CN114720437A - Fe-PDAN-based efficient fluorescence quenching test strip and preparation method and detection method thereof - Google Patents
Fe-PDAN-based efficient fluorescence quenching test strip and preparation method and detection method thereof Download PDFInfo
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Abstract
The invention discloses a Fe-PDAN-based efficient fluorescence quenching test strip and a preparation method and a detection method thereof. The fluorescence quenching test strip takes Fe-PDAN as a beacon carrier. The kit is obtained by sequentially overlapping and pasting a sample pad, a glass fiber pad sprayed with an Fe-PDAN (Fe-Polymer dispersed particles) to-be-detected object antibody complex, a nitrocellulose membrane sprayed with an artificially coupled antigen of a to-be-detected object and a quantum dot fluorescent microsphere as a detection line and sprayed with an anti-mouse antibody or an anti-rabbit antibody as a quality control line, and absorbent paper on a substrate for assembly. During detection, quantitative detection and qualitative judgment are realized by reading fluorescence intensity data of the fluorescence quenching test strip or observing whether a detection line has a fluorescence strip or not under the irradiation of an ultraviolet lamp. The fluorescence quenching test strip prepared by the invention takes Fe-PDAN as a beacon carrier for the first time, compared with PDAN, the fluorescence quenching test strip has stronger fluorescence quenching capability of quantum dot fluorescent microspheres and high stability, and the prepared fluorescence quenching test strip has strong stability and high detection sensitivity.
Description
Technical Field
The invention belongs to the field of food safety detection and medical inspection, and particularly relates to a Fe-PDAN-based efficient fluorescence quenching test strip, and a preparation method and a detection method thereof.
Background
The immunochromatography technology is a detection method based on antigen-antibody specific reaction, has the advantages of high detection speed, good specificity, simple operation, low cost and the like, and can better meet the requirements of field large-scale detection compared with other methods, so the immunochromatography technology is rapidly developed in recent years and is widely applied to the fields of food safety, medical inspection, environmental pollutant monitoring and the like. The colloidal gold immunochromatographic test strip is the most widely applied immunochromatographic product, but the colloidal gold immunochromatographic test strip has poor sensitivity and is easily interfered by matrixes. Therefore, there are three main development directions for the immunochromatography technology in recent years: firstly, separating and concentrating a target substance from a complex matrix by adopting an immunomagnetic separation and enrichment technology to avoid the interference of a sample matrix; secondly, the sensitivity of the immunochromatography method is improved by a signal amplification system (such as a biotin-streptavidin system and the like); thirdly, a novel marker (such as fluorescent microspheres) is adopted to improve the signal output or change the signal output type so as to achieve the purpose of improving the sensitivity.
Biosensors based on fluorescence quenching have been widely used for sensitive detection of various biomolecules in the fields of food safety, clinical diagnosis, environmental monitoring, and the like. The fluorescence quenching ability of the quencher is a key factor in determining the performance of the biosensor. Currently, a commonly used quencher is generally a small molecule with an electron absorption capability, such as a Black Hole Quencher (BHQ) dye, which is a high-efficiency dark quencher, and different BHQ has a significant effect on quenching of specific fluorescence with different wavelengths. However, such small molecule quencher-based probes lack efficient targeting and multimodal sensing capabilities.
At present, various nanomaterials, such as graphene oxide, colloidal gold nanoparticles, colloidal silver nanoparticles, gold nanoflowers, magnetic nanoparticles, manganese dioxide nanosheets, molybdenum disulfide nanosheets and the like, have been widely used for preparing fluorescence quenching biosensors to sensitively detect various biomolecules. Unlike small molecule fluorescence quenchers, nanopenchers have the ability to quench multiple fluorophores of different emission wavelengths, thereby eliminating the cumbersome problem of requiring careful selection of fluorophore quenchers.
Dopamine (DA) is an important neurotransmitter, and is capable of self-polymerizing to form Polydopamine (PDA) by oxidation in an alkaline solution and in an aerobic environment. Dopamine hydrochloride is used as a raw material, and is rapidly polymerized in a sodium hydroxide solution or a Tris buffer solution to prepare the polydopamine microsphere (PDAN) with a certain particle size. In addition, rich surface functional groups of dopamine and polydopamine, such as amino, hydroxyl, catechol and the like, enable the PDAN to have good biocompatibility and coordination capacity to various metal ions, and are suitable for further surface modification. In recent years, polydopamine as an efficient organic dark quencher has been widely applied to fluorescence bioimaging and detection analysis due to its advantages of strong loading capacity, easy surface functionalization, good biocompatibility and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a fluorescence quenching test strip, which specifically adopts the following technical scheme:
a high-efficiency fluorescence quenching test strip based on Fe-PDAN takes Fe-PDAN as a beacon carrier; the Fe-PDAN is a metal polyphenol network microsphere formed by complexing poly dopamine with iron ions, and the preparation process comprises the following steps: adding dopamine hydrochloride solution to FeCl3And in the solution, stirring to react to form a coordination compound, adding a Tris solution, carrying out oxidative polymerization on dopamine hydrochloride under an alkaline condition to form polydopamine, centrifuging after the reaction is finished, discarding a supernatant, cleaning a precipitate, and redissolving the precipitate in a buffer solution to obtain the Fe-PDAN.
The inventor synthesizes a metal polyphenol network microsphere (Fe-PDA N) formed by iron ion coordinated poly dopamine by a one-pot method, compared with PDAN, Fe-PDAN has stronger capability of quenching fluorescence of quantum dot fluorescent microspheres, Fe-PDAN is used as a probe and a nano quencher to be applied to an immunochromatography test strip to establish a fluorescence quenching immunochromatography method, and a traditional 'Turn off' mode output signal for detecting small molecules by a competitive inhibition principle can be converted into a more favorable 'Turn on' mode output signal, so that the detection sensitivity of the test strip is improved. At present, no reports related to the application of Fe-PDAN in fluorescence quenching and the application of Fe-PDAN as a probe in an immunochromatography method exist.
Preferably, the fluorescence quenching test strip comprises a bottom plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped and adhered on the bottom plate; the glass fiber mat is sprayed with an Fe-PDAN to-be-detected object antibody compound, and the Fe-PDAN to-be-detected object antibody compound is prepared from the Fe-PDAN and an object to be detected antibody.
The preparation process of the Fe-PDAN analyte antibody compound is as follows: and adding the antibody of the object to be detected into the Fe-PDAN, uniformly mixing, reacting at room temperature for 2h, then adding a sealing agent, reacting at room temperature for 2h, centrifuging, taking a precipitate, and redissolving to obtain the antibody compound of the object to be detected of the Fe-PDAN. Preferably, before the object antibody to be detected is added to the Fe-PDAN, the Fe-PDAN is pretreated, wherein the pretreatment process comprises the following steps: and (3) carrying out ultrasonic treatment on the Fe-PDAN for 1-10min, and then adjusting the concentration of the Fe-PDAN to 0.01-0.1mg/mL by using 0.01-0.5M, pH ═ 6-8 borate buffer solution.
Preferably, the final concentration of the antibody to be detected after the antibody to be detected is added is 1-100 mug/mL, the final concentration of the blocking agent after the blocking agent is added is 0.1-1%, and the blocking agent is any one of casein, bovine serum albumin, ovalbumin, polyethylene glycol and skimmed milk.
Preferably, the rotation speed of centrifugation is 5000-.
Preferably, the analyte antibody is a monoclonal antibody, a polyclonal antibody, a nanobody or a phage-expressed antibody.
Preferably, the nitrocellulose membrane is coated with an artificial coupling antigen of an object to be detected and a quantum dot fluorescent microsphere as a detection line, and is coated with an anti-mouse antibody or an anti-rabbit antibody as a secondary antibody as a quality control line.
The artificial coupling antigen of the object to be detected is a holoantigen with immunogenicity and reactogenicity, which is prepared by a chemical coupling method of a micromolecule object to be detected and macromolecular protein; wherein, the small molecule object to be detected covers all small molecule substances required to be detected in the fields of medical inspection and food safety detection; the coupling method comprises a diazo method, a carbodiimide method, a glutaraldehyde method, a mixed anhydride method and a succinic anhydride method; the coupling macromolecular protein comprises bovine serum albumin, casein, ovalbumin and keyhole limpet hemocyanin; the coupling ratio is 1:5-1:200, and the artificial coupling antigen is obtained after dialysis and purification after coupling.
The preparation method of the nitrocellulose membrane comprises the following steps:
(1) respectively regulating the coating (the object to be detected is artificially coupled with the antigen and the quantum dot fluorescent microspheres) and the secondary antibody (the anti-mouse antibody or the anti-rabbit antibody) to the concentration of 0.01-5.0 mg/mL by using a PBS (phosphate buffer solution) solution of 0.01-0.5M, pH ═ 6.0-8.0; wherein the secondary antibody is a monoclonal antibody, a polyclonal antibody, a nano antibody or a phage expression antibody;
(2) spraying the artificial coupling antigen of the substance to be detected and the quantum dot fluorescent microspheres after the concentration adjustment on the upper part of the nitrocellulose membrane as a detection line, and spraying the anti-mouse antibody or the anti-rabbit antibody on the lower part of the nitrocellulose membrane as a quality control line; wherein, a certain distance is arranged between the detection line and the quality control line, and the film spraying amount of the detection line and the film spraying amount of the quality control line are both 0.25-0.74 mu L/cm;
(3) and drying the nitrocellulose membrane sprayed with the detection line and the quality control line at 37 ℃ overnight, and storing the nitrocellulose membrane in a room-temperature dry environment for later use.
The invention also provides a preparation method of the fluorescence quenching test strip, which comprises the following steps: and overlapping and pasting the sample pad, the glass fiber pad sprayed with the Fe-PDAN to-be-detected object antibody compound, the nitrocellulose membrane sprayed with the to-be-detected object artificial coupling antigen and the quantum dot fluorescent microspheres as detection lines, and the nitrocellulose membrane sprayed with the anti-mouse antibody or the anti-rabbit antibody as a secondary antibody as a quality control line on a bottom plate in sequence, so as to complete the assembly.
The assembled large test strip plate is cut into required width by a cutter, and the test strip can be directly used or can be put into a plastic card shell for use.
The invention also provides a detection method of the fluorescence quenching test strip, which comprises the following steps: and adding a sample to be detected to a sample pad of the fluorescence quenching test strip, wherein the sample adding volume is 50-200 mu L, the reaction time is 3-30min, the concentration of the sample to be detected is calculated by a built-in standard curve after reading the fluorescence intensity data of the fluorescence quenching test strip to realize quantitative detection, and the qualitative judgment of the detected sample is realized by observing whether a detection line has a fluorescence strip or not by naked eyes under the irradiation of an ultraviolet lamp.
The invention has the beneficial effects that: the fluorescence quenching test strip prepared by the invention takes Fe-PDAN as a beacon carrier for the first time, compared with PDAN, the fluorescence quenching test strip has stronger fluorescence quenching capability of quantum dot fluorescent microspheres and high stability, and the prepared fluorescence quenching test strip has strong stability and high detection sensitivity.
Drawings
FIG. 1 is a schematic structural diagram of a Fe-PDAN-based fluorescence quenching test strip;
FIG. 2 is a schematic diagram of the detection of a fluorescence quenching test strip;
FIG. 3 is a transmission electron microscopy characterization of Fe-PDAN;
FIG. 4 shows the stability evaluation of Fe-PDAN at different pH and NaCl concentrations;
FIG. 5 shows an ultraviolet absorption spectrum of Fe-PDAN, an emission spectrum of quantum dot fluorescent microspheres and a physical diagram;
FIG. 6 is a graph showing a standard curve for detecting Enrofloxacin (ENR) based on PDAN and Fe-PDAN;
FIG. 7 is a diagram showing a Fe-PDAN-based fluorescence quenching test strip for detecting ENR;
FIG. 8 shows Stern-Volmer standard curves for PDAN and Fe-PDAN on nitrocellulose membranes;
FIG. 9 shows the fluorescence quenching constants of PDAN and Fe-PDAN to quantum dot fluorescent microspheres.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments and the accompanying drawings to fully understand the objects, aspects and effects of the present invention.
Example 1: preparation of Fe-PDAN
(1) Solution preparation: tris solution (5 mg/mL);
(2) coordination of iron ions with dopamine hydrochloride: 1mL dopamine hydrochloride (10mg/mL) was added to 7mL Fe Cl3Stirring the solution (0.8mg/mL) to react for 30min, so that iron ions and dopamine hydrochloride form a coordination compound;
(3) oxidative auto-polymerization of dopamine hydrochloride: then adding 2mL of Tris solution (6mg/mL), and carrying out oxidative polymerization on dopamine hydrochloride under an alkaline condition to form Polydopamine (PDA);
(4) washing: centrifuging (rotating speed is 12000r/min, time is 20min) after the reaction is finished, discarding the supernatant, cleaning the precipitate, redissolving the precipitate in ultrapure water to obtain Fe-PDAN, and storing at 4 ℃.
Wherein, the transmission electron microscope representation picture of Fe-PDAN is shown in figure 3; the ultraviolet absorption spectrum, the quantum dot fluorescent microsphere emission spectrum and the physical map of Fe-PDAN are shown in FIG. 5. Meanwhile, stability evaluation tests were performed on Fe-PDAN at different pH and NaCl concentrations, and the results are shown in FIG. 4.
Example 2: preparation of fluorescent quenching test strip for detecting ENR based on Fe-PDAN
Preparation process of fluorescence quenching test strip
1. Preparation of ENR Artificial antigen (ENR-BSA)
The coupling method is a carbodiimide method, the coupling protein is Bovine Serum Albumin (BSA), the coupling ratio is 1:100, and after coupling, dialysis and purification are carried out to obtain ENR-BSA.
2. Preparation of nitrocellulose membranes
ENR-BSA coating onto nitrocellulose membranes: diluting ENR-BSA and the concentration of the quantum dot fluorescent microspheres to be 1mg/mL and 0.1mg/mL respectively by using PBS (phosphate buffer solution) of 0.01M, pH-7.4, mixing the obtained solutions, and spraying the mixed solutions on a membrane to be used as a detection line; the concentration of the diluted anti-mouse antibody is 0.5mg/mL, the obtained solution is sprayed on the membrane after mixing to be used as a quality control line, the spraying amount of two lines is 0.74 mu L/cm, the distance between the detection line and the top edge of the membrane is 10mm, the distance between the middle of the two lines is 5mm, the membrane is dried for 6h at 37 ℃, and the membrane is placed in a drying cabinet for storage.
Preparation of Fe-PDAN antibody composite glass fiber pad
Taking 0.5mg Fe-PDAN (prepared in example 1) and carrying out ultrasonic treatment for 5min, adjusting the concentration of the Fe-PDAN to be 0.05mg/mL by using 0.1M borate buffer solution with the pH value of 7.0, shaking and mixing uniformly, adding 5 mu g ENR monoclonal antibody into 1mL Fe-PDAN, fully mixing, stirring and reacting for 2h at 4 ℃, adding casein with the final concentration of 2.5%, sealing for 2h at room temperature, centrifuging for 10min at 8000r/min, re-dissolving the precipitate into 1/10 with the initial volume by using 0.01M PBS with the pH value of 7.4, spraying the precipitate onto a glass fiber pad according to the volume of 3 mu L/cm, and carrying out vacuum drying for 2 h.
4. Assembled test paper strip
(1) A sample pad with a specification of 0.8 × 30 cm;
(2) a glass fiber mat with the specification of 0.7 multiplied by 30 cm;
(3) spraying a nitrocellulose membrane with a detection line and a quality control line, wherein the specification is 2.5 multiplied by 30 cm;
(4) absorbent paper with the specification of 1.5 multiplied by 30 cm;
(5) the PVC base plate has the specification of 5.5 multiplied by 30 cm.
The materials are sequentially pasted according to the positions of all components in a test strip structural schematic diagram shown in figure 1 (wherein, 1 sample pad, 2 glass fiber pad, 3 nitrocellulose membrane, 4 detection lines, 5 quality control lines, 6 absorbent paper, 7PVC base plate, 8Fe-PDAN antibody compound, 9 quantum dot fluorescent microspheres, 10 artificial coupling antigen of an object to be tested, 11 anti-mouse antibody or anti-rabbit antibody), the materials are assembled and cut into test strips with the size of 4 x 55mm by a cutter, the test strips are loaded into a plastic card shell, the test strips are packed into an aluminum foil bag after being compressed, drying agents are added, the test strips are sealed and stored, and the shelf life of the test strips is 12 months in a room temperature environment.
Second, quantitative determination of ENR in a sample
The detection principle is as follows: as shown in FIG. 2, when ENR is not present in the sample, the Fe-PDAN antibody complex can recognize and bind to ENR-BSA on the T line, thereby causing the fluorescence of the quantum dot fluorescent microspheres on the T line to be quenched by Fe-PDAN. In contrast, when ENR exists in the sample, the Fe-PDAN antibody complex is firstly combined with the ENR, so that the quantum dot fluorescent microspheres emit strong red fluorescence on the T line.
The method for detecting ENR in a sample by using the fluorescence quenching test strip comprises the following steps:
1. adding 100 mu L of milk sample diluted by 10 times of PBS into the sample adding hole of the test strip, and reacting for 10 min;
2. the test strip is inserted into a detection window of a fluorescent test strip reader, the strength of fluorescence of the detection line can be displayed on a display according to the magnitude of a numerical value, the content of ENR in the sample can be calculated according to a standard curve recorded in the reader, and quantitative detection of ENR in the sample is realized.
3. Establishing a standard curve: the ENR concentrations in the standard curve are: 0. 0.005, 0.01, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10ng/mL, and the results are shown in FIG. 6 (the left graph shows a standard curve for detecting ENR by PDAN, and the right graph shows a standard curve for detecting ENR by Fe-PDAN). FIG. 7 is a diagram showing a sample of ENR detection using a Fe-PDAN-based fluorescence quenching test strip (upper panel is taken under UV lamp, and the lower panel is taken under fluorescent lamp).
FIG. 8 shows Stern-Volmer standard curves for PDAN and Fe-PDAN on nitrocellulose membranes (in terms of F)0/F＝1+K[Q](ii) a Wherein F0Is the fluorescence intensity of the quantum dot fluorescent microsphere without the quencher, F is the fluorescence intensity of the quantum dot fluorescent microsphere after the quencher is added, K is the fluorescence quenching constant, [ Q ]]Is the concentration of the quencher). FIG. 9 shows the fluorescence quenching constants of PDAN and Fe-PDAN for quantum dot fluorescent microspheres.
Example 3: preparation of fluorescence quenching test strip for detecting Zearalenone (ZEN) based on Fe-PDAN
Preparation process of fluorescence quenching test strip
1. Preparation of ZEN Artificial antigen (ZEN-BSA):
the coupling method is a mixed anhydride method, the coupling protein is Bovine Serum Albumin (BSA), the coupling ratio is 1:80, and the ZEN-BSA is obtained after coupling and dialysis purification.
2. Preparation of nitrocellulose membrane:
ZEN-BSA and anti-mouse antibody coated onto nitrocellulose membrane: the concentrations of ZEN-BSA and quantum dot fluorescent microspheres diluted by PBS (0.01M, pH) ═ 7.4 are 1.5mg/mL and 0.1mg/mL respectively, and the obtained solutions are mixed and sprayed on a membrane to serve as a detection line; the concentration of the diluted anti-mouse antibody is 0.5mg/mL, the obtained solution is sprayed on the membrane to be used as a quality control line, the spraying amount of two lines is 0.74 mu L/cm, the interval between the detection line and the top edge of the membrane is 10mm, the interval between the two lines is 5mm, the membrane is dried for 12h at 37 ℃, and the membrane is placed in a drying cabinet for storage and standby.
Preparation of Fe-PDAN antibody composite glass fiber pad
Taking 0.5mg Fe-PDAN (prepared in example 1) and carrying out ultrasonic treatment for 5min, adjusting the concentration of Fe-PDAN to 0.05mg/mL by using 0.1M borate buffer solution with the pH value of 7.0, shaking and mixing uniformly, adding 4 mu g ZEN monoclonal antibody into 1mL Fe-PDAN, fully mixing, stirring and reacting for 2h at room temperature, adding casein with the final concentration of 2.5%, sealing for 2h at room temperature, centrifuging for 10min at 8000r/min, re-dissolving precipitates into 1/10 with the initial volume by using 0.01M, pH Phosphate Buffer Solution (PBS) with the concentration of 7.4, spraying onto a glass fiber pad according to the volume of 3 mu L/cm, and carrying out vacuum drying for 2 h.
4. Assembling the test strip:
(1) a sample pad with a specification of 0.8 × 30 cm;
(2) a glass fiber mat with the specification of 0.7 multiplied by 30 cm;
(3) spraying a nitrocellulose membrane with a detection line and a quality control line, wherein the specification is 2.5 multiplied by 30 cm;
(4) absorbent paper with the specification of 1.5 multiplied by 30 cm;
(5) the PVC bottom plate has the specification of 5.5 multiplied by 30 cm.
The materials are sequentially pasted according to the positions of all components in the test strip structural schematic diagram shown in figure 1, the materials are cut into test strips with the size of 4 x 55mm by a cutter after being assembled, the test strips are put into a plastic card shell, the test strips are pressed and then put into an aluminum foil bag, and after a drying agent is added, the test strips are sealed and stored, and the shelf life of the test strips in a room temperature environment is 12 months.
Secondly, quantitatively detecting ZEN in the sample
The method for detecting ZEN in a sample by using the fluorescence quenching test strip comprises the following steps:
1. weighing 2g of feed or grain samples, adding 10mL of extracting solution, shaking for 1min, and taking supernate for detection;
2. adding 100 mu L of sample liquid into the sample adding hole of the test strip, and reacting for 10 min;
3. the test strip is inserted into a detection window of a test strip reader, the intensity of fluorescence of the detection line can be displayed on a display according to the magnitude of a numerical value, the content of ZEN in a sample can be calculated according to a standard curve recorded in the instrument, and quantitative detection of a positive sample is realized.
4. Establishing a standard curve: the ZEN concentrations in the standard curve are: 0. 0.1, 0.5, 1, 2, 4, 8ng/mL, calculating R20.9892, the linear regression equation is: y-0.4011 log (x) + 0.5602.
Example 4: preparation of fluorescent quenching test strip for detecting Sulfamethazine (SMZ) based on Fe-PDAN
Preparation process of fluorescence quenching test strip
1. Preparation of SMZ Artificial antigen (SMZ-OVA):
the coupling method is a glutaraldehyde method, the coupling protein is Ovalbumin (OVA), the coupling ratio is 1:80, and the SMZ-OVA is obtained after coupling, dialysis and purification.
2. Preparation of nitrocellulose membrane:
SMZ-OVA and anti-mouse antibody were coated onto nitrocellulose membranes: the concentrations of SMZ-BSA and quantum dot fluorescent microspheres diluted by PBS (0.01M, pH ═ 7.4) are 0.5mg/mL and 0.1mg/mL respectively, and the obtained solutions are mixed and sprayed on a membrane to be used as a detection line; the concentration of the diluted anti-mouse antibody is 0.5mg/mL, the obtained solution is sprayed on the membrane to be used as a quality control line, the spraying amount of two lines is 0.74 mu L/cm, the interval between the detection line and the top edge of the membrane is 10mm, the interval between the two lines is 5mm, the membrane is dried for 12h at 37 ℃, and the membrane is placed in a drying cabinet for storage and standby.
Preparation of Fe-PDAN antibody complex glass fiber mat:
taking 0.5mg Fe-PDAN (prepared in example 1) and carrying out ultrasonic treatment for 5min, adjusting the concentration of the Fe-PDAN to be 0.05mg/mL by using 0.1M borate buffer solution with the pH value of 7.0, shaking and mixing uniformly, adding 5 mu g of SMZ monoclonal antibody into 1mL Fe-PDAN, fully mixing, stirring and reacting for 2h at room temperature, adding casein with the final concentration of 2.5%, sealing for 2h at room temperature, centrifuging for 10min at 8000r/min, re-dissolving the precipitate into 1/10 with the initial volume by using 0.01M, pH-7.4 Phosphate Buffer Solution (PBS), spraying onto a glass fiber pad according to the volume of 3 mu L/cm, and carrying out vacuum drying for 2 h.
4. Assembling the test strip:
(1) filter paper and sample pad with specification of 10.8 × 30 cm;
(2) a glass fiber mat with the specification of 0.7 multiplied by 30 cm;
(3) spraying a nitrocellulose membrane with a detection line and a quality control line, wherein the specification is 2.5 multiplied by 30 cm;
(4) absorbent paper with the specification of 1.25 multiplied by 30 cm;
(5) the PVC bottom plate has the specification of 5.5 multiplied by 30 cm.
The materials are sequentially pasted according to the positions of all components in the test strip structural schematic diagram shown in figure 1, the materials are cut into test strips with the size of 4 x 55mm by a cutter after being assembled, the test strips are put into a plastic card shell, the test strips are pressed and then put into an aluminum foil bag, and after a drying agent is added, the test strips are sealed and stored, and the shelf life of the test strips in a room temperature environment is 12 months.
Second, quantitative detection of SMZ in a sample
The method for detecting SMZ in a sample by using the fluorescence quenching test strip comprises the following steps:
1. weighing 2g of chicken samples, adding 10mL of extracting solution, shaking for 1min, and taking supernate for detection;
2. adding 100 mu L of sample liquid into the sample adding hole of the test strip, and reacting for 10 min;
3. the test strip is inserted into a detection window of a test strip reader, the intensity of fluorescence of the detection line can be displayed on a display according to the magnitude of a numerical value, the content of ZEN in a sample can be calculated according to a standard curve recorded in the instrument, and quantitative detection of a positive sample is realized.
4. Establishing a standard curve: the negative matrix is labeled with SMZ concentrations in the standard curve: 0. 0.05, 0.1, 0.5, 1, 5, 10ng/mL, calculating R20.991, the linear regression equation is: y ═ 0.3844log (x) + 0.5819.
The above description is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiment, and the present invention shall fall within the protection scope of the present invention as long as the technical effects of the present invention are achieved by the same means. The invention is capable of other modifications and variations in its technical solution and/or its implementation, within the scope of protection of the invention.
Claims (10)
1. A high-efficiency fluorescence quenching test strip based on Fe-PDAN is characterized in that the fluorescence quenching test strip takes Fe-PDAN as a beacon carrier; the Fe-PDAN is a metal polyphenol network microsphere formed by complexing poly dopamine with iron ions, and the preparation process comprises the following steps: adding dopamine hydrochloride solution to FeCl3And stirring the solution to react to form a coordination compound, adding a Tris solution, carrying out oxidative polymerization on dopamine hydrochloride under an alkaline condition to form polydopamine, centrifuging the solution after the reaction is finished, removing supernatant, washing the precipitate, and redissolving the precipitate in a buffer solution to obtain the Fe-PDAN.
2. The fluorescence quenching test strip of claim 1, which comprises a bottom plate, and a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper which are sequentially overlapped and adhered on the bottom plate; the glass fiber mat is sprayed with AN Fe-PD AN to-be-detected object antibody compound, and the Fe-PDAN to-be-detected object antibody compound is prepared from the Fe-PDAN and AN to-be-detected object antibody.
3. The fluorescence quenching test strip of claim 2, wherein the Fe-PDAN analyte antibody complex is prepared as follows: and adding the antibody of the object to be detected into the Fe-PDAN, uniformly mixing, reacting at room temperature for 2h, then adding a sealing agent, reacting at room temperature for 2h, centrifuging, taking a precipitate, and redissolving to obtain the antibody compound of the object to be detected of the Fe-PDAN.
4. The fluorescence quenching test strip of claim 3, wherein the Fe-PDAN is pretreated before the analyte antibody is added to the Fe-PDAN, and the pretreatment comprises: and (3) carrying out ultrasonic treatment on the Fe-PDAN for 1-10min, and then adjusting the concentration of the Fe-PDAN to 0.01-0.1mg/mL by using 0.01-0.5M, pH ═ 6-8 borate buffer solution.
5. The fluorescence quenching test strip of claim 3, wherein the final concentration of the analyte antibody after the analyte antibody is added is 1-100 μ g/mL, the final concentration of the blocking agent after the blocking agent is added is 0.1-1%, and the blocking agent is any one of casein, bovine serum albumin, ovalbumin, polyethylene glycol and skimmed milk.
6. The fluorescence quenching test strip as claimed in claim 3, wherein the rotation speed of centrifugation is 5000-.
7. The fluorescence quenching test strip of claim 2, wherein the analyte antibody is a monoclonal antibody, a polyclonal antibody, a nanobody, or a phage-expressed antibody.
8. The fluorescence quenching test strip of claim 2, wherein the nitrocellulose membrane is coated with an artificial coupling antigen of an object to be tested and a quantum dot fluorescent microsphere as a detection line, and is coated with an anti-mouse antibody or an anti-rabbit antibody as a secondary antibody as a quality control line.
9. The method for preparing the fluorescence quenching test strip of any one of claims 1 to 8, which is characterized by comprising the following steps: and sequentially overlapping and pasting a sample pad, a glass fiber pad sprayed with an Fe-PDAN (Fe-Polymer dispersed optical network) to-be-detected object antibody compound, a nitrocellulose membrane sprayed with an artificially coupled antigen of the to-be-detected object and a quantum dot fluorescent microsphere as a detection line, and a nitrocellulose membrane sprayed with an anti-mouse antibody or an anti-rabbit antibody as a secondary antibody as a quality control line, and absorbent paper on a bottom plate, so that the assembly is completed.
10. The method for detecting a fluorescence quenching test strip of any one of claims 1 to 8, which comprises the following steps: and adding a sample to be detected to the sample pad of the fluorescence quenching test strip, wherein the sample adding volume is 50-200 mu L, the reaction time is 3-30min, the concentration of the sample to be detected is calculated by a built-in standard curve after reading the fluorescence intensity data of the fluorescence quenching test strip to realize quantitative detection, and the qualitative judgment of the detection sample is realized by observing whether the detection line has a fluorescence strip or not by naked eyes under the irradiation of an ultraviolet lamp.
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| CN115856297A (en) * | 2023-01-04 | 2023-03-28 | 吉林大学 | Preparation method of kit for detecting salmonella typhimurium and kit |
| CN115856297B (en) * | 2023-01-04 | 2024-05-28 | 吉林大学 | Preparation method of kit for detecting salmonella typhimurium and kit |
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