Ratiometric fluorescence aptasensor based on structure switching for detection of enrofloxacin and its application

Abstract

The present invention relates to a ratio fluorescence aptamer sensor based on structure conversion for detecting enrofloxacin and its application. The method has the following specific steps: (1) preparing carbon dots by high temperature pyrolysis; (2) synthesizing carbon dot enrofloxacin aptamer conjugates; (3) mixing the carbon dot enrofloxacin aptamer conjugates synthesized in step (2) with two complementary chains (cDNA1 and cDNA2) of enrofloxacin aptamers modified by hemin monomers to construct a structure conversion aptamer sensor; (4) mixing a sample to be tested with the sensor in step (3); (5) mixing the mixture in step (4) with hydrogen peroxide and o-phenylenediamine, measuring the ratio fluorescence intensity thereof, and calculating the enrofloxacin content in the sample by comparing with a standard curve. The method has the advantages that the detection of enrofloxacin has the characteristics of simple operation, good selectivity, high sensitivity, fluorescence visualization and low cost, and can be used to detect the Chinese medicine residue of enrofloxacin in aquatic products.

Description

Technical Field

[0001] The invention relates to a ratiometric fluorescence aptamer sensor based on structure conversion for detecting enrofloxacin and application thereof. Background Art

[0002] Enrofloxacin (ENR), also known as ethylciprofloxacin, belongs to the fluoroquinolone (FQs) family and is a synthetic broad-spectrum antimicrobial agent. Enrofloxacin has been widely used in veterinary medicine for the prevention and treatment of bacterial diseases due to its low price and high efficacy. Although enrofloxacin is still regarded as an effective drug for the treatment of various diseases in aquaculture, its own limitations and overuse may leave residues in aquatic products. Enrofloxacin residues may accumulate in the human body through the food chain, causing various health risks such as irreversible nerve damage, allergic reactions, and liver damage. So far, a variety of methods have been developed, such as high-performance liquid chromatography, capillary electrophoresis, and enzyme-linked immunosorbent assay, to sensitively detect enrofloxacin. However, the above methods often require expensive instruments and complex pretreatment processes. Therefore, it is necessary to develop an analytical method for enrofloxacin that is simple to operate, highly sensitive, and accurate.

[0003] Aptamers (apt) are single-stranded DNA / RNA molecules that are selected by in vitro exponential enrichment technology. Aptamers have been widely used in various fields such as food safety, environmental monitoring and biomedicine due to their good stability, specificity and easy modification. Aptamers have high affinity for target molecules, but lack convenient and effective readable signals. Therefore, fluorescent, colorimetric and electrochemical aptamer sensors have attracted the attention of researchers. A variety of carbon dot-based aptamer sensors have been developed for pesticide and veterinary drug detection. However, the fluorescence response depends only on the conformational changes of specific aptamers, which limits the sensitivity and application of aptamer-based strategies. As a potential aptamer sensing method, the structural conversion strategy can trigger the change of fluorescence signal by utilizing the structural conversion of DNA chains.

[0004] Ratiometric fluorescence sensors are a special type of fluorescence sensors that show two independent emission peaks under a single excitation wavelength. The ratio of these two emission peaks is used to achieve quantitative detection of the target. Therefore, they have the advantages of internal correction, insensitivity to background and instrumentation, and elimination of the effects of excitation wavelength, emission wavelength, environment, and probe concentration. Compared with single-emission fluorescence sensors, ratiometric fluorescence sensors have higher sensitivity. Carbon dots (CDs) are considered to be ideal fluorescent nanomaterials due to their wide excitation band, narrow emission band, good water solubility, low photobleaching, and simple synthesis process, and are widely used in the construction of ratiometric fluorescence sensors.

[0005] At present, there are no reports on the use of structure-converting ratiometric fluorescence aptamer sensors for the detection of enrofloxacin residues in aquatic products. Summary of the invention

[0006] The technical problem to be solved by the present invention is to propose a ratio fluorescence aptamer sensor based on structure conversion for detecting enrofloxacin and its application, using carbon dots and oxidized o-phenylenediamine as dual fluorescence signal elements and specific nucleic acid aptamers as molecular recognition elements to establish a method for detecting enrofloxacin. The method has a simple process, strong anti-interference ability and good application prospects.

[0007] The technical solution of the present invention is:

[0008] A ratiometric fluorescent aptamer sensor based on structure conversion for detecting enrofloxacin. The ratiometric fluorescent aptamer sensor based on structure conversion has three DNA single strands connected to carbon dots, including an enrofloxacin aptamer connected to the carbon dots and two enrofloxacin aptamer complementary strands cDNA1 and cDNA2, and the 3' end of cDNA1 and the 5' end of cDNA2 are respectively modified with hemin monomers.

[0009] As a further preferred embodiment, the nucleic acid sequence of the enrofloxacin aptamer is 5'-CCC ATC AGG GGGCTA GGC TAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-NH2-3'.

[0010] As a further preferred embodiment, the nucleic acid sequences of cDNA1 modified with hemin monomer and cDNA2 modified with hemin monomer are 5'-CCG GGC TCA GAG AGC CGA ACC-hemin-3' and 5'-hemin-GTG TTAGCC TAG CCC CCT GAT-3', respectively.

[0011] As a further preferred solution, the specific steps are as follows:

[0012] (1) placing citric acid in a glass container and heating it at 200° C. to 210° C. for 4 to 6 minutes; then, adding sodium hydroxide solution to adjust the pH value to 7 to obtain a carbon dot (CDs) solution;

[0013] (2) The carbon dot solution prepared in step (1) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution are ultrasonically mixed at room temperature for 30 minutes at a volume ratio of 20:17, wherein the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution is 20 mg / mL; and then mixed with enrofloxacin aptamer, wherein the volume ratio of the enrofloxacin aptamer to the carbon dot solution is 1:20, wherein the concentration of the enrofloxacin aptamer is 20 μmol / L to 100 μmol / L, and the mixture is shaken on a shaker at 25° C. for 4 h to 8 h to obtain a carbon dot-enrofloxacin aptamer conjugate (CDs-apt);

[0014] (3) mixing the carbon dot-enrofloxacin aptamer conjugate synthesized in step (2) with two enrofloxacin aptamer complementary chains cDNA1 and cDNA2 modified with hemin monomer, and shaking the mixture on a shaker at room temperature for 30-60 minutes;

[0015] The volume ratio of the carbon dot-enrofloxacin aptamer conjugate to the two enrofloxacin aptamer complementary chains cDNA1 and cDNA2 modified with hemin monomer is 38:(10-30).

[0016] The concentration of the hemin monomer-modified enrofloxacin aptamer complementary chain cDNA1 is 50 μmol / L; the concentration of the hemin monomer-modified enrofloxacin aptamer complementary chain cDNA2 is 50 μmol / L;

[0017] After the reaction is completed, the solution is removed by centrifugation at 3000-10000 r / min for 5-20 min using a 10-kDa ultrafiltration centrifuge tube, and finally a ratiometric fluorescent aptamer sensor based on structure conversion is obtained.

[0018] Another aspect of the present invention is the use of the above-mentioned ratiometric fluorescence aptasensor based on structure conversion in the detection of enrofloxacin.

[0019] As a further preferred solution, the ratiometric fluorescent aptamer sensor based on structure conversion and the sample to be tested are incubated in a Tris-HCl buffer with a pH of 6 to 9 for 10 to 50 minutes; then o-phenylenediamine solution and hydrogen peroxide are added, and the mixture is shaken and mixed for 3 to 10 minutes;

[0020] The volume ratio of the ratio fluorescence aptamer sensor based on structure conversion to the o-phenylenediamine solution is 1:2, and the volume ratio of the ratio fluorescence aptamer sensor based on structure conversion to hydrogen peroxide is 1:2, wherein the concentration of the o-phenylenediamine solution is 0.2-0.7 mmol / L;

[0021] The fluorescence intensity and fluorescence color of the sensor at 472 nm and 556 nm under 400 nm excitation are recorded at this time; when the fluorescence color is cyan and there is no obvious change in the fluorescence intensity at 472 nm and 556 nm, it means that the sample to be tested does not contain enrofloxacin; when the fluorescence color is orange-yellow and the fluorescence intensity at 472 nm is quenched and the fluorescence intensity at 556 nm is quenched, it means that the sample to be tested contains enrofloxacin.

[0022] As a further preferred solution, 40 μL of the ratiometric fluorescent aptamer sensor based on structural conversion is incubated in a Tris-HCl buffer with a pH of 6 to 9 for 10 to 50 min; then 80 μL of a 0.2 to 0.7 mM o-phenylenediamine solution and 80 μL of a 10 mM hydrogen peroxide solution are added, and the mixture is shaken on a shaker for 3 to 10 min to form a structural ratiometric fluorescent aptamer sensor, and the fluorescence intensity (F) of the sensor at 472 nm and 556 nm under 400 nm excitation is recorded. 472nm )0、(F 556nm )0, calculate the ratio of the two fluorescence peaks (F 472nm / F 556nm )0;

[0023] Then, 40 μL of the ratiometric fluorescent aptamer sensor based on structure conversion was incubated with the sample to be tested in a Tris-HCl buffer with a pH of 6 to 9 for 10 to 50 min. Then, 80 μL of 0.2 to 0.7 mM o-phenylenediamine and 80 μL of 10 mM hydrogen peroxide were added and mixed on a shaker for 3 to 10 min. The fluorescence intensity F of the sensor at 472 nm and 556 nm under 400 nm excitation was recorded. 472nm and F 556nm , calculate the ratio of the two fluorescence peaks at this time F 472nm / F 556nm ;

[0024] Calculate (F 472nm / F 556nm ) / (F 472nm / F 556nm )0 ratio, and bring it into the linear regression equation as the y value to calculate the concentration of enrofloxacin in the sample to be tested;

[0025] The linear regression equation is y=1.1064+0.2663x, wherein x is the concentration of enrofloxacin in the sample to be tested.

[0026] The beneficial effects of the present invention are:

[0027] (1) Using nucleic acid aptamers as the recognition element of the sensor, carbon dots and 2,3-diaminophenazine (oxOPD) as the fluorescence response signal of the sensor, an aptamer structure conversion ratio fluorescence sensor was successfully constructed for the detection of enrofloxacin. The sensor can specifically recognize the detection object enrofloxacin with high selectivity. The sensor is based on the structural change of the hemin chloride dimer caused by the structural change of the aptamer and its complementary chain, and finally triggers the fluorescence response signal by mimicking the enzyme properties of the hemin chloride monomer.

[0028] (2) The detection range of the ratiometric fluorescence aptasensor is 0.1-12 ng / mL, and the minimum detection limit is 40.7 pg / mL. The detection of enrofloxacin has the characteristics of simple operation, good selectivity, high sensitivity, fluorescence visualization and low cost, and can be used to detect enrofloxacin residues in aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a diagram showing the corresponding mechanism of dual fluorescence signals of the structure-converting ratiometric fluorescence aptamer sensor for detecting enrofloxacin according to the present invention;

[0030] Figure 2 1. The nucleic acid electrophoresis (A) and Zeta potential (B) diagrams of the carbon dots and carbon dots-enrofloxacin aptamer conjugates of the present invention;

[0031] Figure 3 is a graph showing the effect of different concentrations of enrofloxacin on the fluorescence intensity of the structure-converting ratiometric fluorescence aptamer sensor of the present invention;

[0032] Figure 4 is a standard curve diagram of the change in fluorescence intensity of the structure-converting ratiometric fluorescent aptamer sensor of the present invention to different concentrations of enrofloxacin;

[0033] Figure 5 Fluorescence color diagram of the structure-converting ratiometric fluorescence aptasensor of the present invention to different concentrations of enrofloxacin (ai: 0, 0.1, 0.5, 1.7, 2.5, 4.0, 6.0, 8.0, 12.0 ng / mL) under 365 nm ultraviolet light;

[0034] Figure 6 This is an evaluation of the specificity of the structure-conversion ratiometric fluorescence aptasensor of the present invention for enrofloxacin detection. DETAILED DESCRIPTION

[0035] Example 1

[0036] (1) 2.5 g of citric acid was placed in a beaker and heated at 200° C. for 4 min. Subsequently, a 4 mg / mL NaOH solution was added dropwise to the beaker containing citric acid until the pH value reached 7, thereby obtaining a carbon dot solution.

[0037] (2) 200 μL of the carbon dot solution prepared in step (1) was ultrasonically mixed with 170 μL of 20 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution at room temperature for 30 min; then mixed with 10 μL of 20 μM enrofloxacin aptamer, wherein the nucleic acid sequence of the enrofloxacin aptamer is 5'-CCC ATC AGG GGG CTA GGC TAACAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-NH2-3', and the mixture was shaken on a shaker at 25° C. for 4 h to obtain a carbon dot-enrofloxacin aptamer conjugate;

[0038] (3) 380 μL of the carbon dot-enrofloxacin aptamer conjugate synthesized in step (2) was mixed with 10 μL of each of two enrofloxacin aptamer complementary chains (cDNA1 and cDNA2) modified with hemin monomers at a concentration of 50 μM, wherein the nucleic acid sequences of the enrofloxacin aptamer complementary chain cDNA1 modified with hemin monomers and the enrofloxacin aptamer complementary chain cDNA2 modified with hemin monomers were 5'-CCG GGC TCA GAG AGC CGA ACC-hemin-3' and 5'-hemin-GTG TTA GCC TAG CCC CCT GAT-3', respectively. The mixture was shaken on a shaker at room temperature for 30 min. The solution (i.e., unbound cDNA1 or cDNA2 single strand) was removed by centrifugation at 3000 r / min for 20 min using a 10-kDa ultrafiltration centrifuge tube to obtain a ratiometric fluorescent aptamer sensor based on structure conversion.

[0039] (4) Take 40 μL of the ratiometric fluorescent aptasensor based on structural conversion in step (3) and incubate with the sample to be tested in Tris-HCl buffer (pH=6) for 10 min; then add 80 μL of 0.2 mM o-phenylenediamine and 80 μL of 10 mM H2O2, and shake on a shaker for 5 min. Then, measure its ratiometric fluorescence intensity, compare with the standard curve, and calculate the enrofloxacin content in the sample.

[0040] Example 2

[0041] (1) Place citric acid in a beaker and heat at 202°C for 5 min. Then, add 4 mg / mL NaOH solution dropwise to the above solution until the pH value of the solution reaches 7, thereby obtaining a carbon dot solution.

[0042] (2) 200 μL of the carbon dot solution prepared in step (1) was ultrasonically mixed with 170 μL of 20 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution at room temperature for 30 min; then mixed with 10 μL of 100 μM enrofloxacin aptamer, wherein the nucleic acid sequence of the enrofloxacin aptamer is 5'-CCC ATC AGG GGG CTA GGCTAA CAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-NH2-3', and the mixture was shaken on a shaker at 25° C. for 6 h to obtain a carbon dot-enrofloxacin aptamer conjugate;

[0043] (3) 380 μL of the carbon dot enrofloxacin aptamer conjugate synthesized in step (2) was mixed with 30 μL of each of two enrofloxacin aptamer complementary chains (cDNA1 and cDNA2) modified with hemin monomers at a concentration of 50 μM, wherein the nucleic acid sequences of the enrofloxacin aptamer complementary chain cDNA1 modified with hemin monomers and the enrofloxacin aptamer complementary chain cDNA2 modified with hemin monomers were: 5'-CCG GGC TCA GAG AGC CGA ACC-hemin-3',: 5'-hemin-GTG TTA GCC TAG CCC CCT GAT-3', respectively. The mixture was shaken on a shaker at room temperature for 60 min. The solution (i.e., unbound single strands of cDNA1 or cDNA2) was removed by centrifugation at 10000 r / min for 5 min using a 10-kDa ultrafiltration centrifuge tube to obtain a ratiometric fluorescent aptamer sensor based on structure conversion.

[0044] (4) Take 40 μL of the ratiometric fluorescent aptasensor based on structural conversion in step (3) and incubate with different concentrations of the sample in Tris-HCl buffer (pH=9) for 50 min. Then add 80 μL of 0.7 mM o-phenylenediamine and 80 μL of 10 mM H2O2, and shake on a shaker for 10 min. Then, measure the ratiometric fluorescence intensity, compare with the standard curve, and calculate the enrofloxacin content in the sample.

[0045] Example 3

[0046] (1) Place citric acid in a beaker and heat at 210°C for 6 min. Then, add 4 mg / mL NaOH solution dropwise to the above solution until the pH value of the solution reaches 7, thereby obtaining a carbon dot solution.

[0047] (2) 200 μL of the carbon dot solution prepared in step (1) was ultrasonically mixed with 170 μL of 20 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution at room temperature for 30 min; then mixed with 10 μL of 50 μM enrofloxacin aptamer, wherein the nucleic acid sequence of the enrofloxacin aptamer is 5'-CCC ATC AGG GGG CTA GGC TAACAC GGT TCG GCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-NH2-3', and the mixture was shaken on a shaker at 25° C. for 8 h to obtain a carbon dot-enrofloxacin aptamer conjugate;

[0048] (3) Mix 380 μL of the carbon dot enrofloxacin aptamer conjugate synthesized in step (2) with 24 μL of each of two enrofloxacin aptamer complementary chains (cDNA1 and cDNA2) modified with hemin monomers at a concentration of 50 μM, wherein the nucleic acid sequences of the enrofloxacin aptamer complementary chain cDNA1 modified with hemin monomers and the enrofloxacin aptamer complementary chain cDNA2 modified with hemin monomers are: 5'-CCG GGC TCA GAG AGC CGAACC-hemin-3', 5'-hemin-GTG TTA GCC TAG CCC CCT GAT-3', respectively, and shake the mixture on a shaker at room temperature for 30 minutes; use a 10-kDa ultrafiltration centrifuge tube to centrifuge at 5000 r / min for 10 minutes to remove the solution (i.e., the unbound cDNA1 or cDNA2 single chain), and obtain a ratiometric fluorescent aptamer sensor based on structure conversion;

[0049] (4) Take 40 μL of the ratiometric fluorescent aptasensor based on structural conversion in step (3) and incubate with different concentrations of the sample in Tris-HCl buffer (pH=7) for 20 min. Then add 80 μL of 0.4 mM o-phenylenediamine and 80 μL of 10 mM H2O2, and shake on a shaker for 3 min. Then, measure the ratiometric fluorescence intensity, compare with the standard curve, and calculate the enrofloxacin content in the sample.

[0050] 1. Mechanism of detection of enrofloxacin based on structure-converting ratiometric fluorescence aptasensor

[0051] The mechanism diagram of the present invention for detecting enrofloxacin based on the structure-conversion ratio fluorescence aptamer sensor is as follows: Figure 1As shown, the ratiometric fluorescent aptamer sensor based on structure conversion of the present invention has three DNA single strands, including an enrofloxacin aptamer connected to carbon dots (CDs) and two enrofloxacin aptamer complementary strands (cDNA1 and cDNA2), and the 3' end of cDNA1 and the 5' end of cDNA2 are modified with hemin monomers (hemin), respectively.

[0052] In the absence of enrofloxacin, cDNA1 and cDNA2 hybridize with CDs-apt to form a fluorescent aptamer sensor. The two adjacent heme molecules on cDNA1 and cDNA2 form a heme dimer, and the dimer has low peroxidase mimetic activity. When enrofloxacin is present, due to its higher affinity, it preferentially binds to the nucleic acid aptamer, resulting in a configuration conversion of the sensor probe, thereby obtaining a heme monomer with high peroxidase mimetic activity. Therefore, Figure 1 It can be seen that after adding hydrogen peroxide (H2O2) and o-phenylenediamine (OPD) in the present invention, o-phenylenediamine is oxidized to generate 2,3-diaminophenazine (oxOPD) with strong yellow fluorescence. At the same time, fluorescence resonance energy transfer occurs between 2,3-diaminophenazine and carbon dots, resulting in a decrease in the fluorescence intensity of the carbon dots. By monitoring the quenching and enhancement of the fluorescence intensity of carbon dots and 2,3-diaminophenazine, we achieve dual signal recognition with good detection accuracy and sensitivity.

[0053] II. Nucleic acid electrophoresis (A) and zeta potential (B) detection of carbon dots and carbon dots-enrofloxacin aptamer conjugates

[0054] The nucleic acid electrophoresis (A) and Zeta potential (B) of the carbon dots and carbon dots-enrofloxacin aptamer conjugates of the present invention are shown in FIG. Figure 2 As shown in the figure, after the carbon dots were coupled with the enrofloxacin nucleic acid aptamer, the nucleic acid electrophoresis speed of the carbon dots-enrofloxacin aptamer conjugate slowed down compared with the pure carbon dots solution, which was due to the increase in the mass-to-charge ratio of the modified carbon dots, indicating that the carbon dots and the enrofloxacin aptamer were successfully bound. In addition, the zeta potential of the carbon dots was -17.23mV, which was due to the presence of negatively charged carboxyl groups on the surface of the carbon dots. The zeta potential of the enrofloxacin aptamer after modification became +0.22mV, which was attributed to the positive charge of the enrofloxacin nucleic acid aptamer, which further indicated that the enrofloxacin nucleic acid aptamer was successfully bound to the carbon dots.

[0055] 3. Detection data of enrofloxacin by ratiometric fluorescence aptasensor based on structure conversion (hereinafter referred to as sensor):

[0056] The sensor prepared by the embodiment of the present invention, taking the sensor prepared by Example 3 as an example, was used to study the detection of enrofloxacin.

[0057] (1) Effect of different concentrations of enrofloxacin on the fluorescence intensity of the structure-converting ratiometric fluorescence aptasensor of the present invention

[0058] Add 0, 0.1, 0.5, 1.7, 2.5, 4.0, 6.0, 8.0, and 12.0 ng / mL of enrofloxacin standard solution into 40 μL of sensor. Figure 3 It can be seen that with the continuous increase of the concentration of enrofloxacin in the sensor solution, the emission peak of 2,3-diaminophenazine (oxOPD) at 556 nm gradually increased, and the emission peak of carbon dots at 472 nm gradually decreased.

[0059] (2) Linear regression equation for enrofloxacin concentration detection

[0060] 40 μL of the structure-converting aptamer sensor was incubated in a Tris-HCl buffer solution with a pH of 9 for 50 min. Then, 80 μL of 0.7 mM o-phenylenediamine and 80 μL of 10 mM hydrogen peroxide were added and shaken for 10 min to form a structure-type ratiometric fluorescent aptamer sensor. The fluorescence intensity (F) of the sensor at 472 nm and 556 nm under 400 nm excitation was recorded. 472nm )0 and (F 556nm )0, calculate the ratio of the two fluorescence peaks (F 472nm / F 556nm )0. In the absence of enrofloxacin in the structure-conversion ratiometric fluorescent aptamer sensing system, cDNA1 and cDNA2 hybridize with the carbon dot-enrofloxacin aptamer conjugate. The two adjacent hemin molecules on cDNA1 and cDNA2 form a heme dimer, and the dimer oxidase mimic activity is low.

[0061] Then, 40 μL of the structure-converting aptamer sensor was incubated with the sample in a Tris-HCl buffer with a pH of 9 for 50 min. Then, 80 μL of 0.7 mM o-phenylenediamine and 80 μL of 10 mM hydrogen peroxide were added and mixed on a shaker for 10 min. The fluorescence intensity F of the sensor at 472 nm and 556 nm under 400 nm excitation was recorded. 472nm and F 556nm Calculate the ratio of the two fluorescence peaks at this time 472nm / F 556nm .

[0062] Finally, we get (F 472nm / F 556nm ) / (F 472nm / F 556nm)0 ratio. When enrofloxacin is present, it will preferentially bind to the aptamer due to its higher affinity, resulting in a sensor configuration conversion, thereby obtaining a heme monomer with high peroxidase mimetic activity. Then, with the increase of enrofloxacin concentration, the fluorescence peak of 2,3-diaminophenazine (oxOPD) at 556nm gradually increased, and the fluorescence peak of carbon dots at 472nm gradually decreased. With the increase of enrofloxacin concentration, (F 472nm / F 556nm ) / (F 472nm / F 556nm )0 ratio also gradually increases. Taking enrofloxacin concentration as the horizontal axis, (F 472nm / F 556nm ) / (F 472nm / F 556nm )0 is used as the ordinate to draw the standard curve, and the linear regression equation is y=1.1064+0.2663x, (R 2 =0.9976). The calculated (F 472nm / F 556nm ) / (F 472nm / F 556nm )0 ratio is substituted into the linear regression equation to calculate the concentration of enrofloxacin in the test sample.

[0063] (3) Draw a limited regression standard curve

[0064] Enrofloxacin standard solutions with concentrations of 0, 0.1, 0.5, 1.7, 2.5, 4.0, 6.0, 8.0, and 12.0 ng / mL were added to the 40 μL sensor. The concentration of enrofloxacin was used as the horizontal axis and (F 472nm / F 556nm ) / (F 472nm / F 556nm )0 is the ordinate, (F 472nm / F 556nm ) and (F 472nm / F 556nm )0 are the fluorescence intensity ratios of the system in the presence and absence of enrofloxacin respectively, and the standard curve is fitted. Figure 4 As shown, the linear range of the standard curve is 0.1-12.0 ng / mL, the equation is y=1.1064+0.2663x, and the correlation coefficient R 2 is 0.9976.

[0065] (4) Fluorescence color changes of the sensor in response to different concentrations of enrofloxacin (ai: 0, 0.1, 0.5, 1.7, 2.5, 4.0, 6.0, 8.0, 12.0 ng / mL)

[0066] like Figure 5As shown in the figure, the concentrations of enrofloxacin in the ai are 0, 0.1, 0.5, 1.7, 2.5, 4.0, 6.0, 8.0, and 12.0 ng / mL, respectively. Under 365nm ultraviolet light, when there is no enrofloxacin, the fluorescence color is cyan, and when there is enrofloxacin, the fluorescence color is orange, and as the concentration of enrofloxacin increases, the orange fluorescence color increases. Enrofloxacin can be qualitatively detected by the fluorescence color of the system, and quantitative detection is expected. The sensing system has the advantage of changing color with the concentration of the analyte.

[0067] (5) Evaluation of sensor specificity for enrofloxacin detection

[0068] Tetracycline (TC), chloramphenicol (Chl), streptomycin (Str), nitrofurazone (Nit), furazolidone (Fur), ciprofloxacin (CIP), norfloxacin (NOR), moxifloxacin (MOX), gatifloxacin (GAT), ofloxacin (OFL) and enrofloxacin (ENR, concentration of 8 ng / mL) were added to the sensor solution at the same concentration (0.8 μg / mL) for detection and analysis. The results are shown in Figure 2. Figure 6 As shown, compared with enrofloxacin, these 10 other representative antibacterial drugs did not cause obvious fluorescence ratio enhancement. Therefore, the aptasensor has strong specificity for the detection of enrofloxacin.

[0069] (6) Actual sample measurement

[0070] Eel, grass carp and green prawn, three common aquatic products in the market, were selected as samples, and sample pretreatment was performed according to the national standard method of the Ministry of Agriculture Announcement No. 783-2-2006 to obtain 5 mL of the sample solution to be tested. 40 μL of the sensor of Example 3 of the present invention was added to the solution to be tested, and actual sample detection was performed. The measurement results are shown in Table 1. The recovery rate ranged from 97.2% to 107.1% at three spiked levels, and the RSD value was 1.1% to 4.4%, all of which were lower than 5%, indicating that the sensor can be used for the detection and analysis of enrofloxacin in actual aquatic samples.

[0071] Table 1 Actual sample analysis (n=5)

[0072]

[0073] The above are only specific embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A ratiometric fluorescence aptasensor based on structure conversion for detecting enrofloxacin, characterized in that: The structure-converting ratiometric fluorescent aptamer sensor has three DNA single strands connected to the carbon dots, including an enrofloxacin aptamer connected to the carbon dots and two enrofloxacin aptamer complementary strands cDNA1 and cDNA2, and the 3' end of cDNA1 and the 5' end of cDNA2 are modified with hemin monomers respectively; The nucleic acid sequence of the enrofloxacin aptamer is 5'-CCC ATC AGG GGG CTA GGC TAA CAC GGT TCGGCT CTC TGA GCC CGG GTT ATT TCA GGG GGA-NH2-3'; The nucleic acid sequences of cDNA1 modified with hemin monomer and cDNA2 modified with hemin monomer are 5'-CCGGGC TCA GAG AGC CGA ACC-hemin-3' and 5'-hemin-GTG TTA GCC TAG CCC CCT GAT-3', respectively.

2. The method for preparing the ratiometric fluorescent aptasensor based on structure conversion for detecting enrofloxacin according to claim 1, characterized in that: The specific steps are as follows: (1) placing citric acid in a glass container and heating it at 200° C. to 210° C. for 4 to 6 minutes; then, adding sodium hydroxide solution to adjust the pH value to 7 to obtain a carbon dot solution; (2) The carbon dot solution prepared in step (1) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution are ultrasonically mixed at room temperature for 30 minutes at a volume ratio of 20:17, wherein the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride solution is 20 mg / mL; and then mixed with enrofloxacin aptamer, wherein the volume ratio of the enrofloxacin aptamer to the carbon dot solution is 1:20, wherein the concentration of the enrofloxacin aptamer is 20 μmol / L to 100 μmol / L, and the mixture is shaken on a shaker at 25° C. for 4 h to 8 h to obtain a carbon dot-enrofloxacin aptamer conjugate; (3) mixing the carbon dot-enrofloxacin aptamer conjugate synthesized in step (2) with two enrofloxacin aptamer complementary chains cDNA1 and cDNA2 modified with hemin monomer, and shaking the mixture on a shaker at room temperature for 30-60 minutes; The volume ratio of the carbon dot-enrofloxacin aptamer conjugate to the two enrofloxacin aptamer complementary chains cDNA1 and cDNA2 modified with the hemin monomer is 38:(10-30); The concentration of the hemin monomer-modified enrofloxacin aptamer complementary chain cDNA1 is 50 μmol / L; the concentration of the hemin monomer-modified enrofloxacin aptamer complementary chain cDNA2 is 50 μmol / L; After the reaction is completed, the solution is removed by centrifugation at 3000-10000 r / min for 5-20 min using a 10-kDa ultrafiltration centrifuge tube, and finally a ratiometric fluorescent aptamer sensor based on structure conversion is obtained.

3. Use of the ratiometric fluorescence aptasensor based on structure conversion as claimed in claim 1 in detecting enrofloxacin.

4. The use of the ratiometric fluorescent aptasensor based on structure conversion in the detection of enrofloxacin according to claim 3, characterized in that: The ratiometric fluorescent aptamer sensor based on structure conversion and the sample to be tested are incubated in a Tris-HCl buffer with a pH of 6 to 9 for 10 to 50 minutes; then, an o-phenylenediamine solution and hydrogen peroxide are added, and the mixture is shaken on a shaker for 3 to 10 minutes; The volume ratio of the ratio fluorescence aptamer sensor based on structure conversion to the o-phenylenediamine solution is 1:2, and the volume ratio of the ratio fluorescence aptamer sensor based on structure conversion to hydrogen peroxide is 1:2, wherein the concentration of the o-phenylenediamine solution is 0.2-0.7 mmol / L; The fluorescence intensity and fluorescence color of the sensor at 472 nm and 556 nm under 400 nm excitation are recorded at this time; when the fluorescence color is cyan and there is no obvious change in the fluorescence intensity at 472 nm and 556 nm, it means that the sample to be tested does not contain enrofloxacin; when the fluorescence color is orange-yellow, and the fluorescence intensity at 472 nm is quenched and the fluorescence intensity at 556 nm is enhanced, it means that the sample to be tested contains enrofloxacin.

5. The use of the ratiometric fluorescent aptasensor based on structure conversion in detecting enrofloxacin according to claim 3, characterized in that: 40 μL of the ratiometric fluorescent aptamer sensor based on structural conversion was incubated in a Tris-HCl buffer with a pH of 6 to 9 for 10 to 50 min; then 80 μL of a 0.2 to 0.7 mM o-phenylenediamine solution and 80 μL of a 10 mM hydrogen peroxide solution were added, and the mixture was shaken and mixed for 3 to 10 min to form a structural ratiometric fluorescent aptamer sensor, and the fluorescence intensity (F) of the sensor at 472 nm and 556 nm under 400 nm excitation was recorded. 472nm )0、(F 556nm )0, calculate the ratio of the two fluorescence peaks (F 472nm / F 556nm )0; Then, 40 μL of the ratiometric fluorescent aptamer sensor based on structure conversion was incubated with the sample to be tested in Tris-HCl buffer with a pH of 6 to 9 for 10 to 50 min. Then, 80 μL of 0.2 to 0.7 mM o-phenylenediamine and 80 μL of 10 mM hydrogen peroxide were added and shaken for 3 to 10 min. The fluorescence intensity F of the sensor at 472 nm and 556 nm under 400 nm excitation was recorded. 472nm and F 556nm Calculate the ratio of the two fluorescence peaks at this time 472nm / F 556nm ; Calculate (F 472nm / F 556nm ) / (F 472nm / F 556nm )0 ratio, and bring it into the linear regression equation as the y value to calculate the concentration of enrofloxacin in the sample to be tested; The linear regression equation is y=1.1064+0.2663x, wherein x is the concentration of enrofloxacin in the sample to be tested.

Images