CN116286836B - Aptamer of enrofloxacin and ciprofloxacin and enzyme-linked aptamer sensor - Google Patents

Aptamer of enrofloxacin and ciprofloxacin and enzyme-linked aptamer sensor Download PDF

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CN116286836B
CN116286836B CN202310574156.XA CN202310574156A CN116286836B CN 116286836 B CN116286836 B CN 116286836B CN 202310574156 A CN202310574156 A CN 202310574156A CN 116286836 B CN116286836 B CN 116286836B
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王赛
王文静
毛相朝
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Abstract

The invention discloses a nucleic acid aptamer and an enzyme-linked aptamer sensor of enrofloxacin and ciprofloxacin, and belongs to the technical field of residual drug detection. The nucleic acid aptamer consists of SEQ3-1 and SEQ3-2, and the nucleotide sequences are shown as SEQ ID NO.2 and SEQ ID NO. 3. The application of the aptamer in preparing a reagent, a kit or a sensor for simultaneously detecting enrofloxacin and ciprofloxacin. An enzyme-linked aptamer sensor comprising the following components: split aptamer SP1, split aptamer SP2, aptamer complementary strand HD1, aptamer complementary strand HD2, and the like. The aptamer disclosed by the invention is a split type aptamer obtained by taking the SEQ3 aptamer as an initial chain and reserving a key binding domain during splitting, and has higher affinity to enrofloxacin and ciprofloxacin. The enzyme-linked aptamer sensor can realize the specific identification of ENR and CIP, and has high sensitivity.

Description

Aptamer of enrofloxacin and ciprofloxacin and enzyme-linked aptamer sensor
Technical Field
The invention relates to a nucleic acid aptamer of enrofloxacin and ciprofloxacin and an enzyme-linked aptamer sensor, belonging to the technical field of residual drug detection.
Background
With the increasing development of intensive cultivation, the high-density cultivation mode enables the use amount of antibacterial agents such as enrofloxacin to be increased dramatically, and the residual antibacterial agents are accumulated in water environment and aquatic products continuously. Enrofloxacin (ENR) is a synthetic quinolone antibacterial drug and is widely used in the prevention and treatment of bacterial infections in aquaculture animals. Ciprofloxacin (CIP) is the major active metabolite after ENR demethylation. Due to the lack of an acquired immune system, ENR and CIP residues are easily accumulated in tissues of aquatic products such as shrimps and crabs, enter food chains, affect human health and ecological environment, and cause potential carcinogenesis and drug resistance of pathogenic bacteria. Therefore, it is important to detect the residue of ENR and CIP, and China and European Union prescribe that the maximum residue limit of the sum of muscle and fat parts ENR and CIP in aquatic products such as shrimps is 100 mug/kg.
The existing detection methods of ENR and CIP mainly comprise ultra-high performance liquid chromatography/high resolution mass spectrometry, fluorescence spectrometry, surface enhanced Raman scattering, lateral flow immunochromatography, capillary electrophoresis and the like, but have the defects in sensitivity, specificity and the like, and how to rapidly, sensitively, efficiently and conveniently detect enrofloxacin and ciprofloxacin simultaneously still faces significant challenges.
The aptamer is a single-stranded oligonucleotide obtained by an exponential enrichment ligand evolution technology, can be specifically identified with a target, and has the advantages of easiness in synthesis, low cost, small batch-to-batch difference and the like, so that the aptamer has great application potential in food analysis and detection. However, insufficient affinity limits the practical use of the aptamer. Various post-SELEX strategies have been developed to increase the affinity of the aptamer, such as sequence optimization, end-immobilization, chemical modification, etc. There is no report on high affinity split-type aptamers to both enrofloxacin and ciprofloxacin.
Aptamers are often combined with various transduction techniques to enable detection of food targets. In recent years, metal-mimic nanoenzymes have received attention because of their ease of synthesis and their ability to exhibit a mimic enzymatic activity to catalyze substance transduction of strong colorimetric signals. Among them, the inherent peroxidase activity of the gold nanoparticles has made them of great interest. Colorimetric aptamer sensor based on AuNPs nano-enzyme (AuNPs) has the advantages of simplicity in operation, low cost, visualization and the like. Unlike proteases, the catalytic reaction of nano-enzymes occurs at the surface, so its catalytic activity can be regulated by surface modification. Single-stranded DNA (ssDNA) can interact with the nanoenzyme to enhance its catalytic activity, as adsorption of ssDNA helps to increase the affinity of the substrate to the nanoenzyme, thereby enhancing the enzymatic activity.
Disclosure of Invention
In view of the above prior art, the present invention provides a nucleic acid aptamer of enrofloxacin and ciprofloxacin, which has high affinity for both enrofloxacin and ciprofloxacin. The invention also provides an enzyme-linked aptamer sensor which is used for simultaneously detecting enrofloxacin and ciprofloxacin.
The invention is realized by the following technical scheme:
the aptamer of enrofloxacin and ciprofloxacin consists of SEQ3-1 and SEQ3-2, and the nucleotide sequences of SEQ3-1 and SEQ3-2 are shown as follows:
SEQ3-1:5'-CAGGGGGACCCAT-3', as shown in SEQ ID NO. 2; a total of 13 bases;
SEQ3-2:5'-GGGGGCTAGGCTAAC-3', as shown in SEQ ID NO. 3; a total of 15 bases.
The application of the aptamer of enrofloxacin and ciprofloxacin in detecting, separating and/or enriching the enrofloxacin and the ciprofloxacin. Further, the detection refers to detecting and/or monitoring enrofloxacin and ciprofloxacin in the aquatic product.
The application of the aptamer of enrofloxacin and ciprofloxacin in preparing a reagent, a kit or a sensor for simultaneously detecting enrofloxacin and ciprofloxacin.
An enzyme-linked aptamer sensor comprising the following components: split-type aptamer SP1, split-type aptamer SP2, aptamer complementary strand HD1, aptamer complementary strand HD2, chitopentaose (COS), nanoenzyme, TMB color-developing solution; the nucleotide sequences of the split aptamer SP1, the split aptamer SP2, the aptamer complementary strand HD1 and the aptamer complementary strand HD2 are as follows:
split aptamer SP1:5'-AACCAGGGGGACCCAT-3', as shown in SEQ ID NO. 7;
split aptamer SP2:5'-GGGGGCTAGGCTAACC-3', as shown in SEQ ID NO. 8;
aptamer complementary strand HD1:5'-GGTTAGCTGGTT-3', as shown in SEQ ID NO. 9;
aptamer complementary strand HD2:5'-ATGGGTGCCCCC-3' as shown in SEQ ID NO. 10.
Further, the nano-enzyme solution is prepared by the following method: adding 1 mL concentration of 1% (mass volume ratio, unit g/mL) tetrachloroauric acid solution into 95 mL ultrapure water, heating to boil, rapidly adding 4 mL concentration of 1% trisodium citrate solution, continuously heating until the solution becomes stable wine red, centrifuging and concentrating to 10 nM for later use.
Further, the enzyme-linked aptamer sensor consists of the following components: 4. Mu.L of split aptamer SP1 solution at a concentration of 25 nM; 4 μl of split aptamer SP2 solution at a concentration of 25 nM; 4. Mu.L of the ligand complementary strand HD1 solution having a concentration of 50 nM; 4. Mu.L of an aptamer complementary strand HD2 solution having a concentration of 50 nM; a chitosan solution with a concentration of 0.4. Mu.g/mL, 10. Mu.L; auNPs solution with concentration of 10 nM, 80. Mu.L; TMB color development, 50. Mu.L.
The enzyme-linked aptamer sensor is applied to simultaneous detection of enrofloxacin and ciprofloxacin.
A method for simultaneously detecting enrofloxacin and ciprofloxacin, comprising the steps of:
(1) Respectively taking a standard solution of ENR and a standard solution of CIP, adding a split type aptamer SP1, a split type aptamer SP2, an aptamer complementary strand HD1 and an aptamer complementary strand HD2, and reacting at room temperature; then adding chito-pentasaccharide and incubating; adding nano enzyme and incubating; adding TMB color developing solution into the reaction mixture, measuring the absorbance value at 650 and nm by using an enzyme-labeling instrument, calculating the signal change rate, and drawing a standard curve of ENR and a standard curve of CIP;
(2) Taking a sample to be detected, adding a split type aptamer SP1, a split type aptamer SP2, an aptamer complementary strand HD1 and an aptamer complementary strand HD2, and reacting at room temperature; then adding chito-pentasaccharide and incubating; adding nano enzyme and incubating; adding TMB color developing solution into the reaction mixture, measuring the absorbance value at 650 and nm by using an enzyme-labeling instrument, and calculating the signal change rate;
(3) Substituting the signal change rate of the sample to be detected into a standard curve, and calculating to obtain the concentration of ENR and the concentration of CIP in the sample to be detected.
Further, the specific operation of the step (1) is as follows:
taking 8 mu L of ENR standard solution, adding 4 mu L of split aptamer SP1 solution and 4 mu L of splitThe solution of the aptamer SP2, the solution of the aptamer complementary strand HD1 in an amount of 4. Mu.L and the solution of the aptamer complementary strand HD2 in an amount of 4. Mu.L are reacted at room temperature for 60 min; then 10 mu L of chitosan solution is added and incubated for 10 min; then adding 80 mu L of AuNPs solution, and incubating for 5 min; finally, 50 mu L of reaction mixture is taken, 50 mu L of TMB color developing solution is added, the absorbance value at 650 and nm is measured by using an enzyme-labeling instrument, and the signal change rate A is calculated 0 -A i /A 0 The method comprises the steps of carrying out a first treatment on the surface of the Detecting ENR standard solutions with different concentration gradients, and drawing a standard curve of the signal change rate along with the variation of the ENR concentration; the concentration gradients of the ENR standard solutions are respectively as follows: 0. 1.4 nM, 3.5 nM, 7 nM, 14 nM, 28 nM, 70 nM, 210 nM, 350 nM, 700 nM, 1400 nM;
taking 8 mu L of CIP standard solution, adding 4 mu L of split-type aptamer SP1 solution, 4 mu L of split-type aptamer SP2 solution, 4 mu L of aptamer complementary strand HD1 solution and 4 mu L of aptamer complementary strand HD2 solution, and reacting for 60 min at room temperature; then 10 mu L of chitosan solution is added and incubated for 10 min; then adding 80 mu L of AuNPs solution, and incubating for 5 min; finally, 50 mu L of reaction mixture is taken, 50 mu L of TMB color developing solution is added, the absorbance value at 650 and nm is measured by using an enzyme-labeling instrument, and the signal change rate A is calculated 0 -A i /A 0 The method comprises the steps of carrying out a first treatment on the surface of the Detecting CIP standard liquids with different concentration gradients, and drawing a standard curve of the signal change rate along with the CIP concentration change; the concentration gradients of the CIP standard solution are respectively as follows: 0. 1.4 nM, 3.5 nM, 7 nM, 14 nM, 28 nM, 70 nM, 210 nM, 350 nM, 700 nM, 1400 nM.
Further, the specific operation of the step (2) is as follows: taking 8 mu L of a sample to be detected, adding 4 mu L of a split type aptamer SP1 solution, 4 mu L of a split type aptamer SP2 solution, 4 mu L of an aptamer complementary strand HD1 solution and 4 mu L of an aptamer complementary strand HD2 solution, and reacting for 60 min at room temperature; then 10 mu L of chitosan solution is added and incubated for 10 min; then adding 80 mu L of AuNPs solution, and incubating for 5 min; finally, 50 mu L of reaction mixture is taken, 50 mu L of TMB color developing solution is added, the absorbance value at 650 and nm is measured by using an enzyme-labeling instrument, and the signal change rate A is calculated 0 -A i /A 0 。
The invention discloses a nucleic acid aptamer of enrofloxacin and ciprofloxacin, which is a pair of split type aptamers (SEQ 3-1 and SEQ 3-2), and is obtained by taking a reported original aptamer SEQ3 of ENR and CIP with certain affinity in the prior art as an initial chain, and optimizing and reforming the aptamer by a novel reforming method (analyzing the combination of the aptamer and a target, finding out a core binding domain and reserving a key binding domain as two parts of the split type aptamer). The modified aptamer SEQ3-1 and SEQ3-2 have higher affinity to the target, and compared with the original aptamer SEQ3, the modified aptamer SEQ3-1 and SEQ3-2 respectively improve 29 times and 55 times, and have remarkable effect. Meanwhile, the invention introduces different numbers of redundant bases on the basis of SEQ3-1 to obtain SEQ3-1a, SEQ3-1b and SEQ3-1c, but compared with SEQ3-1, the affinity is obviously reduced, and the existence of the redundant bases can interfere the binding of an aptamer and a target, so that the affinity is reduced.
The split aptamer has the following advantages in the aspect of constructing a sandwich type biosensor: 1. the non-specific signal is avoided, and the split type aptamer lacks continuous base pairs, so that the split type aptamer cannot spontaneously complement to form a stable heterodimer, and the non-specific signal can be effectively avoided. 2. The detection with high specificity is realized, and the split type aptamer can only recognize the target molecule when two parts are close to each other and form a complex, so that the detection with high specificity can be realized. 3. The split type aptamer is convenient to construct, consists of two smaller fragments, and can be produced and modified in a large scale by chemical synthesis and other technologies. 4. High sensitivity and fast response, when the target molecule exists, two split type aptamer are promoted to attract each other and form a complete aptamer structure, and the structure can start the signal amplification layer to generate strong signals, so that high sensitivity and fast response are realized.
The enzyme-linked aptamer sensor comprises a four-way junction (4 WJ) consisting of a split aptamer SP1, a split aptamer SP2, an aptamer complementary strand HD1 and an aptamer complementary strand HD2 (the traditional 4WJ is either four pieces of complementary DNA or a single aptamer), and the enzyme-linked aptamer sensor based on 4 WJ/AuNPs/COS/nano enzyme is constructed through the high adsorption of the 4WJ to the chitooligosaccharide (the chitooligosaccharide can be pulled to a distance of the metal nano enzyme, so that the peroxidase activity is further enhanced), the specific identification of ENR and CIP can be realized, and the sensitivity is high. The enzyme-linked aptamer sensor and the method for simultaneously detecting enrofloxacin and ciprofloxacin have wide application prospects in actual food safety monitoring. The invention opens up a new way for simultaneously detecting enrofloxacin and ciprofloxacin.
The various terms and phrases used herein have the ordinary meaning known to those skilled in the art.
Drawings
Fig. 1: schematic of the secondary structure of the pro-aptamer SEQ 3.
Fig. 2: molecular docking simulation results of the original aptamer SEQ3 and ENR and CIP are schematically shown.
Fig. 3: the affinity detection results of the pro-aptamer SEQ3 and ENR are schematically shown.
Fig. 4: the affinity detection results of the pro-aptamer SEQ3 and CIP are schematically shown.
Fig. 5: schematic of the secondary structure of SEQ 3-1.
Fig. 6: the secondary structure of SEQ3-2 is schematically shown.
Fig. 7: the affinity detection results of the split-type aptamer SEQ3-1/SEQ3-2 and ENR are schematically shown.
Fig. 8: schematic of affinity detection results of split aptamer SEQ3-1/SEQ3-2 with CIP.
Fig. 9: schematic of the secondary structure of SEQ3-1 a.
Fig. 10: schematic of the secondary structure of SEQ3-1 b.
Fig. 11: schematic of the secondary structure of SEQ3-1 c.
Fig. 12: the affinity detection results of the split-type aptamer SEQ3-1a/SEQ3-2 and ENR are schematically shown.
Fig. 13: the affinity detection results of the split aptamer SEQ3-1b/SEQ3-2 and ENR are schematically shown.
Fig. 14: the affinity detection results of the split aptamer SEQ3-1c/SEQ3-2 and ENR are schematically shown.
Fig. 15: the spectral patterns of ENR standards, wherein 11 curves from top to bottom represent ENR standards with concentrations of 0, 1.4 nM, 3.5 nM, 7 nM, 14 nM, 28 nM, 70 nM, 210 nM, 350 nM, 700 nM, 1400 nM, respectively.
Fig. 16: the spectral diagram of the CIP standard, wherein 11 curves from top to bottom represent CIP standards with concentrations of 0, 1.4 nM, 3.5 nM, 7 nM, 14 nM, 28 nM, 70 nM, 210 nM, 350 nM, 700 nM, 1400 nM, respectively.
Fig. 17: signal response curve of ENR.
Fig. 18: signal response curve of CIP.
Fig. 19: standard curve of ENR.
Fig. 20: CIP standard curve.
Fig. 21: schematic of the rate of change of the signal for 6 antibacterial agents.
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents and materials used in the examples below are conventional instruments, reagents and materials known in the art and are commercially available. The experimental methods and detection methods in the following examples are conventional experimental methods and detection methods in the prior art unless otherwise specified.
The invention utilizes a biomembrane interference molecule interaction instrument to characterize and measure the affinity of the aptamer and the target. The theory of the biological film interference molecular interaction instrument is that the biological film interference technology plays an important role in researching the interaction between biological molecules. The specific measurement method comprises the following steps: fixing the aptamer on the surface of the sensor through the combination of biotin and streptavidin; buffer solution, biotin-labeled aptamer with different concentration and target with different concentration required by reaction are added into a 96-well plate, the instrument is set to have a program of sensor balance 120 s, aptamer fixation 180 s, sensor balance 120 s, target binding 180 s, target dissociation 180 s, temperature 25 ℃ and frequency 2 Hz. The resulting binding-dissociation curves were fitted to give affinity constant Kd values.
Example 1 optimization of the pro-aptamer SEQ3 for ENR and CIP
The nucleotide sequences of the proaptamers of ENR and CIP SEQ3 are shown below as SEQ ID NO. 1:
5'-GTTATTTCAGGGGGACCCATCAGGGGGCTAGGCTAAC-3', a total of 37 bases.
The secondary structure of the pro-aptamer SEQ3 was predicted using the online tool "the mfold web server" as shown in figure 1. The binding of the aptamer and the target is simulated by using molecular docking simulation software Autodock, and key sites for the interaction of the aptamer SEQ3 and the two targets are C17, C18, A19, G24, G25, G26 and G27 based on clustering results and the energy minimum principle, as shown in figure 2.
The experimental results of the biological membrane interference molecular interaction instrument are shown in fig. 3 and 4, and the results show that the dissociation constants (Kd) between the original aptamer SEQ3 and the ENR and CIP are 432.0 nM and 265.6 nM respectively, and the smaller the Kd value is, the higher the affinity is.
To obtain a more optimal aptamer, the aptamer sequence is split, leaving the critical binding domain as two parts of the split aptamer. According to the simulation of the secondary structure of the aptamer and the result of molecular docking, the binding sites of the original aptamer SEQ3 and ENR and CIP are mainly positioned at the stem-loop structure and the G-rich sequence. The stem-loop structure plays an important role in the binding of the aptamer and the target, and GGGGG is a conserved sequence. The aptamer with retained stem loop GGA is called SEQ3-1 (shown as SEQ ID NO. 2), the aptamer with retained stem loop TAG and G-rich sequence is called SEQ3-2 (shown as SEQ ID NO. 3), and the secondary structure is shown as FIG. 5 and FIG. 6. The experimental results of the biological membrane interference molecular interaction instrument are shown in fig. 7 and 8, and the results show that the Kd value of the split aptamer SEQ3-1/SEQ3-2 to ENR is 15.00 nM and the Kd value to CIP is 4.87 nM. Compared with the original aptamer SEQ3, the affinity of the split aptamer to ENR and CIP is improved by 29 times and 55 times respectively, and the effect is obvious.
Meanwhile, policy verification is carried out, SEQ3-2 is kept unchanged, redundant bases with different lengths are respectively introduced into SEQ3-1, and are respectively called SEQ3-1a, SEQ3-1b and SEQ3-1c (shown as SEQ ID NO.3, 4 and 5), and the secondary structure is shown as figure 9, figure 10 and figure 11. The experimental results of the biological film interference molecule interaction instrument are shown in fig. 12, 13 and 14, and the results show that the Kd values of the split type aptamer SEQ3-1a/SEQ3-2, the split type aptamer SEQ3-1b/SEQ3-2 and the split type aptamer SEQ3-1c/SEQ3-2 to ENR are 527.7 nM,850.0 nM and 529.0 nM respectively, which are not different from those of the original aptamer SEQ3, and the existence of redundant bases is inferred to interfere the recognition effect of the aptamer to the ENR and CIP.
Sequence information and affinity values of the original aptamer SEQ3 and the cleaved aptamer are shown in table 1.
Table 1 sequence information and affinity values for aptamers
Example 2 construction of an enzyme-linked aptamer sensor
In order to construct 4WJ, modifying AAC at the 5' end of SEQ3-1 to obtain a split type aptamer SP1, wherein the split type aptamer SP1 is shown as SEQ ID NO.7, and the total number of the split type aptamer SP1 is 16 bases; modification C of the 3' end of SEQ3-2 to obtain split aptamer SP2, which is shown as SEQ ID NO.8, with total 16 bases. The aptamer complementary strand HD1 is complementary to the split aptamer SP1, and is shown as SEQ ID NO.9, for a total of 12 bases; the aptamer complementary strand HD2 is complementary to the split aptamer SP2, as shown in SEQ ID NO.10, for a total of 12 bases. The nucleotide sequences of the split aptamer SP1, the split aptamer SP2, the aptamer complementary strand HD1, and the aptamer complementary strand HD2 are shown in table 2.
TABLE 2 nucleotide sequence of 4WJ
An enzyme-linked aptamer sensor, which consists of the following components (the dosage of each component is the minimum dosage required by single sample detection): 4. Mu.L of split aptamer SP1 solution at a concentration of 25 nM; 4 μl of split aptamer SP2 solution at a concentration of 25 nM; 4. Mu.L of the ligand complementary strand HD1 solution having a concentration of 50 nM; 4. Mu.L of an aptamer complementary strand HD2 solution having a concentration of 50 nM; a chitosan solution with a concentration of 0.4. Mu.g/mL, 10. Mu.L; auNPs solution with concentration of 10 nM, 80. Mu.L; TMB color development, 50. Mu.L.
The solvents of the split-type aptamer SP1 solution, the split-type aptamer SP2 solution, the aptamer complementary strand HD1 solution, the aptamer complementary strand HD2 solution and the chitosan solution are ultrapure water.
The AuNPs solution is prepared by a sodium citrate reduction method: adding 1 mL% tetrachloroauric acid solution into 95 mL ultrapure water, heating to boil, rapidly adding 4 mL% trisodium citrate solution, heating until the solution becomes stable wine red, centrifuging, and concentrating to 10 nM for use.
The chitopentaose was purchased from Qingdao Bozhishui biotechnology Co., ltd (Chitosan oligosaccharide package DP 2-6).
The TMB color development liquid is purchased from 1% TMB solution of Beijing Soy Bao technology Co., ltd., product number: t8120.
Example 3 simultaneous detection of ENR and CIP Using an enzyme-linked aptamer sensor
(1) Detection of standard solution
Taking 8 mu L of ENR standard solution, adding 4 mu L of split-type aptamer SP1 solution, 4 mu L of split-type aptamer SP2 solution, 4 mu L of aptamer complementary strand HD1 solution and 4 mu L of aptamer complementary strand HD2 solution, and reacting for 60 min at room temperature; then 10 mu L of chitosan solution is added and incubated for 10 min; then adding 80 mu L of AuNPs solution, and incubating for 5 min; finally, 50. Mu.L of the reaction mixture was taken, 50. Mu.L of TMB color developing solution was added, and absorbance in the range of 500 to 750 nm was measured by using an ELISA reader.
And (3) detecting the ENR standard solutions with different concentration gradients to obtain a spectrum diagram of the ENR standard product, as shown in figure 15. The concentration gradients of the ENR standard solutions are respectively as follows: 0. 1.4 nM, 3.5 nM, 7 nM, 14 nM, 28 nM, 70 nM, 210 nM, 350 nM, 700 nM, 1400 nM.
The same method was used to test CIP standard solutions with different concentration gradients, and a spectrum of CIP standard was obtained as shown in fig. 16. The concentration gradients of the CIP standard solution are respectively as follows: 0. 1.4 nM, 3.5 nM, 7 nM, 14 nM, 28 nM, 70 nM, 210 nM, 350 nM, 700 nM, 1400 nM.
The ENR standard solution is prepared by the following method: dissolving ENR standard with 1M NaOH solution, adding buffer solution, and preparing into desired concentration.
The CIP standard solution is prepared by the following method: the CIP standard is taken and dissolved by a proper amount of 1M NaOH solution, and buffer solution is added to prepare the required concentration.
The buffer consists of the following components: 20 mM Tris (hydroxymethyl) aminomethane), 100 mM NaCl,5 mM KCl,2 mM MgCl 2 ，1 mM CaCl 2 The balance being water, pH 7.6.
(2) Establishment of a Standard Curve
Calculating the signal change rate A according to the absorbance value of ENR standard solution with different concentration gradients at 650 nm 0 -A i /A 0 A signal response curve is obtained as shown in fig. 17.
Calculating the signal change rate A according to the absorbance value of CIP standard solution with different concentration gradients at 650 nm 0 -A i /A 0 A signal response curve is obtained as shown in fig. 18.
As can be seen from fig. 17 and 18, when the target concentration is 0, no 4WJ is formed, COS is combined with the aptamer, and a small excess of COS is adsorbed on AuNPs, so that the AuNPs is pulled up to generate a local domain limiting effect, and the enzyme activity is enhanced; with the increase of the concentration of the target, after the aptamer is combined with the target, the more 4WJ generated by the self-assembly of the split aptamer SP1 and the split aptamer SP2 are drawn, the more COS is adsorbed, so that no redundant COS is adsorbed on AuNPs, and the enzyme activity is limited. The signal limiting rate of AuNPs increases with increasing target concentration.
Drawing a standard curve of ENR and a standard curve of CIP, wherein the abscissa is the lg value of the concentration of the ENR and the CIP, and the ordinate is the signal change rate A 0 -A i /A 0 As shown in fig. 19 and 20. The linear range of the ELISA aptamer sensor is calculated to be 1.4 to the upper range1400 nM, detection limits of 321 pM (ENR) and 961 pM (CIP), respectively.
(3) Enzyme-linked aptamer sensor specificity evaluation
And selecting Chloramphenicol (CHL), furazolidone (FZD), ofloxacin (OFL), norfloxacin (NOR), enrofloxacin and ciprofloxacin with higher detection rate in aquatic products for carrying out a specificity experiment.
Respectively taking chloramphenicol, furazolidone, ofloxacin, norfloxacin, enrofloxacin and ciprofloxacin to prepare solutions with the concentration of 70 nM.
The mixture solution containing the 6 antibacterial drugs is prepared, and the concentrations of chloramphenicol, furazolidone, ofloxacin, norfloxacin, enrofloxacin and ciprofloxacin are all 70 nM.
When in preparation, firstly, 10 mM mother solution is prepared, and the solvents are all NaOH solutions with the concentration of 1M; and then diluted to 70 nM with the buffer of example 2.
The absorbance values of each solution at 650 nm were measured according to the detection method of step (1): taking 8 mu L of a liquid to be detected, adding 4 mu L of a split type aptamer SP1 solution, 4 mu L of a split type aptamer SP2 solution, 4 mu L of an aptamer complementary strand HD1 solution and 4 mu L of an aptamer complementary strand HD2 solution, and reacting for 60 min at room temperature; then 10 mu L of chitosan solution is added and incubated for 10 min; then adding 80 mu L of AuNPs solution, and incubating for 5 min; finally, 50. Mu.L of the reaction mixture was taken, 50. Mu.L of TMB color developing solution was added, and the absorbance at 650 and nm was measured by using a microplate reader.
Calculating the rate of change A of the signal 0 -A i /A 0 The results are shown in FIG. 21. As can be seen from FIG. 21, only when ENR or CIP is present, there is a clear signal, indicating that the specificity of the ELISA aptamer sensor of the invention is good.
Example 4 recovery evaluation of enzyme-linked aptamer sensor and Simultaneous detection of ENR and CIP in aquatic products
(1) ENR standard solution and CIP standard solution were mixed in different concentration ratios (0:1000 nM, 300 nM:700 nM, 500 nM:500 nM, 700 nM:300 nM, 1000 nM: 0) to obtain 5 kinds of mixed solutions, which were analyzed by using the ELISA aptamer sensor constructed in example 2 (method same as that of example 3), and recovery rate and Relative Standard Deviation (RSD) were calculated, and the results are shown in Table 3. As can be seen from Table 3, the ELISA aptamer sensor has a good recovery rate between [83.95%, 114.6% ], and RSD is lower than 6.373%, which indicates that the ELISA aptamer sensor of the invention has higher accuracy and repeatability.
TABLE 3 recovery evaluation information
(2) Checking Portunus trituberculatus, eriocheir sinensis, prawn, and lobster
Sample preparation: muscle tissue 4 g of each sample (blue crab, eriocheir sinensis, prawn and hawk claw shrimp) is respectively mixed with 20 mL acetonitrile solution containing 25 mM phosphoric acid, homogenized for 5 min, and centrifuged at 4500 rpm for 20 min; collecting supernatant, and centrifuging again at 4500 rpm for 20 min; taking supernatant, and filtering with a 0.22 μm filter membrane to obtain a sample 1, a sample 2, a sample 4 and a sample 6.
To sample 2, ENR mother liquor and CIP mother liquor were added so that the final concentration of ENR was 300 nm and the final concentration of CIP was 700 nM, to obtain sample 3.
To sample 4, ENR mother liquor and CIP mother liquor were added so that the final concentration of ENR was 500 nm and the final concentration of CIP was 500 nM, to obtain sample 5.
To sample 6, ENR mother liquor and CIP mother liquor were added so that the final concentration of ENR was 700 nm and the final concentration of CIP was 300 nM, to obtain sample 7.
The solvents of the ENR mother solution and the CIP mother solution are 1M NaOH solution.
Samples 1 to 7 were analyzed using the enzyme-linked aptamer sensor constructed in example 2 (the method was the same as in example 3), and the sum of ENR and CIP concentrations was calculated, and the results are shown in table 4. As can be seen from Table 4, samples 1, 2, 4 and 6 were all negative, and the labeled samples 3, 5 and 7 were all positive, and the quantitative analysis results were high in accuracy, less in deviation, and consistent with the HPLC test results.
Table 4 results of enzyme-linked sensor and HPLC (n=3)
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.

Claims (6)

1. The aptamer of enrofloxacin and ciprofloxacin is characterized in that: consists of SEQ3-1 and SEQ3-2, the nucleotide sequences of SEQ3-1 and SEQ3-2 are shown below:
SEQ3-1：5’-CAGGGGGACCCAT-3’；
SEQ3-2：5’-GGGGGCTAGGCTAAC-3’。
2. use of the aptamer of enrofloxacin and ciprofloxacin according to claim 1 in the preparation of a reagent, kit or sensor for simultaneous detection of enrofloxacin and ciprofloxacin.
3. An enzyme-linked aptamer sensor, which is characterized by comprising the following components: split aptamer SP1, split aptamer SP2, aptamer complementary strand HD1, aptamer complementary strand HD2, chitooligosaccharide, nanoenzyme, TMB color development liquid;
the nucleotide sequences of the split aptamer SP1, the split aptamer SP2, the aptamer complementary strand HD1, and the aptamer complementary strand HD2 are as follows:
cleavage aptamer SP1:5'-AACCAGGGGGACCCAT-3';
cleavage aptamer SP2:5'-GGGGGCTAGGCTAACC-3';
aptamer complementary strand HD1:5'-GGTTAGCTGGTT-3';
aptamer complementary strand HD2:5'-ATGGGTGCCCCC-3'.
4. The enzyme-linked aptamer sensor of claim 3, wherein the nanoenzyme solution is prepared by: adding 1 mL% tetrachloroauric acid solution into 95 mL ultrapure water, heating to boil, rapidly adding 4 mL% trisodium citrate solution, heating until the solution becomes stable wine red, and centrifuging to concentrate to 10 nM.
5. The enzyme-linked aptamer sensor of claim 3, wherein the enzyme-linked aptamer sensor consists of: 4. Mu.L of split aptamer SP1 solution at a concentration of 25 nM; 4 μl of split aptamer SP2 solution at a concentration of 25 nM; 4. Mu.L of the ligand complementary strand HD1 solution having a concentration of 50 nM; 4. Mu.L of an aptamer complementary strand HD2 solution having a concentration of 50 nM; a chitosan solution with a concentration of 0.4. Mu.g/mL, 10. Mu.L; auNPs solution with concentration of 10 nM, 80. Mu.L; TMB color development, 50. Mu.L.
6. Use of an enzyme-linked aptamer sensor according to claim 3 or 4 or 5 for simultaneous detection of enrofloxacin and ciprofloxacin.
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