CN111876423A

CN111876423A - Aptamer of nitrobenzene, aptamer derivative and application of aptamer derivative

Info

Publication number: CN111876423A
Application number: CN202010777466.8A
Authority: CN
Inventors: 谢丹平; 贾文超; 胡克梅; 刘莹; 刘丽君; 丁紫荣; 付建平
Original assignee: South China Institute of Environmental Science of Ministry of Ecology and Environment
Current assignee: South China Institute of Environmental Science of Ministry of Ecology and Environment
Priority date: 2020-08-05
Filing date: 2020-08-05
Publication date: 2020-11-03
Anticipated expiration: 2040-08-05
Also published as: CN111876423B

Abstract

The invention discloses a nitrobenzene aptamer, a nitrobenzene aptamer derivative and application thereof, wherein the sequence of the nitrobenzene aptamer is a DNA fragment shown as the following. The aptamer can be various similar sequences with higher homology or derivatives obtained by the sequence of the invention. The aptamer and the derivative thereof can be used for detecting, separating, purifying and other products of environmental, food and biological harmful factors, preparing nitrobenzene detection probes and the like. Has the advantages of high specificity and high affinity with nitrobenzene, chemical synthesis, stable property, small molecular weight, good biocompatibility, easy storage and the like.

Description

Aptamer of nitrobenzene, aptamer derivative and application of aptamer derivative

Technical Field

The invention relates to a nucleic acid aptamer, a nucleic acid aptamer derivative and application thereof, in particular to a nucleic acid aptamer capable of being combined with nitrobenzene, a nucleic acid aptamer derivative and application thereof.

Background

Nitrobenzene (NB) is an important raw material for the production of aniline, and its residues are discharged into water with waste during the production process. The benzene ring in the chemical structure of the composite material is stable and difficult to degrade, has a density higher than that of water, is easy to deposit in the water body, and causes pollution to the water body. NB belongs to a highly toxic pollutant, and can invade into the human body through respiratory tract, digestive tract, skin, oral cavity and other modes, causing damage to human blood, liver and central nervous system, so the us national environmental protection agency lists it as one of 128 types of 'priority control toxic organic pollutants'. Therefore, it is important to enhance the analysis and detection of nitrobenzene compounds in water. The state has strict regulations on the discharge of nitrobenzene from water. At present, a chromatography-mass spectrometry and combined detection mode are commonly used in a detection method of nitrobenzene compounds in water, so that the operation is complex and long in time consumption, a large amount of organic solvents are used in the using process, and meanwhile, the treatment cost of organic waste liquid and environmental pollution are increased. Therefore, it has become a great demand to be able to perform green, fast, and efficient detection of nitrobenzene in water.

Aptamers, which are single-stranded DNA or RNA molecules artificially synthesized from bases and having high-specificity and high-affinity recognition ability of antibodies to various types of substances (e.g., ions, small molecules, proteins, bacteria, etc.), are called chemical antibodies. The preparation process of the aptamer is generated by Exponential Enrichment system Evolution (SELEX), and the system is characterized in that a single-stranded random oligonucleotide library is constructed in vitro by combining synthetic chemistry and molecular biology technologies, and the target aptamer capable of being identified with a target is finally obtained through multiple screening and Exponential Enrichment. The method comprises the following specific steps: first, a synthetic resin containing 10¹²～10¹⁵The DNA/RNA oligonucleotide random library has sequence length of 70-100 bases, random sequence in the middle and fixed sequences at two ends. By mixingSeparating the nucleic acid combined with the target from the invalid nucleic acid chain, simultaneously carrying out PCR amplification on the retained nucleic acid molecules, obtaining a library with affinity to the target after multiple screening and PCR amplification, and finally carrying out sequencing analysis to obtain fragment information of the nucleic acid in the library. Compared with the antibody, the preparation period is short, the success rate is high, the storage period is longer, the property is more stable, and large-scale chemical synthesis, modification and marking can be carried out, so that the surface of the sensor is easy to be fixed and modified, the antibody is suitable for being combined with nano, fluorescent and other materials, and the problem of rapid development of the recognition molecule can be solved.

Although the aptamers have the above-mentioned advantages, in particular, for a substance such as nitrobenzene, the aptamers are not always available by performing the above-mentioned procedures, and high-quality aptamers for some substances are extremely difficult to obtain. This is because, compared with small-molecule compounds having a large molecular weight and rich chemical functional groups, nitrobenzene has a weak recognition force when interacting with a nucleic acid library, and thus it is very difficult to obtain an aptamer having high affinity for nitrobenzene.

A large number of researchers have not found aptamers with high affinity to nitrobenzene and low cost for many years. If a nitrobenzene nucleic acid aptamer is obtained, a new means for detecting nitrobenzene and an expanded application range are expected to be provided.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a nitrobenzene aptamer, a nitrobenzene aptamer derivative and application thereof, wherein the nitrobenzene aptamer, the nitrobenzene aptamer derivative and the nitrobenzene aptamer derivative can be combined with nitrobenzene in high specificity and high affinity, can be chemically synthesized, and have good biocompatibility, small molecular weight, stability and easy storage.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the aptamer of nitrobenzene is characterized in that the nucleotide sequence of the aptamer is shown as SEQ ID No.1 or 2 in a sequence table.

Further, a position on the nucleotide sequence of the aptamer is phosphorylated, oxymethylated, methylated, aminated, thiolated, fluorinated, or isotopically esterified.

Furthermore, biotin, digoxin, fluorescent substances, nano materials, polyethylene glycol, peptide fragments, proteins, enzymes or folic acid labels are combined on the nucleotide sequence of the aptamer.

The invention also provides a nucleic acid aptamer derivative of nitrobenzene, which is characterized in that the nucleic acid aptamer derivative is a phosphorothioate skeleton derived from the skeleton of the nucleotide sequence of the nucleic acid aptamer, or is corresponding locked nucleic acid or peptide nucleic acid modified by the nucleic acid aptamer.

The invention also provides application of the aptamer or the aptamer derivative, which is characterized in that the aptamer or the aptamer derivative is used for detecting nitrobenzene in environment, food and organisms.

The invention also provides an application of the aptamer or the aptamer derivative, which is characterized in that nitrobenzene is separated and purified by using the aptamer or the aptamer derivative.

The invention also provides application of the aptamer or the aptamer derivative, which is characterized in that the aptamer or the aptamer derivative is used for preparing a nitrobenzene detection probe.

In another aspect, the present invention provides a nitrobenzene detection method, which is characterized by comprising the following steps:

step (1), preparing the aptamer or the aptamer derivative;

step (2), preparing a detection probe of the nitrobenzene nucleic acid aptamer;

step (3) immobilizing the detection probe on the surface of the SPR chip, wherein the detection probe is immobilized by bovine serum albumin

Sealing the chip of the detection probe;

and (4) carrying out nitrobenzene determination by using the chip on which the detection probe is immobilized.

The nucleotide sequences, whether partially substituted or modified, all have the same or similar molecular structure, physicochemical properties and functions as the original aptamer, and can be used for specific binding with nitrobenzene.

Compared with the prior art, the invention has the advantages that:

(1) the nucleic acid aptamer can be combined with nitrobenzene with high affinity and high specificity, has the advantages of good stability, no toxicity, easy modification and the like, and is beneficial to large-scale industrial production and application.

(2) The nucleic acid aptamer has the characteristic of easy modification, and can convert ultraviolet absorption signals of nitrobenzene into other signals (such as fluorescence) by modifying signal substances, so that high-sensitivity detection of the nitrobenzene is realized.

Drawings

FIG. 1 shows the result of the determination of the affinity between a aptamer probe and nitrobenzene in the examples of the present invention.

FIG. 2 shows the result of the measurement of the specificity of the aptamer probe for nitrobenzene in the examples of the present invention.

FIG. 3 shows the SPR measurement of nitrobenzene using aptamer probe in the present invention.

FIG. 4 shows the result of unlabeled fluorescence detection of aptamer p-nitrobenzene in the examples of the present invention.

FIG. 5 shows fluorescence polarization detection results of aptamer p-nitrobenzene in the examples of the present invention.

Detailed Description

The invention is further described below with reference to the drawings and specific preferred embodiments, without thereby limiting the scope of protection of the invention.

Example (b):

the nucleotide sequence of the aptamer of nitrobenzene of the invention comprises a DNA fragment shown as the following sequence, which can be specifically combined with nitrobenzene:

5’-CACGCATAGGCTCGCCGTAACACACCACACATAGGTCCGCTATTCGCGTCCCTATGCGTG-3’

the nucleotide sequence of the aptamer is selected from naturally occurring or artificially synthesized sequences, or any other source of the same.

It is noted that the scope of the present invention encompasses all sequences comprising the aptamer.

The present invention encompasses such sequences that are phosphorylated, hypermethylated, methylated, fluorinated, aminated, thiolated, or isotopically-modified at a position on the nucleotide sequence of the aptamer.

The invention covers the sequence of the nucleic acid aptamer combined with biotin, digoxigenin, fluorescent substance, nano material, polyethylene glycol, peptide fragment, protein, enzyme or folic acid label (even with radioactive substance connected).

The aptamer can derive other aptamers, and the nucleotide sequence of the derived aptamer can be any one of the following three sequences:

(1) the homology with the nucleotide sequence of the aptamer listed in the embodiment is more than 90% (for example, the nucleotide sequence of the aptamer can be partially complementary by deletion or addition);

(2) a sequence that hybridizes to the nucleotide sequence of the aptamer listed in this example;

(3) this example embodies the RNA sequences transcribed from the nucleotide sequences of the listed aptamers.

The backbone of the nucleotide sequence of the aptamers listed in this example may also be derivatized to a phosphorothioate backbone, and the aptamers described above may also be engineered to lock nucleic acids or peptide nucleic acids accordingly.

The aptamer for nitrobenzene of the present example has the following uses: the application in detecting harmful factors of environment, food and organisms; ② separating and purifying; the usage in the design and development of the medicine; and fourthly, preparing the nitrobenzene detection probe.

The nitrobenzene CAS number used in this example is: 98-95-3.

The aptamer of nitrobenzene in the embodiment is mainly screened by a magnetic bead capture method, and the specific screening process comprises the following steps:

(a) optimizing the nucleic acid library: the structure of the DNA library is divided into three parts. The middle is a random sequence of 40 bases; the random sequence is flanked by two DNA arms, sequences necessary for PCR amplification of 20 fixed bases, while the fixed sequence at the 5' end is complementarily hybridized to the immobilized probe (BDNA). The immobilized probe is a DNA strand which is labeled with biotin at one end and is complementary to the immobilized sequence of the library, and is used for immobilizing the library.

Random library Lib:

5 '-ATTGGCACTCCACGCATAGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTATGCGTGCTACCGTGAA-3' (note: N represents any one of A, T, C, G bases);

5' primer: 5'-ATTGGCACTCCACGCATAGG-3', respectively;

3' primer: 5'-TTCACGGTAGCACGCATAGG-3', respectively;

5' fluorescent primer: 5 '-FAM-ATTGGCACTCCACGCATAGG-3';

3' spacer primer: 5'-AAAAAAAAAAAAAAAAAAAAAAAAA-spacer18-TTCACGGTAGCACGCATAGG-3'

BDNA immobilized probe: 5'-CCTATGCGTGGAGTGCCAAT-biotin-3'

(b) Primary screening: library pre-treatment method: 1.3nmol of the library (about 10)¹⁴DNA strand), 2.9nmol BDNA in 289. mu.L DPBS buffer (0.9mmol/L CaCl)₂、2.7mmol/L KCl、1.5mmol/L KH₂PO₄、0.5mmol/LMgCl₂、136.9mmol/L NaCl、8.1mmol/L Na₂HPO₄) Pre-denaturing at 95 deg.c for 10min, cooling to 60 deg.c at 0.1 deg.c/sec for 1min, and cooling to 25 deg.c at 0.1 deg.c/sec for slow renaturation. The hybridization library was added to a 1mL amount of streptavidin-labeled magnetic bead particles that had been washed 5 times and incubated at room temperature for half an hour in a shaker. The beads were then adsorbed using a magnet and the supernatant was removed, and the beads were suspended and washed 6 times with 400. mu.L of DPBS buffer. The nitrobenzene target solution was then added and placed in a shaker for 40min incubation at room temperature. Adsorbing the magnetic beads with a magnet, and recovering the supernatant to obtain a preliminary screening nucleic acid library containingThere is a complex of DNA and nitrobenzene.

(c) And (3) purification: and (c) carrying out PCR amplification on the primary screening nucleic acid library obtained in the step (b), wherein the amplification primers adopt the primers with the 5 'end and the 3' end which are listed in the step (a) and provided with the spacer arms. And performing single-strand preparative separation on the amplification product by using SDS (sodium dodecyl sulfate) denaturing PAGE (polyacrylamide gel electrophoresis), and then boiling, concentrating by using n-butanol and dialyzing to form a secondary nucleic acid library. Wherein, the PCR reaction amplification conditions are as follows: denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and amplification of N (optimal number of rounds) for several cycles; extension at 72 ℃ for 7 min. And (3) optimizing the annealing temperature by adopting gradient PCR and taking the initial nucleic acid library as a template, wherein the final obtained annealing temperature is 60 ℃. And the optimal number of amplification rounds of each round of screening products is obtained through round number optimization. After round number optimization, the remaining libraries were amplified under the same conditions. Preparing single strand from the PCR later library for the next round of screening, wherein the single strand preparation method comprises the following steps: mixing the PCR product with n-butanol according to the volume ratio of 1: 5 mixing, shaking and mixing for 30s, centrifuging at 6000rpm for 15s, allowing the solution to stratify, removing the upper n-butanol liquid, retaining the lower solution, and allowing the lower solution to be about 50 μ L in volume, adding an equal volume of 2 × TBE solution to the solution, and heating at 95 deg.C for 10 min. The samples were loaded on denaturing gel PAGE while hot, run for 20min using 300V voltage, cut off and collect the fluorescent bands under UV irradiation, and break the gel. Adding 1.2mL of DPBS buffer solution into the crushed gel, boiling for 15min at 95 ℃, centrifuging, collecting supernatant, and repeating for 1-2 times. Adding a mixture of the following components in a volume ratio of 1: 5, shaking for 30s, centrifuging at 6000rpm for 3min, and removing the upper n-butanol layer. Repeating for 2-3 times to make the final sample volume be about 100 μ L. Finally, the samples were dialyzed in DPBS solution overnight at 4 ℃ in a refrigerator.

(d) And (3) circulation: replacing the initial nucleic acid library with the secondary nucleic acid library obtained above and repeating the above steps (b) to (c), using new streptavidin-coated magnetic beads for each cycle, until a nucleic acid library comprising aptamers that bind with high affinity and high specificity to nitrobenzene is obtained.

(e) Evaluation of screening efficiency: screening efficiency was determined by qPCR, i.e.screenedEnrichment of DNA in the process. The detection signal is C of the DNA library in the qPCR process_qValues, converted to the retention of the nitrobenzene pair library. First, a series of concentrations of nucleic acid library solutions were prepared and measured using qPCR, yielding a linear equation of concentration versus Cq value. Then, the concentration of the DNA library in which nitrobenzene competes out is determined again, and the library retention is obtained from the ratio of the measured amount to the input amount. The higher the screening efficiency, the higher the retention. When the retention rate reached plateau, the library was subjected to clonal sequencing.

Investigation 1: the affinity between nitrobenzene and its aptamer in this example is mainly determined by Surface Plasmon Resonance (SPR) method, and the specific determination process includes the following steps:

(1) design of the Probe

5' end of nitrobenzene nucleic acid aptamer is marked with NH₂Group (sensing probe 1):

5'-NH₂-CACGCATAGGCTCGCCGTAACACACCACACATAGGTCCGCTATTCGCGTCCCTATGCGTG-3'

(1) immobilization of aptamer on SPR sensing chip surface

The sensor probe 1 is fixed on the surface of the SPR chip by using an amino coupling mode: a mixed solution of 0.4M (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) EDC and 0.1M NHS (N-hydroxysuccinimide) at the final concentration was continuously reacted and activated for 7min on a sensor chip model CM5 of BIAcore, then a sensor probe 1 labeled with an amino group was introduced at a concentration of 50. mu.M and continuously reacted for 10min, and finally the chip was blocked for 30s using ethanolamine at a concentration of 1M.

(2) Determination of affinity: target nitrobenzene (50nmol/L, 100nmol/L, 200nmol/L, 400nmol/L, 800nmol/L, 1600nmol/L) at different concentrations was added to the flow cell, incubated with the DNA modified on the gold membrane for 3min, washed with binding buffer for 1.5min, and the sensing curve was recorded in real time, as shown in FIG. 1 (A). As shown in FIG. 1(B), the dissociation constant (K) between the probe and nitrobenzene was calculated by plotting the concentration of the target as the abscissa and the response signal caused by the binding of the target to DNA at different concentrations as the ordinate_dValue), measuring K of the probe_dThe value was 167. + -.8 nM. The affinity between small molecule compounds and nucleic acids ranges from 100nM to 50. mu.M, and the affinity between most small molecules and their aptamers ranges from. mu.M. It is demonstrated that the nitrobenzene nucleic acid aptamers we screened were at high affinity levels.

Investigation 2: the specificity between nitrobenzene and its aptamer of this example was mainly determined by fluorescence recovery, and the specific determination procedure included the following steps:

(1) design of the Probe

The 3' -end of the nitrobenzene nucleic acid aptamer is labeled with Cy3 fluorescent dye (aptamer fluorescent probe 1):

5'-CACGCATAGGCTCGCCGTAACACACCACACATAGGTCCGCTATTCGCGTCCCTATGCGTG-Cy3-3'；

the 5' end of the aptamer complementary sequence was labeled with a BHQ-2 fluorescence quenching group (quenching probe):

5'-BHQ-2-CACGCATAGGGACGCGAATAGCGGACCTATGTGTGGTGTGTTACGGCGAGCCTATGCGTG-3'；

(2) determination of specificity: and (3) respectively preparing solutions of the aptamer fluorescent probe and the quenching probe with the concentration of 2 mu M by taking DPBS as a solvent, transferring 75 mu L of solution into each solution, mixing the solutions, shaking and uniformly mixing the solutions, and incubating for 30min at room temperature for later use. The mixed solution was equally divided into three equal parts (50. mu.L each), 50. mu.L of a nitrobenzene solution (nitrobenzene) having a concentration of 2. mu.M, 50. mu.L of an aniline solution (Negative control) having a concentration of 6. mu.M, and 50. mu.L of a screening buffer solution (Blank control) were added to the three parts, and reacted at room temperature for 10min, and then the solutions were transferred to 96-well plates and scanned for fluorescence emission spectra using a multifunction scanner, and fluorescence emission spectra of the above 3 reaction solutions were obtained. As can be seen from fig. 2, for aniline at a concentration 3 times that of nitrobenzene, nitrobenzene produces fluorescence at an intensity of about 42500a.u., whereas aniline at about 7300a.u., the ratio of response values is about 6 times. The aptamer probe is proved to have higher reaction specificity on nitrobenzene, and the aptamer provided by the patent can recognize nitrobenzene with higher specificity.

Investigation 3: the method for measuring nitrobenzene by adopting an SPR sensing competition method comprises the following steps:

detection probe for nitrobenzene nucleic acid aptamer 1:

5’-biotin-CACGCATAGGCTCGCCGTAACACACCACACATAGGTCCGCTATTCGCGTCCCTATGCGTG-3’；

(1) immobilization of nitrobenzene-bovine serum albumin complex on surface of CM5 sensor chip

The sensing probe 1 is fixed on the surface of the SPR chip by means of amino coupling: a mixed solution of 0.4M (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) EDC and 0.1M NHS (N-hydroxysuccinimide) at the final concentration was continuously reacted and activated for 7min on a sensor chip model CM5 of BIAcore, then a solution of a nitrobenzene-bovine serum albumin complex at a concentration of 10. mu.g/mL was introduced and continuously reacted for 3min, and finally the chip was blocked with bovine serum albumin at a concentration of 1mg/mL for 1 min.

(2) Determination of nitrobenzene by SPR competition method

The detection probe 1 and streptavidin (streptavidin, SA) solution are mixed according to a molar ratio of 4: 1 for 30min, the final concentration of the product was 30. mu.M. Different concentrations of target nitrobenzene (10.188nmol/L, 20.375nmol/L, 40.750nmol/L, 81.500nmol/L, 163.00nmol/L, 326.00nmol/L, 652.00nmol/L, 1250.0nmol/L, 2500.0nmol/L, 5000.0nmol/L, 10000nmol/L) were mixed with the product prepared above in a volume ratio of 1: 1 to form 200 mu L of mixed solution, adding the mixed solution into a flow cell respectively, incubating the mixed solution with nitrobenzene-bovine serum albumin modified on a gold membrane for 3min, washing the mixed solution for 1.5min by using a binding buffer solution, and recording a curve. The logarithm of the concentration of the target was plotted as the abscissa and the response signal caused by binding of different concentrations of the target was plotted as the ordinate (see FIG. 3B). It can be seen that as the concentration of nitrobenzene increases, the response value gradually decreases, and a certain inhibition relationship is presented.

Consider 4: the nitrobenzene is measured by a label-free fluorescence method, and the specific measurement process comprises the following steps:

the concentration of the immobilized aptamer was 2. mu.M, and an equal volume of 2 XEventreen dye was added thereto and shaken to form 300. mu.L of a mixed solution. Heating the mixed solution at 95 ℃ for 10min, cooling to room temperature, and adding 50 mu L nitrobenzene solution with final concentration of 195ppb, 390ppb, 781ppb, 1562ppb, 3125ppb, 12500ppb and 25000 ppb. As shown in FIG. 4, it can be seen from the fluorescence emission spectrum result that the fluorescence signal gradually decreases with the increase of the concentration of nitrobenzene, and a good linear relationship is shown between the fluorescence signal and the logarithm of the concentration of nitrobenzene.

Review 5: the nitrobenzene is measured by adopting a fluorescence polarization technology, and the specific measurement process comprises the following steps:

(1) hybrid sequence 1:

5’-SH-AAAAAAAAAACACGCATAGGGACGCGAATAGCGGACCTATGTGTGGTGTGTTACGGCGAGCCTATGCGTG-3’；

the hybridization sequence 2:

5’-SH-AAAAAAAAAACCCCCCCCCCAAAAAACCCCCCCCCCCCCAAAAAAGGGAAAGGAAAACCCCCACCCCCC-3’。

(2) aptamer fluorescent probe 2:

5’-CACGCATAGGCTCGCCGTAACACACCACACATAGGTCCGCTATTCGCGTCCCTATGCGTG-FAM-3’；

DNA control fluorescent probe:

5’-GGGGGGTGGGGGTTTTCCTTTCCCTTTTTTGGGGGGGGGGGGGTTTTTTGGGGGGGGGG-FAM-3’。

preparing a nano silver aptamer fluorescent probe and a DNA contrast probe: (a) the decahedral nano silver is selected as a nano material, 1mL decahedral nano silver solution is transferred, 100 muL of hybridization sequence 1 solution with the concentration of 10 muM and 64 muL of 2M sodium chloride solution are added into the decahedral nano silver solution, the mixture is uniformly shaken and mixed, and the mixture is shaken and reacted for 30min at room temperature and then kept overnight for later use. Centrifuging the prepared nano silver solution at 15000rpm for 10min, removing supernatant, adding 1mL screening buffer, centrifuging at 15000rpm for 10min, removing supernatant, and repeating for 3 times. Then, 100. mu.L of a 100. mu.M fluorescent probe 2 solution was added thereto, and the reaction was carried out at room temperature for 30 min. Then centrifuged, the supernatant removed and resuspended in 500. mu.L of selection buffer for use. (b) And similarly, using decahedral nano silver as a nano material, transferring 1mL decahedral nano silver solution, adding 100 mu L of hybridization sequence 2 solution with the concentration of 10 mu M and 64 mu L of 2M sodium chloride solution, shaking and mixing uniformly, shaking and reacting at room temperature for 30min, and standing overnight for later use. Centrifuging the prepared nano silver solution at 15000rpm for 10min, removing supernatant, adding 1mL screening buffer, centrifuging at 15000rpm for 10min, removing supernatant, and repeating for 3 times. Then, 100. mu.L of a solution of aptamer fluorescent probe 3 at a concentration of 100. mu.M was added thereto, and the reaction was carried out at room temperature for 30 min. Then centrifuged, the supernatant removed and resuspended in 500. mu.L of selection buffer for use.

The nano-silver aptamer fluorescent probe and the DNA contrast probe solution are equally divided into 5 equal parts respectively, the 5 equal parts are added into a 96-well plate, 100 mu L nitrobenzene solution with the final concentration of 40nM, 160nM, 640nM, 2560nM and 10240nM is added respectively, the reaction is carried out for 10min at room temperature, and then the fluorescence polarization detection analysis is carried out. As can be seen from fig. 5(B, C), the fluorescence polarization value of the nitrobenzene aptamer probe gradually decreases with the increase of the concentration of nitrobenzene, and a good linear relationship is presented between the fluorescence polarization signal and the logarithm of the concentration of nitrobenzene; the fluorescence polarization signal generated by the DNA contrast probe has no corresponding relation with nitrobenzene, and the signal is low. Proves that the nucleic acid aptamer detection method provided by the patent can be used for quickly and effectively detecting nitrobenzene.

The above description is only an embodiment of the present invention, and the protection scope of the present invention is not limited to the above-described embodiment. All technical schemes belonging to the idea of the invention belong to the protection scope of the invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention, and such modifications and embellishments should also be considered as within the scope of the invention.

Sequence listing

<110> national institute of environmental science of south China, department of ecological environment

<120> aptamer of nitrobenzene, aptamer derivative and application thereof

<160>7

<170>SIPOSequenceListing 1.0

<210>1

<211>60

<212>DNA

<213> nucleic acid aptamer

<400>1

cacgcatagg ctcgccgtaa cacaccacac ataggtccgc tattcgcgtc cctatgcgtg 60

<210>2

<211>60

<212>DNA

<213> nucleic acid aptamer

<400>2

cacgcatagg gacgcgaata gcggacctat gtgtggtgtg ttacggcgag cctatgcgtg 60

<210>3

<211>20

<212>DNA

<213> primer

<400>3

attggcactc cacgcatagg 20

<210>4

<211>20

<212>DNA

<213> primer

<400>4

ttcacggtag cacgcatagg 20

<210>5

<211>20

<212>DNA

<213> Probe

<400>5

cctatgcgtg gagtgccaat 20

<210>6

<211>70

<212>DNA

<213> hybridization sequence

<400>6

aaaaaaaaaa cacgcatagg gacgcgaata gcggacctat gtgtggtgtg ttacggcgag 60

cctatgcgtg 70

<210>7

<211>70

<212>DNA

<213> hybridization sequence

<400>7

aaaaaaaaaa cccccccccc aaaaaacccc ccccccccca aaaaagggaa aggaaaaccc 60

ccacccccc 69

Claims

1. The aptamer of nitrobenzene is characterized in that the nucleotide sequence of the aptamer is shown as SEQ ID No.1 or 2 in a sequence table.

2. The aptamer of claim 1, wherein a position on the nucleotide sequence of the aptamer is phosphorylated, oxymethylated, methylated, aminated, thiolated, fluorinated, or isotopically modified.

3. The aptamer according to claim 1, wherein the nucleotide sequence of the aptamer is labeled with biotin, digoxigenin, a fluorescent substance, a nanomaterial, polyethylene glycol, a peptide fragment, a protein, an enzyme, or folic acid.

4. An aptamer derivative of nitrobenzene, wherein said aptamer derivative is a phosphorothioate backbone derived from the backbone of the nucleotide sequence of said aptamer of claim 1, 2 or 3, or a corresponding locked nucleic acid or peptide nucleic acid modified from said aptamer of claim 1, 2 or 3.

5. Use of the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4 for the detection of nitrobenzene in environments, foodstuffs and organisms.

6. Use of the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4 for nitrobenzene separation and purification.

7. Use of the aptamer according to any one of claims 1 to 3 or the aptamer derivative according to claim 4 for the preparation of a nitrobenzene detection probe.

8. A nitrobenzene detection method is characterized by comprising the following steps:

a step (1) of preparing an aptamer according to any one of claims 1 to 3 or an aptamer derivative according to claim 4;

step (2), preparing a detection probe of the nitrobenzene nucleic acid aptamer;

fixing the detection probe on the surface of the SPR chip, and sealing the chip on which the detection probe is fixed by using bovine serum albumin;