CN111551724B - 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 thereof, comprising a tetracycline monoclonal antibody and a signal carrier, wherein the signal carrier is an amino-functionalized aluminum nano-sheet, and the particle size of the amino-functionalized aluminum nano-sheet is 520-570 nm. The invention constructs the probe by using the fluorescent metal organic framework material in the immunochromatography test strip detection for the first time, and the work develops a simple, novel, sensitive and quick analysis system for detecting the tetracycline for actual samples such as meat, milk, honey and the like. The lowest limit of detection of tetracycline by the provided test strip is 0.0516ng/mL, which is lower than that reported in many 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 thereof, in particular to a probe which is marked by a fluorescent metal organic framework material and enriches monoclonal antibodies, a method for detecting tetracycline by using a test strip of the probe, and application of the probe in rapid and sensitive detection of tetracycline.
Background
Tetracycline (TET) has been widely used as a feed additive into the food chain as a broad spectrum antibiotic, and long-term ingestion of foods containing tetracycline residues can cause allergic reactions, gastrointestinal discomfort, and even hepatotoxicity in humans. Immunochromatography test strips (LFIA) are the most common immediate detection diagnostic tools for detection of various mycotoxins, proteins, antibiotics, heavy metals, and the like.
Currently, gold nanoparticle (AuNPs) labeled immunochromatographic test strips are mainly used in the method for detecting tetracycline, and the test strips are regarded as gold standards for detecting target objects. However, the conventional gold-labeled immunochromatography method is not effective in 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 (MNCs), are used as signal carriers to improve the sensitivity of LFIA due to their strong fluorescent signals, adjustable optical frequencies, and chemical stability. However, the use of the fluorescent materials described above for signal carriers in the detection of tetracyclines generally requires the use of cross-linker labeled antibodies (Abs), such that current fluorescent test strips (FLIFA) have three limitations in the detection of tetracyclines: (1) The labeling process is quite complex and the fluorescent signal may be affected; (2) Since Abs contain both carboxyl and amino groups, self-crosslinking may occur, leading to wastage of Abs and even possible decrease of its activity. (3) Because of the small size of the fluorescent material, it is difficult to purify and enrich the prepared probe after labeling the antibody, which may cause non-specific binding to cause interference.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the invention constructs a fluorescent carrier to enrich the signal substances in order to solve the problem of complex crosslinking process of the antibody and the signal carrier, and the fluorescent probe has high fluorescence intensity, stable chemical/physical properties and no complex crosslinking agent required for binding the antibody, thus having important significance and application value for monitoring the residual tetracycline in food, namely comprising a fluorescent probe, a method for detecting the tetracycline and application.
In order to achieve the above 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 nano-sheet, and the particle size of the amino-functionalized aluminum nano-sheet is 520-570 nm.
Further, the preparation method of the fluorescent probe comprises the steps of preparing an amino-functionalized aluminum nano-sheet by a hydrothermal method, and then adding a tetracycline monoclonal antibody for adsorption.
Further, the method for preparing the fluorescent probe comprises the following steps:
(1) Preparing an amino-functionalized aluminum nano sheet: mixing aluminum chloride and diamino terephthalic acid for reaction, adding urea solution, stirring, transferring the mixture into a high-pressure reaction kettle, placing the high-pressure reaction kettle into an oven for reaction, washing and centrifuging to obtain the catalyst;
(2) Preparing a fluorescent probe: adding the tetracycline monoclonal antibody into the amino-functionalized aluminum nano-sheet solution in the step (1), mixing, sealing by bovine serum albumin, centrifuging and re-suspending in water to obtain the tetracycline monoclonal antibody.
Specifically, in the step (1), the concentration ratio of aluminum chloride to diamino terephthalic acid is 1:1, and the concentration of urea solution is 0.8-1.0 mol/L.
Specifically, the mixing ratio of the tetracycline monoclonal antibody and the amino-functionalized aluminum nano-sheet is 0.8-1.2 mug, 30-70 mug, and the concentration of the amino-functionalized aluminum nano-sheet 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 immersing a test strip in a fluorescent probe comprising the invention for detection.
Further, the test strip comprises a lining plate, a nitrocellulose membrane is stuck on the lining plate, one end of the nitrocellulose membrane is covered with a water absorption pad, the other end of the nitrocellulose membrane is sequentially covered with a sample pad and a combination pad, a detection line and a control line are transversely arranged on the non-covered surface of the nitrocellulose membrane, and the combination pad and the sample pad are respectively sealed by sealing liquid.
Specifically, the preparation method of the nitrocellulose membrane comprises the following steps: 1mg/mL of tetracycline-bovine serum albumin conjugate is coated on a detection line at a scribing rate of 1 mu L/cm to obtain a detection line, and 1mg/mL of goat anti-mouse immunoglobulin is coated 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 bonding pad comprises the following steps: soaking the glass fiber membrane in 2% sealing liquid, and drying at 36-37 deg.c for 8-10 hr;
the specification of the sample pad bonding pad is 13-18 mm long and 2-4 mm wide, and the specification of the bonding pad is 7-9 mm long and 2-4 mm wide.
The fluorescent probe is used for detecting tetracycline in beef, chicken, milk and honey.
The method for detecting the tetracycline is used for detecting the tetracycline in beef, chicken, milk and honey.
Compared with the prior art, the advantages and positive effects are that:
(1) Breaking the traditional complex crosslinking process. The novel fluorescent probe is prepared by simply adsorbing the fluorescent metal organic framework material and the antibody, so that the traditional complex crosslinking (such as EDC/NHS) method of the antibody and the signal carrier is solved. Avoiding the waste of the antibody and keeping high fluorescence signal intensity.
(2) Novel probes. The probe is constructed by fluorescent metal organic framework materials in the immunochromatography test strip detection for the first time, and a simple, novel, sensitive and rapid analysis system is developed for detecting tetracycline for actual samples such as meat, milk, honey and the like.
(3) The sensitivity is high. The minimum detection limit of the test strip provided by the invention on the tetracycline is 0.0516ng/mL, and the value of the test strip is lower than that reported in a plurality of other documents.
(4) Good practical application. The invention can detect the tetracycline in beef, chicken, milk and honey, has good application prospect, and can be used as a general detection method for detecting various antibiotics.
Drawings
FIG. 1 is a schematic representation of an amino-functionalized aluminum nanomaterial made in accordance with the present invention;
FIG. 2 is a configuration diagram and a detection schematic 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, terramycin, aureomycin, amoxicillin, florfenicol, azithromycin, streptomycin sulfate and erythromycin;
FIG. 4 is a comparison of detection sensitivity of an immunochromatographic test strip prepared by the present invention and a conventional test strip;
FIG. 5 shows the practical application of the immunochromatographic test strip prepared by the present invention;
FIG. 6 is a verification of amino-functionalized aluminum nanoplatelet materials prepared according to the present invention;
FIG. 7 is a verification of a novel fluorescent probe prepared according to the present invention;
the following describes the embodiments of the present invention in further detail with reference to the 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 for enriching the signal substances. 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 nano-sheet of the invention is synthesized by aluminum chloride and diamino terephthalic acid through a one-step hydrothermal method, namely AlNPs, and the amino-functionalized refers to the synthesis through-NH 2 After the aluminum nano-sheet is modified, the water solubility of the aluminum nano-sheet can be effectively improved, the fluorescence stability is enhanced, and the successful connection of Abs is facilitated. Which is a kind ofHas a plurality of excellent properties, and provides a new platform for labeling antibodies (Abs) by virtue of a unique structure (aluminum nano-sheet with large specific surface area, multiple voids and strong adsorption), and the structure can label 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 tetracycline remained in food, and the method is successfully applied to detection of the tetracycline in beef, chicken, milk and honey, and the practicability, the sensitivity and the accuracy are verified.
The AlNPs of the invention is easy to synthesize, has good fluorescence property, hydration property and stability, and has the characteristics of large specific surface area, strong reagent loading capacity, adjustable pore diameter and modifiable outer surface of porous materials, so that the surfaces of the porous materials can contain a large number of amino functional groups, thereby being 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 dispersibility, easy to modify and mark antibodies, and capable of avoiding complex crosslinking processes. Thus, the "fluorescent carrier" in the present invention means that the fluorescent probe is obtained by adsorbing Abs based on fluorescent amino-functionalized aluminum nanoplatelets as carriers for labeling Abs.
The working principle of the test strip is as follows: based on the principle of competitive detection, first, an AlNPs-Ab novel fluorescent probe is added to the sample to be actually detected to capture the target tetracycline (TET), and the bound AlNPs-Ab-TET immune complex moves to the test area of the test paper by capillary action. For positive samples, the AlNPs-Ab probe failed to appear as a visible band on the detection line (when the TET concentration was high enough) or as a band brighter than the blank (when the TET concentration was low). In contrast, for negative samples, the AlNPs-Ab probe will be blocked in the detection line by TET-BSA, displaying a visible fluorescence band under UV light.
The method for preparing the fluorescent probe comprises the following steps:
(1) Preparing an amino-functionalized aluminum nano sheet: mixing aluminum chloride and diamino terephthalic acid for reaction, adding urea solution, stirring, transferring the mixture into a high-pressure reaction kettle, washing and centrifuging to obtain the catalyst; 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 the tetracycline monoclonal antibody into the amino-functionalized aluminum nano-sheet solution in the step (1), mixing, sealing by bovine serum albumin, and centrifugally re-suspending in water to obtain the tetracycline monoclonal antibody, wherein the mixing ratio of the tetracycline monoclonal antibody and the amino-functionalized aluminum nano-sheet is 0.8-1.2 mug to 30-70 mug, and the concentration of the amino-functionalized aluminum nano-sheet solution is 0.8-1.2 mg/mL; the mixing time is 0.8-1.2 h, the final concentration of the bovine serum albumin is 1%, and the sealing time is 20-40 min.
The immunochromatographic test strip is composed of five parts, a nitrocellulose membrane, a sample pad, a binding 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 marked and coated on the nitrocellulose as a detection line (T) and a control line (C) respectively.
The experimental reagents used in the invention are all obtained in the market, are not further processed, and the detection instrument and equipment are all common instruments.
Example 1:
according to the technical scheme, the fluorescent probe AlNPs-Ab and the preparation method are provided in the embodiment, wherein the fluorescent probe AlNPs-Ab is prepared by a hydrothermal method to prepare an amino functionalized aluminum nano-sheet as a signal carrier, and then a tetracycline monoclonal antibody is added for adsorption. The method comprises the following steps:
(1) Preparation of amino-functionalized aluminum nanoplatelets (AlNPs): mixing aluminum chloride and diamino terephthalic acid for reaction, adding urea solution, stirring, transferring the mixture into a high-pressure reaction kettle, washing and centrifuging to obtain the catalyst;
the method comprises the following steps: alCl is added 3 And diamino terephthalic acid (NH) 2 BDC) in a concentration ratio of 1:1 was added to 15mL deionized water and the reaction was continued for 30min. Subsequently, atUrea solution (0.96 mol/L,5 mL) was added dropwise with vigorous stirring, and stirring was continued for 30min. After that, the mixture was transferred to an autoclave and reacted at 150℃for 5 hours, after cooling to room temperature, by centrifugation at 8500r/min for 10min to obtain 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 stirring was continued under the same conditions with methanol instead of DMF. Finally, the prepared AlNPs were centrifuged and resuspended in ultrapure water, dried under vacuum at 70 ℃ overnight, and stored in a refrigerator at 4 ℃.
(2) Preparing a fluorescent probe: adding the tetracycline monoclonal antibody into the amino-functionalized aluminum nano-sheet solution in the step (1), mixing, sealing by bovine serum albumin, centrifuging and re-suspending in water to obtain the tetracycline monoclonal antibody.
mu.L of AlNPs (1.0 mg/mL) and 10. Mu.g of anti-tetracycline Abs (1.0 mg/mL) were added to a 1.5mL centrifuge tube and reacted for 1h to allow sufficient binding. Subsequently, 50. Mu.L of 10% BSA was added and vortexed for 30min. Finally, the AlNPs-Ab probe of the present invention was obtained by centrifugation at 10000r/min for 10min, and then resuspended in 500. Mu.L of ultra pure water and stored at 4℃for further use.
The tetracycline-detecting probes used in examples 2 to 4 below were 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 stuck on the lining plate, one end of the nitrocellulose membrane is covered with a water absorption pad, the other end of the nitrocellulose membrane is sequentially covered with a sample pad and a combination pad, a detection line and a control line are transversely arranged on the non-covered surface of the nitrocellulose membrane, and the combination pad and the sample pad are respectively sealed by sealing liquid.
The preparation method of the nitrocellulose membrane comprises the following steps: 1mg/mL of tetracycline-bovine serum albumin conjugate is coated on a detection line at a scribing rate of 1 mu L/cm to obtain a detection line, and 1mg/mL of goat anti-mouse immunoglobulin is coated on a control line at a scribing rate of 1 mu L/cm to obtain a control line; and then dried at 37 c for later use.
Preparation of sample pad: cutting glass fiber membrane into 15mm long and 3mm wide, soaking in sealing solution (2% BSA), drying at 37deg.C for 8 hr to obtain sample pad, and storing in a refrigerator at 4deg.C.
Preparation of the bond pad: cutting glass fiber membrane into 8mm wide and 3mm wide, soaking in sealing solution (2% BSA), taking out, drying at 37deg.C for 8 hr to obtain sample pad, and storing in refrigerator at 4deg.C.
Cutting the water absorbing paper into a specification of 18mm and 3mm wide to obtain the water absorbing pad.
Assembling a test strip: firstly, attaching a nitrocellulose membrane to a lining plate, then pressing a sample pad by 2mm, pressing the nitrocellulose membrane by 2mm, and sequentially attaching a water-absorbing pad by 2mm to the lining plate to obtain the immunochromatography test strip for rapidly detecting tetracycline.
Example 3: sensitivity determination of test strip for rapid detection of tetracycline
Performance evaluation of immunochromatographic test strips for rapid detection of tetracycline as described above.
The detection process comprises the following steps: the tetracycline standard was dissolved in ultrapure water, which was used as a blank, and the test solutions were serially diluted to give different concentrations ranging from 0 to 12.0 ng/mL. The 0.5. Mu.L AlNPs-Ab probe was incubated with 100. Mu.L tetracycline standard solution and the sample pad of the test strip was immersed in 100. Mu.L test solution, and the mixture migrated toward the absorbent pad by capillary action. After 15min of reaction, the strip was scanned using a fluorescent strip reader to obtain Fluorescence Intensity (FI). With FI ratio (FI) of the test line (T line) to the control line (C line) T /FI C ) For quantitative analysis, this can both counteract the variability between test strips and minimize environmental factors affecting FI.
Detection result: when the T line is seen by the naked eye to be significantly shallower than the negative control strip, the corresponding minimum concentration of tetracycline is defined as the Visual Detection Limit (VDL), and when the T line is completely absent, the corresponding minimum concentration is defined as the threshold concentration. Competitive inhibition rate IC 10 Defined as the limit of detection (LOD).
See FIG. 3, with tetracycline concentrationThe degree is increased, the fluorescence of the test strip T line is shallower and shallower, FI T /FI C The value was continuously decreased with increasing tetracycline concentration. The VDL was 0.375ng/mL and the threshold concentration at which the T line was completely eliminated was 6ng/mL. IC by calculation of AlNPs-FLFIA 50 The value was 0.163.+ -. 0.029ng/mL, while the LOD was 0.0516.+ -. 0.053ng/mL. The sensitivity is higher than that of other literature reports (see Table 1), and is 29 times that of the traditional test strip (see FIG. 4). Therefore, the method can detect the tetracycline with high sensitivity, and can be used as a general method for rapidly and conveniently detecting the residue of antibiotics in food. Table 1 shows that the immunochromatographic test strip prepared by the invention has a lower limit of detection (LOD) of 0.0516 compared with other existing detection methods.
Table 1 shows the comparison of the detection sensitivity of the immunochromatography test strip prepared by the invention
Example 4: specific determination of test strip for rapidly detecting salmonella enteritidis
Performance evaluation of immunochromatographic test strips for rapid detection of tetracycline as described above.
The detection process comprises the following steps: respectively diluting Oxytetracycline (OXY), aureomycin (CTE), amoxicillin (AMX), florfenicol (FLO), azithromycin (AZM), streptomycin Sulfate (STR) and Erythromycin (EM) to the concentration of 100ng/mL by using ultrapure water, respectively taking 100 mu L of solution as a detection solution, mixing and incubating with 0.5 mu L of AlNPs-Ab probe, immersing a sample pad of the test strip in 100 mu L of test solution, and simultaneously taking 100 mu L of ultrapure water as a blank control solution. After 15min, the strip was scanned using a fluorescent strip reader to obtain the Fluorescent Intensity (FI), and the FI ratio (FI) of the detection line (T line) to the control line (C line) T /FI C ) For specificity analysis.
As shown in FIG. 3, the sample doped with tetracycline alone resulted in a significant decrease in fluorescence intensity of the detection line, and the addition of oxytetracycline and aureomycin resulted in a slight decrease in fluorescence intensity of the detection line due to the oxytetracycline and aureomycin belonging to the tetracycline class, whereas a strong fluorescence band was observed on the detection line for other common antibiotics. The invention can identify the tetracycline with high specificity and has good specificity.
Example 5: application of test strip for rapidly detecting tetracycline
Performance evaluation of immunochromatographic test strips for rapid detection of tetracycline as described above.
The detection process comprises the following steps: beef, chicken, milk and honey samples (confirmed to be free of tetracycline by High Performance Liquid Chromatography (HPLC)) were labeled with tetracycline. For beef and chicken samples, 1g each was placed in a 15mL centrifuge tube containing 2mL 3% aqueous trichloroacetic acid, vortexed for 5min, and centrifuged (10,000 rpm,10 min). Subsequently, the supernatant was neutralized with NaOH (1M) solution. Finally, the obtained supernatant was diluted 20-fold with ultrapure water.
For milk and honey samples, 1mL of the mixture was placed in a 15mL centrifuge tube containing 1mL of 3% trichloroacetic acid solution, vortexed for 5min, and centrifuged (10,000 rpm,10 min). Subsequently, the supernatant was neutralized with a NaOH (1M) solution and diluted 10-fold with ultrapure water.
The above treated actual sample solutions were diluted to different multiples (tetracycline concentration 0-12 ng/mL), 100. Mu.L of each solution was used as the detection solution, mixed with 0.5. Mu.L of AlNPs-Ab probe for incubation, and then the sample pad of the test strip was immersed in 100. Mu.L of the test solution, while 100. Mu.L of ultrapure water was used as the blank solution. After 15min, the strip was scanned using a fluorescent strip reader to obtain the Fluorescent Intensity (FI), and the FI ratio (FI) of the detection line (T line) to the control line (C line) T /FI C ) For specificity 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 a tetracycline labeling sample, which strongly indicates that the method can specifically detect tetracycline. For the honey sample, its VDL was 0.75ng/mL, indicating that the honey matrix had little effect on the sensitivity of the technique. In conclusion, the sensitivity of beef, chicken and milk samples to AlNPs-LFIA is consistent with that of the tetracycline labeling sample, and the good practical application value of the beef, chicken and milk samples is reflected.
Example 6: characterization of amino-functionalized aluminum nanoplatelet materials
In order to demonstrate the good properties of the amino-functionalized aluminum nanoflakes prepared by the present invention, the present inventors have also made the following experiments (see fig. 6):
(1) Scanning electron microscope and transmission electron microscope: the image A and the image B are respectively a scanning electron microscope image and a transmission electron microscope image of AlNPs, are diamond-shaped nanometer plates, have complete morphology and an average size of 545.97nm, and can be seen that the specific surface area is large, and the modification and the activation are easy.
(2) X-ray diffraction (XRD) pattern and X-ray photoelectron spectroscopy (XPS): panel C, panel D shows XRD and XPS spectra, respectively, for AlNPs, it can be seen that AlNPs have NH 2 Several characteristic peaks of MOF of MIL-53 type and illustrate that AlNPs consist mainly of O, N, C and Al elements.
(3) Fourier transform infrared (FT-IR) spectrum: FIG. E is a Fourier transform infrared spectrum of AlNPs at 1000-1100cm -1 Two Al-O peaks were observed, which confirm the oxygen atom and Al 3+ Has been successfully connected to NH 2 -BDC; in addition, due to symmetrical and asymmetrical stretching vibrations of N-H bonds, peaks of AlNPs appear at 3384 and 3500cm -1 Where it is located. Meanwhile, since hydroxyl groups in AlNPs are abundant and amino groups have good preservability, they have remarkable water dispersibility.
(4) Fluorescence spectrum (FL) and Excitation Emission Matrix (EEM) spectrum: graphs F and G are fluorescence spectrum (FL) and Excitation Emission Matrix (EEM) spectrum of AlNPs, respectively, it can be seen that AlNPs emits peak at 430nm and excitation peak at 340nm, and exhibits blue fluorescence under ultraviolet lamp.
(5) Condition optimization of fluorescence intensity: panel H shows the fluorescence intensity of AlNPs at different pH values, it can be seen that the fluorescence intensity is maintained at an almost constant level at pH values of 1.0 to 6.0, and at pH values of 7.0, the fluorescence intensity is highest, and thereafter there is a significant tendency to decrease, so that pH value of 7.0 is selected as the optimum pH value.
(6) Stability test: FIG. I shows the stability test results of AlNPs, and it can be seen that the fluorescence intensity of AlNPs does not change significantly after being left at room temperature for one month, thus indicating that the AlNPs has good stability.
Example 7: characterization of novel fluorescent probes
To demonstrate the success of the present invention in preparing a novel fluorescent probe, the present inventors have also made the following experiments (see fig. 7):
(1) Ultraviolet visible spectrum diagram: panel A is an ultraviolet-visible spectrum of AlNPs, alNPs-Abs and Abs, and it can be observed that the peak of the Abs is changed from 280nm to 285nm after AlNPs modification, indicating successful labeling of Abs with AlNPs.
(2) zeta potential: panel B is a zeta potential plot of AlNPs and AlNPs-Ab, and it can be seen that there is a significant change in the zeta potential of AlNPs (17.3 mv) and AlNPs-Ab (3.62 mv), indicating that Abs were successfully labeled on the surface of AlNPs.
(3) Fourier transform infrared (FT-IR) spectrum: FIG. C is a Fourier transform infrared spectrum of AlNPs, alNPs-Ab and Abs, and it can be seen that AlNPs are at 1640-1650cm -1 And 1520-1530cm -1 There appears a distinct characteristic absorption peak, 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 after AlNPs were linked to BSA, alNPs-BSA exhibited a characteristic protein band at 60kDa, whereas it was not present in AlNPs. The results indicate that proteins can be successfully loaded onto AlNPs.
Claims (4)
1. A method for detecting tetracycline is characterized by comprising the steps of immersing a test strip in a solution containing a fluorescent probe for detection;
the fluorescent probe comprises a tetracycline monoclonal antibody and a signal carrier, wherein the signal carrier is an amino-functionalized aluminum nano-sheet, and the particle size of the amino-functionalized aluminum nano-sheet is 545.97 nm;
the preparation method of the fluorescent probe comprises the steps of preparing amino-functionalized aluminum nano-sheets by a hydrothermal method, and then adding a tetracycline monoclonal antibody for adsorption to obtain the fluorescent probe
The method for preparing the fluorescent probe comprises the following steps:
(1) Preparing an amino-functionalized aluminum nano sheet: aluminum chloride and diamino terephthalic acid are mixed according to the concentration ratio of 1:1 adding into 15mL deionized water for mixing reaction, then adding 5mL urea solution with the concentration of 0.96mol/L, stirring, transferring the mixture into a high-pressure reaction kettle, placing in an oven for reaction at 150 ℃ for 5h, washing and centrifuging to obtain the catalyst;
(2) Preparing a fluorescent probe: 500 mu L of AlNPs and 10 mu g of tetracycline monoclonal antibody are added into a 1.5mL centrifuge tube, reaction is carried out for 1h, 50 mu L of 10% BSA is added, vortex oscillation is carried out for 30min, centrifugation and resuspension are carried out in water, the concentration of AlNPs is 1.0mg/mL, and the concentration of tetracycline monoclonal antibody is 1.0 mg/mL.
2. The method for detecting tetracycline according to claim 1, wherein the test strip comprises a lining board, a nitrocellulose membrane is attached to the lining board, one end of the nitrocellulose membrane is covered with a water absorbing pad, the other end of the nitrocellulose membrane is sequentially covered with a sample pad and a bonding pad, a detection line and a control line are transversely arranged on the non-covered surface of the nitrocellulose membrane, and the bonding pad and the sample pad are respectively sealed by sealing liquid.
3. The method for detecting tetracycline of claim 2, wherein said nitrocellulose membrane is prepared by a method comprising: 1mg/mL tetracycline-bovine serum albumin conjugate is coated on a detection line at a scribing rate of 1 mu L/cm to obtain a detection line, and 1mg/mL goat anti-mouse immunoglobulin is coated 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 bonding pad comprises the following steps: soaking the glass fiber membrane in 2% sealing liquid, and drying for 8-10 hours at 36-37 ℃;
the sample pad has the specification of 13-18 mm long and 2-4 mm wide, and the bonding pad has the specification of 7-9 mm long and 2-4 mm wide.
4. Use of the method for detecting tetracycline of any one of claims 1-3 for detecting tetracycline in beef, chicken, milk and honey.
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| CN112505012A (en) * | 2020-12-23 | 2021-03-16 | 郑州轻工业大学 | Application and preparation method of fusiform nano-sheet Al-MOF fluorescent probe material |
| CN113433322A (en) * | 2021-07-01 | 2021-09-24 | 武汉钟瑞生物科技有限公司 | High-sensitivity tetracycline quantum dot detection test paper and preparation method thereof |
| CN113698621B (en) * | 2021-09-15 | 2022-09-16 | 中国农业大学 | Application of aluminum metal organic framework material in aflatoxin B1 detection |
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009012528A1 (en) * | 2007-07-24 | 2009-01-29 | Adelaide Research & Innovation Pty Ltd | Optical fiber sensor |
| WO2011017691A2 (en) * | 2009-08-07 | 2011-02-10 | Northwestern University | Enhanced molecular detection of targeted intracellular mrna transcription using polynucleotide functionalized nanoparticles |
| CN103030179A (en) * | 2013-01-08 | 2013-04-10 | 江苏大学 | Tungsten trioxide nano-sheet prepared by hydrothermal method and application of tungsten trioxide nano-sheet |
| RU2583940C1 (en) * | 2014-12-17 | 2016-05-10 | государственное бюджетное образовательное учреждение высшего профессионального образования "Московский государственный медико-стоматологический университет имени А.И. Евдокимова" Министерства здравоохранения Российской Федерации | Method of determining composition of individual extracellular vesicles in human blood |
| WO2017052474A1 (en) * | 2015-09-23 | 2017-03-30 | Nanyang Technological University | A metal-organic framework nanosheet |
| CN107290316A (en) * | 2017-06-27 | 2017-10-24 | 宁波工程学院 | A kind of tetracycline fluorescence new detecting method based on zirconium base MOF |
| CN107655864A (en) * | 2016-07-25 | 2018-02-02 | 上海溯源生物技术有限公司 | A kind of method using nanoparticle time-resolved fluorescence probe in detecting tetracycline |
| CN109060787A (en) * | 2018-08-28 | 2018-12-21 | 福建出入境检验检疫局检验检疫技术中心 | A method of tetracycline antibiotics are detected based on nano enzyme |
| CN109490549A (en) * | 2018-10-19 | 2019-03-19 | 迪瑞医疗科技股份有限公司 | The chemical luminescent analysis reagent kid and preparation method thereof of creatine kinase isozyme in a kind of detection serum/plasma |
| CN110133258A (en) * | 2019-02-28 | 2019-08-16 | 上海健耕医药科技股份有限公司 | A kind of time-resolved fluoroimmunoassay chromatography reagent strip of quick detection cyclosporin |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN201518027U (en) * | 2009-07-24 | 2010-06-30 | 杭州南开日新生物技术有限公司 | Tetracycline immune colloidal gold quick test reagent board |
| CN110887964A (en) * | 2019-11-07 | 2020-03-17 | 西北农林科技大学 | Sensitive probe, method for detecting tetracycline and application |
-
2020
- 2020-04-03 CN CN202010258389.5A patent/CN111551724B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009012528A1 (en) * | 2007-07-24 | 2009-01-29 | Adelaide Research & Innovation Pty Ltd | Optical fiber sensor |
| WO2011017691A2 (en) * | 2009-08-07 | 2011-02-10 | Northwestern University | Enhanced molecular detection of targeted intracellular mrna transcription using polynucleotide functionalized nanoparticles |
| CN103030179A (en) * | 2013-01-08 | 2013-04-10 | 江苏大学 | Tungsten trioxide nano-sheet prepared by hydrothermal method and application of tungsten trioxide nano-sheet |
| RU2583940C1 (en) * | 2014-12-17 | 2016-05-10 | государственное бюджетное образовательное учреждение высшего профессионального образования "Московский государственный медико-стоматологический университет имени А.И. Евдокимова" Министерства здравоохранения Российской Федерации | Method of determining composition of individual extracellular vesicles in human blood |
| WO2017052474A1 (en) * | 2015-09-23 | 2017-03-30 | Nanyang Technological University | A metal-organic framework nanosheet |
| CN107655864A (en) * | 2016-07-25 | 2018-02-02 | 上海溯源生物技术有限公司 | A kind of method using nanoparticle time-resolved fluorescence probe in detecting tetracycline |
| CN107290316A (en) * | 2017-06-27 | 2017-10-24 | 宁波工程学院 | A kind of tetracycline fluorescence new detecting method based on zirconium base MOF |
| CN109060787A (en) * | 2018-08-28 | 2018-12-21 | 福建出入境检验检疫局检验检疫技术中心 | A method of tetracycline antibiotics are detected based on nano enzyme |
| CN109490549A (en) * | 2018-10-19 | 2019-03-19 | 迪瑞医疗科技股份有限公司 | The chemical luminescent analysis reagent kid and preparation method thereof of creatine kinase isozyme in a kind of detection serum/plasma |
| CN110133258A (en) * | 2019-02-28 | 2019-08-16 | 上海健耕医药科技股份有限公司 | A kind of time-resolved fluoroimmunoassay chromatography reagent strip of quick detection cyclosporin |
Non-Patent Citations (1)
| Title |
|---|
| 有机金属框架材料在环境检测中的应用;杨帆;;绿色科技(02);全文 * |
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