CN113341143A - Fluorescent marking test strip for rapidly detecting food-borne pathogenic bacteria and preparation method thereof - Google Patents
Fluorescent marking test strip for rapidly detecting food-borne pathogenic bacteria and preparation method thereof Download PDFInfo
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- CN113341143A CN113341143A CN202110655622.8A CN202110655622A CN113341143A CN 113341143 A CN113341143 A CN 113341143A CN 202110655622 A CN202110655622 A CN 202110655622A CN 113341143 A CN113341143 A CN 113341143A
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Abstract
The invention discloses a fluorescence labeling test strip for rapidly detecting food-borne pathogenic bacteria and a preparation method thereof. Connecting the food-borne pathogenic bacteria specific antibody with a fluorescent substance to form a signal probe, and spraying the signal probe on a specific glass fiber membrane; then taking the food-borne pathogenic bacteria specific antibody and a second antibody corresponding to the food-borne pathogenic bacteria antibody as capture probes, and scribing on a nitrocellulose membrane to form a detection line (T line) and a quality control line (C line); and assembling the treated glass fiber membrane, the nitrocellulose membrane and the PVC base plate to obtain the fluorescence labeling test strip. The object to be tested dripped on the test strip sample pad can be combined with the signal probe, and can be combined with the corresponding antibody and the second antibody on the T line and the C line again in the lateral flowing process, and the strip display condition of the corresponding position is observed through a fluorescence imaging device to judge whether the food-borne pathogenic bacteria exist, so that the rapid diagnosis of the food-borne pathogenic bacteria is realized.
Description
Technical Field
The invention belongs to the technical field of pathogen detection, and particularly relates to a test strip which is constructed by using more than one conversion nanoparticles as fluorescent markers and is used for realizing rapid, sensitive and high-specificity detection of food-borne pathogens.
Background
Food-borne diseases have become a global public health safety issue, often caused by ingestion of food contaminated with pathogenic microorganisms and their toxins or other chemicals. Wherein food affected by food-borne bacteria can lead to illness and death in severe cases.
People are suitable hosts of food-borne pathogenic bacteria, and children, the old and people with immunodeficiency are more susceptible. About 14000 cases of shigellasis are reported annually, and the bacillary dysentery caused by shigella is mainly transmitted through the digestive tract. In order to monitor the occurrence and spread of relevant food-borne diseases, food needs to be checked regularly to ensure public health safety, and therefore, rapid and sensitive screening of these food-borne pathogens using rapid, highly sensitive tools is of paramount importance.
In the traditional detection method, selective culture and biochemical detection are mainly used for detecting food-borne pathogenic bacteria. These methods typically require a significant amount of time and labor. Due to the development of molecular biology techniques, a real-time quantitative PCR method based on deoxyribonucleic acid (DNA) molecules has been developed to detect food-borne pathogenic bacteria. However, PCR-based methods require complex cell disruption and nucleic acid extraction protocols. Therefore, rapid and real-time detection of food-borne pathogenic bacteria is limited to a certain extent.
On the whole, although the traditional detection method has low technical requirements and low cost, the detection method is time-consuming, labor-consuming and inaccurate, and the real-time quantitative PCR method overcomes some defects of the traditional method, but has high cost, high operation technical requirements and long time consumption.
Disclosure of Invention
In order to solve the defects in the prior art, the invention provides a fluorescent labeling test strip for rapidly detecting food-borne pathogenic bacteria and a preparation method thereof, which can realize rapid, high-specificity and high-sensitivity detection of the food-borne pathogenic bacteria in food.
The technical scheme adopted by the invention is as follows:
a preparation method of a fluorescent labeling test strip for rapidly detecting food-borne pathogenic bacteria comprises the following steps:
step 1, synthesizing rare earth element doped up-conversion fluorescent nanoparticles by adopting a high-temperature thermal decomposition method;
step 2, carboxyl modification is carried out on the surface of the upconversion fluorescent nanoparticle synthesized in the step 1 by using nitrosonium tetrafluoroborate ion, so that the upconversion fluorescent nanoparticle has water solubility and antibody connection stability;
step 3, carrying out particle initial washing, activation, coupling and sealing in an aqueous solution by using an acylation catalyst to form a stable connection structure of the food-borne pathogenic bacterium specific antibody and the upconversion nanoparticles; the stable linking structure is an antibody up-conversion conjugate;
step 4, processing the sample pad by using the glass fiber membrane processing liquid, and cutting the sample pad into proper sizes for splicing after the sample pad is dried;
step 5, treating the combination pad by using glass fiber membrane treatment liquid, drying, and spraying the combination pad by using the antibody up-conversion conjugates prepared in the step 3 in different proportions;
step 6, diluting the specific antibodies of the food-borne pathogenic bacteria and the specific secondary antibody solution for resisting the food-borne pathogenic bacteria antibodies by using a coating solution, and then scribing on a nitrocellulose membrane at the spraying amount of 0.5 mu L/cm and the speed of 30mm/s to form a T line and a C line, wherein the distance between the T line and the C line is constant; specific antibodies of food-borne pathogenic bacteria and specific secondary antibodies of the food-borne pathogenic bacteria antibodies are used as capture probes;
step 7, respectively sticking the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on a PVC fluorescent rubber plate at a constant distance and an overlapping thickness so as to ensure that the chromatography process is smoothly carried out;
step 8, preparing bacterial liquids with different concentration gradients, detecting the bacterial liquids by using the test strips prepared in the step 7, measuring fluorescence intensity values of a T line and a C line by using a fluorescence spectrometer, and drawing a standard curve;
step 9, preparing a sample to be detected, measuring fluorescence values of a T line and a C line after the sample to be detected is added, obtaining the number of actual target bacteria in the sample to be detected, and realizing quantitative detection;
and step 10, correcting the test strip according to the results of the step 8 and the step 9, adding a known amount of sample liquid to be detected, and judging the fluorescence of the T line and the C line on the test strip so as to realize rapid detection.
Further, the rare earth doped up-conversion fluorescent nano material is a lanthanide doped up-conversion fluorescent nano particle, and the up-conversion fluorescent nano particle has a strong fluorescence value at 548nm under 980nm excitation light, and can be used for labeling an antibody.
Further, the rare earth element doped up-conversion fluorescent nano material is synthesized by adopting a high-temperature thermal decomposition method, and the specific operation is as follows: chloride of rare earth activator and sensitizer, NaOH and NH4F、YCl3Adding the rare earth element into an organic solvent, reacting at different stages, washing with ethanol, and dissolving in cyclohexane to obtain the rare earth element doped up-conversion fluorescent nano material.
Further, the method is characterized in that carboxyl modification is carried out on the surface of the upconversion fluorescent nanoparticle by nitrosation of nitroso tetrafluoroborate ions, so that the surface of the upconversion fluorescent nanoparticle has water solubility and antibody connection stability.
Further, the operation process of nitrosation by using nitroso-ion of tetrafluoroborate is as follows: BF mixing4Dissolving and dispersing NO and up-conversion particles in an N, N-dimethylformamide solution; adding toluene and cyclohexane, centrifuging, adding a polyacrylic acid aqueous solution and acetone, and stirring to obtain the upconversion fluorescent nanoparticle with the surface subjected to carboxyl modification.
Further, the operation process of preparing the stable connection structure of the antibody and the upconverting particle by amidation reaction in step 3 is: adding NHS, EDC, a primary washing buffer solution, a coupling buffer solution, a blocking buffer solution, an antibody and upconversion particles to obtain an antibody upconversion conjugate;
further, the preparation method of the sample pad and the bonding pad is characterized in that: soaking the sample pad in glass fiber membrane treatment solution, and drying with adsorption force of 19-23 μ L/cm2(ii) a Soaking the bonding pad in glass fiber membrane treatment solution, air-drying at room temperature, and oven-drying; the sample pad and the conjugate pad were cut in equal proportions.
Further, the method for preparing the nitrocellulose membrane T line and the C line in step 6 is characterized in that: the climbing speed of the nitrocellulose membrane is 89-90 seconds, the nitrocellulose membrane is cut to a set length, and the nitrocellulose membrane is stuck on a fluorescent PVC rubber plate; diluting related antibodies by using coating solution of T line and C line, and scribing on a nitrocellulose membrane to form a detection area and a quality control area; the distance between the T line and the C line is constant; then placing the mixture in an air-blast drying oven at 37 ℃ for drying for 48-50h, and sealing and storing.
Further, the operation of splicing the test paper strips in the step 7 is as follows: the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to a PVC rubber plate, the connection part of the sample pad and the combination pad is overlapped by 1-1.5mm, and the connection part of the nitrocellulose membrane and the water absorption pad is overlapped by 2mm, so that the chromatographic process is ensured to be carried out smoothly, and the spliced test strip is stored at room temperature.
Furthermore, the method is characterized in that the fluorescence labeling test strip is prepared by utilizing the up-conversion luminescent material with adjustable luminescent property, so that the fluorescence labeling test strip has obvious light stability, is used for monitoring the food-borne pathogenic bacteria for a long time, has no obvious loss of signal intensity, and has more reliable and accurate result.
Further, the detection method in step 10 is characterized in that: the calibrated test strip is placed into portable intelligent reading equipment, light emitted by a near-infrared excitation light source of the equipment passes through a focusing lens, a dichroic mirror, the focusing lens and a light filter and then is captured by a rear camera of the smart phone, and a fluorescence labeling test strip image is obtained so as to rapidly distinguish the food-borne pathogenic bacteria pollution condition of the sample.
Further, a fluorescence image of the fluorescence labeling test strip is obtained through a portable fluorescence imaging device, and quantitative detection of the food-borne pathogenic bacteria is achieved by combining image processing and chemometrics technologies.
The fluorescent marking test strip for rapidly detecting the food-borne pathogenic bacteria is prepared by adopting the fluorescent marking test strip for rapidly detecting the food-borne pathogenic bacteria and the preparation method thereof, and is used for detecting the food-borne pathogenic bacteria in food.
The invention has the beneficial effects that:
the method of the invention modifies the up-conversion nano particles on the antibody to be used as fluorescence signals, and sprays corresponding specific primary antibody solution and specific secondary antibody solution on the nitrocellulose membrane. After the target is added, the target can be combined with the up-conversion antibody conjugate, in the flowing process of the test strip, the object to be tested can be combined with the T-line antibody on the nitrocellulose membrane, after the test strip is irradiated by infrared exciting light, the T-line shows fluorescence, and the higher the concentration of the object to be tested is, the stronger the fluorescence intensity of the T-line is. Meanwhile, the C line emits light all the time and can be used as a basis for judging whether the test strip has detectability. According to different fluorescence intensities, the quantitative detection of the target bacteria can be realized. In addition, in portable test paper strip reading equipment, the accessible smart mobile phone formation of image judges whether contain the sample that awaits measuring in the sample fast, realizes quantitative determination simultaneously. The method has the advantages of high sensitivity, strong specificity, simple operation and high detection speed, can realize the detection of different target objects by changing the connection of different antibodies, and has important significance in the field of food analysis and detection.
Drawings
FIG. 1 is a schematic diagram of the structure and detection of a fluorescence-labeled test strip of the present invention;
in FIG. 2, A is the T-line fluorescence spectrum of an actual milk sample; and B is the imaging result of the actual milk sample.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a preparation method of a fluorescent marking test strip for rapidly detecting food-borne pathogenic bacteria, which comprises the following steps:
step 1, respectively preparing chloride, NaOH and NH of a rare earth activator and a rare earth sensitizer by adopting a high-temperature thermal decomposition method4F、YCl3Adding the mixture into an organic solvent mixed with 1-octadecene and oleic acid, reacting at 3 stages of temperature, rapidly heating to 300 ℃, maintaining the temperature for 1 hour, and repeatedly washing with ethanol and cyclohexane to obtain the rare earth element-doped up-conversion fluorescent nano material; the rare earth doped up-conversion fluorescent nano material in the embodiment is up-conversion fluorescent nano particles doped with lanthanide, for example, doped NaYF obtained by Er4Yb/Er upconversion fluorescent nanoparticles; and the upconversion fluorescent nanoparticle has a strong fluorescence value at 548nm under 980nm excitation light, and can be used for labeling an antibody.
Step 2, carboxyl modification is carried out on the surface of the upconversion fluorescent nano-particles synthesized in the step 1 by using nitrosonium tetrafluoroborate, and BF is weighed4Dissolving NO solid in N, N-dimethylformamide solution, adding the upconversion nanoparticles synthesized in the step 1 into the solution to uniformly disperse the upconversion nanoparticles in the solution, adding toluene and cyclohexane, and centrifuging; an aqueous polyacrylic acid solution was added and heated. Finally adding acetone, and centrifuging to obtain carboxyl modified upconversion nanoparticles; the surface of the up-conversion fluorescent nano-particle has water solubility and antibody connection stability.
And 3, forming a stable connector of the antibody and the upconversion nanoparticles in an aqueous solution by using an acylation catalyst, wherein the operation process comprises the following steps: preparing a primary washing buffer solution, a coupling buffer solution, a blocking buffer solution, a storage buffer solution and the like to connect the upconversion nanoparticles with the antibody, and realizing the combination of the antibody and the upconversion particles through EDC and sulfo-NHS mediated amidation reaction;
step 4, taking the milk and the dairy products as samples in the embodiment, soaking the glass fiber sample pad for applying the milk and dairy products samples in the glass fiber membrane treatment solution, soaking in a rotary manner on a rotary oscillator, airing at room temperature, and then drying by air blowing. Cutting the dried sample pad into 4mm × 15mm, putting into an aluminum foil bag, vacuumizing, sealing and storing;
and 5, soaking the sample pad for loading the particle-labeled antibody in glass fiber treatment solution, performing rotary soaking on a rotary oscillator, airing at room temperature, and then performing air blast drying. The dried conjugate pad was cut to a size of 4mm x 3 mm. Diluting the prepared UCNP-antibody marker with a microsphere diluent, spraying the diluted UCNP-antibody marker onto a bonding pad, and then drying by air blowing. And after drying, putting the bonding pad into an aluminum foil bag, vacuumizing, sealing and storing. After drying, the conjugate pad is assembled with the sample pad to ensure that the solution migrates properly through the test strip during the test.
And 6, diluting the specific antibody against the shigella flexneri and the specific secondary antibody solution against the shigella flexneri with a coating solution, cutting the nitrocellulose membrane, attaching the cut nitrocellulose membrane to a PVC (polyvinyl chloride) rubber plate, and scribing the diluted specific secondary antibody solution on the nitrocellulose membrane at the spraying amount of 0.5 mu L/cm and the speed of 30mm/s to form a T line (detection line) and a C line (quality control line) which are in flat distribution, namely forming a detection area and a quality control area, wherein the distance between the detection area and the quality control area is 5 mm. And after the line is drawn, the whole PVC rubber plate is placed into a blast drying oven for drying, and after drying, the PVC rubber plate is placed into an aluminum foil bag for vacuumizing, sealing and storing.
Step 7, adhering the sample pad, the combination pad and the water absorption pad on a PVC fluorescent rubber plate at a constant distance and with an overlapping thickness of 1mm so as to ensure that the chromatography process is smoothly carried out; before the test paper strip is assembled, each part needs to be processed, and the method comprises the following specific operations:
sample pad: a glass fiber membrane is adopted, and the glass fiber membrane is firstly soaked in glass fiber membrane treatment fluid (20mM Tris-base containing 0.5% (w/v) Tetronic 1307, 0.05% (w/v) casein, 0.3% (w/v) PVP-K30, 0.05% (v/v) Tween-20 and the pH value being 8.0), is soaked for 2 hours in a rotary mode, is dried in the air at room temperature, is placed in a blast drying oven, is dried for 48 hours at the temperature of 30 ℃, and is then placed in an aluminum foil bag to be vacuumized and sealed for storage.
Combining the pads: a glass fiber membrane is adopted, and the membrane is soaked in glass fiber treatment solution (20mM Tris-base containing 0.5% (w/v) Tetronic 1307, 0.05% (w/v) casein, 0.3% (w/v) PVP-K30, 0.05% (v/v) Tween-20 and the pH value being 8.0) and is soaked in a rotary mode for 2h, the membrane is dried at room temperature and then placed in an air-blast drying oven, and the membrane is dried at 30 ℃ for 48 h. The prepared UCNPs-antibody markers were diluted with microsphere diluent (20nM Tris-base containing 15% (w/v) sucrose, 0.5% (w/v) BSA, 0.5% (v/v) tween-20, 0.5% (w/v) PVP, 0.05% (v/v) P-300, pH 8.4), and then sprayed on a conjugate pad, and then placed in an air-drying oven, and dried at 30 ℃ for 48h in an air-drying oven at 30 ℃ for 48 h. And putting the dried mixture into an aluminum foil bag, vacuumizing, sealing and storing.
Cellulose nitrate membrane: cutting and sticking on a fluorescent PVC rubber plate. The corresponding antibodies were used to draw a T-line and a C-line on the nitrocellulose membrane, respectively. The relevant antibodies were diluted with coating solution (10mM sodium citrate, containing 1% (w/v) sucrose, pH 7.4) before streaking, and streaked onto nitrocellulose membranes at a distance of 5mM, and dried for 1 hour after streaking.
Assembling the test strip: stacking the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad in a certain sequence, and paying attention to the overlapping distance of each part, for example, the combination pad is pasted on the upper left of the nitrocellulose membrane, the sample pad is pasted on the upper left of the combination pad, meanwhile, the connection part of the sample pad and the combination pad is ensured to be overlapped by 1mm, and the connection part of the combination pad and the nitrocellulose membrane is ensured to be overlapped by 2 mm; a water absorption pad is attached to the right upper side of the nitrocellulose membrane, and the connection part of the nitrocellulose membrane and the water absorption pad is overlapped by 1mm to ensure that the chromatography process is smoothly carried out. Finally, the stacked parts are glued to a PVC base plate as shown in figure 1.
Step 8, preparing bacterial liquids with different concentration gradients, inoculating the shigella flexneri bacterial liquids into a beef extract peptone medium, performing plate counting after culturing at the constant temperature of 37 ℃ for 24 hours, calculating the number of shigella flexneri per milliliter, adding PBS buffer solution to dilute bacterial suspensions with different concentration gradients, sampling, adding the samples into the test paper strip prepared in the step 7, detecting the test paper strip, measuring the fluorescence intensity values of a T line and a C line by using a fluorescence spectrometer, and drawing a standard curve;
step 9, preparing a sample to be detected, adding the test strip prepared in the step 7 into the sample to be detected with known bacteria concentration, measuring the fluorescence values of a T line and a C line after the sample to be detected is added by using a portable fluorescence spectrometer, and obtaining the number of actual target bacteria in the sample to be detected according to a standard curve;
The following further explains the detection process of the test strip prepared by the method: when a non-to-be-detected object is dripped into the test strip sample pad, the signal probe in the combination pad is not combined with the non-to-be-detected object, the signal probe is only combined with the corresponding second antibody on the C line in the lateral flow process, and the C line generates a fluorescence signal only under the excitation of 980nm near infrared light. When an object to be detected is dripped into a test strip sample pad, a signal probe in the combination pad is combined with the object to be detected, and is combined with corresponding antibodies and secondary antibodies on a T line and a C line again in the lateral flow process, so that the strip display condition of the corresponding position is observed through a fluorescence imaging device to judge whether food-borne pathogenic bacteria exist, and meanwhile, the quantitative detection of the food-borne pathogenic bacteria is realized through the acquired fluorescence image in combination with the image processing and chemometrics technology; spectral signals are collected through self-made portable fluorescence detection, and a quantitative detection model of the food-borne pathogenic bacteria is established.
To further test the specificity of the detection system: staphylococcus aureus, escherichia coli and bacillus cereus with the quantity far higher than that of food-borne pathogenic bacteria are added into the specific test strip prepared in the step 7, and under the imaging of intelligent equipment, the test strip can be seen to have T-line fluorescence reaction only on target bacteria;
through the example, the method has the advantages of high sensitivity, strong specificity, simple operation and high detection speed, can realize the detection of other target objects by replacing related antibodies, and has very important significance in the field of food safety detection.
The above embodiments are only used for illustrating the design idea and features of the present invention, and the purpose of the present invention is to enable those skilled in the art to understand the content of the present invention and implement the present invention accordingly, and the protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes and modifications made in accordance with the principles and concepts disclosed herein are intended to be included within the scope of the present invention.
Claims (13)
1. A preparation method of a fluorescent labeling test strip for rapidly detecting food-borne pathogenic bacteria is characterized by comprising the following steps:
step 1, synthesizing rare earth element doped up-conversion fluorescent nanoparticles by adopting a high-temperature thermal decomposition method;
step 2, carboxyl modification is carried out on the surface of the upconversion fluorescent nanoparticle synthesized in the step 1 by using nitrosonium tetrafluoroborate ion, so that the upconversion fluorescent nanoparticle has water solubility and antibody connection stability;
step 3, carrying out particle initial washing, activation, coupling and sealing on an acylation catalyst in an aqueous solution to form a stable connection structure between the specific antibody of the food-borne pathogenic bacteria and the upconversion nanoparticles, thus obtaining an antibody upconversion conjugate which is used as a signal probe;
step 4, processing the sample pad by using the glass fiber membrane processing liquid, and cutting the sample pad into proper sizes for splicing after the sample pad is dried;
step 5, treating the combination pad by using glass fiber membrane treatment liquid, drying, and spraying the combination pad by using the antibody up-conversion conjugates prepared in the step 3 in different proportions;
step 6, diluting the specific antibodies of the food-borne pathogenic bacteria and the specific secondary antibody solution for resisting the food-borne pathogenic bacteria antibodies by using a coating solution, and then scribing on a nitrocellulose membrane at the spraying amount of 0.5 mu L/cm and the speed of 30mm/s to form a T line and a C line, wherein the distance between the T line and the C line is constant; specific antibodies of food-borne pathogenic bacteria and specific secondary antibodies of the food-borne pathogenic bacteria antibodies are used as capture probes;
step 7, respectively sticking the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad on a PVC fluorescent rubber plate at a constant distance and an overlapping thickness so as to ensure that the chromatography process is smoothly carried out;
step 8, preparing bacterial liquids with different concentration gradients, detecting the bacterial liquids by using the test strips prepared in the step 7, measuring fluorescence intensity values of a T line and a C line by using a fluorescence spectrometer, and drawing a standard curve;
step 9, preparing a sample to be detected, measuring fluorescence values of a T line and a C line after the sample to be detected is added, obtaining the number of actual target bacteria in the sample to be detected, and realizing quantitative detection;
and step 10, correcting the test strip according to the results of the step 8 and the step 9, adding a known amount of sample liquid to be detected, and judging the fluorescence of the T line and the C line on the test strip so as to realize rapid detection.
2. The preparation method of the fluorescence labeling test strip for the rapid detection of the food-borne pathogenic bacteria as claimed in claim 1, wherein the rare earth doped up-conversion fluorescence nano material is a lanthanide doped up-conversion fluorescence nano particle, and the up-conversion fluorescence nano particle has a strong fluorescence value at 548nm under 980nm excitation light, and can be used for labeling an antibody.
3. The preparation method of the fluorescence labeling test strip for the rapid detection of the food-borne pathogenic bacteria according to claim 2, characterized in that the rare earth element doped up-conversion fluorescence nano material is synthesized by a high-temperature thermal decomposition method, and the specific operations are as follows: chloride of rare earth activator and sensitizer, NaOH and NH4F、YCl3Adding the rare earth element into an organic solvent, reacting at different stages, washing with ethanol, and dissolving in cyclohexane to obtain the rare earth element doped up-conversion fluorescent nano material.
4. The preparation method of the fluorescence labeling test strip for the rapid detection of the food-borne pathogenic bacteria according to claim 1, characterized in that carboxyl modification is carried out on the surface of the upconversion fluorescence nanoparticles by nitrosation of tetrafluoroborate nitrosoions, so that the surface of the upconversion fluorescence nanoparticles has water solubility and stability of antibody connection.
5. The preparation method of the fluorescence labeling test strip for the rapid detection of the food-borne pathogenic bacteria according to claim 4, characterized in that the nitrosation of the nitrosonium tetrafluoroborate is carried out by the following steps: BF mixing4Dissolving and dispersing NO and up-conversion particles in an N, N-dimethylformamide solution; adding toluene and cyclohexane, centrifuging, adding a polyacrylic acid aqueous solution and acetone, and stirring to obtain the upconversion fluorescent nanoparticle with the surface subjected to carboxyl modification.
6. The method for preparing a fluorescence labeling test strip for rapidly detecting food-borne pathogenic bacteria according to claim 1, wherein the operation process of preparing the stable connection structure of the antibody and the upconversion particles by amidation reaction in the step 3 is as follows: adding NHS, EDC, a primary washing buffer solution, a coupling buffer solution, a blocking buffer solution, an antibody and upconversion particles to realize the connection of the antibody and upconversion fluorescent nanoparticles to obtain an antibody upconversion conjugate; and used as a signaling probe.
7. The method for preparing a fluorescence labeling test strip for rapidly detecting food-borne pathogenic bacteria according to claim 1, wherein the sample pad and the bonding pad are prepared by the following steps: soaking the sample pad in glass fiber membrane treatment solution, and drying with adsorption force of 19-23 μ L/cm2(ii) a Soaking the bonding pad in glass fiber membrane treatment solution, air-drying at room temperature, and oven-drying; the sample pad and the conjugate pad were cut in equal proportions.
8. The method for preparing a fluorescence labeling test strip for rapidly detecting food-borne pathogenic bacteria according to claim 1, wherein the method for preparing the nitrocellulose membrane T line and the nitrocellulose membrane C line in step 6 comprises the following steps: the climbing speed of the nitrocellulose membrane is 89-90 seconds, the nitrocellulose membrane is cut to a set length, and the nitrocellulose membrane is stuck on a fluorescent PVC rubber plate; diluting related antibodies by using coating solution of T line and C line, and scribing on a nitrocellulose membrane to form a detection area and a quality control area; the distance between the T line and the C line is constant; then placing the mixture in an air-blast drying oven at 37 ℃ for drying for 48-50h, and sealing and storing.
9. The method for preparing a fluorescence labeling test strip for rapidly detecting food-borne pathogenic bacteria according to claim 1, wherein the splicing operation of the test strip in the step 7 is as follows: the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to a PVC rubber plate, the connection part of the sample pad and the combination pad is overlapped by 1-1.5mm, and the connection part of the nitrocellulose membrane and the water absorption pad is overlapped by 2mm, so that the chromatographic process is ensured to be carried out smoothly, and the spliced test strip is stored at room temperature.
10. The method for preparing a fluorescence labeling test strip for rapidly detecting food-borne pathogenic bacteria according to claim 1, characterized in that the fluorescence labeling test strip is prepared by utilizing an up-conversion luminescent material with adjustable luminescent property, so that the fluorescence labeling test strip has remarkable light stability, is used for monitoring the food-borne pathogenic bacteria for a long time without remarkable signal intensity loss, and has more reliable and accurate result.
11. The fluorescence labeling test strip for rapidly detecting food-borne pathogenic bacteria and the preparation method thereof as claimed in claim 1, wherein the detection method in step 10 is: the calibrated test strip is placed into portable intelligent reading equipment, light emitted by a near-infrared excitation light source of the equipment passes through a focusing lens, a dichroic mirror, the focusing lens and a light filter and then is captured by a rear camera of the smart phone, and a fluorescence labeling test strip image is obtained so as to rapidly distinguish the food-borne pathogenic bacteria pollution condition of the sample.
12. The fluorescence labeling test strip for rapidly detecting food-borne pathogenic bacteria and the preparation method thereof as claimed in claim 1, characterized in that the quantitative detection of the food-borne pathogenic bacteria is realized by acquiring the fluorescence image of the fluorescence labeling test strip through a portable fluorescence imaging device and combining with image processing and chemometrics technology.
13. A fluorescent-labeled test strip for rapidly detecting food-borne pathogenic bacteria, which is characterized by being prepared by the fluorescent-labeled test strip for rapidly detecting food-borne pathogenic bacteria and the preparation method thereof in claim 1 and used for detecting the food-borne pathogenic bacteria in food.
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