CN111551724A - Fluorescent probe, method for detecting tetracycline and application - Google Patents
Fluorescent probe, method for detecting tetracycline and application Download PDFInfo
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Abstract
The invention discloses a fluorescent probe, a method for detecting tetracycline and application of the fluorescent probe, and the fluorescent probe comprises a tetracycline monoclonal antibody and a signal carrier, wherein the signal carrier is an amino-functionalized aluminum nanosheet, and the particle size of the amino-functionalized aluminum nanosheet is 520-570 nm. The invention firstly constructs the probe by using the fluorescent metal organic framework material in the immunochromatographic test strip detection, and develops a simple, novel, sensitive and rapid analysis system for detecting tetracycline by using actual samples such as meat, milk, honey and the like. The provided test strip has the lowest detection limit of 0.0516ng/mL for tetracycline, and the value is lower than that reported by other documents.
Description
Technical Field
The invention belongs to the field of biological detection, and relates to a fluorescent probe, a tetracycline detection method and application, in particular to a probe which is marked by a fluorescent metal organic framework material and is enriched with a monoclonal antibody, a tetracycline detection method by using a test strip of the probe, and application of the probe in rapid and sensitive tetracycline detection.
Background
Tetracycline (TET), a broad spectrum antibiotic, has been widely used as a feed additive into the food chain, and long-term ingestion of foods containing tetracycline residues can cause allergic reactions in humans, gastrointestinal discomfort, and even liver toxicity. An immunochromatographic test strip (LFIA) is the most common instant detection and diagnosis tool and is used for detecting various mycotoxins, proteins, antibiotics, heavy metals and the like.
Currently, gold nanoparticle (AuNPs) labeled immunochromatographic test strips are mainly used in methods for detecting tetracycline, and the test strips are considered as gold standards for detecting targets. However, the traditional gold-labeled immunochromatography method is unable to be applied to ultra trace analysis due to limitations such as low sensitivity. In addition, various Fluorescent Materials (FMs), such as magnetic Quantum Dots (QDs), Carbon Dots (CDs), fluorescent dyes, and Metal Nanoclusters (MNC), are used as signal carriers to improve sensitivity of LFIA due to their characteristics of strong fluorescence signal, adjustable optical frequency, and chemical stability. However, when the fluorescent material is used as a signal carrier for tetracycline detection, a cross-linking agent is usually required to label an antibody (Abs), so that the current fluorescent test strip (FLIFA) has three limitations in tetracycline detection: (1) the labeling process is rather complex and the fluorescence signal may be affected; (2) since Abs contains both carboxyl and amino groups, self-crosslinking may be induced, resulting in waste of Abs and possibly even reduced activity. (3) Due to the small size of the above fluorescent materials, it is difficult to purify and enrich the prepared probes after labeling the antibodies, which may cause interference due to non-specific binding.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the invention aims to solve the problem of complex crosslinking process of the antibody and the signal carrier, and constructs a fluorescent carrier to enrich the signal substance, the fluorescent probe has high fluorescence intensity and stable chemical/physical properties, and the conjugated antibody does not need complex crosslinking agent, so that the fluorescent probe has important significance and application value for monitoring residual tetracycline in food, namely the fluorescent probe, the method for detecting tetracycline and the application.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
a fluorescent probe comprises a tetracycline monoclonal antibody and a signal carrier, wherein the signal carrier is an amino-functionalized aluminum nanosheet, and the particle size of the amino-functionalized aluminum nanosheet is 520-570 nm.
Further, the preparation method of the fluorescent probe comprises the steps of preparing an amino-functionalized aluminum nanosheet by a hydrothermal method, and then adding a tetracycline monoclonal antibody for adsorption to obtain the tetracycline fluorescent probe.
Further, the method for preparing the fluorescent probe comprises the following steps:
(1) preparing an amino-functionalized aluminum nanosheet: mixing aluminum chloride and diamino terephthalic acid for reaction, then adding a urea solution, stirring, transferring the mixture into a high-pressure reaction kettle, placing the high-pressure reaction kettle in an oven for reaction, and then washing and centrifuging to obtain the product;
(2) preparing a fluorescent probe: and (2) adding the tetracycline monoclonal antibody into the amino-functionalized aluminum nanosheet solution obtained in the step (1), mixing, sealing with bovine serum albumin, and centrifugally suspending in water to obtain the tetracycline nano-antibody.
Specifically, in the step (1), the concentration ratio of the aluminum chloride to the diamino terephthalic acid is 1:1, and the concentration of the urea solution is 0.8-1.0 mol/L.
Specifically, the mixing ratio of the tetracycline monoclonal antibody to the amino-functionalized aluminum nanosheet is 0.8-1.2 mug: 30-70 muL, and the concentration of the amino-functionalized aluminum nanosheet solution is 0.8-1.2 mg/mL;
in the step (2), the mixing time is 0.8-1.2 h, the final concentration of bovine serum albumin is 1%, and the sealing time is 20-40 min.
A method for detecting tetracycline comprises the step of immersing a test strip into a fluorescent probe containing the tetracycline for detection.
Further, the test paper strip include the welt, the welt is pasted with the cellulose nitrate membrane, the one end cover of cellulose nitrate membrane absorbs water the pad, the other end of cellulose nitrate membrane covers sample pad and combination pad in proper order, set up detection line and control line along transversely on the non-cover face of cellulose nitrate membrane, combination pad and sample pad are respectively through confining liquid sealing treatment.
Specifically, the preparation method of the nitrocellulose membrane comprises the following steps: coating the 1mg/mL tetracycline-bovine serum albumin conjugate on a detection line at a scribing rate of 1 mu L/cm to obtain a detection line, and coating the 1mg/mL goat anti-mouse immunoglobulin on a control line at a scribing rate of 1 mu L/cm to obtain a control line;
the preparation method of the sample pad and the combined pad comprises the following steps: soaking the glass fiber membrane in 2% of sealing liquid, and drying for 8-10 h at 36-37 ℃;
the specification of sample pad combination pad be length 13 ~ 18mm, wide 2 ~ 4mm, the specification of combination pad be length 7 ~ 9mm, wide 2 ~ 4 mm.
The fluorescent probe is applied to detecting tetracycline in beef, chicken, milk and honey.
The method for detecting tetracycline is applied to detecting tetracycline in beef, chicken, milk and honey.
Compared with the prior art, its advantage lies in with positive effect:
(1) breaking the traditional complex cross-linking process. The invention only carries out simple adsorption on the fluorescent metal organic framework material and the antibody to prepare the novel fluorescent probe, and solves the problem of the traditional complex crosslinking (such as EDC/NHS) method of the antibody and a signal carrier. Avoid the waste of antibody and maintain high fluorescence signal intensity.
(2) A novel probe. The probe is constructed by using a fluorescent metal organic framework material in the immunochromatographic test strip detection for the first time, and a simple, novel, sensitive and rapid analysis system is developed for detecting tetracycline by using actual samples such as meat, milk, honey and the like.
(3) The sensitivity is high. The test strip provided by the invention has the lowest detection limit of 0.0516ng/mL for tetracycline, and the value is lower than that reported in other documents.
(4) Good practical application. The method can be used for detecting the tetracycline in the beef, the chicken, the milk and the honey, has good application prospect, and can be used as a universal detection method for detecting various antibiotics.
Drawings
FIG. 1 is a schematic representation of an amino-functionalized aluminum nanoplatelet material prepared in accordance with the present invention;
FIG. 2 is a structural diagram of the immunochromatographic test strip and a detection principle diagram of the immunochromatographic test strip of the present invention;
FIG. 3 shows the detection sensitivity and specificity of the immunochromatographic test strip prepared by the present invention, wherein Control, OXY, CTE, AMX, FLO, AZM, STR and EM respectively represent blank, oxytetracycline, aureomycin, amoxicillin, florfenicol, azithromycin, streptomycin sulfate and erythromycin;
FIG. 4 is a comparison of the detection sensitivity of the immunochromatographic test strip prepared in the present invention and a conventional test strip;
FIG. 5 shows the practical application of the immunochromatographic test strip prepared in the present invention;
FIG. 6 is a validation of an amino-functionalized aluminum nanoplatelet material prepared in accordance with the present invention;
FIG. 7 is a verification of the novel fluorescent probe prepared by the present invention;
the following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Detailed Description
In order to solve the problem of complex cross-linking process of the antibody and the signal carrier, the invention constructs a novel fluorescent carrier to enrich the signal substance. The novel fluorescent probe has high fluorescence intensity and stable property, and has important significance and application value for monitoring residual tetracycline in food.
The amino-functionalized aluminum nanosheets of the present invention are synthesized from aluminum chloride and diaminoterephthalic acid by a one-step hydrothermal process, i.e., AlNPs, and the amino-functionalized aluminum nanosheets are synthesized by-NH2After the aluminum nano sheet is modified, the water solubility of the aluminum nano sheet can be effectively improved, the stability of fluorescence is enhanced, and the successful connection of the Abs is facilitated. The fluorescent probe has many excellent performances, a new platform is provided for labeling antibodies (Abs) by virtue of a unique structure (large specific surface area, multiple pores and strong adsorption of aluminum nanosheets), and the structure labels Abs by a simple adsorption method to obtain the fluorescent probe, thereby replacing a complex crosslinking process.
In order to obtain the best assay performance, the inventors optimized the concentration of AlNPs, the amount of Abs used, the volume of the novel fluorescent probe and the immunization time, and determined the optimal system conditions. The finally prepared test strip is used for detecting the residual tetracycline in food, and the method is successfully applied to the detection of the tetracycline in beef, chicken, milk and honey, so that the practicability, sensitivity and accuracy of the method are verified.
The AlNPs are easy to synthesize, have good fluorescence characteristics, hydration and stability, and have the characteristics of large specific surface area of porous materials, strong reagent loading capacity, adjustable pore size and modifiable outer surface, so that the surfaces of the AlNPs can contain a large number of amino functional groups to be beneficial to activation and modification. Compared with other fluorescent substances such as magnetic quantum dots, carbon dots, fluorescent dyes, metal nanoclusters and the like, the AlNPs are simple and stable to synthesize, good in dispersity, easy to modify and mark antibodies, and capable of avoiding complex cross-linking processes. Therefore, the "fluorescent carrier" in the present invention refers to an aluminum nanosheet functionalized with a fluorescent amino group as a carrier for labeling Abs, and the fluorescent probe is obtained after the Abs is adsorbed.
The working principle of the test strip is as follows: based on the competitive detection principle, firstly, a novel AlNPs-Ab fluorescent probe is added to a sample to be actually detected to capture target tetracycline (TET), and the bound AlNPs-Ab-TET immune complex moves to a test area of a test strip through capillary action. For positive samples, the AlNPs-Ab probe failed to show a visible band on the detection line (when the TET concentration was high enough) or a band brighter than the blank control band (when the TET concentration was low). In contrast, for negative samples, the AlNPs-Ab probe will be intercepted by TET-BSA in the detection line, showing a visible fluorescent band under UV light.
The method for preparing the fluorescent probe comprises the following steps:
(1) preparing an amino-functionalized aluminum nanosheet: mixing aluminum chloride and diamino terephthalic acid for reaction, then adding a urea solution, stirring, transferring the mixture into a high-pressure reaction kettle, washing and centrifuging to obtain the product; the concentration ratio of the aluminum chloride to the diamino terephthalic acid is 1:1, and the concentration of the urea solution is 0.8-1 mol/L.
(2) Preparing a fluorescent probe: adding a tetracycline monoclonal antibody into the amino-functionalized aluminum nanosheet solution obtained in the step (1), mixing, sealing with bovine serum albumin, centrifuging, and suspending in water to obtain the tetracycline nano-antibody-amino-functionalized aluminum nanosheet solution, wherein the mixing ratio of the tetracycline monoclonal antibody to the amino-functionalized aluminum nanosheet is 0.8-1.2 mug: 30-70 muL, and the concentration of the amino-functionalized aluminum nanosheet solution is 0.8-1.2 mg/mL; the mixing time is 0.8-1.2 h, the final concentration of bovine serum albumin is 1%, and the sealing time is 20-40 min.
The immunochromatographic test strip is composed of five parts, wherein a nitrocellulose membrane, a sample pad, a combination pad and an absorption pad are sequentially attached to a lining plate, wherein tetracycline-bovine serum albumin conjugate (TET-BSA) and goat anti-mouse immunoglobulin (IgG) are coated on the nitrocellulose in a marking manner and are respectively used as a detection line (T) and a control line (C).
The experimental reagents used in the invention are all obtained from markets, no further treatment is carried out, and detection instruments and equipment and the like are all common instruments.
Example 1:
according to the technical scheme, the fluorescent probe AlNPs-Ab and the preparation method thereof are provided, and the preparation method comprises the steps of preparing an amino-functionalized aluminum nanosheet serving as a signal carrier by a hydrothermal method, and then adding a tetracycline monoclonal antibody for adsorption to obtain the tetracycline monoclonal antibody. The method comprises the following steps:
(1) preparation of amino-functionalized aluminum nanoplates (AlNPs): mixing aluminum chloride and diamino terephthalic acid for reaction, then adding a urea solution, stirring, transferring the mixture into a high-pressure reaction kettle, washing and centrifuging to obtain the product;
the method specifically comprises the following steps: mixing AlCl3And diaminoterephthalic acid (NH)2-BDC) at a concentration ratio of 1:1 was added to 15mL of deionized water and mixed thoroughly and the reaction was continued for 30 min. Subsequently, a urea solution (0.96mol/L, 5mL) was added dropwise with vigorous stirring, and stirring was continued for 30 min. Then, the mixture was transferred to an autoclave and reacted at 150 ℃ for 5 hours, and after cooling to room temperature, it was centrifuged at 8500r/min for 10 minutes to obtain a yellow powder, which was washed with ultrapure water. Thereafter, the product was redispersed in 20mL of N, N-Dimethylformamide (DMF) and stirred at room temperature under dark conditions for 12h, then under the same conditions methanol was used instead of DMF for further 12 h. Finally, the prepared AlNPs were centrifuged and resuspended in ultrapure water, vacuum dried overnight at 70 ℃, and stored in a 4 ℃ refrigerator.
(2) Preparing a fluorescent probe: and (2) adding the tetracycline monoclonal antibody into the amino-functionalized aluminum nanosheet solution obtained in the step (1), mixing, sealing with bovine serum albumin, and centrifugally suspending in water to obtain the tetracycline nano-antibody.
Add 500. mu.L of AlNPs (1.0mg/mL) and 10. mu.g of anti-tetracycline Abs (1.0mg/mL) to a 1.5mL centrifuge tube and react for 1h to allow sufficient binding. Subsequently, 50. mu.L of 10% BSA was added and vortexed for 30 min. Finally, the AlNPs-Ab probe of the invention was obtained by centrifugation at 10000r/min for 10min and then resuspended in 500. mu.L of ultrapure water and stored at 4 ℃ for further use.
The tetracycline detecting probe used in examples 2-4 described below was prepared in example 1.
Example 2:
the embodiment provides a high sensitivity immunochromatographic test strip for rapidly detecting tetracycline, which comprises a lining plate, wherein a nitrocellulose membrane is pasted on the lining plate, one end of the nitrocellulose membrane covers a water absorption pad, the other end of the nitrocellulose membrane covers a sample pad and a combination pad in sequence, a detection line and a control line are transversely arranged on a non-covering surface of the nitrocellulose membrane, and the combination pad and the sample pad are respectively subjected to sealing treatment by a sealing liquid.
The preparation method of the nitrocellulose membrane comprises the following steps: coating the 1mg/mL tetracycline-bovine serum albumin conjugate on a detection line at a scribing rate of 1 mu L/cm to obtain a detection line, and coating the 1mg/mL goat anti-mouse immunoglobulin on a control line at a scribing rate of 1 mu L/cm to obtain a control line; then dried at 37 ℃ for use.
Preparation of sample pad: cutting the glass fiber membrane into pieces with length of 15mm and width of 3mm, soaking in blocking solution (2% BSA), drying at 37 deg.C for 8 hr to obtain sample pad, and storing in refrigerator at 4 deg.C.
Preparation of the bonding pad: cutting the glass fiber membrane into pieces with length of 8mm and width of 3mm, soaking in blocking solution (2% BSA), taking out, drying at 37 deg.C for 8 hr to obtain sample pad, and storing in refrigerator at 4 deg.C.
Cutting the absorbent paper into pieces with the length of 18mm and the width of 3mm to obtain the absorbent pad.
Assembling the test strip: firstly, attaching the nitrocellulose membrane to a lining plate, pressing the sample pad by 2mm, pressing the nitrocellulose membrane by 2mm, and sequentially attaching the nitrocellulose membrane by 2mm through the water absorption pad to the lining plate, thereby obtaining the immunochromatographic test strip for rapidly detecting tetracycline.
Example 3: sensitivity determination of test strip for rapid detection of tetracycline
The performance evaluation of the immunochromatographic test strip for rapidly detecting tetracycline is described above.
And (3) detection process: tetracycline standards were dissolved in ultrapure water and serially diluted to varying concentrations ranging from 0 to 12.0ng/mL test solutions, with the ultrapure water being the blank. 0.5. mu.L of AlNPs-Ab probe was mixed with 100. mu.L of tetracycline standard solution and incubated, and thenThe sample pad of the test strip was immersed in 100 μ L of the test solution and the mixture migrated by capillary action toward the absorbent pad. After 15min of reaction, the strip was scanned using a fluorescent strip reader to obtain the Fluorescence Intensity (FI). Using FI ratio (FI) of detection line (T line) to control line (C line)T/FIC) The method is used for quantitative analysis, and can be used for offsetting the difference between test strips and minimizing environmental factors influencing FI.
And (3) detection results: when the T-line was visibly lighter than the negative control bar, the corresponding minimum concentration of tetracycline was defined as the Visual Detection Limit (VDL), and when the T-line disappeared completely, the corresponding minimum concentration was taken as the threshold concentration. Competitive inhibition ratio IC10Defined as limit of detection (LOD).
As shown in FIG. 3, as the concentration of tetracycline increases, the fluorescence of the T line of the test strip becomes shallower and FIT/FICThe values decrease with increasing tetracycline concentration. VDL was 0.375ng/mL and the threshold concentration at which the T-line disappeared completely was 6 ng/mL. IC by calculation of AlNPs-FLFIA50The value was 0.163. + -. 0.029ng/mL, while the LOD was 0.0516. + -. 0.053 ng/mL. The sensitivity is higher than that reported in other literatures (see table 1) and is 29 times higher than that of the traditional test strip (see fig. 4). Therefore, the method can detect tetracycline with high sensitivity, and can be used as a general method for quickly and conveniently detecting the antibiotic residue in food. Table 1 shows the comparison of the detection sensitivity of the immunochromatographic test strip prepared by the invention, and it can be seen that the immunochromatographic test strip (AlNPs-FLFIA) prepared by the invention has the lowest limit of detection (LOD) of 0.0516, and has better effect than other existing detection methods.
Table 1 shows the comparison of the detection sensitivity of the immunochromatographic test strip prepared in the present invention
Example 4: specificity determination of test strip for rapidly detecting salmonella enteritidis
The performance evaluation of the immunochromatographic test strip for rapidly detecting tetracycline is described above.
And (3) detection process: respectively mixing Oxytetracycline (OXY) and chlortetracycline (A), (B), (C)CTE), Amoxicillin (AMX), Florfenicol (FLO), Azithromycin (AZM), streptomycin Sulfate (STR) and Erythromycin (EM) are diluted to the concentration of 100ng/mL with ultrapure water, 100 μ L of the solution is taken as a detection solution respectively, mixed and incubated with 0.5 μ L of AlNPs-Ab probe, then the sample pad of the test strip is immersed in 100 μ L of the test solution, and 100 μ L of ultrapure water is taken as a blank control solution at the same time. After 15min, the strip was scanned using a fluorescent strip reader to obtain the Fluorescence Intensity (FI), using the FI ratio (FI) of the detection line (T line) to the control line (C line)T/FIC) For specific analysis.
As shown in FIG. 3, the sample only doped with tetracycline causes a significant decrease in fluorescence intensity of the detection line, and the sample added with oxytetracycline and chlortetracycline causes a slight decrease in fluorescence intensity of the detection line due to the fact that oxytetracycline and chlortetracycline belong to tetracycline antibiotics, whereas a strong fluorescence band can be observed on the detection line for other common antibiotics. The invention can recognize tetracycline with high specificity and has good specificity.
Example 5: application of test strip for rapidly detecting tetracycline
The performance evaluation of the immunochromatographic test strip for rapidly detecting tetracycline is described above.
And (3) detection process: beef, chicken, milk and honey samples (tetracycline free as confirmed by High Performance Liquid Chromatography (HPLC)) were spiked with tetracycline. For beef and chicken samples, 1g each was placed in a 15mL centrifuge tube containing 2mL of a 3% aqueous solution of trichloroacetic acid, vortexed for 5min, and then centrifuged (10,000rpm, 10 min). Subsequently, the supernatant was adjusted to neutrality with NaOH (1M) solution. Finally, the obtained supernatant was diluted 20 times with ultrapure water.
For milk and honey samples, 1mL of this was placed in a 15mL centrifuge tube containing 1mL of 3% trichloroacetic acid solution, vortexed for 5min, and then centrifuged (10,000rpm, 10 min). Subsequently, the supernatant was adjusted to neutrality with a NaOH (1M) solution and diluted 10-fold with ultrapure water.
Diluting the treated actual sample solution to different times (tetracycline concentration is 0-12ng/mL), taking 100 μ L of solution as detection solution, mixing and incubating with 0.5 μ L of AlNPs-Ab probe,the sample pad of the test strip was then immersed in 100. mu.L of the test solution, while 100. mu.L of ultrapure water was taken as a blank control solution. After 15min, the strip was scanned using a fluorescent strip reader to obtain the Fluorescence Intensity (FI), using the FI ratio (FI) of the detection line (T line) to the control line (C line)T/FIC) For specific analysis.
See FIG. 5: for beef, chicken and milk samples, the Visual Detection Limit (VDL) under ultraviolet light is 0.375ng/mL, and the detection result is consistent with that of tetracycline labeling samples, which strongly indicates that the method can specifically detect tetracycline. For the honey samples, the VDL was 0.75ng/mL, indicating that the sensitivity of the technique was minimally affected by the honey matrix. In conclusion, the sensitivity of the beef, chicken and milk samples to AlNPs-LFIA is consistent with that of the tetracycline labeling samples, and the good practical application value of the samples is reflected.
Example 6: characterization of amino-functionalized aluminum nanoplatelet material
In order to prove the good properties of the amino-functionalized aluminum nanosheet material prepared by the present invention, the present inventors also performed the following experiments (see fig. 6):
(1) scanning electron microscope and transmission electron microscope: and the images A and B are respectively a scanning electron microscope image and a transmission electron microscope image of AlNPs, are in a diamond nano plate shape, are complete in shape, have the average size of 545.97nm, and show that the AlNPs are large in specific surface area and easy to modify and activate.
(2) X-ray diffraction (XRD) spectrum and X-ray photoelectron spectrum (XPS): panel C, D are XRD and XPS spectra of AlNPs, respectively, and it can be seen that AlNPs have NH2Several characteristic peaks of MOF form MIL-53 and states that AlNPs are mainly composed of O, N, C and Al elements.
(3) Fourier transform infrared (FT-IR) spectroscopy: FIG. E is a Fourier transform infrared spectrum of AlNPs at 1000--1Two Al-O peaks were observed, which confirmed the oxygen atom and Al3+Has been successfully connected to NH2-BDC; in addition, due to the symmetric and asymmetric stretching vibrations of the N-H bond, the peaks of AlNPs appear at 3384 and 3500cm-1To (3). Meanwhile, since hydroxyl groups in the AlNPs are abundant and amino groups have good preservability, they have remarkable water dispersibility.
(4) Fluorescence spectrum (FL) and Excitation Emission Matrix (EEM) spectrum: FIGS. F and G are fluorescence spectrum (FL) and Excited Emission Matrix (EEM) spectrum of AlNPs, respectively, and it can be seen that AlNPs have an emission peak at 430nm and an excitation peak at 340nm, and exhibit blue fluorescence under an ultraviolet lamp.
(5) And (3) optimizing the condition of fluorescence intensity: the graph H shows the fluorescence intensity of AlNPs at different pH values, and it can be seen that the fluorescence intensity is kept at an almost constant level when the pH value is 1.0-6.0, and the fluorescence intensity is highest when the pH value is 7.0, and then has a significant tendency to decrease, so that the pH value of 7.0 is selected as the optimized pH value.
(6) And (3) stability testing: FIG. I shows the stability test results of AlNPs, and it can be seen that the fluorescence intensity of AlNPs has no significant change after being placed at room temperature for one month, indicating that AlNPs have good stability.
Example 7: characterization of novel fluorescent probes
In order to prove that the present invention successfully prepares a novel fluorescent probe, the present inventors also performed the following experiment (see fig. 7):
(1) ultraviolet-visible spectrum: panel A is a UV-visible spectrum of AlNPs, AlNPs-Ab and Abs, and it can be observed that the peak of the Abs changed from 280nm to 285nm after AlNPs modification, indicating successful labeling of Abs with AlNPs.
(2) zeta potential: panel B is a zeta potential diagram of AlNPs and AlNPs-Ab, and it can be seen that significant changes in zeta potential occurred in AlNPs (17.3mv) and AlNPs-Ab (3.62mv), indicating that Abs were successfully labeled on the surface of AlNPs.
(3) Fourier transform infrared (FT-IR) spectroscopy: FIG. C is a Fourier transform infrared spectrum of AlNPs, AlNPs-Ab and Abs, which shows that AlNPs are 1640--1And 1520 and 1530cm-1A distinct characteristic absorption peak is shown, indicating successful binding of Abs to AlNPs.
(4) Polyacrylamide gel electrophoresis: panel D shows the results of gel electrophoresis of AlNPs and AlNPs-BSA. It was found that when AlNPs were linked to BSA, a characteristic protein band appeared at 60kDa in AlNPs-BSA, but not in AlNPs. The results show that the protein can be successfully loaded on the AlNPs.
Claims (10)
1. The fluorescent probe is characterized by comprising a tetracycline monoclonal antibody and a signal carrier, wherein the signal carrier is an amino-functionalized aluminum nanosheet, and the particle size of the amino-functionalized aluminum nanosheet is 520-570 nm.
2. The fluorescent probe of claim 1, wherein the preparation method of the fluorescent probe comprises the steps of preparing an amino-functionalized aluminum nanosheet by a hydrothermal method, and then adding a tetracycline monoclonal antibody for adsorption.
3. The fluorescent probe of claim 1 or 2, prepared by a method comprising:
(1) preparing an amino-functionalized aluminum nanosheet: mixing aluminum chloride and diamino terephthalic acid for reaction, then adding a urea solution, stirring, transferring the mixture into a high-pressure reaction kettle, placing the high-pressure reaction kettle in an oven for reaction, and then washing and centrifuging to obtain the product;
(2) preparing a fluorescent probe: and (2) adding the tetracycline monoclonal antibody into the amino-functionalized aluminum nanosheet solution obtained in the step (1), mixing, sealing with bovine serum albumin, and centrifugally suspending in water to obtain the tetracycline nano-antibody.
4. The fluorescent probe according to claim 3, wherein in the step (1), the concentration ratio of the aluminum chloride to the diaminoterephthalic acid is 1:1, and the concentration of the urea solution is 0.8 to 1.0 mol/L.
5. The fluorescent probe according to claim 3, wherein the mixing ratio of the tetracycline monoclonal antibody to the amino-functionalized aluminum nanosheets is 0.8-1.2 μ g: 30-70 μ L, and the concentration of the amino-functionalized aluminum nanosheet solution is 0.8-1.2 mg/mL;
in the step (2), the mixing time is 0.8-1.2 h, the final concentration of bovine serum albumin is 1%, and the sealing time is 20-40 min.
6. A method for detecting tetracycline, which comprises immersing a test strip in a fluorescent probe according to any one of claims 1 to 5.
7. The method for detecting tetracycline according to claim 6, wherein the test strip comprises a backing plate, a nitrocellulose membrane is attached to the backing plate, one end of the nitrocellulose membrane covers a water absorbent pad, the other end of the nitrocellulose membrane sequentially covers a sample pad and a binding pad, a detection line and a control line are transversely arranged on a non-covered surface of the nitrocellulose membrane, and the binding pad and the sample pad are respectively subjected to sealing treatment by a sealing solution.
8. The method of detecting tetracycline of claim 7, wherein the method of making the nitrocellulose membrane comprises: coating the 1mg/mL tetracycline-bovine serum albumin conjugate on a detection line at a scribing rate of 1 mu L/cm to obtain a detection line, and coating the 1mg/mL goat anti-mouse immunoglobulin on a control line at a scribing rate of 1 mu L/cm to obtain a control line;
the preparation method of the sample pad and the combined pad comprises the following steps: soaking the glass fiber membrane in 2% of sealing liquid, and drying for 8-10 h at 36-37 ℃;
the specification of sample pad combination pad be length 13 ~ 18mm, wide 2 ~ 4mm, the specification of combination pad be length 7 ~ 9mm, wide 2 ~ 4 mm.
9. Use of the fluorescent probe of any one of claims 1 to 5 for the detection of tetracycline in beef, chicken, milk and honey.
10. Use of the method of detecting tetracycline according to any one of claims 6-8 for detecting tetracycline in beef, chicken, milk and honey.
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