Nucleic acid aptamers and derivatives that specifically bind risperidone, uses, kits

Abstract

The application discloses a nucleic acid aptamer and derivatives, application and kit capable of specifically combining with risperidone, comprising a nucleotide sequence shown in SEQ ID No. 1, or a nucleotide sequence with high homology with the nucleotide sequence shown in SEQ ID No. 1 and capable of specifically combining with risperidone, or a nucleotide sequence derived from the nucleotide sequence shown in SEQ ID No. 1 and capable of specifically combining with risperidone. The application provides a nucleic acid aptamer and derivatives capable of combining with risperidone, which have high specificity, stable chemical properties, are easy to preserve and label, and also correspondingly provides a screening method and application of the nucleic acid aptamer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology, and relates to aptamer, in particular to aptamer specifically binding to risperidone and derivatives, application and kit. BACKGROUND

[0002] Risperidone is a phenylpiperazine isoxazole derivative, with a molecular formula of C23H27FN4O2, a molecular weight of 410.484, and a chemical name of 3-[2-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidyl]ethyl]-6,7,8,9-tetrahydro-2-methyl-4H-pyrido[1,2-a]pyrimidin-4-one. It is a psychotropic drug used for treating acute and chronic schizophrenia. In particular, it has good effects on positive and negative symptoms and associated affective symptoms (such as anxiety, depression, etc.), and can also reduce affective symptoms associated with schizophrenia. For patients who are effective in acute phase treatment, risperidone can continue to exert its clinical effects in maintenance phase treatment.

[0003] However, due to the wide pharmacokinetic variation of risperidone among patients, monitoring the drug concentration in blood and adjusting it to a target level are useful for improving drug effectiveness and minimizing toxicity. It is reported that the pharmacokinetic variation of risperidone and its derivatives in different individuals and among individuals is 13-fold, which is affected by many factors. The result of these differences is that the same amount of the same drug can lead to significantly different clinical results in different individuals. According to the clearance rate of individual drugs in patients and the final drug concentration in blood, the effects of risperidone at the same dose are also significantly different. Therefore, it is of great significance to establish a risperidone detection method with high specificity and sensitivity for developing individualized dose regimen of risperidone, evaluating its clinical efficacy and safety.

[0004] At present, the determination of risperidone blood concentration is mostly carried out by radioimmunoassay, high performance liquid chromatography and immunological detection method. However, radioimmunoassay has the disadvantages of high cost, easy radiation pollution and low instrument popularization rate. High performance liquid chromatography needs multiple repeated extraction, and has the disadvantages of complicated operation, complexity, time-consuming and laborious, low throughput, etc., and cannot realize rapid and convenient detection, which is not conducive to clinical promotion. Immunological detection method is dependent on antibody detection kit, although some can achieve rapid and simple detection, but the antibody preparation process is relatively complex, and there are differences between batches, so there are certain defects.

[0005] The main defects are that other methods are high in cost, instruments are expensive, and immunological detection methods using antibodies as recognition elements have certain shortcomings. For example, antibody production relies on animals, has large batch differences, is high in cost, is strongly dependent on temperature, pH value and salt concentration, is not reversibly denatured, and the like, resulting in problems such as high price, difficulty in reuse, difficulty in ensuring stable results and high detection cost.

[0006] Aptamer refers to a DNA or RNA molecule obtained by screening and separation through SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can be combined with other targets such as proteins, metal ions, small molecules, polypeptides or even entire cells with high affinity and specificity, and thus has broad prospects in biochemical analysis, environmental monitoring, basic medicine and new drug synthesis.

[0007] Compared with antibodies, aptamers have small molecular weight, better stability, easy modification, no immunogenicity, short production cycle, and can be synthesized artificially, thus avoiding a series of processes such as animal immunization, feeding, protein extraction and purification, and thus aptamers are an ideal molecular probe.

[0008] Based on the SELEX method, an aptamer that binds to a specific small molecule is screened, and the aptamer is used for detection of the small molecule, which is also widely studied. However, no one has published or applied an aptamer for risperidone, and thus there is a need in the art for an aptamer with high binding affinity for risperidone. SUMMARY

[0009] In order to overcome the deficiencies of the prior art, the purpose of the present application is to provide an aptamer and derivatives thereof capable of binding risperidone, which has high specificity, stable chemical properties, is easy to store and label, and also correspondingly provides a screening method, a kit and an application of the aptamer.

[0010] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0011] In a first aspect, the present application provides an aptamer that specifically binds to risperidone, comprising a nucleotide sequence as shown in SEQ ID No. 1, or a nucleotide sequence having high homology with the nucleotide sequence of SEQ ID No. 1 and capable of specifically binding to risperidone, or a nucleotide sequence derived from the nucleotide sequence as shown in SEQ ID No. 1 and capable of specifically binding to risperidone.

[0012] As a preferred solution of the present application, the high homology refers to at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the nucleotide sequence shown in SEQ ID No. 1.

[0013] As a preferred solution of the present application, the nucleic acid aptamer comprises a nucleotide sequence complementary to the nucleotide sequence and retains the affinity.

[0014] As a preferred solution of the present application, the nucleotide sequence of the nucleic acid aptamer comprises a base modification and retains the affinity.

[0015] As a preferred solution of the present application, the base modification is a thio modification, a phosphorylation modification, a methylation modification, an amination modification, a sulfydryl modification, a selenium substitution oxygen modification, or a connection isotope modification.

[0016] Those skilled in the art should understand that, as an improvement to the above technical solution, a modification can be made to a certain position on the nucleotide sequence of the above nucleic acid aptamer, for example, phosphorylation, methylation, amination, sulfydryl, substitution of oxygen with sulfur, substitution of oxygen with selenium, or connection of isotopes, provided that the nucleic acid aptamer sequence obtained after such modification has desirable properties, for example, can have an affinity for binding risperidone equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or although the affinity is not significantly improved but has higher stability.

[0017] As a preferred solution of the present application, the nucleotide sequence of the nucleic acid aptamer comprises a label and retains the affinity.

[0018] As a preferred solution of the present application, the label is a fluorescent label, a radioactive label, a therapeutic label, a biotin label, a digoxin label, a nanoluminescent material label, a small peptide label, a siRNA label, or an enzyme label.

[0019] Those skilled in the art should understand that, as an improvement to the above technical solution, a fluorescent substance, a radioactive substance, a therapeutic substance, biotin, digoxin, a nanoluminescent material, a small peptide, siRNA, or an enzyme label, etc. can be connected to the nucleotide sequence of the above nucleic acid aptamer, provided that the nucleic acid aptamer sequence obtained after such modification has desirable properties, for example, can have an affinity for binding risperidone equal to or higher than that of the parent nucleic acid aptamer sequence before modification, or although the affinity is not significantly improved but has higher stability.

[0020] In other words, the above nucleic acid aptamer sequence, whether partially substituted or modified, has substantially the same or similar molecular structure, physical and chemical properties and functions as the original nucleic acid aptamer, and can be applied to the binding of risperidone.

[0021] As a general technical concept, the nucleic acid aptamer described in the present application can also comprise any one of the following three sequences:

[0022] (1) a nucleotide sequence having more than 60% homology with the nucleotide sequence of the nucleic acid aptamer described in all the foregoing technical solutions (for example, the foregoing nucleic acid aptamer sequence can be deleted or supplemented with partially complementary nucleotides), preferably, the homology with the nucleotide sequence shown in SEQ ID No. 1 can be more than 70%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 99%;

[0023] (2) a nucleotide sequence capable of hybridizing with the nucleotide sequence of the nucleic acid aptamer described in all the foregoing technical solutions under stringent conditions;

[0024] (3) an RNA sequence transcribed from the nucleotide sequence of the nucleic acid aptamer described in all the foregoing technical solutions;

[0025] Among the above (1)-(3), the nucleotide sequence can specifically bind to risperidone.

[0026] In a second aspect, the present application also provides a nucleic acid aptamer derivative, which is a phosphorothioate backbone derived from the backbone of the nucleotide sequence of the nucleic acid aptamer described in all the foregoing technical solutions, or is a corresponding peptide nucleic acid modified from the nucleic acid aptamer described in all the foregoing technical solutions.

[0027] In a third aspect, the present application also provides an application of the foregoing nucleic acid aptamer or nucleic acid aptamer derivative. For example, the nucleic acid aptamer or its derivative of the present application can be used for the detection of risperidone, and the nucleic acid aptamer or its derivative of the present application can be used to detect the content of risperidone in the blood of a subject.

[0028] The above derived nucleic acid aptamer or other derivative has substantially the same or similar molecular structure, physical and chemical properties and functions as the original nucleic acid aptamer.

[0029] In a fourth aspect, the present application provides a kit for detecting risperidone, which comprises the nucleic acid aptamer of the first aspect of the present application.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] 1) Compared with antibodies, aptamers have small molecular weight, better stability, easy modification, no immunogenicity, short production cycle, and can be synthesized by artificial synthesis, which avoids a series of processes such as animal immunization, feeding, protein extraction and purification, so that the aptamer is an ideal molecular probe. There is no one to publish and no one to apply the aptamer for risperidone, therefore, there is a demand in the art for aptamers with high binding affinity for risperidone.

[0032] 2) Compared with antibodies, aptamers are more stable, and since they are chemically synthesized, there is almost no batch difference; and the aptamer is successfully used for the detection of risperidone small molecules.

[0033] 3) The present application provides an aptamer capable of binding risperidone and its derivatives, which has high specificity, stable chemical properties, is easy to store and label, and also provides a screening method and application of the aptamer. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is the simulated secondary structure of the sequence shown in SEQ ID No. 1 of the present application.

[0035] Figure 2 is the ITC titration graph of the sequence of the aptamer of the present application.

[0036] Figure 3 is the ITC titration graph of the control sequence of the present application.

[0037] Figure 4 is the fluorescence detection graph of the present application.

[0038] Figure 5 is the linear curve fitting of the present application. DETAILED DESCRIPTION

[0039] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0040] Example 1

[0041] Screening of ssDNA aptamer specific to risperidone:

[0042] 1. Synthesize the random single-stranded DNA library shown in the following sequence and the corresponding primer:

[0043] Random single-stranded DNA library: 5'-GTTGGCACTCCACGCATAGG(36N)CCTATGCGTGCTACCGTGAA-3'.

[0044] wherein "36N" represents a sequence of 36 arbitrary nucleotide bases. The library was synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.

[0045] The primer information is shown in Table 1, which was synthesized by General Biosystems.

[0046] Table 1. Primers and their sequences

[0047]

[0048]

[0049] wherein S in the primer name represents a forward primer, A in the primer name represents a reverse primer, the complementary sequences of S1 are denoted as S1CS, -biotin is a streptavidin modification, 20 A in the sequence represents a polyA tail composed of 20 adenosine (A), and "Spacer 18" represents a six-ethyleneglycol spacer of 18 atoms. The structure of "Spacer 18" is shown in formula I. The structure of "Spacer 18" used in the above A2-polyA primer is shown in formula I.

[0050]

[0051] The primers were prepared into 100 μM stock solutions with DPBS buffer (NaCl: 8 g / L, KCl: 0.2 g / L, Na2HPO4: 1.15 g / L, KH2PH4: 0.2 g / L, CaCl2: 0.1 g / L, MgCl2·6H2O: 0.1 g / L; PH 7.4) and stored at -20 °C for standby use.

[0052] 2. Magnetic beads and graphene oxide immobilized library screening of risperidone

[0053] The library was immobilized by magnetic beads and graphene oxide, and screened by small molecule competition binding method, a total of 10 rounds of screening, wherein rounds 1-7 were screened by magnetic bead immobilized library method, and rounds 8-10 were screened by graphene oxide immobilized library method. The specific screening methods are as follows, taking the first round and the eighth round as examples.

[0054] 2.1 Library dissolution: Take out the library powder biosynthesized by Shengong, centrifuge at 12000 rpm for 10 min. Add DPBS buffer to dilute the library to 10000 nM 130 μL, vortex to mix, then centrifuge at 12000 rpm for 5 min. Take 100 μM 26 μL from the S1CS primer dissolved in DPBS and add to the diluted library. After mixing the primer and library well, centrifuge at 12000 rpm for 2 min.

[0055] 2.2. Library and primer matching: The mixture of library and complementary primer is dispensed into PCR tubes and placed in a PCR instrument for slow renaturation. The following PCR instrument settings are used for the renaturation program: 95°C for 10 min, slow cooling to 60°C at a rate of 0.1°C / s; 60°C for 1 min; slow cooling to 25°C at a rate of 0.1°C / s. After the renatured library and complementary primer mixture is placed at room temperature for 5 min, a small amount is taken to determine the concentration C1 by UV (A260).

[0056] 2.3. Take 1000 μL of streptavidin magnetic beads (SA magnetic beads, purchased from Invitrogen, Dynabeads MyOne Streptavidin, item number: 65001), wash the magnetic beads 4 times with DPBS, each time using 500 μL of DPBS. Specifically, use a magnet to fish the magnetic beads, remove the supernatant, add DPBS buffer for washing, and finally store the magnetic beads in a small amount of DPBS to prevent the magnetic beads from drying out. (Hereafter, the library amount is reduced to 800 nM 100 μL for the second to seventh rounds, the magnetic bead amount is 100 μL, and the DPBS volume used for washing the magnetic beads is reduced to 200 μL.) TM MyOne TM Streptavidin, item number: 65001), wash the magnetic beads 4 times with DPBS, each time using 500 μL of DPBS. Specifically, use a magnet to fish the magnetic beads, remove the supernatant, add DPBS buffer for washing, and finally store the magnetic beads in a small amount of DPBS to prevent the magnetic beads from drying out. (Hereafter, the library amount is reduced to 800 nM 100 μL for the second to seventh rounds, the magnetic bead amount is 100 μL, and the DPBS volume used for washing the magnetic beads is reduced to 200 μL.)

[0057] 2.4. Add the renatured library and complementary primer mixture to the magnetic beads from which the supernatant has been removed in 2.3, mix well, shake on a rotary instrument at room temperature for 60 min, use a magnet to fish the magnetic beads, recover the supernatant, and take a small amount of the supernatant to determine the UV (A260) concentration to obtain the value C2. According to the determined concentration, calculate the immobilization efficiency of the library coupled to the magnetic beads. Library immobilization efficiency = (C1-C2) / C1. To ensure the success rate of screening, the first round of library immobilization efficiency is greater than 50%, and the second to seventh rounds of library immobilization efficiency is greater than 85% to continue screening.

[0058] 2.5. Wash the magnetic beads from the previous step by adding 500 μL of wash buffer (DPBS) to the beads, mix to resuspend the beads, and incubate at room temperature for 1 minute. Then, use a strong magnet to attract the beads and remove the supernatant. Repeat this wash step four more times, and note that a new EP tube is needed for each wash. Next, perform a long wash by adding 200 μL of wash buffer to the beads, mix to resuspend the beads, and incubate at room temperature for 20 minutes on a shaker. Then, use a strong magnet to attract the beads and remove the supernatant. (From the second to seventh rounds of screening, use only 200 μL of wash buffer for this step.)

[0059] 2.6. Elute the target: Dissolve risperidone (molecular formula C23H27FN4O2, molecular weight 410.484, molecular structure shown in Formula II) in methanol to 5 mM, and add 4 μL to 196 μL of DPBS, which is diluted 50-fold to 100 μM. Mix well and add the entire mixture to the SA magnetic beads from step 2.5, and incubate at room temperature for 20 minutes on a shaker. Use a strong magnet to attract the beads and recover the supernatant into an EP tube, which is labeled as Elution.

[0060]

[0061] 3. Preparation of secondary library

[0062] 3.1 Amplify the double-stranded DNA: Use the nucleic acid molecules in Elution as a template for PCR (ePCR) amplification. The method is as follows: add all of the template Elution to 2 mL of PCR mix, mix well, and then divide the mixture into 100 μL / tube and add to PCR tubes. Place the tubes in a PCR instrument (Bio-Rad, gradient PCR instrument T100) for amplification, with the following conditions: 95°C pre-denaturation for 2 minutes, 95°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 30 seconds, for a total of 25 cycles, and 4°C storage.

[0063] The formula of the PCR mix is shown in Table 2.

[0064] Table 2. Formula of ePCR mix

[0065] Reagents Total volume 1000 μL ddH2O 866 μL 10*pfu enzyme buffer 100 μL dNTP mix (10 mM) 20 μL Forward primer S1-FAM (100 μM) 5 μL Reverse primer A2-polyA (100 μM) 5 μL Pfu enzyme 4 μL (20 U)

[0066] 3.2 Concentrate the amplified product with n-butanol: Collect all of the ePCR product into a 15 mL sharp-bottomed centrifuge tube, add 2 volumes of n-butanol, and shake well on a vortex mixer. Centrifuge at 9000 rpm (revolutions per minute) at room temperature for 10 minutes. After centrifugation, obtain a layered liquid, and remove the upper phase (n-butanol) to obtain the concentrated PCR amplification product.

[0067] 3.3 Preparation of single strand: The concentrated PCR product was added to TBE / urea denaturation buffer at a volume ratio of 1:1, mixed and denatured by boiling for 10 minutes, then the sample was subjected to urea denaturation polyacrylamide gel electrophoresis, and the extended FAM-labeled strand was separated from the reverse strand by electrophoresis at a voltage of 400 V until the bromophenol blue reached the bottom of the gel. The 7M urea denaturation polyacrylamide gel formula is shown in Table 3.

[0068] Table 3. Denaturation polyacrylamide gel formula

[0069] Ingredients Amount Urea 3.78g 40% polyacrylamide 1.8 mL 5*TBE 1.8 mL ddH2O 2.25 mL 10% APS 60 μL TEMED 15 μL

[0070] Cutting and recovering the FAM-labeled strand: The gel was taken out and placed on a plastic film, Ex (nm): 495, Em (nm): 517 was used to detect the FAM-labeled ssDNA we needed; a clean blade was used to cut the target band directly, the gel strip was transferred to a 1.5 mL EP tube and crushed, 1.2 mL buffer was added and boiled in a water bath for 15 minutes, the ssDNA in the gel was transferred to the solution, i.e. centrifuged at 12000 rpm for 2 minutes, the supernatant was recovered and transferred to a 15 mL centrifuge tube, 1 mL buffer was added to the crushed gel and boiled and centrifuged again, and the supernatant was all transferred to the same 15 mL centrifuge tube. To the 15 mL centrifuge tube, 12 mL of n-butanol was added, mixed well by shaking up and down, and then centrifuged at 9000 rpm for 5 min. After centrifugation, the solution was layered, the upper layer was removed, and the lower layer with fluorescent FAM single strand library was recovered. The obtained DNA single strand was dialyzed overnight at 4°C using a 3.5KD dialysis bag, which could be used as the library for the next round of screening.

[0071] 4. Multiple rounds of screening: In the next 2-7 rounds of screening, the secondary library obtained in the previous operation was used as the starting nucleic acid library each time, and the following concentrations and volumes were used for library fixation: library 800 nM, 100 μL; complementary primer CS-biotin: 1600 nM, 100 μL; SA magnetic beads 100 μL.

[0072] 5. After the seventh round of screening, the obtained single-stranded nucleic acid product was used as the eighth round library for screening, and graphene oxide was used to fix the library for screening to further enrich the library. Graphene oxide can adsorb ssDNA through π-π stacking, but it is not easy to adsorb ssDNA that has been conformationally folded after binding with the target molecule. Based on this principle, graphene oxide was used to fix the library for screening for 8-10 rounds, and the eighth round is taken as an example as follows:

[0073] 5.1 The single-stranded nucleic acid product obtained by the seventh round of screening was used as the library, and the library was diluted to 500 nM*100 μL, and the library was quickly denatured and renatured using a PCR instrument with the following settings: 95°C, 10 min; 4°C, 5 min, cooling rate 4°C / s.

[0074] 5.2 Dilute Risperidone to 50uM, 100uL, and add the denatured library from last step, mix well, and incubate at room temperature for 2h.

[0075] 5.3 Take 1000uL of graphene oxide, centrifuge at 12000rpm for 5min at 4C, remove the supernatant, and wash the graphene oxide with DPBS for 3 times, each time with 500uL of DPBS, and centrifuge to remove the supernatant.

[0076] 5.4 Add the target library mixture from 5.2 to the graphene oxide from 5.3, mix well, and incubate at room temperature in the dark for 30min, centrifuge at 12000rpm for 10min at 4C, take the supernatant and load into an EP tube, mark as Elution+, and use the nucleic acid molecules in Elution+ as templates for amplification by PCR (ePCR). Prepare single-stranded according to 3 steps, as the library for the next round of screening. In the treatment of graphene oxide, pay attention not to suck the graphene oxide when centrifuging to remove the supernatant, and the same when centrifuging to remove the supernatant.

[0077] 6. Multiple rounds of screening: In the next 9-10 rounds of screening, the secondary library obtained in the previous operation is used as the starting nucleic acid library each time, the library usage, the graphene oxide and risperidone input are consistent, and the screening method is consistent.

[0078] 7. After 10 rounds of screening, the obtained product is analyzed by high-throughput sequencing, and the nucleic acid aptamer is finally selected.

[0079] In the screening method, the screening pressure can be increased round by round to improve the enrichment degree of the screened nucleic acid aptamer and shorten the screening process. The increase of the screening pressure includes reducing the amount of input single-stranded DNA library, risperidone and incubation time of the library fixed magnetic beads, increasing the washing time in step 2.5, and the number of washing times.

[0080] 8. After the enriched library product is analyzed by high-throughput sequencing, a number of sequences are selected by homology comparison, synthesized by General Biosystems, and the affinity is detected and verified.

[0081] In the subsequent detection, one sequence with strong binding capacity is determined, which is named risperidone No. 5 (LPT-5).

[0082] The specific sequence is SEQ ID No. 1:

[0083] 5'-GTTGGCACTCCACGCATAGGGGCGATGGCACGATCAGCACGCATTGGCTATCGCTGCCTATGCGTGCTACCGTGAA-3'. Its secondary structure is shown in Figure 1

[0084] Example 2

[0085] ITC was used to detect the affinity between risperidone aptamer and risperidone:

[0086] ITC is based on the principle of heat detection to detect the interaction

[0087] Basic experimental model: "ligand" is placed in the titration needle, and "macromolecule" is placed in the sample cell to measure the reaction heat. The test group solution and the control group solution are titrated with risperidone diluent respectively, and the heat change during titration is detected. The instrument used is PEAQ-ITC of Malvern Instruments Ltd., UK.

[0088] 1. The nucleic acid aptamer (LPT-5) synthesized by General Biosystems and the control sequence (LPT-CTL) were diluted to 20 μM, 200 μL respectively, and then 192 μL DPBS and 8 μL methanol were added respectively, and mixed thoroughly. The final concentration of the aptamer was 10 μM.

[0089] The sequence of LPT-CTL (SEQ ID No. 5) (control sequence) is:

[0090] 5'-ATTGAAACTCCACGCATAGGGCCACAAGGAACACCCGCACGAGTGATCATACAGCACCTATGCGTGCTACCCCGAC-3'.

[0091] 2. Risperidone was diluted to 109 μM with DBPS. The dilution method was 2 μL of 5.45 mM risperidone (dissolved in methanol) added to 98 μL of DPBS, and mixed thoroughly.

[0092] 3. Titration was performed. The diluted solution was placed in the instrument to start titration, and risperidone was titrated with nucleic acid aptamer.

[0093] As shown in Figure 3 , the heat change during titration between the control sequence LPT-CTL and risperidone was low and there was no obvious heat change, and the instrument could not fit the parameters; as shown in Figure 2 , the titration process between LPT-5 and risperidone had obvious heat change, and the two were determined to have binding. The specific binding parameters were automatically fitted by PEAQ-ITC instrument according to the heat of titration.

[0094] ​Example 3

[0095] Application of Risperidone Aptamer:

[0096] Graphene oxide can not only adsorb ssDNA, but also has very good fluorescence quenching effect, which can quench the fluorescence group adsorbed on its surface. The high specificity and high affinity risperidone aptamer can be modified with FAM fluorescence. After adsorbed by graphene oxide, the fluorescence is quenched. After the addition of risperidone target, because the aptamer and the target bind, the conformation of the aptamer changes, which cannot be adsorbed to the surface of graphene oxide, so that the fluorescence quenching effect of graphene oxide is inactivated, and the FAM fluorescence appears. In order to make the aptamer and the target better bind, the FAM modified aptamer and the target can be combined first, and then graphene oxide is added for incubation. After incubation, fluorescence detection is carried out.

[0097] Specific steps:

[0098] 1. The FAM modified single-stranded DNA sequence shown below was synthesized by General Biosystems:

[0099] 5'-FAM-SEQ ID No. 1 specific sequence:

[0100] FAM-GTTGGCACTCCACGCATAGGGGCGATGGCACGATCAGCACGCATTGGCTATCGCTGCCTATGCGTGCTACCGTGAA.

[0101] 2. Dissolution: Take out the modified primer dry powder synthesized by General Biosystems, centrifuge at 12000 rpm for 10 min. Dissolve by adding DPBS buffer, and then dilute to a final concentration of 500 nM 500 μL with DPBS, vortex mix, and wait for use.

[0102] 3. Denaturation treatment: put the aptamer diluent obtained in step 2 into a pcr instrument for denaturation, and the pcr program is set as: 95℃ 10min, 4℃ 5min; so as to form the required structure of the aptamer. After mixing the denatured aptamer, it is divided into 100 μL / tube, divided into 5 tubes, and labeled as 0nM, 50nM, 125nM, 250nM, 500nM for use.

[0103] 4. Dilute risperidone of different concentrations with 10% serum-containing DPBS buffer: 0nM, 50nM, 100nM, 200nM, 400nM, each 100 μL.

[0104] 5. Add the different concentrations of risperidone obtained by dilution in the above step to the 5 tubes of aptamer in step 3 respectively, and incubate at room temperature in the dark for 1 h.

[0105] 6. To the mixture obtained in the last step, 1000 μL of cleaned graphene oxide was added to adsorb and quench the unbound fluorescently labeled aptamer. After 30 min incubation at room temperature in the dark, the mixture was subjected to fluorescence detection.

[0106] 7. The fluorescence intensity of each incubated mixture was detected using a F-7000 fluorescence spectrometer at an excitation wavelength of 490 nm, an emission wavelength of 520 nm, and a photomultiplier voltage of 800 V. The data was collected and the results are shown in Figure 4

[0107] The obtained data was consistent with the experimental prediction. The fluorescence signal was fitted linearly with the amount of risperidone added, as shown in Figure 5 , which was good, and the R 2 tended to 1, which was consistent with the experimental expectation. This example used a standard of risperidone and modified the aptamer fluorescence of Example 1 to react with it. The reaction product was then added to graphene oxide, which quenched the fluorescence of the aptamer that had not bound to risperidone, thereby converting the concentration of risperidone into a fluorescence signal. The main purpose was to use this aptamer for the detection of small molecules, and to use it as a template for subsequent application kits.

[0108] The above description is only the preferred embodiment of the present application, and is not any form of and substantial limitation on the present application. It should be noted that for ordinary skilled persons in the art, without departing from the method of the present application, a number of improvements and supplements can also be made, which should be considered as the protection scope of the present application. For those skilled in the art, without departing from the spirit and scope of the present application, some changes, modifications and equivalent variations made by using the disclosed technical content are equivalent embodiments of the present application; at the same time, any equivalent changes, modifications and variations made according to the essential technology of the present application are still within the scope of the technical solutions of the present application.​

Claims

1. A nucleic acid aptamer that specifically binds risperidone, characterized in that, The nucleotide sequence of the aptamer is shown as SEQ ID No.

1.

2. The nucleic acid aptamer that specifically binds to risperidone according to claim 1, characterized in that, The nucleotide sequence of the nucleic acid aptamer is connected with a label.

3. The nucleic acid aptamer that specifically binds to risperidone according to claim 2, characterized in that, The label is a fluorescent label, a radioactive label, a therapeutic label, a biotin label, a digoxin label, a nanoluminescent material label, a small peptide label, a siRNA label or an enzyme label.

4. Use of the nucleic acid aptamer specifically binding to risperidone according to any one of claims 1 to 3, characterized in that, The nucleic acid aptamer is used for detecting risperidone.

5. A kit for the detection of risperidone, characterized in that The nucleic acid aptamer specifically binding to risperidone according to any one of claims 1-3.

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