Enrofloxacin visual rapid detection method based on magnetic bead magnetic separation-signal output dual function

Abstract

The application discloses a kind of based on magnetic bead magnetic separation-signal output dual function enrofloxacin visual rapid detection method.The specific method of the application is as follows: 1) the surface of carboxylated magnetic bead with particle size of 100-1000nm is modified enrofloxacin monoclonal antibody, and immune magnetic bead dispersion is obtained after blocking;2) enrofloxacin-bovine serum albumin complex is coupled with carboxylated magnetic bead with particle size of 10-50nm, and labeled magnetic bead dispersion is obtained after blocking;3) immune magnetic bead dispersion is mixed with sample solution, and mixed dispersion is obtained after reaction;4) labeled magnetic bead dispersion is mixed with mixed dispersion, and after magnetic separation, supernatant is obtained, and the absorbance of supernatant at 405nm is detected;5) the absorbance is substituted into standard curve, and the concentration of enrofloxacin is obtained.The method of the application is simple, rapid, high sensitivity, and has the capacity of visual qualitative / semi-quantitative, can be on-site rapid detection enrofloxacin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of visual detection, in particular to a visual rapid detection method of enrofloxacin based on magnetic separation-signal output dual function of magnetic beads. BACKGROUND

[0002] Enrofloxacin (ENR), also known as ethyl ciprofloxacin, is a fluoroquinolone antibiotic that can achieve bactericidal effect by inhibiting the activity of DNA gyrase, and has the characteristics of wide antibacterial spectrum, strong antibacterial activity, strong tissue penetration, etc., and is widely used in the treatment of diseases of aquatic animals, livestock and poultry animals. However, this drug can cause potential side effects on the human body, and large-scale use can induce bacterial resistance, endangering human health. The maximum limit of the total amount of enrofloxacin and ciprofloxacin in muscle tissue of animal products such as aquatic products is 100 μg / kg. Therefore, it is particularly important to realize rapid and sensitive detection of enrofloxacin.

[0003] The traditional detection methods of enrofloxacin residues mainly include microbial inhibition method and instrument analysis method represented by liquid chromatography-mass spectrometry. Instrument analysis method has the advantages of high sensitivity and low detection limit, and is the standard method for detecting enrofloxacin at present, but its pretreatment is complex and requires professional operation and expensive precision instruments, which cannot realize on-site detection; the microbial inhibition method is time-consuming and unstable, and lacks timeliness. In recent years, rapid detection methods represented by enzyme-linked immunosorbent assay (ELISA) and lateral flow immunochromatography (LFIA) have developed rapidly. However, ELISA needs repeated washing in the detection process, and the operation is relatively cumbersome; LFIA is simple to operate, but lacks in detection precision and accuracy. SUMMARY

[0004] The purpose of the present application is to explore an enrofloxacin visual rapid detection method integrating magnetic bead (MB) magnetic separation-signal output dual function in view of the deficiencies of the prior art. The present application provides an enrofloxacin visual rapid detection method based on magnetic bead magnetic separation-signal output dual function. The method of the present application is sensitive, rapid, specific, simple to operate, can realize qualitative / semi-quantitative detection through visual observation of color change, and can realize quantitative detection through measurement of absorbance value, and can realize on-site rapid detection of enrofloxacin.

[0005] The technical scheme adopted by the present application is as follows:

[0006] (I) An enrofloxacin visual rapid detection method based on magnetic bead magnetic separation-signal output dual function

[0007] The enrofloxacin visual rapid detection method comprises the following steps:

[0008] 1) activating the carrier magnetic beads to obtain activated carrier magnetic beads, then modifying enrofloxacin monoclonal antibody on the surface of the activated carrier magnetic beads, and obtaining an immunomagnetic bead dispersion after blocking with bovine serum albumin; the carrier magnetic beads are carboxylated magnetic beads with a particle size of 100-1000 nm; the mass ratio of the activated carrier magnetic beads to enrofloxacin monoclonal antibody is 1:0.02-0.1; the mass ratio of the activated carrier magnetic beads to bovine serum albumin is 1:30-50; the phosphate buffer solution can further contain a 0.01% Tween-20 solution and a 0.1% bovine serum albumin solution in terms of mass fraction, which are used to reduce the non-specific adsorption of non-target objects; the enrofloxacin monoclonal antibody is of murine origin, IgG2b, protein G purified, with a purity greater than 95% and a concentration of 1 mg / mL;

[0009] The step 1) comprises the following steps:

[0010] 1.1) washing the carrier magnetic beads with a MES buffer solution (2-(N-morpholine) ethanesulfonic acid), repeating the washing 1-3 times, and then dispersing the washed carrier magnetic beads in an activation solution to obtain an activated carrier magnetic bead dispersion after reaction at room temperature for 30 min-1 h;

[0011] 1.2) separating the activated carrier magnetic beads from the activated carrier magnetic bead dispersion by magnetic separation, washing the activated carrier magnetic beads with a borate buffer solution, repeating the washing 1-3 times, and then dispersing the washed activated carrier magnetic beads in the borate buffer solution to obtain a borate dispersion of the activated carrier magnetic beads;

[0012] 1.3) mixing the borate dispersion of the activated carrier magnetic beads with an enrofloxacin monoclonal antibody solution, and then reacting at room temperature for 1-4 h to obtain an antibody-carrier magnetic bead borate dispersion;

[0013] 1.4) adding a bovine serum albumin solution with a mass concentration of 1%-5% to the antibody-carrier magnetic bead borate dispersion, and reacting at room temperature for 1-4 h to obtain an immunomagnetic bead borate dispersion; separating the immunomagnetic beads from the immunomagnetic bead borate dispersion by magnetic separation, washing the immunomagnetic beads with a phosphate (PBS) buffer solution, repeating the washing 1-3 times, and then dispersing the washed immunomagnetic beads in the phosphate buffer solution to obtain an immunomagnetic bead dispersion.

[0014] 2) coupling enrofloxacin with bovine serum albumin to obtain an enrofloxacin-bovine serum albumin complex; activating the pre-labeled magnetic beads to obtain activated pre-labeled magnetic beads, then coupling the enrofloxacin-bovine serum albumin complex with the activated pre-labeled magnetic beads, and obtaining a labeled magnetic bead dispersion after blocking with bovine serum albumin; the pre-labeled magnetic beads are carboxylated magnetic beads with a particle size of 10-50 nm;

[0015] The mass ratio between the activated pre-labeled magnetic beads and the enrofloxacin-bovine serum albumin complex in the step 2) is 4:3; the mass ratio between the activated pre-labeled magnetic beads and the bovine serum albumin is 1:150-250.

[0016] The step 2) comprises the following steps according to mass fraction:

[0017] 2.1) 2 parts of enrofloxacin, 1 part of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 1.25 parts of N-hydroxysuccinimide sodium sulfonate are dissolved in N,N-dimethylformamide to react for 8-16 hours at room temperature to obtain A liquid; 5 parts of bovine serum albumin are dissolved in a phosphate buffer solution with pH of 6-8 to obtain B liquid; the A liquid is added dropwise into the B liquid, and after stirring and reacting for 2-4 hours at room temperature, the mixture is treated by dialysis and centrifugation, and then the supernatant is taken to obtain a solution of enrofloxacin-bovine serum albumin complex;

[0018] The ratio between the mass of enrofloxacin and the volume of N,N-dimethylformamide is 10-30 mg:0.5-1.5 mL; the ratio between the mass of bovine serum albumin and the volume of phosphate buffer solution is 30-70 mg:2-4 mL; and the volume ratio between the A liquid and the B liquid is 0.5-1.5:2-4;

[0019] In the step 2.1), the dialysis treatment is as follows: the mixture of the A liquid and the B liquid after stirring and reaction is loaded into a dialysis bag with a molecular weight cut-off of 8000-14000 D, and dialysis is performed at 4°C for 3 days, with the dialysis solution being changed 3 times a day. In the step 2.1), the centrifugal treatment is performed at a centrifugal speed of 3000 rpm or above for 5-10 minutes.

[0020] 2.2) The pre-labeled magnetic beads are washed with MES buffer solution (2-(N-morpholino) ethanesulfonic acid), and after being washed for 1-3 times, the washed pre-labeled magnetic beads are dispersed in an activation solution, and reacted for 30 minutes to 1 hour at room temperature to obtain an activated pre-labeled magnetic bead dispersion;

[0021] 2.3) The activated pre-labeled magnetic beads are separated from the activated pre-labeled magnetic bead dispersion by magnetic separation, and washed with a borate buffer solution, and after being washed for 1-3 times, the washed activated pre-labeled magnetic beads are dispersed in the borate buffer solution to obtain a borate dispersion of activated pre-labeled magnetic beads;

[0022] 2.4) The borate dispersion of activated pre-labeled magnetic beads is mixed with the enrofloxacin-bovine serum albumin complex solution, and reacted for 1-4 hours at room temperature to obtain a borate dispersion of unblocked labeled magnetic beads;

[0023] 2.5) adding a bovine serum albumin solution with a mass concentration of 1% to 5% to the borate dispersion liquid of the unblocked labeled magnetic beads, and reacting at room temperature for 1 to 4 hours to obtain a labeled magnetic bead borate dispersion liquid; separating the labeled magnetic beads from the labeled magnetic bead borate dispersion liquid through magnetic separation, washing the labeled magnetic beads with a phosphate (PBS) buffer, and after repeating the washing for 1 to 3 times, dispersing the washed labeled magnetic beads in the phosphate buffer to obtain a labeled magnetic bead dispersion liquid.

[0024] 3) mixing the immunomagnetic bead dispersion liquid with the sample solution to be detected, and then rotating and mixing uniformly at room temperature to react, after the reaction is completed, magnetic separation and washing are performed, and then the mixture is dispersed in a phosphate (PBS) buffer to obtain a mixed dispersion liquid, wherein the components of the mixed dispersion liquid of the capture magnetic beads include immunomagnetic beads with captured enrofloxacin on the surface and magnetic beads without captured enrofloxacin on the surface; the concentration of enrofloxacin in the sample solution to be detected is 1 to 100 ng / mL, and the detection limit is 0.18 ng / mL.

[0025] The step 3) is specifically: 40 parts by volume of the immunomagnetic bead dispersion liquid with a concentration of 2 mg / mL (calculated based on the concentration of the magnetic nanoparticles) is mixed with 160 parts by volume of the sample solution to be detected; then rotating and mixing uniformly at room temperature for at least ten minutes; after the reaction is completed, magnetic separation and washing are performed, and then the mixture is dispersed in 178 parts by volume of a phosphate (PBS) buffer to obtain a mixed dispersion liquid.

[0026] 4) mixing the labeled magnetic bead dispersion liquid with the mixed dispersion liquid obtained in the step 3) and rotating and mixing uniformly at room temperature to react, after the reaction is completed, using a Thermo Fisher DynaMag TM -2 magnetic stand to separate the supernatant, and detecting the absorbance value of the supernatant at a specific wavelength, wherein the absorbance value of the supernatant at the specific wavelength is the detection value of the sample solution to be detected; the specific wavelength is 405 nm. In this process, enrofloxacin is detected by taking advantage of the difference in magnetic separation speed of the immunomagnetic beads combined with different numbers of labeled magnetic beads, which avoids the use of other signal markers and a complicated washing process, and simplifies the detection process.

[0027] The step 4) is specifically: 22 parts by volume of the labeled magnetic bead dispersion liquid with a concentration of 0.4 mg / mL (calculated based on the concentration of the magnetic nanoparticles) is mixed with 178 parts by volume of the mixed dispersion liquid; rotating and mixing uniformly at room temperature for at least ten minutes;

[0028] In the step 4), after the reaction is completed, 1-2 enrichment treatments are carried out, and then the clear liquid is obtained through magnetic separation; the process of the enrichment treatment is as follows: after magnetic separation is carried out until the clear liquid is clear and free of impurities, the clear liquid is not discarded, and the precipitate is resuspended in the clear liquid by mixing; the purpose of the enrichment treatment (several times of magnetic separation and re-mixing) is to increase the difference in the magnetic separation speed during detection, and to improve the detection sensitivity.

[0029] The mass ratio between the immunomagnetic beads contained in the immunomagnetic bead dispersion liquid in the step 3) and the labeled magnetic beads contained in the labeled magnetic bead dispersion liquid in the step 4) is 100:5-15; within the above mass ratio range, the sensitivity of the detection method is the highest; in specific implementation, the mass ratio can be selected from the preferred range of the mass ratio by screening the competitive inhibition rate under different concentration conditions and the like.

[0030] 5) The detection value is substituted into the standard curve prepared in advance to obtain the concentration of enrofloxacin in the sample solution to be detected.

[0031] In the step 1) and the step 2), the pH of the activation solution is 6.0, and the activation solution is a MES buffer solution containing 10 mmol / L of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 15 mmol / L of N-hydroxysuccinimide sulfonate sodium salt (NHSS).

[0032] In the step 1) and the step 2), the pH of the MES buffer solution is 5-7; the pH of the borate buffer solution is 7-9; and the pH of the phosphate buffer solution is 6-8.

[0033] The establishment process of the standard curve prepared in advance is as follows: different concentrations of enrofloxacin standard solutions are respectively treated according to the step 3) to the step 4) to obtain the detection values corresponding to each enrofloxacin standard solution, and the concentration of the enrofloxacin standard solution and the corresponding detection value are linearly fitted to obtain the standard curve.

[0034] The present application fully utilizes the dual functions of magnetic separation and signal output of magnetic beads, and competitively binds the binding sites on the surface of the immunomagnetic beads by the labeled magnetic beads and the target enrofloxacin, and innovatively utilizes the difference in the magnetic separation speed when the immunomagnetic beads bind different amounts of labeled magnetic beads to detect the enrofloxacin residue.

[0035] (B) An enrofloxacin visual rapid detection kit based on the dual functions of magnetic separation and signal output of magnetic beads

[0036] The kit comprises an immune magnetic bead dispersion liquid and a labeled magnetic bead dispersion liquid; in the immune magnetic bead dispersion liquid, the immune magnetic bead is mainly formed by surface modification of carrier magnetic beads by enrofloxacin monoclonal antibody and blocking by bovine serum albumin; the labeled magnetic bead dispersion liquid is mainly formed by coupling of pre-labeled magnetic beads and enrofloxacin-bovine serum albumin complex and blocking by bovine serum albumin; the carrier magnetic beads adopt carboxylated magnetic beads with a particle size of 100-1000 nm; the pre-labeled magnetic beads adopt carboxylated magnetic beads with a particle size of 100-1000 nm.

[0037] The method mainly relates to an immune magnetic bead IMB modified by enrofloxacin monoclonal antibody 大 and a labeled magnetic bead MB functionalized by bovine serum albumin-enrofloxacin (BSA-ENR) 小 -BSA-ENR. By using the high separation efficiency and excellent optical properties of the magnetic beads, a simple and stable nanobiosensor is constructed to realize rapid, sensitive and specific detection of enrofloxacin.

[0038] The beneficial effects of the present application are:

[0039] (1) The present application uses magnetic beads with a larger particle size as a separation and enrichment tool to improve the detection sensitivity, and uses magnetic beads with a smaller particle size as an output signal tool to improve the detection stability. The method of the present application detects enrofloxacin residues by utilizing the difference in magnetic separation speed of the immune magnetic beads IMB 大 combined with different numbers of labeled magnetic beads MB 小 -BSA-ENR, and the detection result is stable and accurate.

[0040] (2) The detection method of the present application is simple and fast, does not require precise instruments, reduces the detection threshold, can realize on-site rapid detection of enrofloxacin residues, and has a wide range of applications.

[0041] (3) The detection kit of the present application only needs to use the naked eye to observe the color change, without the need for precise instruments, so that qualitative or semi-quantitative detection can be realized, and on-site quantitative or semi-quantitative rapid detection of the target can be realized.

[0042] (4) The detection method of the present application avoids the use of other signal markers and complicated washing processes, and simplifies the detection process.

[0043] (5) The detection time of the detection method of the present application is much less than that of enzyme-linked immunosorbent assay and colloidal gold immunofiltration method, and the detection limit is also lower. The linear detection range can reach 1-100 ng / mL, and the detection limit can reach 0.18 ng / mL. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The schematic diagram of the principle of the method of the present application.

[0045] Figure 2 The standard curve of absorbance value of large magnetic beads at different concentrations at 405 nm wavelength.

[0046] Figure 3 The standard curve of absorbance value of small magnetic beads at different concentrations at 405 nm wavelength.

[0047] Figure 4 The schematic diagram of the absorbance value of immune magnetic beads at 450 nm wavelength in the method of the present application.

[0048] Figure 5 The ultraviolet absorption spectrum diagram of the labeled magnetic beads in the method of the present application.

[0049] Figure 6 The standard curve diagram of the method of the present application in buffer.

[0050] Figure 7 The standard curve diagram of the method of the present application in fish meat extract solution.

[0051] Figure 8 The standard curve diagram of the method of the present application in pond water sample. DETAILED DESCRIPTION

[0052] The present application will be further described in detail below in conjunction with the accompanying drawings and specific examples, but is not limited to the present application

[0053] As Figure 1 shown, the detection principle of the present application is that enrofloxacin in the sample solution to be detected is first captured by immune magnetic beads (IMB 大 ) and occupies part of the binding sites on the surface of IMB 大 After magnetic separation and washing, the labeled magnetic beads (MB 小 -BSA-ENR) as a competitor are combined with the remaining sites on the surface of IMB 大 , and finally the quantitative detection of enrofloxacin is realized by detecting the absorbance value of the supernatant after magnetic separation.

[0054] Among them, the immune magnetic beads IMB 大 combined with different numbers of labeled magnetic beads MB 小 -BSA-ENR have different magnetic separation speeds. The more the immune magnetic beads IMB 大 combined with the labeled magnetic beads MB 小 -BSA-ENR, the faster the separation speed in the magnetic separation process.

[0055] The specific implementation of the present application is as follows:

[0056] In this embodiment, the carrier magnetic beads (carboxylated large magnetic beads) are carboxylated magnetic beads with a particle size of about 130 nm, and the pre-labeling magnetic beads (carboxylated small magnetic beads) are carboxylated magnetic beads with a particle size of about 20 nm. As shown in Figure 2 and Figure 3 The pre-experiment shows that the absorbance values of the two kinds of magnetic beads at different concentrations have a good linear relationship at 405 nm.

[0057] 1) Preparation of enrofloxacin monoclonal antibody modified IMB 大

[0058] 1.1) Prepare a mixed solution containing 10 mmol / L EDC and 15 mmol / L NHSS in 400 μL;

[0059] 1.2) Disperse 20 μL of carboxylated large magnetic beads (Shanghai Aurun Micro-nano New Material Technology Co., Ltd., particle size about 130 nm) with a concentration of 10 mg / mL in a 2-(N-morpholine) ethanesulfonic acid (MES) buffer with a pH of 6.0, and perform sequential magnetic separation and removal of the washing supernatant using the MES buffer, and repeat the process 3 times. Resuspend the treated product in 400 μL of the mixed solution of EDC and NHSS obtained in step 1.1), and rotate to mix at room temperature for 30 min to obtain an activated carrier magnetic bead solution. Perform sequential magnetic separation and removal of the washing supernatant using a borate buffer with a pH of 7.4, and repeat the process 3 times. Then disperse the borate buffer dispersion liquid containing the activated large magnetic beads in 396 μL of borate buffer;

[0060] 1.3) Mix 396 μL of the borate buffer dispersion liquid containing 0.2 mg of activated carrier magnetic beads with 4 μL of a solution containing 0.004 mg of enrofloxacin monoclonal antibody (purchased from Wuhan Huamei Biological Engineering Co., Ltd.), and shake to react at room temperature for 2.5 h to obtain a borate buffer dispersion liquid of enrofloxacin monoclonal antibody modified IMB 大 ;

[0061] 1.4) Add 400 μL of a 2% bovine serum albumin solution to the borate buffer dispersion liquid of enrofloxacin monoclonal antibody modified IMB 大 obtained in step 1.3), and shake to react at room temperature for 1 h. Perform sequential magnetic separation and removal of the washing supernatant using a phosphate buffer with a pH of 7.4, and repeat the process 3 times. Then disperse the phosphate dispersion liquid containing the bovine serum albumin blocked, enrofloxacin monoclonal antibody modified IMB 大 in 100 μL of phosphate buffer (the final concentration of IMB 大 is 2 mg / mL), and store at 4°C for later use.

[0062] Figure 4 The figure shows the absorbance value of immunomagnetic beads at 450 nm wavelength in the method of the present application. Specifically, the absorbance value of carboxylated large magnetic beads (MB 大 -COOH), large magnetic beads-bovine serum albumin complex (MB 大 -BSA) and immunomagnetic beads (IMB 大 ) after binding with enzyme-labeled secondary antibody.

[0063] As can be seen from the figure, the OD 大 values of MB 大 -COOH and MB 450 -BSA only have a slight difference. The OD 大 value of IMB 450 is much higher than the other two, which is due to the immunoreaction between enzyme-labeled secondary antibody and the antibody on the surface of IMN 大 catalyzing the formation of oxidation product of 3,3',5,5'-tetramethylbenzidine (TMB) substance and generating strong absorbance at 450 nm. Therefore, the result can prove that IMB 大 is successfully prepared.

[0064] 2) Preparation of MB 小 -BSA-ENR

[0065] 2.1) 20 mg of enrofloxacin, 10 mg of NHSS and 12.5 mg of EDC were dissolved in 1 mL of N,N-dimethylformamide (DMF) and shaken at room temperature overnight to prepare solution A; bovine serum albumin (2.5 mg of bovine serum albumin per mg of enrofloxacin) was dissolved in phosphate buffer solution with pH of 7.4 to prepare solution B; 1 mL of solution A was added dropwise to 3 mL of solution B while stirring, then stirred at room temperature for 3 h, then the mixture was loaded into a dialysis bag (8000-14000 D), dialyzed at 4°C for 3 days, the dialysis solution was changed 3 times a day, the supernatant was obtained by centrifugation (3000 rpm, 5 min) and a dispersion liquid containing BSA-ENR complex was obtained, which was diluted 10 times for use (the concentration of bovine serum albumin was 0.6 mg / mL).

[0066] 2.2) Prepare 400 μL of a mixed solution of 10 mmol / L EDC and 15 mmol / L NHSS; disperse 20 μL of carboxylated small magnetic beads (Xi'an Ruishi Biological Technology Co., Ltd., particle size about 20 nm) with a concentration of 2 mg / mL in a 2-(N-morpholino)ethanesulfonic acid (MES) buffer with a pH of 6.0, and perform a magnetic separation and removal of the washing supernatant in sequence with the MES buffer and repeat the process 3 times, resuspend in 400 μL of the mixed solution of EDC and NHSS, rotate and mix at room temperature for 30 min to obtain an activated carrier magnetic bead solution; perform a magnetic separation and removal of the washing supernatant in sequence with a borate buffer with a pH of 7.4 and repeat the process 3 times, and then disperse in 350 μL of the borate buffer to obtain a borate dispersion of activated pre-labeled magnetic beads;

[0067] 2.3) Mix 350 μL of the borate dispersion of activated pre-labeled magnetic beads with 50 μL of a dispersion containing 0.03 mg of BSA-ENR complex (0.75 mg of BSA-ENR complex per mg of MB 小 -BSA-ENR), and shake and react at room temperature for 2.5 h to obtain a borate buffer dispersion containing MB 小 -BSA-ENR;

[0068] 2.4) Add 400 μL of a 2% bovine serum albumin solution to the borate buffer dispersion containing MB 小 -BSA-ENR of step 3.3), shake and react at room temperature for 1 h, perform a magnetic separation and removal of the waste liquid in sequence with a phosphate buffer with a pH of 7.4 and repeat the process 3 times, and then disperse in 100 μL of the phosphate buffer to obtain a phosphate buffer dispersion containing BSA-blocked MB 小 -BSA-ENR (the final concentration of MB 小 -BSA-ENR is 0.4 mg / mL in terms of the concentration of small magnetic beads), and store at 4°C for standby use.

[0069] Figure 5 UV-vis absorption spectra of the bovine serum albumin-enrofloxacin complex (BSA-ENR), the carboxylated small magnetic beads (MB 小 -COOH), and the small magnetic bead-bovine serum albumin-enrofloxacin complex (MB 小 -BSA-ENR). As can be seen from the figure, BSA-ENR has a protein characteristic absorption peak at 278 nm, MB 小 -BSA-ENR also has a characteristic peak at 278 nm, and there is no such peak in the determination result of MB 小 -COOH, so it can be shown that the MB 小 -BSA-ENR complex is successfully prepared.

[0070] 3) Detection of enrofloxacin in buffer

[0071] 3.1) Establishing standard curve model between enrofloxacin concentration and OD 405 ;

[0072] 3.1.1) Prepare a series of enrofloxacin solutions with known concentrations (10 -2 ~ 10 4 ng / mL) in phosphate buffer with pH of 6.0.

[0073] 3.1.2) Mix 40 μL of phosphate dispersion of IMB 大 containing 0.08 mg of bovine serum albumin blocking (concentration of 2 mg / mL) obtained in step 1.4) with 160 μL of solution containing different enrofloxacin concentrations (10 -2 ~ 10 4 ng / mL), and place on a rotary mixer for reaction at room temperature for 10 min. After reaction, the mixed solution is subjected to magnetic separation and removal of washing supernatant in sequence with pH 6.0 phosphate buffer and repeated for 3 times, and then dispersed in 178 μL of phosphate buffer to obtain a mixed solution of surface-modified enrofloxacin immune magnetic nanoparticles (IMB 大 -ENR) and unmodified enrofloxacin magnetic nanoparticles (IMB 大 -Non ENR);

[0074] 3.1.3) Take the mixed solution obtained in step 3.1.2), and add 22 μL of phosphate buffer dispersion of MB 小 -BSA-ENR containing bovine serum albumin blocking with a concentration of 0.4 mg / mL, and place on a rotary mixer for reaction at room temperature for 10 min. After magnetic separation and resuspension (without removing supernatant), immediately take the supernatant after magnetic separation at room temperature for 10 s, and measure OD 405 value.

[0075] 3.1.4) Establish standard curve by fitting the OD 405 value obtained in the above step 3.1.3) with the known enrofloxacin concentration in the sample.

[0076] The results of the detection of 11 known different enrofloxacin concentrations in this example and the sub-example are shown in the following table, and the detection parameters and performance of the application applied to 10 concentration examples are shown in the following table:

[0077]

[0078] The linear detection range of the present embodiment is 1-100 ng / mL, the linear regression equation is y=0.3822logx+1.0808 (r=0.992), and the detection limit is 0.18 ng / mL (as shown in (A) of FIG. 6 and (B) of FIG. 7). 2 Figure 6 Figure 6

[0079] As can be seen from (B) of FIG. 6, the color depth of the supernatant increases with the increase of the enrofloxacin concentration, and therefore, the method of the present embodiment can be used for visual detection of the enrofloxacin concentration. Figure 6

[0080] 4) Detection of enrofloxacin in fish tissue extract

[0081] 4.1) Pretreatment of fish sample

[0082] 4.1.1) Weigh 3 g of chopped sample into a centrifuge tube;

[0083] 4.1.2) Add 6 g of anhydrous sodium sulfate and 18 mL of acetonitrile-1% FA (formic acid) solution in two portions to the sample of step 4.1.1), vortex for 10 minutes to obtain a mixed solution containing the aquatic product tissue;

[0084] 4.1.3) Centrifuge the mixed solution containing the aquatic product tissue obtained in step 4.1.2) at 8000 rpm at room temperature for 8 min to obtain a supernatant;

[0085] 4.1.4) Take 6 mL of the supernatant obtained in step 4.1.3), dry it under nitrogen at 50°C, then add 1 mL of phosphate buffer with pH 6.0, vortex for 2 min, and filter it through a microporous filter membrane with a pore size of 0.22 μm to obtain an aquatic product tissue extract, which is stored at 4°C for standby use;

[0086] 4.2) Establish a standard curve of enrofloxacin concentration in fish tissue extract versus OD 405 ;

[0087] 4.2.1) Prepare a series of enrofloxacin solutions with known concentrations (10 -2 -10 4 ng / mL) in fish tissue extract.

[0088] 4.2.2) Mix 40 μL of the IMB 大 containing 0.08 mg of bovine serum albumin blocking solution with 160 μL of the phosphate dispersion containing different enrofloxacin concentrations (10 -2 -10 4 ​​​​ng / mL) of fish tissue extract, placed on a rotary mixer for reaction at room temperature for 10 min, the reaction mixture was subjected to sequential magnetic separation and removal of the washing supernatant using a phosphate buffer solution with a pH of 6.0, and the treatment was repeated three times, and then dispersed in 178 μL of phosphate buffer solution to obtain immunomagnetic nanoparticles (IMB) surface-modified with enrofloxacin. 大 -ENR) and unmodified enrofloxacin magnetic nanoparticles (IMB 大 -Non ENR) composed of a mixed solution;

[0089] 4.2.3) Take the mixture obtained in step 4.2.2) and add 22 μL of MB containing bovine serum albumin blocking 小 -BSA-ENR phosphate buffer dispersion was placed on a rotary mixer for 10 min at room temperature, magnetically separated and resuspended (without removing the supernatant), and magnetically separated at room temperature for 10 s and the supernatant was immediately removed to measure OD 405 value.

[0090] 4.2.4) The OD obtained in step 4.2.3) above was 405 The standard curve was established by fitting the values ​​with the known concentrations of enrofloxacin in the samples; the obtained standard curve is shown in Figure 7 ;

[0091] 4.3) After the aquatic product sample to be tested is treated according to step 4.1), an aquatic product extract is obtained. Enrofloxacin solution of known concentration is added to the aquatic product extract (the final enrofloxacin concentrations in the aquatic product extract are: 4 ng / mL, 40 ng / mL, and 80 ng / mL, respectively). Then, the sample is treated according to steps 4.2.1) to 4.2.4) to obtain absorbance values, which are compared with the standard curve model ( Figure 7 ) to obtain the enrofloxacin concentration in the aquatic product sample to be tested. The test results and accuracy are shown in the following table:

[0092]

[0093] The average recovery rate of the method of the present invention in fish samples ranged from 81.9% to 94.7% (an average recovery rate of 100% is considered the most accurate, while those skilled in the art consider a recovery rate between 80% and 120% to be good). This demonstrates that the nanobiosensor of the present invention, which utilizes the dual functions of magnetic nanoparticle magnetic separation and signal output, is well suited for the detection of enrofloxacin in aquatic product samples.

[0094] 5) Detection of Enrofloxacin in Pond Water Samples

[0095] 5.1) Pond water sample pretreatment

[0096] 5.1.1) The pond water sample was precipitated for 48h, filtered by micropore filter membrane with pore size of 0.22 μm, then 10% volume of 100 mmol / L phosphate buffer was added to obtain the extract of the pond water sample, which was stored at 4℃ for standby.

[0097] 5.2) The standard curve between the concentration of enrofloxacin in the extract of the pond water sample and OD 405 was established

[0098] 5.2.1) A series of enrofloxacin solutions with known concentrations (10 -2 ~ 10 4 ng / mL) were prepared in the extract of the pond water sample;

[0099] 5.2.2) The standard curve was prepared according to the steps of steps 4.2.2)~4.2.4), and the model of the standard curve obtained is shown in Figure 8 .

[0100] 5.3) The extract of the pond water sample was obtained by treating the pond water sample to be detected according to step 5.1), and a known concentration of enrofloxacin solution was added to the extract of the pond water sample (the final concentration of enrofloxacin in the extract of the pond water sample was 4 ng / mL, 40 ng / mL and 80 ng / mL, respectively), then the absorbance value was obtained by treating according to steps 4.2.1)~4.2.4), and the concentration of enrofloxacin in the extract of the pond water sample to be detected was obtained by comparing with the model of the standard curve obtained in step 5.2) Figure 8 , as shown in the following table:

[0101]

[0102] The average recovery rate of the method of the present application in fish meat sample detection is between 85.9% and 95.7% (the average recovery rate of 100% is the most accurate, and the skilled person in the art considers that 80%~120% is good). Therefore, it can be illustrated that the nano-biosensor based on magnetic nanoparticle magnetic separation-signal output dual function of the present application can be better applied to the detection of enrofloxacin in pond water samples.

[0103] 6) Compared with the existing rapid detection method of enrofloxacin, the results are as follows:

[0104]

[0105] The detection time of the method is much less than that of enzyme-linked immunosorbent assay and colloidal gold immunofiltration method, and the detection limit is much lower than that of Raman spectroscopy.

[0106] From the above examples, the application provides a kind of enrofloxacin visual rapid detection method based on magnetic bead magnetic separation-signal output dual function, sensitive, fast, simple operation, without complex precision instrument, with the potential of on-site rapid detection enrofloxacin, it is expected to become a kind of on-site rapid detection means, with good development prospect.

[0107] The above detailed description is used to explain and illustrate the present application, rather than limit the present application, any modification and change made to the present application within the spirit and protection scope of the claims, fall into the protection scope of the present application. The above is only the preferred embodiment of the present application, therefore, equivalent changes or modifications made to the structure, features and principles described in the scope of the present application are included in the scope of the present application.

Claims

1. A visual rapid detection method for enrofloxacin based on the dual functions of magnetic separation and signal output of magnetic beads, characterized by: The following steps are involved: 1) activating the carrier magnetic beads to obtain activated carrier magnetic beads, then modifying the surface of the activated carrier magnetic beads with enrofloxacin monoclonal antibodies, and blocking to obtain an immunomagnetic bead dispersion; The carrier magnetic beads are carboxylated magnetic beads with a particle size of 100-1000 nm; 2) activating the pre-labeled magnetic beads to obtain activated pre-labeled magnetic beads, then coupling the enrofloxacin-bovine serum albumin complex to the activated pre-labeled magnetic beads, and blocking to obtain a labeled magnetic bead dispersion; The pre-labeled magnetic beads are carboxylated magnetic beads with a particle size of 10 to 50 nm; 3) mixing the immunomagnetic bead dispersion with the sample solution to be tested, rotating and mixing at room temperature, and after the reaction is completed, performing magnetic separation and washing, and then dispersing in phosphate buffer to obtain a mixed dispersion; 4) mixing the labeled magnetic bead dispersion and the mixed dispersion, rotating and mixing at room temperature to react. After the reaction is completed, the supernatant is obtained by magnetic separation and the absorbance of the supernatant is detected at a specific wavelength. The absorbance of the supernatant at the specific wavelength is the detection value; the specific wavelength is 405 nm; 5) Substituting the detected value into the pre-drawn standard curve, the concentration of enrofloxacin in the sample solution to be detected is obtained.

2. The method for rapid visualization of enrofloxacin based on the dual functions of magnetic separation and signal output of magnetic beads according to claim 1, characterized in that: In the step 4), after the reaction is completed, one or two enrichment treatments are performed, and then magnetic separation is performed to obtain a clear liquid; the process of the enrichment treatment is: after magnetic separation, the precipitate is resuspended in the clear liquid.

3. The method for rapid visualization of enrofloxacin based on the dual functions of magnetic separation and signal output of magnetic beads according to claim 1, characterized in that: In the step 1), the mass ratio of the activated carrier magnetic beads to the enrofloxacin monoclonal antibody is 1:0.02-0.1; in the step 2), the mass ratio of the activated pre-labeled magnetic beads to the enrofloxacin-bovine serum albumin complex is 4:

3.

4. The method for rapid visualization detection of enrofloxacin based on the dual functions of magnetic separation and signal output of magnetic beads according to claim 1, characterized in that: The mass ratio between the immunomagnetic beads in step 3) and the labeled magnetic beads in step 4) is 100:5-15.

5. The method for rapid visualization detection of enrofloxacin based on the dual functions of magnetic separation and signal output of magnetic beads according to claim 1, characterized in that: The concentration of enrofloxacin in the sample solution to be detected is 1-100 ng / mL, and the detection limit is 0.18 ng / mL.

6. The method for rapid visualization detection of enrofloxacin based on the dual functions of magnetic separation and signal output of magnetic beads according to claim 1, characterized in that: The step 1) comprises the following steps: 1.1) washing the carrier magnetic beads with MES buffer solution, dispersing the washed carrier magnetic beads in an activation solution, and reacting at room temperature to obtain an activated carrier magnetic bead dispersion; 1.2) separating the activated carrier magnetic beads from the activated carrier magnetic bead dispersion by magnetic separation, washing the activated carrier magnetic beads with a borate buffer, and dispersing the washed activated carrier magnetic beads in a borate buffer to obtain a borate dispersion of the activated carrier magnetic beads; 1.3) Mixing the borate dispersion of activated carrier magnetic beads with the enrofloxacin monoclonal antibody solution, followed by reaction at room temperature for 1-4 hours to obtain a borate dispersion of antibody-carrier magnetic beads; 1.4) adding a 1% to 5% by mass bovine serum albumin solution to the borate dispersion of the antibody-carrier magnetic beads and reacting at room temperature for 1 to 4 hours to obtain a borate dispersion of immunomagnetic beads; separating the immunomagnetic beads from the borate dispersion of the immunomagnetic beads by magnetic separation, washing the immunomagnetic beads with a phosphate buffer, and dispersing the washed immunomagnetic beads in a phosphate buffer to obtain an immunomagnetic bead dispersion.

7. The method for rapid visualization detection of enrofloxacin based on the dual functions of magnetic separation and signal output of magnetic beads according to claim 1, characterized in that: According to parts by mass, the step 2) comprises the following steps: 2.1) 2 parts of enrofloxacin, 1 part of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.25 parts of N -Hydroxysuccinimide sulfonic acid sodium salt is dissolved in N , N -dimethylformamide, react at room temperature to obtain solution A; dissolve 5 parts of bovine serum albumin in phosphate buffer to obtain solution B; add solution A dropwise to solution B, stir and react at room temperature, dialyze and centrifuge, and then collect the supernatant to obtain an enrofloxacin-bovine serum albumin complex solution; 2.2) washing the pre-labeled magnetic beads with MES buffer solution, dispersing the washed pre-labeled magnetic beads in an activation solution, and reacting them at room temperature to obtain an activated pre-labeled magnetic bead dispersion; 2.3) separating the activated pre-labeled magnetic beads from the activated pre-labeled magnetic bead dispersion by magnetic separation, washing the activated pre-labeled magnetic beads with a borate buffer, and dispersing the washed activated pre-labeled magnetic beads in a borate buffer to obtain a borate dispersion of the activated pre-labeled magnetic beads; 2.4) mixing the activated pre-labeled borate dispersion of magnetic beads with the enrofloxacin-bovine serum albumin complex solution, and reacting them at room temperature to obtain a borate dispersion of unblocked labeled magnetic beads; 2.5) adding a 1% to 5% by mass bovine serum albumin solution to the borate dispersion of the unblocked labeled magnetic beads, and reacting at room temperature to obtain a borate dispersion of the labeled magnetic beads; separating the labeled magnetic beads from the borate dispersion of the labeled magnetic beads by magnetic separation, washing the labeled magnetic beads with a phosphate buffer solution, washing for times, and dispersing the washed labeled magnetic beads in a phosphate buffer solution to obtain a labeled magnetic bead dispersion.

8. The method for rapid visualization of enrofloxacin based on the dual functions of magnetic separation and signal output of magnetic beads according to claim 1, characterized in that: The process of establishing the pre-drawn standard curve is as follows: according to step 3) to step 4), enrofloxacin standard solutions of different concentrations are treated respectively to obtain the detection value corresponding to each enrofloxacin standard solution, and the concentration of the enrofloxacin standard solution and the corresponding detection value are linearly fitted to obtain a standard curve.

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