CN116083433A

CN116083433A - Nucleic acid aptamer capable of specifically recognizing vancomycin and application of nucleic acid aptamer

Info

Publication number: CN116083433A
Application number: CN202211100909.5A
Authority: CN
Inventors: 方晓娜; 何磊; 仇志锌; 罗昭锋
Original assignee: Anhui Province Angpumai Biotechnology Co ltd
Current assignee: Anhui Province Angpumai Biotechnology Co ltd
Priority date: 2022-09-09
Filing date: 2022-09-09
Publication date: 2023-05-09

Abstract

The invention discloses a nucleic acid aptamer capable of specifically recognizing vancomycin, wherein the nucleotide sequence of the nucleic acid aptamer comprises at least one of the following three sequences: (1) a DNA sequence shown in any one of SEQ ID NOs 1 to 3; (2) A DNA sequence which has more than 60% homology with the DNA sequence shown in any one of SEQ ID NO. 1-3 and specifically binds to vancomycin; (3) RNA sequences transcribed from the DNA sequences shown in any one of SEQ ID NO 1-3 and specifically bind to vancomycin. The invention also discloses application of the nucleic acid aptamer in detecting vancomycin. The aptamer has the characteristics of high specificity, stable chemical property, easy preservation and marking, has the capability of specifically binding vancomycin, and can be applied to detection of the vancomycin.

Description

Nucleic acid aptamer capable of specifically recognizing vancomycin and application of nucleic acid aptamer

Technical Field

The invention relates to the technical field of biology, in particular to a nucleic acid aptamer for specifically recognizing vancomycin and application thereof.

Background

Vancomycin (Vancomycin) alias: gu Meisu; vancomycin; vancomycin hydrochloride, a glycopeptide antibiotic, kills bacteria by inhibiting their growth and reproduction, and is used for treating bacterial infections. The medicine can interfere the synthesis of cell walls by interfering with one key component in the cell wall structure of bacteria, inhibit the generation of phospholipids and polypeptides in the cell walls, and is an antibiotic with definite and safer curative effects for treating severe infections caused by methicillin-resistant staphylococcus aureus, methicillin-resistant coagulase-resistant staphylococcus and enterococcus, including septicemia, lung infection and skin soft tissue infection. Vancomycin is the preferred drug for the resistance to infection of the lungs in methicillin-resistant staphylococcus aureus hospital. Vancomycin is an antibiotic with definite and safer curative effects for treating severe infections caused by MRSA/MRCON and enterococci, including septicemia, pulmonary infection and skin soft tissue infection, has strong bactericidal effect on gram-positive cocci, and has excellent curative effect on treating clostridium difficile pseudomembranous colitis by oral administration.

Vancomycin is a glycopeptide antibiotic produced by streptomycete, has a complex structure, has strong potency, has skin reaction after intravenous drip and can cause thrombophlebitis after concentration is too high; intramuscular injection can cause severe pain, so it is impossible to perform intramuscular injection; there are serious ototoxicity and nephrotoxicity, so other antibiotics are only used for rescuing in a short period of time when they are ineffective against germs.

There are various methods for detecting vancomycin at present, for example: immunoscreening; the chromatographic methods have high detection sensitivity and accurate detection results, but expensive instruments and equipment are required, the requirements on detection materials are high, purification treatment and the like are required, and rapid and convenient detection cannot be realized. The existing immunoscreening method is a detection kit which is dependent on antibodies, and although some methods can achieve rapid and simple detection, the preparation process of the antibodies is complex, the batches have differences and certain defects.

Aptamer refers to DNA or RNA molecules obtained by screening and separating by an exponential enrichment ligand system evolution (SELEX) technology, and can be combined with other targets such as proteins, metal ions, small molecules, polypeptides and even whole cells with high affinity and specificity, so that the aptamer has wide prospects in biochemical analysis, environmental monitoring, basic medicine, new drug synthesis and the like. Compared with an antibody, the nucleic acid aptamer has the advantages of small molecular weight, better stability, easy transformation and modification, no immunogenicity, short preparation period, capability of avoiding a series of processes of animal immunization, feeding, protein extraction, purification and the like by artificial synthesis and the like, so that the nucleic acid aptamer is a very ideal molecular probe.

The SELEX-based method, which screens out aptamer binding to a specific small molecule and uses the aptamer for detection of the small molecule, is also widely studied at present. Nucleic acid aptamers to vancomycin have not been published, and therefore there is a need in the art for nucleic acid aptamers to vancomycin that have high binding affinity.

Disclosure of Invention

Based on the technical problems in the background technology, the invention provides a nucleic acid aptamer for specifically recognizing vancomycin and application thereof.

The invention provides a nucleic acid aptamer capable of specifically recognizing vancomycin, wherein the nucleotide sequence of the nucleic acid aptamer comprises at least one of the following three sequences:

(1) A DNA sequence shown in any one of SEQ ID NOs 1 to 3;

the nucleotide sequence shown in SEQ ID NO. 1 is as follows:

5’TAGGACCCGCCTGGGAGAGTACGCCTTCTCGACCCGTGGAATCCTA 3’

the nucleotide sequence shown in SEQ ID NO. 2 is as follows:

5’ATAGCTCGACACGAGGGCTCTCCGAGTGGAGTACGTGAGTCCTAGCCTAT 3’

the nucleotide sequence shown in SEQ ID NO. 3 is as follows:

5’CGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCG 3’；

(2) A DNA sequence which has a homology of 60% or more with the DNA sequence shown in any one of SEQ ID NO. 1-3 and specifically binds to vancomycin, for example, a DNA sequence which has a homology of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the DNA sequence shown in any one of SEQ ID NO. 1-3 and specifically binds to vancomycin;

(3) RNA sequences transcribed from the DNA sequences shown in any one of SEQ ID NO 1-3 and specifically bind to vancomycin.

Preferably, the nucleotide sequence of the nucleic acid aptamer is modified and the modified nucleic acid aptamer specifically binds to vancomycin, the modification being selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopicalization; provided that the nucleic acid aptamer sequence so modified has desirable properties, e.g., may have an affinity for binding vancomycin equal to or greater than the parent nucleic acid aptamer sequence prior to modification, or may have greater stability although the affinity is not significantly improved.

In other words, the above nucleic acid aptamer sequence, whether partially substituted or modified, has substantially the same or similar molecular structure, physicochemical properties and functions as the original nucleic acid aptamer, and can be used for binding to vancomycin.

The invention also provides a conjugate of the nucleic acid aptamer, which is obtained by connecting at least one of fluorescent markers, radioactive substances, therapeutic substances, biotin, digoxin, nano luminescent materials, small peptides, siRNA and enzyme markers on the nucleotide sequence of the nucleic acid aptamer; provided that the nucleic acid aptamer sequence so modified has desirable properties, e.g., may have an affinity for binding vancomycin equal to or greater than the parent nucleic acid aptamer sequence prior to modification, or may have greater stability although the affinity is not significantly improved.

The invention also provides a derivative of the nucleic acid aptamer, which is obtained by modifying the skeleton of the nucleotide sequence of the nucleic acid aptamer or the conjugate of the nucleic acid aptamer into a phosphorothioate skeleton, or is peptide nucleic acid modified by the nucleic acid aptamer or the conjugate of the nucleic acid aptamer, and the derivative of the nucleic acid aptamer specifically binds to vancomycin.

The above aptamer, whether derived or other derived derivatives, has substantially the same or similar molecular structure, physicochemical properties and functions as the original aptamer.

The invention also provides application of the nucleic acid aptamer specifically recognizing vancomycin, the conjugate of the nucleic acid aptamer or the derivative of the nucleic acid aptamer in detecting vancomycin.

Fluorescence detection is one of the common and effective methods in aptamer biosensors, and fluorescence is highly sensitive and compatible with organic sample solutions, thus being widely applied to biosensor design. The aptamer is non-fluorescent per se, and fluorescent groups are required to be introduced into a reaction system to trigger optical signal change so as to detect. The common method is to design a Molecular Beacon (MB) system for target detection, and the method has high sensitivity, quick response and multiple selectable marker types. Molecular beacon fluorescence detection of nucleic acid aptamers is based on fluorescence resonance energy transfer phenomena (Fluorescence Resonance Energy Transfer, FRET) with conformational changes upon binding to the target. When the molecular beacon is in a free state, the fluorescent group and the quenching group are closely spaced (about 7-10 nm) so that FRET phenomenon occurs, fluorescence emitted by the fluorescent group is absorbed by the quenching group and emitted in a thermal form, and the fluorescence is almost completely quenched and not detected. When the molecular beacon is combined with the target molecule, the structure of the sequence is changed, so that the hairpin of the fluorescence quenching group is opened or two complementary chains are separated, the distance between the fluorescence group and the quenching group is increased, the fluorescence intensity is changed, and the fluorescence of the fluorescence group is recovered. The principle is utilized to calculate the detected fluorescence intensity and the target content in the solution.

Based on the above principle, the present invention also provides a biosensor for detecting vancomycin, comprising an aptamer chain modified with a fluorescent group and a quenching chain modified with a quenching group, the aptamer chain comprising the nucleic acid aptamer specifically recognizing vancomycin according to

claim

1 or 2, the conjugate of the nucleic acid aptamer according to claim 3 or the derivative of the nucleic acid aptamer according to claim 4;

the end of the aptamer strand has a complementary pairing sequence capable of forming the aptamer strand into a hairpin structure, and the quenching strand is capable of complementarily binding to the end of the aptamer to quench the fluorescent group of the aptamer strand.

Preferably, the sequence of the aptamer chain is shown as SEQ ID NO. 4, the sequence of the quenching chain is shown as SEQ ID NO. 5, the 5 'end of the sequence of the aptamer chain is modified with a fluorescent group, and the 3' end of the sequence of the quenching chain is modified with a quenching group;

the nucleotide sequence shown in SEQ ID NO. 4 is as follows:

5’CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA 3’

the nucleotide sequence shown in SEQ ID NO. 5 is as follows:

5’AGCCGAACTGAG 3’。

preferably, the fluorescent group is FAM and the quenching group is Dabcyl.

The principle of detecting vancomycin based on the biosensor is shown in fig. 5, firstly, after hybridization is carried out on an aptamer chain modified with a fluorescent group and a quenching chain modified with a quenching group, fluorescence is quenched, fluorescence in a system is in a closed state, when vancomycin exists in a detection system, the structure of a sequence can be changed, the hairpin of the fluorescent quenching group is opened or two complementary chains are separated, so that the distance between the fluorescent group and the quenching group is increased, the fluorescence intensity is changed along with the increase, and the fluorescence of the fluorescent group is recovered.

The invention also provides a method for detecting the vancomycin content in a sample based on the biosensor, which comprises the following steps:

adding an aptamer chain and a quenching chain into a buffer solution according to the molar ratio of (2-3) to obtain a solution containing an aptamer chain-quenching chain complex, then adding a sample to be detected to obtain a reaction system, incubating the reaction system, and performing fluorescence measurement after incubation is finished.

Preferably, the incubation temperature is room temperature and the incubation time is 10-30min.

Preferably, the concentration of the aptamer chain in the reaction system is 60-100nM.

The beneficial effects of the invention are as follows:

the inventor designs and synthesizes a random single-stranded DNA library and a corresponding primer, screens and obtains a nucleic acid aptamer which has high specificity, stable chemical property, easy preservation and marking and can be combined with vancomycin, and the nucleic acid aptamer has the capability of specifically combining with the vancomycin and can be applied to detection of the vancomycin.

Drawings

Fig. 1 is a schematic flow chart of an experimental method for screening vancomycin according to the invention.

FIG. 2 shows the result of detecting the affinity of the nucleotide sequence shown in SEQ ID NO. 1 with vancomycin by an isothermal titration microcalorimeter.

FIG. 3 shows the result of detecting the affinity of the nucleotide sequence shown in SEQ ID NO. 2 with vancomycin by an isothermal titration microcalorimeter.

FIG. 4 shows the result of detecting the affinity of the nucleotide sequence shown in SEQ ID NO. 3 with vancomycin by an isothermal titration microcalorimeter.

Fig. 5 is a schematic diagram of the principle of detecting vancomycin by the biosensor according to the present invention.

FIG. 6 is a schematic diagram of the secondary structure of an aptamer chain used in example 3 of the present invention.

FIG. 7 shows the results of tests performed by the biosensor in example 3 of the present invention for detecting vancomycin at different concentrations.

FIG. 8 shows the results of a specific assay for detecting different antibiotics using the biosensor in example 3 of the present invention.

Detailed Description

The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below, unless otherwise specified, are all conventional biochemical reagents and are commercially available.

The technical scheme of the invention is described in detail through specific embodiments.

Example 1: screening of ssDNA nucleic acid aptamers that specifically bind to vancomycin

1. Random single stranded DNA libraries and primers shown in the following sequences were synthesized:

random single-stranded DNA library:

5’-TTCAGCACTCCACGCATAGC-40N-CCTATGCGTGCTACCGTGAA-3’

wherein "40N" represents a sequence of 40 arbitrary nucleotide bases linked. The library was synthesized by the division of biological engineering (Shanghai).

Primer information is shown in Table 1.

Table 1 primers and sequences thereof

Wherein S in the primer name represents a forward primer, A in the primer name represents a reverse primer, 19A in the sequence represent a polyA tail consisting of 19 adenylates (A), and "Spacer 18" represents an 18-atom hexaethyleneglycol Spacer. The structural formulas of the three "Spacer 18" are shown in formulas I-III below. The structural formula of "Spacer 18" used in the A2-ployA primer is shown in the formula I.

The primers were each prepared with DPBS buffer (NaCl: 8g/L, KCl:0.2g/L, na) ₂ HPO ₄ ：2.99g/L，KH ₂ PO ₄ :0.2g/L，CaCl ₂ :0.1g/L，MgCl ₂ ·6H ₂ O is 0.1g/L; PH 7.4) to prepare 100uM stock solution, and store at-20 ℃ for standby.

2. Vancomycin screening of fixed library by magnetic bead method

The library is immobilized by magnetic beads, and is screened by a small molecule competition combination method, wherein the total screening is six rounds, and the screening flow is shown in figure 1. The specific screening method is as follows:

2.1 library lysis: the biosynthesized library dry powder was taken and centrifuged at 12000rpm for 10min. 260ul of DPBS buffer was added, the library diluted to 5. Mu.M, vortexed, and centrifuged at 8000rpm for 30s. The dissolved S1CS primer was added to the library by pipetting 29ul, the final concentration of S1CS primer was about 10. Mu.M, and the primer and library were thoroughly mixed and centrifuged at 8000rpm for 30S.

2.2 library matches with primers: subpackaging the mixture of the library and the complementary primer into a PCR tube, and setting the following procedures by using a PCR instrument, wherein the temperature is 95 ℃ for 10min, and the temperature is slowly reduced to 60 ℃ at the speed of 0.1 ℃/s;60 ℃ for 1min; slowly cooling to 25 ℃, wherein the cooling rate is 0.1 ℃/s. The library with good renaturation and the complementary primer mixture are taken to measure the concentration as C1 by ultraviolet (A260).

2.3. 1mL of streptavidin magnetic beads (Invitrogen, dynabeads) were pipetted ^TM MyOne ^TM Strepitavidin C1, cat: 65001 The beads were washed 6 times with DPBS, each 400uL in volume, and recovered with a strong magnet. (magnetic beads were temporarily stored in a small amount of DPBS at the time of the last washing, and drying of the magnetic beads was prevented.)

2.4. Adding the library after renaturation and the complementary primer mixed solution into 2.3 magnetic beads, uniformly mixing, shaking on a room temperature rotator for 50min, fishing the magnetic beads by a magnet, recovering the supernatant, and taking a small amount of supernatant to measure the concentration of ultraviolet (A260) to obtain a value C2. From the concentrations measured, the efficiency of coupling the library to the magnetic beads can be calculated. Library fixation efficiency= (C1-C2)/C1, the first round of library fixation efficiency was greater than 50%, after which each round of library fixation efficiency was greater than 80%, continuing the screening.

2.5 washing of library: the beads obtained in the previous step were rinsed, 400ul of rinsing buffer (DPBS containing 2% methanol) was added to each bead, and after suspending the beads, they were allowed to stand at room temperature for 2 minutes, and then the beads were attracted by a strong magnet. The rinsing operation was repeated 4 times. The new EP tube was replaced with each pass of washing the beads. (the rinse buffer volume for each wash was reduced to 200ul. From the second round of screening) immediately after the beads were rinsed once, i.e., 400ul of rinse buffer was added, the beads were suspended and then incubated for 20 min in a shaker, and then the beads were attracted by a strong magnet and the supernatant was removed. (also, only 200ul. Of rinse buffer was used at this stage since the second round of screening.)

2.6 target elution: vancomycin (formula C) ₆₆ H ₇₆ Cl ₃ N ₉ O ₂₄ ) Dissolving into 5mM with methanol, sucking 4ul, adding into 196ul DPBS, namely diluting 50 times to 100uM, mixing, adding into SA magnetic beads obtained in 2.5 steps, and incubating for 45min in a shaking table. The magnet was used to fish the beads and the supernatant was recovered in an EP tube and recorded as Elutation. Wherein, the molecular structure of vancomycin is as follows:

3. secondary library preparation

3.1 amplification of double strand: amplification was performed by emulsion PCR (ePCR) using the nucleic acid molecules in Elutation as templates. The method comprises the following steps: all templates were added to 2ml PCR mix and mixed well, 4 volumes of ePCR microdroplets were added to generate oil, and vortexed for 2 minutes to prepare an emulsion. The emulsion was divided into 100 ul/tube and added to the PCR tube under the following amplification conditions: pre-denaturation at 95℃for 2 min, denaturation at 95℃for 60 sec, annealing at 60 sec, elongation at 72℃for 60 sec, 30 cycles total, and storage at 4 ℃. ePCR microdroplet generation oil was purchased from Agropmai (Aptamy) Biotechnology Inc. (product number: EPO 100) of Anhui province and the formulation of PCR mix is shown in Table 2.

TABLE 2 ePCR mix formulation

Reagent(s)	Total volume of 1000ul
		ddH ₂ O	866ul
Pfu enzyme buffer 10 ×	100ul
		dNTPmix(10mM)	20ul
Forward primer S1-FAM (100 uM)	5ul
		Reverse primer A2-polyA (100 uM)	5ul
Pfu enzyme	4ul(20U)

3.2 amplification products were concentrated with n-butanol: collecting all the ePCR products in a 15ml sharp bottom centrifuge tube, adding n-butanol with the volume of 2 times, and vibrating on a vortex mixer to fully mix; a bench centrifuge, at 7000rpm (revolutions per minute) for 2 minutes at room temperature; the upper phase (n-butanol) was removed to give a concentrated PCR amplification product.

3.3 preparation of single strands: the concentrated PCR product was prepared in a volume ratio of 1:1 adding TBE/urea denaturation buffer (Anhui, biotechnology Co., ltd., cat# TLB-5), boiling denaturation for 15 min to denature DNA, then ice-bath for 1min, subjecting all samples to urea-denatured polyacrylamide gel electrophoresis, electrophoresis at 300V voltage until bromophenol blue reaches the bottom of the gel, separating the lengthened FAM-labeled chain from the reverse chain, and 7M urea-denatured polyacrylamide gel formulation as shown in Table 3.

TABLE 3 modified polyacrylamide gel formulations

Cutting gel to recover FAM marked chain: the gel was taken out and placed on a plastic film, ex (nm): 495, em (nm): 517 detecting the required FAM-labeled ssDNA; the target band was cut directly with a clean blade, the strips were transferred to a 1.5ml EP tube and centrifuged to break the gel, and 1ml ddH was added ₂ After O, ssDNA in the gel was transferred to the solution by 10 minutes of boiling water bath, centrifuged at 12000rpm for 2 minutes, the supernatant was recovered, transferred to a 15mL centrifuge tube, 1mL of ultrapure water was again taken into the crushed gel, and the centrifugation was repeated by boiling and transferring the supernatant to the same 15mL centrifuge tube. To a 15ml centrifuge tube, 12ml of n-butanol was added, and the mixture was centrifuged upside down, at 9000rpm for 5min. After centrifugation, the solution was delaminated, the supernatant was aspirated and the lower single stranded library was recovered. The obtained DNA single strand was dialyzed overnight at 4℃with a 3.5KD dialysis bag to obtain a library for the next round of screening.

4. Multiple rounds of screening: in the next 2-5 rounds of screening, each operation uses the secondary library obtained in the previous operation as a starting nucleic acid library, and the following concentrations and volumes are adopted for the fixation of the library: library 700nm x 100 μl; the complementary primer CS-biotin is as follows: 1400nm x 100 μl; the SA beads were 70. Mu.L. And after the fifth round of screening, carrying out high-throughput sequencing analysis on the obtained product to finally obtain the nucleic acid aptamer.

In the screening method, the screening pressure can be increased round by round so as to improve the enrichment degree of the screening nucleic acid aptamer and shorten the screening process. The increase in screening pressure includes a decrease in the amount of single-stranded DNA library that is put into, vancomycin, and incubation time of the library-immobilized magnetic beads, and an increase in the washing time in step 2.5, washing times.

5. And (3) after high-throughput sequencing analysis of the obtained enriched library product, selecting a plurality of sequences to be synthesized by Shanghai engineering, and detecting affinity.

In the subsequent detection, 3 sequences with strong binding capacity were determined and designated SEQ ID NOs 1 to 3, respectively.

Example 2: isothermal titration microcalorimetry (ITC) for detecting affinity of vancomycin aptamer and vancomycin

1. Diluting the nucleic acid aptamer SEQ ID NO 1-3 synthesized by Shanghai with DPBS to 10uM respectively, taking 200ul respectively, then adding 192ul of DPBS and 8ul of methanol, and fully mixing to obtain a nucleic acid aptamer solution, wherein the final concentration of the aptamer is 5uM.

2. Vancomycin was diluted to 100uM with DBPS to give a solution of vancomycin by adding 2ul of vancomycin to 98ul of DPBS.

3. Titration was performed and the aptamer solution was titrated with vancomycin solution. The results of the measurement are shown in figures 2-4, and the results show that heat is released in the titration process between SEQ ID NO 1-3 and vancomycin, so that the sequence of SEQ ID NO 1-3 is combined with the vancomycin.

Example 3: specificity test for detecting vancomycin by biosensor

The following biosensors were used for detection:

the biosensor comprises an aptamer chain modified with a fluorescent group and a quenching chain modified with a quenching group, the sequences of which are shown in table 4:

table 4 the sensor includes a sequence

The principle of detecting vancomycin is as follows: when the quenching strand and the aptamer strand are hybridized in a mixed mode, the 5' -end of the aptamer strand and the quenching strand in complementary pairing form a partial double-stranded helix, dabcyl approaches FAM, and fluorescence intensity of FAM is quenched. After vancomycin is added, the structure of the sequence is changed, so that the hairpin of the fluorescence quenching group is opened or two complementary chains are separated, the distance between the fluorescence group and the quenching group is increased, the fluorescence intensity is changed along with the increase, and the fluorescence of the fluorescence group is recovered.

The sensor is adopted to detect vancomycin with different concentrations, and the specific method comprises the following steps: adding the quenching chain and the aptamer chain into a DPBS buffer solution to form an aptamer chain-quenching chain complex, obtaining a detection solution, wherein the final concentration of the aptamer chain and the quenching chain in the detection solution is 50nM and 100nM respectively, and then adding 20 mu L of a DPBS buffer solution of vancomycin into 180 mu L of the detection solution to obtain a reaction system, wherein the final concentration of the vancomycin in the reaction system is 90 mu M, 46 mu M, 23 mu M, 11.5 mu M, 7.7 mu M, 5.8 mu M, 2.9 mu M, 0.8 mu M, 0.3 mu M and 0 mu M respectively; after incubation for 10 minutes at room temperature, the emission spectrum of each sample at 500-600 nm under 488nm excitation is detected by a fluorescence spectrometer, the width of the excitation and emission slits is set to be 5nm, the fluorescence intensity at the wavelength of 520nm is counted as F', and F is the fluorescence intensity of the sensor in the absence of vancomycin. The test results are shown in fig. 7, wherein fig. 7a shows the fluorescence intensity of the sensor in the presence of vancomycin at different concentrations, and fig. 7b shows the fluorescence value in the presence of vancomycin at different concentrations/the fluorescence value without vancomycin. It can be seen that the fluorescence intensity of the sensor increases with the concentration of vancomycin added, when the concentration of vancomycin is 45 μm, the fluorescence intensity of the hybridization chain is recovered to 45% of the maximum possible fluorescence response, and the recovery of the fluorescence signal tends to be slow with a further increase in the concentration of vancomycin; the resulting data were fitted using the nonlinear fit equation y=b×x/(k+x) for Graph Pad Prism, and the result showed that the curve of fig. 7B was well-linear. The above results indicate that the aptamer chain modified with a fluorescent group can be used as a probe to develop a fluorescence sensor for detecting vancomycin, and the fluorescence intensity of the sensor increases with the concentration of the vancomycin.

To determine the specificity of the sensor, the fluorescence intensity after incubation with several other antibiotics (ciprofloxacin, sulfadiazine, clothianidin, neomycin, tetracycline) was measured and the resulting fluorescence values were compared with the fluorescence response of vancomycin in the presence and in a blank test. The specific method comprises the following steps: adding the quenching chain and the aptamer chain into a DPBS buffer solution to form an aptamer chain-quenching chain complex, obtaining a detection solution, wherein the final concentration of the aptamer chain and the quenching chain in the detection solution is 50nM and 100nM respectively, and adding 20 mu L of the DPBS buffer solution of the antibiotics into 180 mu L of the detection solution to obtain a reaction system, and the final concentration of the antibiotics in the reaction system is 100uM; after incubation at room temperature for 10 minutes, fluorescence values at 520nm were detected with a microplate reader. The test was repeated three times. The results are shown in fig. 8, which shows an increase in fluorescence after vancomycin incubation, while no recovery of fluorescence values occurred after incubation of several other antibiotics at 100uM concentration, indicating that the sensor exhibited excellent selectivity.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Claims

1. A nucleic acid aptamer specifically recognizing vancomycin, characterized in that the nucleotide sequence of the nucleic acid aptamer comprises at least one of the following three sequences:

(1) A DNA sequence shown in any one of SEQ ID NOs 1 to 3;

the nucleotide sequence shown in SEQ ID NO. 1 is as follows:

5’TAGGACCCGCCTGGGAGAGTACGCCTTCTCGACCCGTGGAATCCTA 3’

the nucleotide sequence shown in SEQ ID NO. 2 is as follows:

5’ATAGCTCGACACGAGGGCTCTCCGAGTGGAGTACGTGAGTCCTAGCCTAT 3’

the nucleotide sequence shown in SEQ ID NO. 3 is as follows:

5’CGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCG 3’；

(2) A DNA sequence which has more than 60% homology with the DNA sequence shown in any one of SEQ ID NO. 1-3 and specifically binds to vancomycin;

2. The aptamer specifically recognizing vancomycin according to claim 1, wherein the nucleotide sequence of the aptamer is modified and the modified aptamer specifically binds to vancomycin, the modification being selected from at least one of phosphorylation, methylation, amination, sulfhydrylation, substitution of oxygen with sulfur, substitution of oxygen with selenium, and isotopicization.

3. A conjugate of a nucleic acid aptamer, characterized in that it is obtained by ligating at least one of a fluorescent label, a radioactive substance, a therapeutic substance, biotin, digoxin, a nano luminescent material, a small peptide, siRNA and an enzyme label to the nucleotide sequence of the nucleic acid aptamer according to claim 1 or 2.

4. A derivative of a nucleic acid aptamer, characterized in that it is obtained by modifying the backbone of the nucleotide sequence of the nucleic acid aptamer of claim 1 or 2 or the conjugate of the nucleic acid aptamer of claim 3 into a phosphorothioate backbone, or a peptide nucleic acid modified by the nucleic acid aptamer of claim 1 or 2 or the conjugate of the nucleic acid aptamer of claim 3, and the derivative of the nucleic acid aptamer specifically binds to vancomycin.

5. Use of a nucleic acid aptamer specifically recognizing vancomycin according to claim 1 or 2, a conjugate of a nucleic acid aptamer according to claim 3 or a derivative of a nucleic acid aptamer according to claim 4 for detecting vancomycin.

6. A biosensor for detecting vancomycin, comprising an aptamer chain modified with a fluorescent group and a quenching chain modified with a quenching group, the aptamer chain comprising the nucleic acid aptamer specifically recognizing vancomycin according to claim 1 or 2, the conjugate of the nucleic acid aptamer according to claim 3, or the derivative of the nucleic acid aptamer according to claim 4;

7. The biosensor for detecting vancomycin according to claim 6, wherein the sequence of the aptamer chain is shown as SEQ ID NO. 4, the sequence of the quenching chain is shown as SEQ ID NO. 5, the 5 '-end of the sequence of the aptamer chain is modified with a fluorescent group, and the 3' -end of the sequence of the quenching chain is modified with a quenching group;

the nucleotide sequence shown in SEQ ID NO. 4 is as follows:

5’CTCAGTTCGGCTCAGTGACCCCACAGGAGACTGTAGGTTGACCTCTTGTAGCCGAA 3’

the nucleotide sequence shown in SEQ ID NO. 5 is as follows:

5’AGCCGAACTGAG3’。

8. the biosensor for detecting vancomycin according to claim 7, wherein the fluorescent moiety is FAM and the quenching moiety is Dabcyl.

9. A method for detecting the content of vancomycin in a sample based on the biosensor according to any one of claims 6-8, comprising the steps of:

adding an aptamer chain and a quenching chain into a buffer solution according to the molar ratio of (2-3) to obtain a detection solution, then adding a sample to be detected to obtain a reaction system, incubating the reaction system, and performing fluorescence measurement after incubation is finished.

10. The method for detecting the vancomycin content in a sample according to claim 9, wherein the incubation temperature is room temperature and the incubation time is 10-30min;