result of the sample to be tested is inversely proportional to the theanine content in the sample to be tested, such as Fig.12 As shown, this test can detect the content of theanine in the solution, and the theanine aptamer has a high affinity with theanine.

Claims

1. A theanine nucleic acid aptamer, characterized in that: The nucleotide sequence of the nucleic acid aptamer is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The nucleic acid aptamer according to claim 1, characterized in that The nucleotide sequence of the nucleic acid aptamer is modified, and the modification is selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, replacement of oxygen with sulfur, replacement of oxygen with selenium, and assimilation.

3. The nucleic acid aptamer according to claim 1, characterized in that The nucleotide sequence of the nucleic acid aptamer is connected with a substance for labeling or detection, and the substance is selected from at least one of a fluorescent marker, a radioactive substance, biotin, streptavidin, digoxin, a nanoluminescent material and an enzyme.

4. A detection probe or detection kit for theanine, characterized in that: The invention comprises the nucleic acid aptamer according to any one of claims 1 to 3.

5. Use of the nucleic acid aptamer according to any one of claims 1 to 3 or the detection probe or detection kit according to claim 4 in in situ detection of theanine.

6. Application of aptamer-based immunohistochemistry in detecting theanine, characterized in that: The nucleic acid aptamer is the nucleic acid aptamer according to any one of claims 1 to 3.

7. The use according to claim 6, characterized in that: The nucleic acid aptamer is labeled with digoxigenin, and the signal molecule is alkaline phosphatase.

8. Application of aptamer-based enzyme-linked immunosorbent assay in detecting theanine, characterized in that: The nucleic acid aptamer is the nucleic acid aptamer according to any one of claims 1 to 3.

9. Use of the nucleic acid aptamer described in any one of claims 1 to 3 in the separation and enrichment of theanine.

10. Use of the nucleic acid aptamer according to any one of claims 1 to 3 in the preparation of a theanine targeting agent.

Images