CN116466090B - Double-function DNA tweezers nano biosensor and preparation method and application thereof - Google Patents
Double-function DNA tweezers nano biosensor and preparation method and application thereof Download PDFInfo
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Classifications
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- 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
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- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- 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
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
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- Biotechnology (AREA)
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- Nanotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a double-function DNA tweezers nano biosensor and a preparation method and application thereof. According to the double-function DNA tweezer nanometer machine, two ends of the double-function DNA tweezer nanometer machine are connected with specific split aptamer sequences, a specific ternary complex is formed by the split aptamer and a target, antibiotic residues in food matrixes are respectively identified at two ends of the tweezer structure, and false positive signals are effectively reduced. The method can realize the rapid detection of two antibiotics enrofloxacin and kanamycin, and has strong specificity and high sensitivity. Solves the single target recognition problem of the traditional single DNA tweezer biosensor.
Description
Technical Field
The invention relates to a double-function DNA tweezers nano biosensor and a preparation method and application thereof, belonging to the technical field of food detection.
Background
Antibiotics are commonly used for preventing and treating animal diseases and promoting the yield of livestock and poultry foods. However, they are often stored and accumulated in animal tissues, cells or organs, and remain in ecological environment, resulting in exceeding of drug residues. Kanamycin (kanamycin, KANA) is an aminoglycoside antibiotic suitable for treating various diseases such as gonorrhea, salmonella, phthisis and the like, and is originally isolated from soil by scientist Okami and Umezawa. Kanamycin affects both gram positive and gram negative bacteria by interfering with the protein synthesis process. Enrofloxacin (Enrofloxacin, ENR) is a fluoroquinolone drug, is generally considered to be targeted by DNA gyrase and topoisomerase IV, inhibits the replication and transcription process of prokaryotes, has strong antibacterial performance, and is used for treating various intracellular bacterial infections, septicemia and other diseases in the veterinary field. Excessive use of antibiotic drugs can cause accumulation in the environment and soil, enter food chains to cause nephrotoxicity and neuromuscular blocking effects, and the drug resistance of pathogenic microorganisms and environmental microorganisms is gradually improved, so that a series of side effects are brought to human health. Therefore, almost every country has made specific regulations for the maximum residual amount of veterinary drugs in animal-derived foods, wherein, with reference to the European veterinary drug residual limit standard, the maximum residual limit of kanamycin in milk is 150 μg/kg and the maximum residual limit of enrofloxacin is 100mg/kg. National standards in China prescribe a maximum residual amount of kanamycin of 200 μg/kg, enrofloxacin varying from 100 μg/kg to 300 μg/kg depending on the animal product (GB 31650-2019).
Currently, the detection methods of enrofloxacin and kanamycin mainly comprise high performance liquid chromatography, gas chromatography-mass spectrometry (LC-MS), a microbiological method, an immunological method and the like. The method can realize qualitative and quantitative detection of antibiotics, but usually requires expensive and large-scale experimental equipment and professional operators, or has short quality guarantee period of reagents and time and labor-consuming detection procedures. Detection by biosensors is also an emerging detection technology that has been popular in recent years, such as fluorescence, colorimetry, electrochemical methods, and aptamer sensor methods. Typically, these methods are combined or applied simultaneously, and finally sensitive, efficient or low-cost detection is achieved.
Nucleic acid aptamers (aptamers) are small molecule DNA or RNA fragments that bind specifically to a target substance, screened from a library of in vitro synthesized random oligonucleotides by the systematic evolution of exponentially enriched ligands (SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT, SELEX) technique. Because of the high biological affinity, the detection method by means of the aptamer sensor is one of the most sensitive and best-selectivity biosensor methods at present, and the aptamer serving as a novel nano probe is widely applied to the fields of biological sensing, food detection, drug delivery and the like. However, conventional small molecule aptamers typically have fewer binding epitopes or unstable specificity, resulting in false positive or non-specific signals. Stojanovic et al split the complete single-stranded aptamer into two or more fragments for the first time, forming a specific ternary complex with the target, effectively reducing false positive signals. When the target is not present, the two cleavage aptamers are independent and nonfunctional. Therefore, the unique advantage of the split aptamer has wider application value in the aspects of identification and detection of small molecules such as ATP, cocaine, 17 beta-estradiol and the like.
DNA tweezers belong to one of DNA nanomachines, are the simplest and most basic DNA mechanical device form, are usually assembled by three or four single-stranded DNA sequences through a base complementary pairing principle, can realize micro-or nano-scale mechanical movement, can capture, clamp and release small molecules, have the advantages of programmability, biocompatibility and high specificity, and can react under the external stimulus of nucleic acid, PH, metal ions, enzymes, proteins and the like, and are used for drug delivery, biosensing, food detection and the like. To date, there are few cleavage aptamers that combine the structure of double DNA tweezers for detection of antibiotic residues in food products.
Disclosure of Invention
In order to solve the technical problems, the invention provides a rapid and effective dual-function DNA tweezers nano biosensor which is used for simultaneously detecting enrofloxacin and kanamycin through the change of fluorescent signals. The two ends of the double-function DNA tweezer nanometer machine are connected with specific split aptamer sequences, a specific ternary complex is formed by the split aptamer and a target, antibiotic residues in food matrixes are respectively identified at the two ends of the tweezer structure, and false positive signals are effectively reduced. The method can realize the rapid detection of two antibiotics enrofloxacin and kanamycin, and has strong specificity and high sensitivity. Solves the single target recognition problem of the traditional single DNA tweezer biosensor.
A first object of the present invention is to provide a dual function DNA tweezer nanosensor for simultaneous detection of two targets, comprising a dual DNA tweezer nanostructure, a split aptamer chain of the two targets attached to the dual DNA tweezer nanostructure, and two sets of fluorescent and quenching groups; the double-DNA tweezers nanostructure consists of four chains of a long chain S1, a long chain S2, a short chain S3 and a short chain S4, wherein the 5 'end of the S1 and the 3' end of the S2 are respectively connected with two split aptamer chains of a first target, the 3 'end of the S1 and the 5' end of the S2 are respectively connected with two split aptamer chains of a second target, the two ends of the S3 are respectively marked with a fluorescent group and a quenching group, and the two ends of the S4 are respectively marked with another group of fluorescent groups and quenching groups; wherein, the middle segment of S1 and S2 has a sequence complementary to S3 and S4, and four chains form 4 support arms.
Further, the nucleotide sequence of the long-chain S1 is shown as SEQ ID NO.1 ：GCTAAGCCGAATCATC ACGGAACATTCTCGCTGGACTAATTGTTACCAATCACCGACCGCTGAATAATATAAATCA CCCATCAGGGGGCTAGGCTAACACGGTTCGGC.
Further, the nucleotide sequence of the long-chain S2 is shown as SEQ ID NO.2 ：TCTCTGAGCCCGGGTT ATTTCAGGGGGAACTATACATTAACCGCCTCCTGCCACTATGGTAACAATATCATGGACC TTACGAAGCCTACGACTATGGGGGTTGAG.
Further, the nucleotide sequence of the short-chain S3 is shown as SEQ ID NO. 3: TTATATTATTCAGCGGT CGGACTAGGCAGGAGGCGGTTAATGTA.
Further, the nucleotide sequence of the short-chain S4 is shown as SEQ ID NO. 4: CGTAGGCTTCGTAAGG TCCAATCACCAGCGAGAATGTTCCGTGA.
Further, the fluorescent and quenching groups are two groups of ROX and BHQ 2, FAM and BHQ 1, HEX and TAMRA, cy3 and BHQ 2, cy5 and BHQ 3.
Further, the first target is enrofloxacin, and the nucleotide sequences of two split aptamer chains of the enrofloxacin are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7.
Further, the second target is kanamycin, and the nucleotide sequences of two split aptamer chains of the kanamycin are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.
The second object of the present invention is to provide a method for preparing the dual-function DNA tweezer nano-biosensor, comprising the steps of: 1-2 of long chain S1 and long chain S2 of split aptamer chains connected with two targets and short chain S3 and short chain S4 marked with fluorescent groups and quenching groups: 1-2: 2-3: 2-3, heating for 4-6 min at 85-95 ℃, and slowly cooling to 20-30 ℃ to obtain the double-function DNA tweezer nano-biosensor.
A third object of the present invention is to provide the use of the dual function DNA tweezer nanosensor in simultaneous detection of two antibiotic targets.
Further, the application is that the double-function DNA tweezer nano-biosensor is mixed with a solution prepared by a target to be detected to obtain a mixed solution, and the mixed solution is incubated for 1-1.5h at 35-40 ℃; and detecting two fluorescence intensities of the incubated solution by adopting a fluorescence spectrophotometer, and analyzing the detection result according to a standard curve.
Further, the mixed solution comprises 100-300 nM of the double-function DNA tweezer nano-biosensor, 10-30 mM MgCl 2, 400-600 mM NaCl and 0.04-0.06% by volume of Tween-20.
Further, the standard curve is the quantitative relation between the fluorescence intensity signal and the target content measured according to the method according to the standard solution of the target to be measured.
In the present invention, for the detection of enrofloxacin and kanamycin, the fluorophore and quencher are selected from the group consisting of ROX and BHQ 2, FAM and BHQ 1, respectively.
Wherein, the parameter of the FAM adopting the F-7000 fluorescence spectrophotometer is that the excitation wavelength is 465nm, and the emission wavelength is 480-560 nm. The ROX adopts F-7000 fluorescence spectrophotometer with excitation wavelength of 550nm and emission wavelength of 580-660 nm.
The quantitative relationship between the fluorescence intensity signal and the enrofloxacin concentration is as follows: f= 2091.32-90.09C ENR;
The quantitative relationship between fluorescence intensity signal and kanamycin concentration is: f= 3280.27-27.21C KANA;
Wherein F is fluorescence intensity; the concentration of C ENR is enrofloxacin; the concentration of C KANA was kanamycin.
The mechanism of the invention:
The invention combines the split aptamer and the DNA tweezers for the first time, and constructs a simple and novel DNA tweezer biosensor capable of realizing multifunctional identification and double detection of two antibiotics. The DNA tweezers are assembled by 4 chains of strand1 (S1), strand 2 (S2), strand 3 (S3) and strand 4 (S4), and split aptamer chains with enrofloxacin and kanamycin respectively connected to the 5 'and 3' ends of the two long chains of S1 and S2 are used as two free arms of the tweezers for identifying and combining targets. The two long chains are respectively provided with 18 bases for complementary pairing with the short chains S3 and S4 to form 4 support arm regions, wherein a fluorescent group ROX is marked at the 5 'end of the S3, a quenching group BHQ2 is marked at the 3' end, a fluorescent group FAM is marked at the 5 'end of the S4, and a quenching group BHQ1 is marked at the 3' end. When enrofloxacin and kanamycin exist in the sensing system, the split aptamer and the target form a ternary complex of a sandwich structure, the free two arms are pulled close, and the tweezer structures at the two ends are closed, so that fluorescence is reduced. The concentrations of enrofloxacin and kanamycin can be quantitatively detected by two fluorescence intensity changes.
The beneficial effects of the invention are as follows:
1. The split aptamer which has the unique advantages of high affinity and specificity recognition, capability of forming a ternary complex with a specific target, good stability, low cost, long shelf life, easiness in modification and the like is selected as the recognition element.
2. The invention provides a double-function DNA tweezers nano biosensor, and split aptamer which is skillfully connected with a specific target at two ends of tweezers is used as a recognition element, so that the stability of the aptamer structure is improved, a ternary complex can be formed only under the condition that the target exists, and the specificity of the recognition element is greatly improved.
3. The method of the invention can not depend on the traditional large-scale instrument method, and avoids expensive test equipment and professional operators. The method can realize high-sensitivity detection of antibiotics enrofloxacin and kanamycin, and the detection limits of enrofloxacin and kanamycin are respectively 0.18ng/mL and 0.36ng/mL.
Description of the drawings:
FIG. 1 is a schematic diagram of a dual function DNA tweezer nanosensor of the present invention for detecting enrofloxacin and kanamycin;
FIG. 2 is a representation of polyacrylamide electrophoresis of a dual-function DNA tweezer;
FIG. 3 is a feasibility verification of a dual function DNA tweezer;
FIG. 4 is a graph of fluorescence spectra of dual function DNA tweezers at different concentrations of enrofloxacin and kanamycin;
FIG. 5 is a graph showing the fit and linear correlation of enrofloxacin and kanamycin at various concentrations for a dual function DNA tweezer;
FIG. 6 is an optimized graph of fluorescence signal changes before and after incubation of bifunctional DNA tweezers with targets at different concentrations;
FIG. 7 is an optimized view of incubation time of bifunctional DNA tweezers with a target;
FIG. 8 is an optimized view of incubation temperatures of bifunctional DNA tweezers with a target;
FIG. 9 is the specificity of the bifunctional DNA tweezers for a target.
Detailed Description
The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.
In the following examples, primers were synthesized by Shanghai Biotechnology services Co., ltd:
The nucleotide sequence of Strand 1 (S1) is (as shown in SEQ ID No. 1):
5'-GCTAAGCCGAATCATCACGGAACATTCTCGCTGGACTAATTGTTACCAATCACCGACCGCTGAATAATATAAATCACCCATCAGGGGGCTAGGCTAACACGGTTCGGC-3'
The nucleotide sequence of Strand 2 (S2) is (as shown in SEQ ID No. 2):
5'-TCTCTGAGCCCGGGTTATTTCAGGGGGAACTATACATTAACCGCCTCCTGCCACTATGGTAACAATATCATGGACCTTACGAAGCCTACGACTATGGGGGTTGAG-3'
the nucleotide sequence of Strand 3 (S3) is (as shown in SEQ ID No. 3):
5’-ROX-TTATATTATTCAGCGGTCGGACTAGGCAGGAGGCGGTTAATGTA-BHQ2-3’
the nucleotide sequence of Strand 4 (S4) is (as shown in SEQ ID No. 4):
5’-FAM-CGTAGGCTTCGTAAGGTCCAATCACCAGCGAGAATGTTCCGTGA-BHQ1-3’
the nucleotide sequence of Strand 5 (S5) is (as shown in SEQ ID No. 5):
5'-GACTAGCCGAATCATCACGGAACATTCTCGCTGGACTAATTGTTACCAATCACCGACCGCTGAATAATATAAATCACCCATCAGGGGGCTAGGCTAACACGGTACTAC-3'
the nucleotide sequence of the cleavage aptamer 1 of enrofloxacin is (as shown in SEQ ID No. 6):
5’-CCCATCAGGGGGCTAGGCTAACACGGTTCGGC-3’
The nucleotide sequence of the cleavage aptamer 2 of enrofloxacin is (as shown in SEQ ID No. 7):
5’-TCTCTGAGCCCGGGTTATTTCAGGGGGA-3’
The nucleotide sequence of the kanamycin cleavage aptamer 1 is (as shown in SEQ ID No. 8):
5’-TGGGGGTTGAG-3’
The nucleotide sequence of the kanamycin cleavage aptamer 2 is (as shown in SEQ ID No. 9):
5’-GCTAAGCCGA-3’
Enrofloxacin (enrofloxacin, ENR), kanamycin (kanamycin, KANA), danofloxacin (danofloxacin, DAN), ciprofloxacin (ciprofloxacin, CIP), streptomycin (streptomycin, STR), and standard stock solutions of dihydrostreptomycin (dihydrostreptomycin, DI-STR) (100 μg/mL) used in the present invention were purchased from the company of the tendril allta science, china.
Example 1: preparation of double-function DNA tweezers nano biosensor
The bifunctional DNA tweezers were composed of sequences 1-4 at 2:2:3:3 in a PBS (pH=7.4) solution, and vortexing for 1min to give a final concentration of 200nM. The mixed solution was heated at 90 ℃ for 5min and then slowly cooled to room temperature to form a tweezer-like structure.
The synthetic properties of the DNA tweezers were verified by polyacrylamide Gel electrophoresis (PAGE), the concentration of added DNA strand in each lane was 1.5. Mu.M, and the results after staining with Gel red are shown in FIG. 2, which were performed in 1 XTBE buffer at room temperature for 35 min. Since the conformation of the DNA tweezers has a significant effect on the migration efficiency of electrophoresis, bands of dsDNA strands used to construct the DNA tweezers are generated in lanes 1-4. The new bands generated in lanes 5 and 6 demonstrate that when 2 long chains (S1 and S2) and 1 short chain (S3 or S4) are present in the system, the tweezer structure can still self-assemble. In lane 7, which contains four strands, a new band region was also observed and the movement speed was significantly slower, indicating that the DNA tweezer structure had formed, but that a portion of the free single strand was not involved in the formation of the tweezer structure. Lanes 5,6 and 7 show no significant differences in migration compared to lanes 1-4. This is probably due to the relatively few bases (40 bp) of S3 and S4, which have little effect on the structural conformation of the DNA tweezer.
Example 2: double-function DNA tweezers nano biosensor for detecting enrofloxacin, kanamycin and feasibility characterization
The test procedure for a typical dual function DNA tweezer nanosensor is as follows: as shown in FIG. 5, enrofloxacin and kanamycin standard solutions of different concentrations were prepared, 10. Mu.L of the mixed solution containing enrofloxacin and kanamycin was added to 380. Mu.L of the mixed solution containing 200nM DNA tweezers, 20mM MgCl 2, 500mM NaCl, 0.05% (v/v) Tween-20 for incubation, and incubated at about 37℃for 90min.
In order to further verify the feasibility of the proposed nanosensor. The progress of the operation of the nano-biosensor under different conditions was studied using the change of the fluorescence signal, as shown in fig. 3. The blank sample (sample 1) showed higher fluorescence intensity, with no presence of both antibiotics. In sample 2, which has only ENR, the ROX fluorescence signal is significantly reduced, since ENR binds to its cleavage aptamer only closing one end of the DNA tweezer. Similarly, in sample 3 KANA closes the other end of the DNA tweezer with its cleaving aptamer 2, resulting in a decrease in the fluorescence signal of FAM. When the sequence of one of the cleavage aptamers of both targets was replaced (strand 5) (sample 4), the decrease in fluorescence was not significant, similar to sample 1, indicating that DNA tweezers were not able to specifically recognize both targets at this time. Sample 5, with both targets ENR and KANA, showed high fluorescence signals in both the ROX and FAM channels, indicating that both ends of the double DNA tweezer were "closed" simultaneously. When the incubation time was 45min, the fluorescence signal of ROX and FAM was partially reduced, but there was still some fluorescence signal, indicating that part of the target and cleavage aptamer had formed a sandwich structure as shown in sample 6, and not all DNA tweezers were "closed". These changes in fluorescence signal indirectly indicate that experimental principles are feasible.
Example 3: performance characterization of dual-function DNA tweezers nanosensor for detecting enrofloxacin and kanamycin
The dual-function DNA tweezers nano-biosensor was used to detect targets at different concentrations (enrofloxacin concentrations of 0ng/mL,0.05ng/mL,0.5ng/mL,1ng/mL,10ng/mL,100ng/mL,1000ng/mL; kanamycin concentrations of 0ng/mL,0.05ng/mL,0.5ng/mL,1ng/mL,5ng/mL,50ng/mL,100ng/mL,1000ng/mL, respectively).
380. Mu.L of enrofloxacin and kanamycin with different concentrations are added into 380. Mu.L of forceps containing double functions DNA, and the mixed solution contains 200nM of forceps containing DNA, 20mM of MgCl 2, 500mM of NaCl and 0.05% (v/v) of Tween-20 and is incubated for 90min at about 37 ℃. FAM fluorescence spectrum excited by a Hitachi F-7000 fluorescence spectrophotometer at 465nm in 480-560 nm scanning range and ROX fluorescence spectrum excited at 550nm in 580-660 nm scanning range.
The results are shown in FIG. 4: the concentrations of enrofloxacin and kanamycin increased from 0ng/mL to 1000ng/mL, with a gradual decrease in fluorescence intensity. The linear equation for enrofloxacin, f= 2091.32-90.09C ENR(R2 =0.996, in the range of 0.2-10ng/mL, and for kanamycin, f= 3280.27-27.21C KANA(R2 =0.993), in the range of 0.5-50 ng/mL, where F is fluorescence signal intensity and C is target concentration. The detection limits for enrofloxacin and kanamycin were 0.18ng/mL and 0.36ng/mL, respectively, according to the 3-fold signal-to-noise principle (lod=3δ/κ, where δ is the standard deviation of the blank parallel assay and κ is the slope of the calibration curve). The relative standard deviation of 5ng/mL enrofloxacin was 3.8% for 11 replicates and 2.5% for 11 replicates of 5ng/mL kanamycin.
Example 4: specificity characterization of double-function DNA nano-biosensor for detecting enrofloxacin and kanamycin
Some fluoroquinolones and aminoglycosides with similar structures are selected to evaluate the anti-interference capability and superiority of the dual-function DNA tweezers. When only enrofloxacin or kanamycin exists in the system, the fluorescence is reduced, and the fluorescence change of other veterinary medicines is not obvious. In mixed sample 1 containing enrofloxacin and kanamycin, fluorescence decreased. In contrast, in mixed sample 2 without enrofloxacin and kanamycin, the fluorescence change was negligible, as shown in figure 9. The results show that the DNA tweezers have high specificity for specific drugs.
Example 5: actual sample marking recovery
In order to evaluate the practical application performance of the bifunctional DNA nano-biosensor in a complex environment, the method provided by the invention is adopted to analyze labeled practical samples (milk and chicken) containing enrofloxacin and kanamycin with different concentrations.
Pretreatment of milk samples: first 2g of milk was adjusted to ph=4.4 with 20% acetic acid solution, shaken 30min, centrifuged at 5000r/min for 20min to remove fat, and then filtered through a 0.22 μm filter to blank.
Pretreatment of solid samples: cutting chicken samples, and homogenizing thoroughly. 1% trichloroacetic acid was used: after ultrasonic extraction of acetonitrile (9:1, V/V) for 10min, centrifugation is carried out at 5000r/min for 10min to remove interfering substances, and supernatant is taken. The pH of the supernatant was adjusted to neutral using 2M NaOH solution, filtered through a 0.22 μm filter and subjected to a blank.
The results are shown in Table 1, with enrofloxacin recovery ranging from 95.4% to 102.7%, relative Standard Deviation (RSD) ranging from 3.7% to 5.5%, kanamycin recovery ranging from 92.6% to 100.6%, and Relative Standard Deviation (RSD) ranging from 1.4% to 6.1%. The result shows that the double-function DNA nano biosensor constructed by the invention can effectively resist the interference of complex matrixes in actual samples, and has good actual analysis potential.
TABLE 1 labelling recovery Performance of bifunctional DNA nanosensors in real samples
ND:not detected
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (5)
1. A dual-function DNA tweezer nanosensor for simultaneously detecting two targets, characterized by comprising a dual-DNA tweezer nanostructure, a split aptamer chain of the two targets connected with the dual-DNA tweezer nanostructure, and two groups of fluorescent groups and quenching groups; the double-DNA tweezers nanostructure consists of four chains of a long chain S1, a long chain S2, a short chain S3 and a short chain S4, wherein the 5 'end of the S1 and the 3' end of the S2 are respectively connected with two split aptamer chains of a first target, the 3 'end of the S1 and the 5' end of the S2 are respectively connected with two split aptamer chains of a second target, the two ends of the S3 are respectively marked with a fluorescent group and a quenching group, and the two ends of the S4 are respectively marked with another group of fluorescent groups and quenching groups; wherein, the middle sections of S1 and S2 are respectively provided with a section of sequence which is complementarily paired with S3 and S4, and four chains form 4 support arms;
The nucleotide sequence of the long-chain S1 is shown as SEQ ID NO.1 ：GCTAAGCCGAATCATCACGGAACATTCTCGCTGGACTAATTGTTACCAATCACCGACCGCTGAATAATATAAATCACCCATCAGGGGGCTAGGCTAACACGGTTCGGC;
The nucleotide sequence of the long-chain S2 is shown as SEQ ID NO.2 ：TCTCTGAGCCCGGGTTATTTCAGGGGGAACTATACATTAACCGCCTCCTGCCACTATGGTAACAATATCATGGACCTTACGAAGCCTACGACTATGGGGGTTGAG;
The nucleotide sequence of the short chain S3 is shown in SEQ ID NO. 3: TTATATTATTCAGCGGTCGGACTAGGCAGGAGGCGGTTAATGTA;
The nucleotide sequence of the short chain S4 is shown in SEQ ID NO. 4: CGTAGGCTTCGTAAGGTCCAATCACCAGCGAGAATGTTCCGTGA;
The 5 'end of the short chain S3 is marked with a fluorescent group ROX, and the 3' end is marked with a quenching group BHQ2;
the 5 'end of the short chain S4 is marked with a fluorescent group FAM, and the 3' end is marked with a quenching group BHQ1;
The first target is enrofloxacin, and the nucleotide sequences of two split aptamer chains of the enrofloxacin are respectively shown as SEQ ID NO.6 and SEQ ID NO. 7;
The second target is kanamycin, and the nucleotide sequences of two split aptamer chains of the kanamycin are respectively shown as SEQ ID NO.8 and SEQ ID NO. 9.
2. The method for preparing the dual-function DNA tweezer nano-biosensor according to claim 1, which is characterized by comprising the following steps: the method comprises the steps of (1) mixing a long chain S1 and a long chain S2 of a split aptamer chain connected with two targets with a short chain S3 and a short chain S4 marked with a fluorescent group and a quenching group, wherein the ratio of the long chain S1 to the long chain S2 to the short chain S3 and the short chain S4 is 1-2: 1-2: 2-3: and (3) mixing and dissolving the mixture in a molar ratio of 2-3 in a buffer solution, heating the mixture at 85-95 ℃ for 4-6 min, and slowly cooling the mixture to 20-30 ℃ to obtain the double-function DNA tweezer nano biosensor.
3. Use of the dual function DNA tweezer nanosensor of claim 1 for simultaneous detection of two antibiotic targets.
4. The application according to claim 3, wherein the application is that the dual-function DNA tweezer nano-biosensor is mixed with a solution prepared by a target to be detected to obtain a mixed solution, and the mixed solution is incubated for 1-1.5h at 35-40 ℃; and detecting two fluorescence intensities of the incubated solution by adopting a fluorescence spectrophotometer, and analyzing the detection result according to a standard curve.
5. The use according to claim 4, wherein the mixed solution comprises 100-300 nM of the bifunctional DNA tweezer nano-biosensor, 10-30 mM MgCl 2, 400-600 mM NaCl and 0.04-0.06% by volume of Tween-20.
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| DNA纳米机器;杨洋;柳华杰;刘冬生;;化学进展;20080324(第Z1期);第197-207页 * |
| Programmed dual-functional DNA tweezer for simultaneous and recognizable fluorescence detection of microRNA and protein;Yang W T等;《Analytical Chemistry》;20190528;第91卷(第12期);第7743-7749页 * |
| Simultaneous detection of aflatoxin B1 and ochratoxin A in food samples by dual DNA tweezers nanomachine;Xiong Z W等;《Food Chemistry》;20200929;第338卷;第128122页 * |
| 基于劈裂适配体识别的DNA生物传感器检测恩诺沙星和卡那霉素的研究;尚滋萱;《中国优秀硕士学位论文全文数据库 (农业科学辑)》;20240415(第04期期);D050-615 * |
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