CN110257383B - Aptamer for specifically recognizing di (2-ethyl) hexyl phthalate as well as screening method and application thereof - Google Patents
Aptamer for specifically recognizing di (2-ethyl) hexyl phthalate as well as screening method and application thereof Download PDFInfo
- Publication number
- CN110257383B CN110257383B CN201910547712.8A CN201910547712A CN110257383B CN 110257383 B CN110257383 B CN 110257383B CN 201910547712 A CN201910547712 A CN 201910547712A CN 110257383 B CN110257383 B CN 110257383B
- Authority
- CN
- China
- Prior art keywords
- aptamer
- nucleic acid
- ethyl
- nucleotide sequence
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Urology & Nephrology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a nucleic acid aptamer for specifically recognizing di (2-ethyl) hexyl phthalate, which is one of the following nucleotide sequences: has a nucleotide sequence shown as SEQ ID NO. 1; has the nucleotide sequence shown in SEQ ID NO. 2. The invention also provides a screening method and application of the nucleic acid aptamer. The aptamer can rapidly and conveniently detect di (2-ethyl) hexyl phthalate, has strong specificity, and does not have cross reaction with the di (2-ethyl) hexyl phthalate analogue; high affinity, and an affinity constant of 2.26+/-0.06 nM; the sensitivity is high: the linear detection range was 7.629 pg/mL-2. Mu.g/mL, and the lowest limit of detection (LOD) at which di (2-ethyl) hexyl phthalate could be detected was 0.103pg/mL.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a nucleic acid aptamer for specifically recognizing di (2-ethyl) hexyl phthalate, a screening method and application thereof.
Background
Di (2-ethylhexyl) phthalate, dioctyl phthalate, DOP, DEHP, dioctyl phthalate, of formula (formula) C 24 H 38 O 4 Is an ester compound formed by phthalic acid and 2-ethylhexanol. DEHP is one of plasticizers used in large amounts and in a wide range of applications, and can cause liver tumors in animals, and is considered as one of possible carcinogens for humans. Therefore, rapid monitoring of DEHP is of great importance for protecting human health.
Currently, the methods for detecting DEHP fall into two broad categories: instrument detection techniques and rapid detection techniques. Wherein, instrument detection technique includes: high performance liquid chromatography (High performance liquid chromatography, HPLC) with a minimum detection limit of 0.05-10mg/L; high performance liquid chromatography-mass spectrometry (High performance liquid chromatography-Mass spectrometry, HPLC-MS), the minimum detection limit of the method is 0.01-0.1ng/mL; gas chromatography-mass spectrometry (Gas chromatography-mass spectrometry, GC-Ms) with a minimum detection limit of 0.05mg/L to 0.01mug/mL; raman spectroscopy (Raman spectroscopy, SER), which has a minimum detection limit of 0.9x10-9M; mass spectrometry (mass spectrometry, MS) with a minimum detection limit of 0.21. Mu.g/L. The instrument methods have the defects of high detection cost, high detection limit, professional operation requirement and the like, and cannot meet the requirements of basic units on rapid DEHP monitoring. The rapid detection technology developed based on DEHP recognition molecules can realize complementation to large instrument detection technology, including: enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) with a minimum limit of detection of 4.2pg/mL; a multi-residue detection method based on aptamer, wherein the detection limit of the method is 3.9pg/mL. The obvious advantage of these two rapid detection techniques is the high sensitivity, which allows for the monitoring of trace amounts of DEHP in the sample. In the development of rapid detection techniques, recognition of molecules is critical. Compared with an antibody, the aptamer has the advantages of short preparation period, wide target range (no modification is needed for a small molecular target), easiness in marking and transformation and the like.
The nucleic acid aptamer is a single-or double-stranded DNA or RNA obtained by SELEX technology (Systematic Evolution of Ligands byExponential Enrichment, systematic evolution of adaptation systems for systematic exponential enrichment). The aptamer can specifically identify various target molecules including proteins, small molecules, cells and tissues, has high stability, is easy to synthesize and modify, has low cost, and has wide application prospects in the fields of biosensing, imaging, drug research and development and the like.
At present, a lot of nucleic acid aptamers are obtained through in vitro screening technology, and a lot of nucleic acid aptamers are detected rapidly and sensitively on line, but few reports are related to specific recognition of phthalate plasticizer nucleic acid aptamers. Comparative document 1: the application number is CN201610949635.5, the invention is named as a phthalate plasticizer single-stranded DNA aptamer, a screening and characterization method thereof and an electrochemical sensor, and target molecules need to be modified in advance, so that the cost, complexity and difficulty of experiments are increased. And affinity (affinity constant up to 100+5nM) is still to be improved; the detection interval of the aptamer is 160 ppt-1.6 ppm, and the lowest detection limit value is higher.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a nucleic acid aptamer capable of specifically recognizing di (2-ethyl) hexyl phthalate, which can recognize the di (2-ethyl) hexyl phthalate with high specificity and high affinity; the invention also provides a screening method of the nucleic acid aptamer, which uses a systematic evolution technology of exponential enrichment ligand, uses magnetic beads as a separation method, does not need to fix target molecules on a carrier, uses a complete structure of di (2-ethyl) hexyl phthalate as a target, and screens to obtain the aptamer specifically combined with the target molecules with high affinity; and (3) carrying out rapid and high-throughput identification on a large number of nucleic acid aptamers after enrichment by adopting a nano gold biosensing method.
The invention is realized in the following way:
in a first aspect of the invention, there is provided a nucleic acid aptamer specifically recognizing di (2-ethyl) hexyl phthalate, which is one of the following nucleotide sequences:
has a nucleotide sequence shown as SEQ ID NO. 1;
has the nucleotide sequence shown in SEQ ID NO. 2: both ends of the nucleotide sequence of SEQ ID NO.1 are truncated by 11bp.
Preferably, the nucleic acid aptamer further comprises a nucleic acid aptamer that is phosphorylated, oxymethylated, methylated, aminated, sulfhydrylated or isotopically substituted at a position on the nucleotide sequence of the nucleic acid aptamer.
Preferably, the nucleic acid aptamer further comprises a nucleotide sequence to which biotin, digoxin, a fluorescent substance, a nano luminescent material, polyethylene glycol or folic acid markers are bound.
Specifically, the secondary structure of the nucleic acid aptamer with the nucleotide sequence shown in SEQ ID NO.1 has a protruding loop, and the Gibbs free energy dG= -19.81; the secondary structure of the aptamer with the nucleotide sequence shown in SEQ ID No.2 has a protruding loop, and the Gibbs free energy dG= -17.56.
In a second aspect of the present invention, there is provided a method for screening for said nucleic acid aptamer, comprising the steps of:
step 1, artificially synthesized ssDNA library;
step 2, fixing the library on the surface of magnetic beads, incubating a target substance DEHP with the library, and capturing nucleic acid aptamer on the magnetic beads by using free small molecules;
step 3, screening and separating ssDNA combined with a target substance DEHP;
step 4, PCR amplification, denaturation to single chain, and preparation of a secondary library;
step 5, enrichment screening;
step 6, high-throughput sequencing;
and 7, identifying the binding activity of the nucleic acid aptamer by a nano gold biosensing method, and screening to obtain the nucleic acid aptamer with high affinity.
In a third aspect of the invention, there is provided the use of said nucleic acid aptamer in the identification of di (2-ethyl) hexyl phthalate or in the preparation of a kit for the detection of di (2-ethyl) hexyl phthalate.
The invention has the beneficial effects that:
1. the nucleic acid aptamer for specifically recognizing di (2-ethyl) hexyl phthalate provided by the invention can be used for rapidly and conveniently detecting the di (2-ethyl) hexyl phthalate, and the nucleic acid aptamer for recognizing the di (2-ethyl) hexyl phthalate is determined to have strong specificity through cross reaction: there is no cross-reaction with di (2-ethyl) hexyl phthalate analogues; high affinity, and an affinity constant of 2.26+/-0.06 nM; the sensitivity is high: the linear detection range is 7.629pg/mL-2 mug/mL, and the lowest detection Limit (LOD) of the di (2-ethyl) hexyl phthalate is 0.103pg/mL;
2. the nucleic acid aptamer specifically recognizing di (2-ethyl) hexyl phthalate provided by the invention has unique sequence composition, and can be a short sequence of a full-length sequence (shown as SEQ ID NO. 1) or a truncated sequence (shown as SEQ ID NO. 2) (after the fixed regions of 11bp at two ends are removed), so that the synthesis cost can be saved.
3. The screening method for specifically recognizing the nucleic acid aptamer of di (2-ethyl) hexyl phthalate provided by the invention can screen more nucleic acid aptamers with strong specificity. In the prior art, the aptamer which is easy to screen is not used for identifying the small molecule, but is used for identifying the derivative. In order to solve the problem, the method of fixing the library on the surface of the magnetic beads is adopted, and free small molecules are utilized to capture the nucleic acid aptamer on the magnetic beads; the method not only can realize simultaneous screening of multiple targets, but also can screen out the nucleic acid aptamer for identifying the small molecular hazard.
4. The screening method of the nucleic acid aptamer specifically recognizing di (2-ethyl) hexyl phthalate provided by the invention combines high-throughput sequencing with nano-gold primary screening and LSPR confirmation, so that the sequence information of the enrichment library is large and is not repeated. In order to save the synthesis cost of the aptamer, the fixed region is removed when the monoclonal aptamer is screened. Meanwhile, by combining a nano gold biosensing method, a large number of (unrepeated aptamer) identification activities are initially identified. Thereby rapidly determining the nucleic acid aptamer with recognition activity.
Drawings
FIG. 1 is a flow chart of a screening method for specifically recognizing nucleic acid aptamer of di (2-ethyl) hexyl phthalate according to an embodiment of the present invention;
FIG. 2 is a SELEX screening retention rate plot;
FIG. 3 is a diagram of a nano-gold primary screen;
FIG. 4 shows the secondary structure of aptamer 1 (shown in SEQ ID NO. 1) specifically recognizing di (2-ethyl) hexyl phthalate according to the present invention;
FIG. 5 shows the secondary structure of aptamer 2 (shown in SEQ ID NO. 2) specifically recognizing di (2-ethyl) hexyl phthalate according to the present invention;
FIG. 6 is a schematic diagram of a carboxyl-modified DEHP;
FIG. 7 is a cross-reaction diagram of aptamer 2 specifically recognizing di (2-ethyl) hexyl phthalate provided in experimental examples of the present invention;
FIG. 8 is a graph showing the DEHP detection criteria for aptamer 2 specifically recognizing di (2-ethyl) hexyl phthalate provided in experimental examples of the present invention.
Detailed Description
Example 1 screening of nucleic acid aptamers specifically recognizing di (2-ethyl) hexyl phthalate
1. A random single-stranded DNA library shown in Table 1 and a Biotin primer were synthesized, and SA magnetic beads (streptavidin-modified magnetic beads) were prepared and purchased from the company Thermofiser.
TABLE 1
2. Incubation of target DEHP with ssDNA:
binding buffer for ssDNA library (0.1 g CaCl) 2 ，0.2g KCl，0.2g KH 2 PO 4 ，0.1g MgCl 2 .6H 2 O，8g NaCl，1.15g Na 2 HPO 4 1L) was dissolved, and Biotin primer (molar ratio to library 2:1) was added to perform slow renaturation. The mixture was added to SA beads (the beads were washed with binding buffer 4 times before use) and immobilized for 30-45min, and the immobilization efficiency was measured. Washing with binding buffer for 6 times, adding 100-200 μL DEHP with final concentration of 100 μM, and incubating at room temperature for 60-90min.
3. Screening and isolating ssDNA that binds to the target substance DEHP:
the magnetic beads are fished by a magnet, the eluent is recovered, 100-200 mu L of DEHP with the final concentration of 100 mu M is added for elution, and the eluates are combined. At the end of each round of screening, 2. Mu.L of eluent was used for monitoring library enrichment by fluorescent quantitative PCR.
4. PCR amplification, denaturation to single strand, preparation of secondary library:
all eluents were added to emulsion PCR-mix (FAM labeled upstream primer and polyA downstream primer as shown in Table 1), the reaction system was shown in Table 2, mixed well, added with 8mL of emulsifier (1 mL of EM90, 25. Mu.L of Triton X-100, 49mL of Mineral oil), vortexed for 2min to prepare emulsion, and left for 5min (emulsification with emulsifier followed by PCR amplification to make the amplified template into a plurality of droplets, avoiding preferential amplification as much as possible, and making enrichment to nucleic acid aptamer recognizing target molecule easier during enrichment). PCR amplification (95 ℃ C. 2min,95 ℃ C. 1min,60 ℃ C. 1min,72 ℃ C. 1min,25 cycles) was performed. The PCR products were concentrated by n-butanol method, denatured, and then electrophoretically separated by SDS-PAGE, and the ssDNA library was recovered by gel cutting. Dialysis was performed overnight in binding buffer.
It should be noted that: the Spacer in the polyA downstream primer provides the necessary spacing for oligonucleotide labeling to achieve long and short strand amplification, thereby facilitating the preparation of a secondary single stranded library. Spacer 18 is commonly used to introduce a strongly hydrophobic group.
TABLE 2
5. The next round of enrichment screening:
the next day the library concentration was measured for the next round of screening, with the 1 st-4 th round of screening steps consistent, and direct positive screening. Rounds 5-8 of screening were performed with either the analog or screening buffer followed by positive screening with DEHP target. The amount of ssDNA library screened in the first round was 1.3nM, and the amount of ssDNA library screened in each subsequent round was 80-100pM.
6. And during enrichment screening, the real-time fluorescence quantitative PCR technology is simultaneously adopted to monitor the enrichment condition of the library. The reaction system shown in Table 3 was subjected to Q-PCR. The reaction procedure is: 2min at 95 ℃, 0.5min at 60 ℃, 0.5min at 72 ℃,25cycles. Wherein, the Q-PCR quantitative standard curve uses a random single-stranded DNA initial library as a template, and the template concentration is 16000pM, 1600pM, 160pM, 16pM and 1.6pM respectively.
TABLE 3 Table 3
Q-PCR quantitative Standard curve Y= -0.269 x+4.547, R 2 0.9935. The retention results are shown in figure 2. Starting from 1-4 rounds, the retention rate gradually increased as the number of enrichment rounds increased. After the reverse screening was started from run 5-8, the retention was reduced compared to run 4, indicating that non-specifically bound molecules were elutriated during the reverse screening. However, with the increase of the number of enrichment rounds, the retention rate gradually increases, and the retention rate is obviously improved by the time of 8 th round screening, which indicates that the enrichment effect is obvious.
6. High throughput sequencing:
the final round of library enrichment was carried out by the company for high throughput sequencing analysis. For the sequenced sequences, the sequences with the changed fixed areas are removed, 300 sequences are selected in sequence from large to small according to the enrichment times, and 129 sequences are selected through ClustalX2 and phylogenetic tree analysis. These sequences were aligned with a truncated 22bp total sequence for a secondary structure analysis, and 86 total sequences were selected with unchanged structure and loop regions greater than 1. Finally, 10 sequences with the largest occurrence number are added, and 96 truncated non-labeled aptamer sequences (58 bp) with 22bp total are synthesized for affinity preliminary screening.
7. Identification of binding Activity of nucleic acid aptamer by nanogold biosensing method
The gold nanoparticles are synthesized by adopting a method of reducing tetra-chloroauric acid by trisodium citrate, and after the gold nanoparticles are synthesized, supernatant is removed by centrifugation, and ultra-pure water is redissolved. The final concentration of aptamer 2 (aptamer after removal of 11 bases before and after the constant region) was 0.4. Mu.M, and incubated with 1. Mu.g/mL DEHP for 30min, respectively. Nano Jin Fuyo min was added and 0.9M NaCl was added. Calculating A according to the color change condition of the nano-gold 620nm And A 520nm Thereby screening out the aptamer with highest affinity. The results are shown in FIG. 3. After analysis by homology alignment and secondary structure analysis, the selected aptamer was assayed by nanogold colorimetric method (A) 620nm /A 520nm )/(A 0 620nm /A 0 520nm ) The ratios are all higher than 1, indicatingThe aptamer has binding activity to the target. Wherein, aptamer 31, 123, 203, 281 (A 620nm /A 520nm )/(A 0 620nm /A 0 520nm ) The ratios were all higher than 1.2, indicating high binding activity of the aptamer to the target.
Example 2 Secondary Structure of nucleic acid aptamer
1. Nucleic acid aptamer with nucleotide sequence shown in SEQ ID NO.1
The secondary structure of the aptamer having the nucleotide sequence shown in SEQ ID No.1 was analyzed using an M-fold platform. The result shows that the full-length sequence (80 bp) secondary structure (4A) has a prominent loop, and the Gibbs free energy dG= -19.81 shows that the structure has higher stability. The secondary structure is shown in figure 4.
2. Nucleic acid aptamer with nucleotide sequence shown in SEQ ID NO.2
The secondary structure of the aptamer having the nucleotide sequence shown in SEQ ID No.2 was analyzed using an M-fold platform. The result shows that the secondary structure (4B) of each 11 base sequence (58 bp) before and after the removal of the constant region has a protruding loop, and the Gibbs free energy dG= -17.56 shows that the structure has higher stability. The secondary structure is shown in fig. 5.
Experimental example aptamer performance analysis
1. Detection of aptamer affinity using localized surface plasmon resonance
Detection of aptamer affinity using Localized Surface Plasmon Resonance (LSPR) techniques, comprising the specific steps of: DEHP was first carboxyl modified (structure as shown in FIG. 6) and then fixed to NH 2 And sealing the chip by using sealing liquid. The baseline was equilibrated with buffer and then loaded at different concentrations (2.5 nM,5nM,10nM,20nM or 240s for ligand binding time for aptamer 31. The assay used in this experiment was the TraceDrawer (Ridgeview Instruments ab, sweden) and the assay was an One To One assay model. The results are shown in Table 4 where the affinity of aptamer 31 was highest, reaching 2.26+ -0.06 nM, far above the reported affinity constant for aptamer To target, laying foundation for further application.
TABLE 4 local surface plasmon resonance determination affinity constants
2. Specificity analysis of aptamer-Cross reaction experiments
Under the same conditions, different phthalate esters (diphenyl phthalate (DPHP), dihexyl phthalate (DHXP, DNHP), dicyclohexyl phthalate (DCHP), dimethyl phthalate (Dimethyl phthalate), dibutyl phthalate (Dibutyl phthalate), diisobutyl phthalate (Diisobutyl phthalate), diethyl phthalate (diethyl phthalate), di-n-octyl phthalate (DOP), butyl Benzyl Phthalate (BBP)) were experimentally selected as cross analogues, and the specificity of the nucleic acid aptamer 31 (having the nucleotide sequence shown in SEQ ID NO. 2) was examined. The same concentration (30.518 pg/mL) of phthalate solution was added to the test solution and impedance testing was performed after the aptamer sensor reaction was completed. And comparing electrochemical impedance response values of the aptamer sensor after the aptamer sensor reacts with various analogues, and analyzing the specificity. As a result, as shown in FIG. 7, the nucleic acid aptamer 31 (having the nucleotide sequence shown in SEQ ID NO. 2) was highly specific, and its response signal was significantly higher than that of the similar substance.
3. Sensitivity analysis of nucleic acid aptamer-establishment of electrochemical sensor label curve manufacturing method
And (3) performing sulfhydryl modification on the aptamer 31 (removing a 22bp constant region), and modifying the aptamer on the surface of a gold electrode by a gold-sulfur bond self-assembly method to construct the electrochemical sensor. DEHP was formulated with PBS into standard solutions of varying concentrations (7.629 pg/mL, 30.518pg/mL, 122.07pg/mL, 0.488ng/mL, 1.953ng/mL, 7.813ng/mL, 31.25ng/mL, 0.125. Mu.g/mL, 0.5. Mu.g/mL, 2. Mu.g/mL), and 10mM K was contained in 10mM PBS 3 [Fe(CN) 6 ]/K 4 [Fe(CN) 6 ](1:1) and containing 0.5M KCl as supporting electrolyte under optimized conditions (aptamer mass concentration 0.01. Mu.M, solution pH=7.0, incubation time 30 min) to give impedance (Z is notResponse value, Z, of standard solution of the same concentration 0 For bare electrode response values), linear regression equations are obtained by linear fitting. As a result, as shown in FIG. 8, the linear range was 7.629 pg/mL-2. Mu.g/mL, and the lowest limit of detection (LOD) was 0.103pg/mL. The minimum detection limit is far lower than that of the prior report.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Sequence listing
<110> university of Hubei
<120> aptamer specifically recognizing di (2-ethyl) hexyl phthalate, screening method and application thereof
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 80
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
attggcactc cacgcatagg gtgcgaccac atacgcccca tgtatgtccc ttggttgtgc 60
cctatgcgtg ctaccgtgaa 80
<210> 2
<211> 58
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
acgcataggg tgcgaccaca tacgccccat gtatgtccct tggttgtgcc ctatgcgt 58
<210> 3
<211> 80
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (21)..(60)
<223> n=a or c or g or t
<400> 3
attggcactc cacgcatagg nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60
cctatgcgtg ctaccgtgaa 80
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
cctatgcgtg gagtgccaat 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
attggcactc cacgcatagg 20
<210> 6
<211> 40
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
aaaaaaaaaa aaaaaaaaaa ttcacggtag cacgcatagg 40
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
attggcactc cacgcatagg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ttcacggtag cacgcatagg 20
Claims (4)
1. A nucleic acid aptamer specifically recognizing di (2-ethyl) hexyl phthalate, characterized in that it is one of the following nucleotide sequences:
has a nucleotide sequence shown as SEQ ID NO. 1;
has the nucleotide sequence shown in SEQ ID NO. 2: truncating 11bp at both ends on the nucleotide sequence of SEQ ID NO. 1; is phosphorylated, oxymethylated, methylated, aminated, sulfhydrylated or isotopically substituted at a position on the nucleotide sequence of the nucleic acid aptamer.
2. The aptamer of claim 1, further comprising a nucleotide sequence to which biotin, digoxigenin, a fluorescent substance, a nano-luminescent material, polyethylene glycol, or a folic acid label is bound.
3. The nucleic acid aptamer of claim 1, wherein the secondary structure of the nucleic acid aptamer having the nucleotide sequence shown in SEQ ID No.1 has a protruding loop, gibbs free energy dg= -19.81; the secondary structure of the aptamer with the nucleotide sequence shown in SEQ ID No.2 has a protruding loop, and the Gibbs free energy dG= -17.56.
4. Use of a nucleic acid aptamer according to any one of claims 1 to 3 for the recognition of di (2-ethyl) hexyl phthalate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910547712.8A CN110257383B (en) | 2019-06-24 | 2019-06-24 | Aptamer for specifically recognizing di (2-ethyl) hexyl phthalate as well as screening method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910547712.8A CN110257383B (en) | 2019-06-24 | 2019-06-24 | Aptamer for specifically recognizing di (2-ethyl) hexyl phthalate as well as screening method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110257383A CN110257383A (en) | 2019-09-20 |
| CN110257383B true CN110257383B (en) | 2023-05-26 |
Family
ID=67920721
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910547712.8A Active CN110257383B (en) | 2019-06-24 | 2019-06-24 | Aptamer for specifically recognizing di (2-ethyl) hexyl phthalate as well as screening method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110257383B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111118015B (en) * | 2020-01-19 | 2022-07-29 | 华侨大学 | Preparation and application of potassium perfluorooctane sulfonate-resistant aptamer |
| CN111876423B (en) * | 2020-08-05 | 2021-10-22 | 生态环境部华南环境科学研究所 | Aptamer of nitrobenzene, aptamer derivative and application of aptamer derivative |
| CN112852823B (en) * | 2021-02-02 | 2023-02-28 | 江南大学 | Aptamer with capability of identifying acyclovir analogues in broad spectrum |
| CN113025617B (en) * | 2021-04-30 | 2023-05-23 | 福建农林大学 | Nucleic acid aptamer for specifically recognizing quinclorac and application thereof |
| CN115044587B (en) * | 2022-05-23 | 2023-04-07 | 江南大学 | Aptamer capable of specifically recognizing levamisole, and dual-mode nanoprobe and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2760687A1 (en) * | 2009-05-01 | 2010-11-04 | Ophthotech Corporation | Methods for treating or preventing ophthalmological diseases |
| WO2015086604A1 (en) * | 2013-12-11 | 2015-06-18 | Qiagen Gmbh | Nucleic acid detection and quantification |
| CN106399317A (en) * | 2016-10-26 | 2017-02-15 | 首都师范大学 | DNA (deoxyribonucleic acid) aptamer of phthalate type plasticizer and screening and expression method and electro-chemical sensor thereof |
| WO2018090414A1 (en) * | 2016-11-15 | 2018-05-24 | 河南省农业科学院 | Nucleic acid aptamer screening method based on localized surface plasmon resonance technology |
| KR20180089974A (en) * | 2017-02-02 | 2018-08-10 | 한양대학교 산학협력단 | Single strand DNA aptamer for detection of phthalate |
| CN108977448A (en) * | 2018-08-03 | 2018-12-11 | 湖北师范大学 | For detecting aptamer and its screening technique and the application of clenobuterol hydrochloride |
-
2019
- 2019-06-24 CN CN201910547712.8A patent/CN110257383B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2760687A1 (en) * | 2009-05-01 | 2010-11-04 | Ophthotech Corporation | Methods for treating or preventing ophthalmological diseases |
| WO2015086604A1 (en) * | 2013-12-11 | 2015-06-18 | Qiagen Gmbh | Nucleic acid detection and quantification |
| CN106399317A (en) * | 2016-10-26 | 2017-02-15 | 首都师范大学 | DNA (deoxyribonucleic acid) aptamer of phthalate type plasticizer and screening and expression method and electro-chemical sensor thereof |
| CN109609507A (en) * | 2016-10-26 | 2019-04-12 | 首都师范大学 | A kind of phthalic ester plasticizer single stranded DNA nucleic acid aptamers and its screening and characterizing method and electrochemical sensor |
| WO2018090414A1 (en) * | 2016-11-15 | 2018-05-24 | 河南省农业科学院 | Nucleic acid aptamer screening method based on localized surface plasmon resonance technology |
| KR20180089974A (en) * | 2017-02-02 | 2018-08-10 | 한양대학교 산학협력단 | Single strand DNA aptamer for detection of phthalate |
| CN108977448A (en) * | 2018-08-03 | 2018-12-11 | 湖北师范大学 | For detecting aptamer and its screening technique and the application of clenobuterol hydrochloride |
Non-Patent Citations (2)
| Title |
|---|
| 基于核酸适配体建立的小分子危害物检测技术研究进展;刘细霞;《 食品安全质量检测学报》;全文 * |
| 庆大霉素特异性单链DNA适配体的筛选、表征和应用;巫朦朦;韩旭艳;蔡蓉凤;李茂林;田亚平;周楠迪;;中国科学:生命科学(第05期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110257383A (en) | 2019-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110257383B (en) | Aptamer for specifically recognizing di (2-ethyl) hexyl phthalate as well as screening method and application thereof | |
| CN104508150B (en) | Aptamer-based multiplex assays | |
| KR20040047971A (en) | Multiparameter analysis of comprehensive nucleic acids and morphological features on the same sample | |
| US9983203B2 (en) | Method for protein analysis | |
| CN111172166B (en) | Nucleic acid aptamer for specifically recognizing beta-lactoglobulin and application thereof | |
| CN104911186B (en) | A kind of single strand dna oligonucleotide aptamer of specific recognition aflatoxin B1 | |
| CN108103174A (en) | For the plasmid standards for quantitation and its clone method of two generation of qPCR accurate quantification Illumina platforms sequencing sample | |
| EP1431398A1 (en) | A method for detecting in a mixture an amount of analytes | |
| CN108841828B (en) | Single-stranded DNA aptamer for specifically recognizing tobramycin and application thereof | |
| CN111122847A (en) | Method for rapidly detecting aflatoxin B1 on site based on aptamer | |
| CN103243101B (en) | One group of aptamers for specifically recognizing staphylococcus aureus enterotoxin C1 | |
| CN112083159B (en) | Evanescent wave aptamer sensor and method for detecting small molecules by applying evanescent wave aptamer sensor | |
| CN110988325A (en) | Blocking agent and kit containing same | |
| CN113567685B (en) | HGFR (human liver factor receptor) identification method based on nucleic acid aptamer probe and kit for detecting HGFR | |
| CN115786466A (en) | CRISPR/Cas regulated DNA silver nanocluster sensor and method and application thereof for detecting meat adulteration | |
| KR102403628B1 (en) | Method for Obtaining Profiles for Population of Target Molecules of a Sample | |
| CN111060576B (en) | Electrochemical sensor and method for detecting aflatoxin B1 | |
| KR100898623B1 (en) | Method for Immobilzing Biomolecules on Substrate Using Gold Nanoparticles | |
| CN101929997B (en) | Method for improving sensitivity of immune detection | |
| US20140248715A1 (en) | Method for detecting biomolecules | |
| CN113897419B (en) | Kit and method for capturing extracellular vesicles or quantitatively analyzing extracellular vesicle contents | |
| CN114410637B (en) | Screening and application of nucleic acid aptamer specifically binding to chlorpromazine | |
| CN112961859B (en) | Aptamer for specifically recognizing amantadine and application thereof | |
| CN113999890B (en) | VEGF (vascular endothelial growth factor) identification method based on aptamer probe and kit for detecting VEGF | |
| CN113340864B (en) | Aptamer sensor for secondary amplification of INS signal based on MEF effect and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |