CN110618273B - Preparation method of fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins - Google Patents

Preparation method of fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins Download PDF

Info

Publication number
CN110618273B
CN110618273B CN201910799967.3A CN201910799967A CN110618273B CN 110618273 B CN110618273 B CN 110618273B CN 201910799967 A CN201910799967 A CN 201910799967A CN 110618273 B CN110618273 B CN 110618273B
Authority
CN
China
Prior art keywords
fluorescence
enterotoxin
test
fluorescent
quality control
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910799967.3A
Other languages
Chinese (zh)
Other versions
CN110618273A (en
Inventor
邹小波
胡雪桃
石吉勇
李艳肖
甘子玉
徐艺伟
李亚惠
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu University
Original Assignee
Jiangsu University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu University filed Critical Jiangsu University
Priority to CN201910799967.3A priority Critical patent/CN110618273B/en
Publication of CN110618273A publication Critical patent/CN110618273A/en
Application granted granted Critical
Publication of CN110618273B publication Critical patent/CN110618273B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex

Abstract

The invention belongs to the field of rapid detection of food safety, and relates to a preparation method of a fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins; the method comprises the following steps: firstly, preparing two fluorescent carbon quantum dots with good fluorescence characteristics, good water solubility and good biocompatibility, and preparing a series of fluorescent coding microspheres with different fluorescence characteristics by adjusting the doping ratio of the two carbon quantum dots; using carbon quantum dots to perform fluorescence coding on the microspheres and correspondingly marking the antibody of the enterotoxin to obtain a plurality of fluorescent antibody probes; finally, dissolving the multiple fluorescent antibody probes in a phosphate buffer solution containing sucrose and Tween 20 in equal mass to obtain a mixed solution containing the multiple fluorescent antibody probes; then uniformly spraying the test paper strip on a glass fiber membrane to be used as a bonding pad, so as to prepare the fluorescent microsphere immunochromatographic test paper strip, and realize simultaneous qualitative and quantitative detection of various enterotoxins; the invention can improve the detection efficiency and accuracy and realize the rapid detection of a large number of food samples.

Description

Preparation method of fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins
Technical Field
The invention belongs to the field of rapid detection of food safety, and particularly relates to a preparation method of a fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins.
Background
Staphylococcus aureus is a common food-borne pathogenic bacterium, widely existing in nature, and the secreted staphylococcus aureus enterotoxin (SE) is a main cause of staphylococcus aureus food poisoning. Studies have shown that most S.aureus produces many enterotoxins, A, B, C, D, E and F. In the food processing process, enterotoxin produced by the external secretion of the thalli has extremely strong resistance to heat, can not be completely destroyed after being heated to 100 ℃ for 30 minutes, can still resist the decomposition of protease in stomach after being taken into a human body, and causes symptoms of vomiting, diarrhea and the like of the human body. At present, methods for detecting enterotoxin mainly comprise animal infection experiments, molecular biology methods and enzyme-linked immunosorbent assay. These conventional methods are long in time consumption, cumbersome to operate, high in cost, and not suitable for rapid screening of food samples, so that a method for rapidly detecting enterotoxin in food needs to be researched.
The immunochromatographic detection technology is a rapid detection method developed by combining an immunological technology and a chromatographic technology of antigen-antibody immunological reaction, wherein the main labeled materials comprise radioactive materials, enzymes, colloidal gold, fluorescein and the like, and the main immunochromatographic test paper in the market at present is mainly a colloidal gold labeled immunochromatographic test paper strip and can only realize the detection and analysis of one substance or only realize the colorimetric detection and analysis; with the rapid development of novel nano materials, the fluorescent coding microspheres show great application potential in the fields of marking, tracing, detection and the like by virtue of stable morphological structures and stable and efficient luminous efficiency; therefore, there is a need to develop an immunochromatographic test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins, so as to greatly improve the detection efficiency and detection accuracy of food samples.
Disclosure of Invention
Aiming at the defects of the prior detection technology, the invention aims to solve one of the problems; the carbon quantum dot fluorescence coding microsphere immunochromatography test strip capable of simultaneously detecting various staphylococcus aureus enterotoxins is provided, so that the staphylococcus aureus enterotoxins can be efficiently, quickly and accurately detected; the invention firstly prepares two fluorescent carbon quantum dots with good fluorescence characteristic, good water solubility and good biocompatibility, and prepares a series of fluorescent coding microspheres with different fluorescence characteristics by adjusting the doping ratio of the two carbon quantum dots; and then preparing a fluorescent microsphere immunochromatographic test strip to realize simultaneous qualitative and quantitative detection of multiple enterotoxins.
In order to achieve the above purpose, the method specifically comprises the following steps:
step 1, preparing carbon quantum dot fluorescent coding microspheres;
adding sucrose A into phosphoric acid A, stirring, and then placing in a microwave oven to heat A to obtain carbon quantum dots, which are marked as CQD 1; adding sucrose B into phosphoric acid B, stirring, and heating in a microwave oven to obtain carbon quantum dots (CQD 2);
then mixing CQD1 and CQD2 in m different proportions to obtain a mixture of m carbon quantum dots, respectively dispersing the mixture in chloroform, respectively adding poly (maleic anhydride-alt-1-octadecene) (PMAO), polymethyl methacrylate (PMMA) and Sodium Dodecyl Sulfate (SDS) to perform first ultrasonic dispersion, finally adding sodium hydroxide (NaOH) to perform second ultrasonic dispersion, and centrifuging and washing to obtain m carbon quantum dot fluorescence encoding microspheres, wherein the m carbon quantum dot fluorescence encoding microspheres are marked as QB 1 、QB 2 、QB 3 、……、QB m ；
Step 2, marking the antibody probe by the fluorescent coding microsphere;
taking m staphylococcus aureus enterotoxins as SE1, SE2, SE3, … … and SEm respectively, and taking antibodies corresponding to the m staphylococcus aureus enterotoxins as Ab respectively 1 、Ab 2 、Ab 3 、……、Ab m ；
Fluorescent coding microspheres QB 1 Bovine Serum Albumin (BSA) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were added to phosphate buffer for a first stirring reaction, followed by the addition of antibody Ab to SE1 1 And N-hydroxy thiosuccinimide (NHS) for the second stirring reaction, and centrifuging after the reaction is finished to obtain a fluorescent coding microsphere labeled antibody probe QB 1 @Ab 1 (ii) a In the same way, QB is processed according to the steps 2 Ab antibody to SE2 2 Combining, QB 3 Ab antibody to SE3 3 In combination, … …, QB m Ab antibody to SEm m Binding to obtain QB 1 @Ab 1 、QB 2 @Ab 2 、QB 3 @Ab 3 、……、QB m @Ab m A fluorescent antibody probe; namely, the m carbon quantum dot fluorescent coding microspheres obtained in the step 1 are used for correspondingly marking the enterotoxin antibodies one by one to obtain m fluorescent antibody probes; finally, dissolving m kinds of fluorescent antibody probes in equal mass in a phosphate buffer solution containing sucrose and Tween 20 to obtain a mixed solution containing m kinds of fluorescent antibody probes;
step 3, preparing the fluorescent coding microsphere test strip;
s1, preparing a bonding pad, a test line and a quality control line;
uniformly spraying the fluorescent antibody probe mixed solution prepared in the step 2 onto a glass fiber membrane to serve as a bonding pad, and drying and packaging for later use;
then marking m test lines T and 1 quality control line on the nitrocellulose membrane, wherein the test lines are respectively marked as T1, T2, T3, … … and Tm, the quality control lines are marked as C1, the lines of the test lines and the quality control lines keep certain width, and certain intervals are kept between the lines;
taking m staphylococcus aureus enterotoxin antigens, and respectively marking as SE1-BSA, SE2-BSA, SE3-BSA, … … and SEm-BSA; then, spraying SE1-BSA to a test line T1, spraying SE2-BSA to a test line T2, and spraying … … and SEm-BSA to a test line Tm by using a film spraying machine to finish the preparation of the test line; spraying the goat anti-mouse IgG immunoglobulin to the position of a quality control line C1 to complete the preparation of the quality control line; drying and packaging the obtained nitrocellulose membrane for later use;
s2, assembling an immunochromatographic test strip;
the test strip takes a PVC lining plate as a substrate, and a sample pad, a combination pad, a nitrocellulose membrane with m test lines T and 1 quality control line C1 and an absorption pad are respectively stuck on the PVC lining plate substrate from left to right; the two adjacent stickers are mutually overlapped, the right end of the sample pad is overlapped with the left end of the combination pad, the right end of the combination pad is overlapped with the left end of the nitrocellulose membrane, and the right end of the nitrocellulose membrane is overlapped with the left end of the absorption pad; after the bonding is finished, cutting the test paper appropriately to obtain a fluorescent coding microsphere test paper strip, and storing the test paper strip at 4 ℃;
preferably, in the step 1, the power of the microwave oven is 700-900W; the heating time A is 60-120 s; the time for heating B is 20-50 s.
Preferably, in the step 1, the dosage ratio of the sucrose A to the phosphoric acid A is 0.1-2 g: 5-15 mL; the dosage ratio of the sucrose B to the phosphoric acid B is 0.1-2 g: 5-15 mL.
Preferably, in the step 1, the mass ratio of the mixed CQD1 and CQD2 is 0-3: 0-2.5, and CQD1 and CQD2 are not 0 at the same time.
Preferably, in the step 1, the amount ratio of the carbon quantum dot mixture, chloroform, PMAO, PMMA, SDS and NaOH is 0.2 to 0.8 g: 5-10L: 1-2 g: 1-2 g: 0.1-0.5 g: 0.1 to 1 g.
Preferably, in the step 1, the time for the first ultrasonic dispersion is 5-20 min, and the time for the second ultrasonic dispersion is 15-30 min.
Preferably, in the step 2, the first stirring reaction is carried out for 30-60 min; the time of the second stirring reaction is 60 min-120 min.
Preferably, in step 2, the fluorescently encoded microspheres QB 1 The dosage ratio of BSA, EDC, phosphate buffer, antibody and NHS is 1-10 mug: 10-100 mg: 1-2 mg: 10-20 mL: 10-20 μ g: 0.5-1 mg.
Preferably, in the step 2, the dosage ratio of the fluorescent antibody, the sucrose, the tween 20 and the phosphate buffer is 3-30 μ g: 5-20 mg: 0.1-2 mL: 5-15 mL; the concentration of the phosphate buffer solution is 0.1mol/L, and the pH value is 7.4.
Preferably, in step 3, in S1, the test line and the quality control line have a certain width of 2mm to 5mm, and a certain distance of 3mm to 8mm is maintained between the lines.
Preferably, in step 3, in S1, the drying temperature is 35 to 40 ℃.
Preferably, in step 3, in S1, the spraying amount of the fluorescent antibody probe mixed solution uniformly sprayed on the glass fiber is 1 to 20 μ L/cm; the spraying amount of the staphylococcus aureus enterotoxin antigen sprayed to the test line is 5-20 mu L/cm; the spraying amount of the goat anti-mouse IgG immunoglobulin to the quality control line C1 is 5-20 mu L/cm.
Preferably, in S2 of step 3, the width of the overlapping portion is 2 to 4 mm.
Preferably, in the step 1-3, m is less than or equal to 6 and is a positive integer;
method for detecting staphylococcus aureus enterotoxin by using fluorescent coding microsphere test strip
(1) Preparing an enterotoxin solution, dropwise adding the enterotoxin solution to the sample pad of the immunochromatography test strip prepared in the step 3, standing for reaction for a period of time, placing the test strip in a portable fluorescence imager, and recording the change conditions of the fluorescence color and the fluorescence intensity of the test line and the quality control line; if the test strip does not show fluorescence at the position of the quality control line C1, the test result of the test strip is invalid, and if the test strip shows fluorescence, the test result is valid;
the higher the concentration of enterotoxin in the enterotoxin solution is, the weaker the fluorescence intensity of the test line on the test strip is; correspondingly arranging different fluorescence pictures of the test lines according to the enterotoxin concentration from low to high; establishing a fluorescence colorimetric card for detecting enterotoxin SE1 by arranging fluorescence pictures of a test line T1, and recording the fluorescence colorimetric card as K1; arranging the fluorescence pictures of the test line T2, establishing a fluorescence colorimetric card for detecting enterotoxin SE2, and recording as K2; arranging the fluorescence pictures of the test line T3, establishing a fluorescence colorimetric card for detecting enterotoxin SE3, and recording as K3; … …, arranging fluorescence pictures of the test line Tm, and establishing a fluorescence colorimetric card for detecting the enterotoxin SEm, and recording the colorimetric card as Km;
further using a fluorescence immunochromatography reading instrument to read the fluorescence signal intensity of the test line and the quality control line C1, and establishing a standard curve by using the ratio of the fluorescence signal intensity of the test line and the quality control line C1 and the linear relation of the corresponding enterotoxin concentration: y is a + BX, where Y is the ratio of the fluorescence signal intensity of the test line to the quality control line C1, X is the concentration of enterotoxin, a is a constant term, and B is the coefficient of the equation;
(2) simultaneous detection of m enterotoxins in a food sample;
the pretreatment of the food sample refers to standard 'detection method of staphylococcus aureus enterotoxin A in import and export food electrophoresis and immunoblotting' to obtain a food sample solution;
the detection is the same as the step (1), the difference is that the enterotoxin solution is replaced by the food sample solution, the food sample solution is dripped on the sample pad, after standing reaction, whether the test strip quality control line C1 has fluorescence or not is firstly observed, if no fluorescence exists, the test strip is invalid, and the food sample solution needs to be re-detected by using a new test strip; if fluorescence exists, continuously observing the fluorescence colors of the sample test line T1, the test line T2, the test lines T3, … … and the test line Tm, respectively comparing the fluorescence colors with the fluorescence color comparison cards K1, K2, K3, … … and Km constructed in the step (1), and preliminarily judging the concentration range of the enterotoxin through the fluorescence colors, so that the visualization and semi-quantitative detection of the enterotoxin are realized;
obtaining fluorescence signal values of test lines T1, T2, T3, … … and Tm through a fluorescence immunochromatography reading instrument, and respectively recording the fluorescence signal values as T1s, T2s, T3s, … … and Tms; and (3) recording the fluorescence signal value of C1 as C1s, and calculating the ratio of the fluorescence signal intensity of the test line to the fluorescence signal intensity of the quality control line C1, substituting the ratio into the quantitative standard curve established in the step (1), and calculating the content of the m enterotoxins respectively, thereby realizing the rapid and accurate detection of the m enterotoxins in the food sample.
Preferably, in the step (1), the concentration of the enterotoxin solution is 0-1 mg/mL; the reaction time is 5-10 min.
The invention has the beneficial effects that:
(1) the invention firstly prepares two fluorescent carbon quantum dots with different emission properties, synthesizes a plurality of fluorescent coding microspheres with different fluorescence properties by adjusting the proportion of the two carbon quantum dots, and compared with other fluorescent materials, the fluorescent coding microspheres have the advantages of stable structure, high fluorescent quantum yield, stable fluorescence properties, uniform particle size distribution and the like.
(2) The immunochromatographic test strip prepared based on the carbon quantum dot fluorescent coding microspheres can meet the requirement of simultaneous detection of various staphylococcus aureus enterotoxins, can improve the detection efficiency, and can realize rapid detection of a large number of food samples.
(3) The established detection method can realize qualitative and quantitative detection of staphylococcus aureus enterotoxin; the prepared fluorescent colorimetric card can visually and visually detect the content of the staphylococcus aureus enterotoxin, can realize qualitative or semi-quantitative detection of the staphylococcus aureus enterotoxin and realize quick screening of unqualified foods; the established quantitative detection method can realize the rapid and simultaneous detection of various staphylococcus aureus enterotoxins; to a certain extent, the invention only needs one portable fluorescence imaging analyzer and one portable fluorescence immunochromatographic reading instrument, has simple and convenient operation and high sensitivity, and can realize real-time, rapid and visual detection of various staphylococcus aureus enterotoxins.
Drawings
FIG. 1 (a) is a schematic representation of two fluorescent carbon quantum dots prepared in example 1; (b) schematic diagrams of three fluorescence-encoded microspheres and corresponding fluorescence spectrum curves thereof are shown.
FIG. 2 is a schematic diagram of the immunochromatographic test strip prepared in example 1; wherein, 1-PVC lining board, 2-sample pad, 3-combination pad, 4-nitrocellulose membrane, 5-test line T1, 6-test line T2, 7-test line T3, 8-quality control line C1, 9-absorption pad.
FIG. 3 is a fluorescent colorimetric card for detecting enterotoxins A, B and C in example 1.
FIG. 4 is the immunochromatographic test strip for detecting a food sample in example 1.
Detailed Description
The invention will be described in further detail with reference to the drawings and the detailed description, but the scope of the invention is not limited thereto.
Staphylococcus aureus enterotoxin antibodies and antigens were purchased from North Noro Biotech, Inc. of Shanghai, and Staphylococcus aureus enterotoxin was purchased from Toxin Technology, USA.
The prepared carbon quantum dot fluorescence coding microsphere immunochromatography test strip can realize simultaneous, rapid and visual detection of various staphylococcal enterotoxins, and is beneficial to ensuring the quality safety of foods, agricultural products and the like. Therefore, the present embodiment further illustrates the present invention by taking the preparation of a fluorescence-encoded microsphere immunochromatographic test strip for simultaneously detecting 3 enterotoxin carbon quantum dots, the preparation of a fluorescence-encoded microsphere immunochromatographic test strip for detecting 1 enterotoxin carbon quantum dot, and the preparation of a fluorescence-encoded microsphere immunochromatographic test strip for simultaneously detecting 6 enterotoxin carbon quantum dots as specific examples:
example 1:
the invention relates to a carbon quantum dot fluorescence coding microsphere immunochromatography test strip for simultaneously detecting 3 staphylococcus aureus enterotoxins, wherein the 3 staphylococcus aureus enterotoxins are enterotoxin A, enterotoxin B and enterotoxin C, and the preparation method specifically comprises the following steps:
step 1, preparing carbon quantum dot fluorescent coding microspheres;
firstly, 1g of cane sugar and 10mL of phosphoric acid are mixed and stirred and then placed in a microwave oven (750W) to be heated for 1min and 30s respectively, 2 fluorescent carbon quanta are obtained and marked as CQD1 and CQD2, emission peaks are 440nm and 650nm respectively, and blue fluorescence and red fluorescence are displayed respectively; according to the following steps of 1: 0. 3: 1 and 0: 1, 3 kinds of CQD1 and CQD2 are premixed in different proportions, and 1mg of poly (maleic anhydride-alt-1-octadecene), 1.2mg of polymethyl methacrylate and 0.5mg of proportioned carbon quantum dots are dispersed in 10mL of chloroform; then, 0.3mg of sodium dodecylsulfate was added to the above chloroform dispersion and dispersed by ultrasound for 10 min. Then adding 0.2mg of sodium hydroxide and carrying out ultrasonic treatment for 25 min; finally, the reaction mixed solution is centrifuged and washed by ultrapure water, and the carbon quantum dot fluorescent coding microspheres (blue QB) with 3 different fluorescent colors are obtained 1 Blue-violet QB 2 And red QB 3 ) (ii) a As shown in fig. 1 (a), is a schematic diagram of two kinds of carbon quantum dots, which respectively display blue and red fluorescence; fig. 1 (b) is a schematic diagram of the prepared 3 kinds of carbon quantum dot fluorescence-encoded microspheres, and it can be seen from the schematic diagram that the usage ratios of the blue carbon quantum dots and the red carbon quantum dots are respectively 1: 0. 3: 1 and 0: 1. the proportion is 1: 0 fluorescent microsphere QB 1 The emission peak is 440nm, and blue fluorescence is developed; the proportion is 3: 1 fluorescent microspheres QB 2 Emission peaks are 440nm and 650nm, and blue-violet fluorescence is developed; the ratio is 0: 1 fluorescent microspheres QB 3 The emission peak is 650nm, and the color is developed into red fluorescence; therefore, the invention successfully prepares the 3-color fluorescence coding microsphere, blue QB 1 Blue-violet QB 2 And red QB 3 ；
Step 2, marking the antibody probe by the fluorescent coding microsphere;
taking 3 staphylococcus aureus enterotoxin A, B and C, and numbering as SE1, SE2 and SE 3; the antibodies corresponding to the 3 staphylococcus aureus enterotoxins are respectively marked as Ab 1 、Ab 2 、Ab 3 ；
Carrying out fluorescence encoding on the QB microspheres of the three carbon quantum dots obtained in the step 1 1 、QB 2 And QB 3 3 antibodies (Ab1, Ab2 and Ab3, forming QB) are respectively marked 1 @Ab1、QB 2 @ Ab2 and QB 3 @ Ab3 fluorescent antibody probe, and mixing the obtained 3 fluorescent antibody probes to form a fluorescent antibody probe capable of specifically and simultaneously detecting SE1, SE2 and SE 3;
the specific steps are 5 mu g of carbon quantum dot fluorescence coding microspheres QB 1 Adding 20mg bovine serum albumin and 1mg 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide into 15mL phosphate buffer solution (0.2mol/L, pH 7.4) in sequence, stirring for reaction for 45min, then adding 10 mu g of detection SE1 antibody (Ab1) and 0.5mg N-hydroxy thiosuccinimide, stirring, mixing and reacting for 1.5h, and centrifuging after the reaction is finished to obtain a fluorescence coding microsphere labeled antibody probe QB 1 @ Ab1, QB 2 @ Ab2 and QB 3 @ Ab3 fluorescent antibody probe; finally, the three fluorescent antibodies were mixed in equal amounts and redissolved in 10mL of phosphate buffer (0.1mol/L, pH 7.4) containing 10mg of sucrose and 1mL of Tween 20 for use.
Step 3, preparing an immunochromatographic test strip;
(1) preparing a bonding pad, a test line and a quality control line: uniformly spraying the three fluorescent antibody probe mixed solutions prepared in the step 2 on glass fibers to prepare a bonding pad, wherein the spraying amount is 10 mu L/cm; marking 3 test lines (T1, T2 and T3) and 1 quality control line C1 on the nitrocellulose membrane by using a pencil, wherein the width of the line is 2mm, and the interval between the adjacent lines is 4 mm;
taking 3 staphylococcus aureus enterotoxin A, B and C antigens, respectively marking as SE1-BSA, SE2-BSA and SE 3-BSA; then, respectively spraying the antigens SE1-BSA, SE2-BSA and SE3-BSA of staphylococcus aureus enterotoxin A, B and C to the positions of test lines T1, T2 and T3 by using a film spraying machine to obtain test lines T1, T2 and T3, wherein the spraying amount is 8 mu L/cm; spraying goat anti-mouse IgG immunoglobulin to the position of a quality control line C1 to obtain a quality control line C1, wherein the spraying amount is 12 mu L/cm; drying and packaging the prepared combined pad and the nitrocellulose membrane at 37 ℃ for later use;
(2) assembling the test strip: the assembled immunochromatographic test strip is shown in fig. 2, the test strip takes a PVC lining plate as a substrate, and a sample pad, a combination pad, a nitrocellulose membrane with a test line T1, a test line T2, a test line T3 and a quality control line C1 and an absorption pad are respectively stuck on the PVC lining plate substrate from left to right; overlapping two adjacent stickers, such as overlapping the right end of the sample pad and the left end of the combination pad, overlapping the right end of the combination pad and the left end of the nitrocellulose membrane, overlapping the right end of the nitrocellulose membrane and the left end of the absorption pad, wherein the width of the overlapping part is 3mm, cutting the substances into test strips with the width of 3.5mm by a cutting machine after the adhesion is finished, sealing the test strips to obtain the immunochromatography test strip, and storing the test strip at 4 ℃; thus obtaining the immunochromatographic test strip which can simultaneously detect enterotoxins 1, 2 and 3;
a method for detecting enterotoxin;
(1) preparing 9 mixed solutions containing enterotoxin 1, 2 and 3 with different concentrations, wherein the concentrations are 0, 0.05, 0.1, 0.3, 0.5, 0.8, 1, 2 and 3 ng/mL; wherein the concentration of enterotoxin 1, 2, and 3 in each mixture is the same; respectively dropwise adding the mixed solution with different concentrations onto a sample pad of an immunochromatography test strip, wherein one concentration corresponds to one test strip; after 5min, placing the test strip in a portable fluorescence imager to observe the change conditions of the fluorescence color and the fluorescence intensity of the test line and the quality control line (as shown in figure 3); if the test strip does not show fluorescence at the position of the quality control line C1, the test strip is invalid, and if the test strip shows fluorescence, the test result is valid;
when the prepared mixed solution does not contain enterotoxin, the test line T1, the test line T2, the test line T3 and the quality control line C1 on the test strip have obvious fluorescence, and the fluorescence intensity of the test line T1, the test line T2 and the test line T3 in the test strip is gradually weakened along with the increase of the concentration of the enterotoxin; the fluorescence of the test line is weakened because the fluorescent antibody probe on the bonding pad is not bonded with the antigen in the test line after being bonded with the enterotoxin in the mixed solution; the fluorescence colorimetric card K1 for detecting the enterotoxin 1 is established according to a test line T1 arranged from low to high in the concentration of the enterotoxin 1, the fluorescence colorimetric card K2 for detecting the enterotoxin 2 is established according to a test line T2 arranged from low to high in the concentration of the enterotoxin 2, and the fluorescence colorimetric card K3 for detecting the enterotoxin C is established according to a test line T3 arranged from low to high in the concentration of the enterotoxin 3. As shown in FIG. 3, the intensity of blue fluorescence of the test line T1 continuously decreases with the increase of SE1 concentration; the blue-violet fluorescence intensity of the test line T2 continuously weakens with the increase of the concentration of SE 2; the intensity of red fluorescence of test line T3 decreased with increasing concentration of SE 3. The visual detection of the enterotoxin can be realized by utilizing the prepared fluorescence colorimetric cards K1, K2 and K3, and the enterotoxin 1, 2 and 3 can be detected qualitatively or semi-quantitatively;
the fluorescence signal intensity of the test line and the quality control line is read by using a fluorescence immunochromatography reading instrument, and a standard curve y (1.0209-0.2988 x) is established by utilizing the linear relation between the fluorescence signal ratio (T1/C1) of the test line T1 and the quality control line C1 and the concentration of enterotoxin 1 (correlation coefficient R) 2 0.9943, y is the fluorescence signal ratio of T1/C1, and x is the concentration of enterotoxin 1 (ng/mL)); a standard curve y (1.2657-0.4761 x) is established by utilizing the linear relation between the fluorescence signal ratio (T2/C1) of a T2 test line and a quality control line C1 and the concentration of enterotoxin 2 (correlation coefficient R) 2 0.9851, y is the fluorescence signal ratio of T2/C1, x is the concentration of enterotoxin B). The linear relation between the fluorescence signal ratio (T3/C1) of the test line T3 and the quality control line C1 and the enterotoxin C concentration is utilized to establish a standard curve y of 2.6419-0.8289x (correlation coefficient R) 2 0.9926, y is the fluorescence signal ratio of T3/C1, and x is the concentration of enterotoxin C). Therefore, the invention establishes a method for quantitatively detecting the enterotoxin A, B and C according to the fluorescence signal of the immunochromatographic test paper.
(2) Simultaneous detection of enterotoxin A, B and C in the food sample;
the pretreatment of the milk sample refers to standard 'detection method electrophoresis and immunoblotting of staphylococcus aureus enterotoxin A in import and export foods', and the pretreatment steps are as follows: measuring 1mL of milk sample, adding 10mL of normal saline, and homogenizing to obtain a sample preparation solution; detecting enterotoxin in the food sample according to the step 4, dropwise adding the preparation solution of the pretreated sample to the sample pad, and standing for reaction for 5 min; after the reaction is finished, putting the test strip into a portable fluorescence imager, putting the test strip into the portable fluorescence imager, firstly observing whether a quality control line C1 has fluorescence or not, if no fluorescence exists, indicating that the test strip is invalid, if the quality control line C1 has fluorescence, continuously observing the fluorescence colors of test lines T1, T2 and T3 of the sample through naked eyes (as shown in figure 4), and comparing the fluorescence colors with fluorescence colorimetric cards K1, K2 and K3 respectively, and finding that the concentration of enterotoxin A is about 0.1ng/mL, the concentration of enterotoxin B is about 0.1-0.3 ng/mL, and the concentration of enterotoxin C is about 0.3ng/mL, so that the visualization and semi-quantitative detection of the enterotoxin A, B and C are realized;
reading fluorescence signal values of a test line T1, a test line T2, a test line T3 and a quality control line C1 by a fluorescence immunochromatography reading instrument, and respectively calculating the content of enterotoxin according to 3 quantitative standard curves established in the step 4; the method specifically comprises the following steps: substituting the fluorescence signal ratio T1s/C1s of enterotoxin A into the standard curve y of 1.0209-0.2988x, so that the concentration of the enterotoxin A is QA (0.989-1.0209)/-0.2988 of 0.107 ng/mL; substituting the fluorescence signal ratio T2s/C1 s-1.072 of enterotoxin B into the standard curve y-1.2657-0.4761 x, so that the concentration of enterotoxin B is QB (1.172-1.2657)/-0.4761-0.197 ng/mL; substituting the fluorescence signal ratio T3s/C1 s-2.41 of enterotoxin C into the standard curve y-2.6419-0.8289 x; the concentration of enterotoxin C is QC (2.41-2.6419)/0.8289 (0.280 ng/mL), thereby enabling quantitative, rapid and accurate detection of enterotoxin A, B and C in milk samples.
Example 2:
the preparation of the carbon quantum dot fluorescence coding microsphere immunochromatography test strip and the detection of staphylococcus aureus enterotoxin B (SEB) in the embodiment specifically comprises the following steps:
step 1, preparing carbon quantum dot fluorescent coding microspheres;
firstly, 1g of cane sugar and 10mL of phosphoric acid are mixed and stirred and then placed in a microwave oven (750W) to be heated for 1min and 30s respectively, 2 fluorescent carbon quanta are obtained and marked as CQD1 and CQD2, emission peaks are 440nm and 650nm respectively, and blue fluorescence and red fluorescence are displayed respectively; 1, according to 1 mixture ratio: 1 premixing CQD1 and CQD2, and dispersing 1mg of poly (maleic anhydride-alt-1-octadecene), 1.2mg of polymethyl methacrylate and 0.5mg of proportioned carbon quantum dots in 10mL of chloroform; then adding 0.3mg of sodium dodecyl sulfate into the chloroform dispersion liquid for ultrasonic dispersion for 10 min; then adding 0.2mg of sodium hydroxide and carrying out ultrasonic treatment for 25 min; finally, centrifuging the reaction mixed solution and washing the reaction mixed solution with ultrapure water to obtain carbon quantum dot fluorescent coding microspheres with 1 fluorescent color, namely QB;
step 2, marking the antibody probe by the fluorescent coding microsphere;
marking staphylococcus aureus enterotoxin B (SEB) antibody (Ab) by the carbon quantum dot fluorescence coding microspheres (QB) in the step 1 to form a QB @ Ab fluorescent antibody probe;
the method comprises the specific steps of sequentially adding 5 mu g of carbon quantum dot fluorescence coding microspheres QB, 20mg of bovine serum albumin and 1mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide) into 15mL of phosphate buffer solution (PBS, 0.2mol/L and pH 7.4) for stirring reaction for 45min, then adding 10 mu g of detection SEB antibody (Ab) and 0.5mg of N-hydroxy thiosuccinimide for stirring and mixing reaction for 1.5h, and centrifuging after the reaction is finished to obtain a fluorescence coding microsphere labeled antibody probe QB @ Ab so as to obtain a fluorescence antibody probe QB @ Ab capable of specifically detecting staphylococcus aureus enterotoxin B;
step 3, preparing an immunochromatographic test strip;
(1) preparing a bonding pad, a test line and a quality control line: uniformly spraying the fluorescent antibody probe QB @ Ab prepared in the step 2 onto a glass fiber membrane to prepare a binding pad, wherein the spraying amount is 10 mu L/cm; marking 1 test line T and 1 quality control line C on the nitrocellulose membrane by using a pencil, wherein the line width is 2mm, and the interval between adjacent lines is 5 mm; then, spraying a staphylococcus aureus enterotoxin B antigen (SEB-BSA) to a test line T by using a film spraying machine to obtain the test line T, wherein the spraying amount is 8 mu L/cm; spraying goat anti-mouse IgG immunoglobulin to the position of a quality control line C to obtain the quality control line C, wherein the spraying amount is 12 mu L/cm; drying and packaging the prepared combined pad and the nitrocellulose membrane at 37 ℃ for later use;
(2) assembling the test strip: the test strip takes a PVC lining plate as a substrate, and a sample pad, a combination pad, a nitrocellulose membrane with 1 test line T and 1 quality control line C and an absorption pad are respectively stuck on the PVC lining plate substrate from left to right; the two adjacent stickers are mutually overlapped, for example, the right end of the sample pad is overlapped with the left end of the combination pad, the right end of the combination pad is overlapped with the left end of the nitrocellulose membrane, the right end of the nitrocellulose membrane is overlapped with the left end of the absorption pad, the width of the overlapped part is 3mm, after the adhesion is finished, the sample pad is cut into test strips with the width of 4mm by a cutting machine and sealed to obtain immunochromatographic test strips, and the immunochromatographic test strips are stored at 4 ℃; therefore, the fluorescent coding microsphere immunochromatographic test strip capable of detecting staphylococcus aureus enterotoxin SEB is prepared in the embodiment.
A method for detecting enterotoxin;
(1) preparing 9 solutions containing enterotoxin B, wherein the concentrations of the solutions are 0, 0.05, 0.1, 0.3, 0.5, 0.8, 1, 2 and 3ng/mL respectively; dripping the solution on a sample pad of an immunochromatography test strip, wherein one concentration corresponds to one test strip; and after 5min, placing the test strip in a portable fluorescence imager to observe the change conditions of the fluorescence color and the fluorescence intensity of the test line and the quality control line. If the test strip does not show fluorescence at the position of the quality control line C1, the test strip is invalid, and if the test strip shows fluorescence, the test strip is valid, and the detection result is valid. When the mixed solution does not have the concentration of the enterotoxin B, the test line T and the quality control line C on the test strip have obvious fluorescence, and the fluorescence intensity of the test line T in the test strip is gradually weakened along with the increase of the concentration of the enterotoxin; this is because the fluorescent antibody probe on the conjugate pad is not bound to the antigen in the test line after binding to enterotoxin, which results in the decrease of fluorescence of the test line, and the purple fluorescence intensity of the test line T is continuously decreased with the increase of the concentration of SEB. The method comprises the following steps of establishing a fluorescence colorimetric card K for detecting the enterotoxin B according to a T test line arranged from low to high in the concentration of the enterotoxin B, wherein the prepared fluorescence colorimetric card K can be used for realizing visual detection of the enterotoxin and can be used for qualitatively or semi-quantitatively detecting the enterotoxin B;
the fluorescence signal intensity of the test line and the quality control line is read by using a fluorescence immunochromatography reading instrument, and a standard curve y is established as 1.5627-0.5141x (correlation coefficient R) by utilizing the linear relation between the fluorescence signal ratio (T/C) of the test line T and the quality control line C and the concentration of the enterotoxin B 2 0.9851, y is the fluorescence signal ratio of T/C, and x is the concentration of enterotoxin B (ng/mL)); therefore, the method for quantitatively detecting the enterotoxin B is established according to the fluorescent signal of the immunochromatographic test paper.
(2) Detection of enterotoxin in a food sample;
the pork sample is pretreated according to standard 'detection method electrophoresis and immunoblotting method of staphylococcus aureus enterotoxin A in import and export food', and the pretreatment steps are as follows: a pork sample of 1g is weighed, and then 10mL of physiological saline is added and homogenized to obtain a sample preparation solution. And (4) detecting enterotoxin in the food sample according to the step 4, dropwise adding the preparation solution of the pretreated sample to the sample pad, and standing for reaction for 5 min. After the reaction is finished, the test strip is placed into a portable fluorescence imaging instrument, the test strip is placed into the portable fluorescence imaging instrument, whether the quality control line C1 has fluorescence or not is firstly observed, if no fluorescence exists, the test strip is invalid, if the quality control line C1 has fluorescence, the fluorescence color of the test line T of the sample is continuously observed through naked eyes and is compared with the fluorescence colorimetric card K, the concentration of the enterotoxin B is approximately 0.5ng/mL, and therefore the visualization and semi-quantitative detection of the enterotoxin B are achieved.
And (4) reading fluorescence signal values of the test line T and the quality control line C by a fluorescence immunochromatography reading instrument, and calculating the content of the enterotoxin B according to the quantitative standard curve established in the step 4. The method specifically comprises the following steps: and substituting the fluorescence signal ratio Ts/Cs of the enterotoxin B into the standard curve y of 1.5627-0.5141x, so that the concentration of the enterotoxin B is QB (1.312-1.5627)/-0.5141 of 0.488ng/mL, thereby realizing the quantitative, rapid and accurate detection of the enterotoxin B in the pork sample.
Example 3:
the embodiment 3 of the invention prepares the carbon quantum dot fluorescence coding microsphere immunochromatography test strip for simultaneously detecting 6 staphylococcus aureus enterotoxins, wherein the 6 staphylococcus aureus enterotoxins are A, B, C, D, E and F, and the method comprises the following specific steps:
step 1, preparing carbon quantum dot fluorescent coding microspheres;
firstly, 1g of sucrose and 10mL of phosphoric acid are mixed and stirred and then placed in a microwave oven (750W) to be heated for 1min and 30s respectively, 2 kinds of fluorescent carbon quanta are obtained, which are marked as CQD1 and CQD2, emission peaks are 440nm and 650nm respectively, and blue fluorescence and red fluorescence are displayed respectively; according to the following steps of 1: 0. 1: 2. 1: 3. 0: 1. 2: 1 and 3: 1, 6 CQD1 and CQD2 are premixed in different proportions, and 1mg of poly (maleic anhydride-alt-1-octadecene), 1.2mg of polymethyl methacrylate and 0.5mg of proportioned carbon quantum dots are dispersed in 10mL of chloroform; then adding 0.3mg of sodium dodecyl sulfate into the chloroform dispersion liquid for ultrasonic dispersion for 10 min; then adding 0.2mg of sodium hydroxide and carrying out ultrasonic treatment for 25 min; finally, the reaction mixture is centrifuged and mixed with the ultra-high-pressure waterWashing with pure water to obtain 6 carbon quantum dot fluorescence encoding microspheres with different fluorescence colors, which are marked as QB 1 、QB 2 、QB 3 、QB 4 、QB 5 And QB 6 。
Step 2, marking the antibody probe by the fluorescent coding microsphere;
taking 6 staphylococcus aureus enterotoxin A, B, C, D, E and F, wherein the serial numbers are SE1, SE2, SE3, SE4, SE5 and SE 6; antibodies corresponding to 6 staphylococcus aureus enterotoxins are respectively marked as Ab 1 、Ab 2 、Ab 3 、Ab 4 、Ab 5 And Ab 6 ；
The carbon quantum dots in the step 1 are subjected to fluorescence encoding to obtain QB microspheres 1 、QB 2 、QB 3 、QB 4 、QB 5 And QB 6 Antibody Ab for respectively labeling 6 staphylococcus aureus enterotoxins 1 、Ab 2 、Ab 3 、Ab 4 、Ab 5 And Ab 6 Form QB 1 @Ab 1 、QB 2 @Ab 2 、QB 3 @Ab 3 、QB 4 @Ab 4 、QB 5 @Ab 5 、QB 6 @Ab 6 Fluorescent antibody probes, and mixing the 6 obtained fluorescent antibody probes to form the fluorescent antibody probes capable of specifically and simultaneously detecting staphylococcus aureus enterotoxin A, B, C, D, E and F.
The specific steps are 5 mu g of carbon quantum dot fluorescence coding microspheres QB 1 20mg of bovine serum albumin and 1mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide were added to 15mL of phosphate buffer solution (PBS, 0.2mol/L, pH 7.4) in sequence, stirred and reacted for 45min, and then 10. mu.g of antibody Ab for detecting SE1 was added 1 Stirring and mixing the probe and 0.5mg of N-hydroxy thiosuccinimide for reaction for 1.5h, and centrifuging after the reaction is finished to obtain a fluorescent coding microsphere labeled antibody probe QB 1 @Ab 1 QB can be obtained by the same method 2 @Ab 2 、QB 3 @Ab 3 、QB 4 @Ab 4 、QB 5 @Ab 5 、QB 6 @Ab 6 A fluorescent antibody probe; finally, 50ng of 6 kinds of fluorescent antibodies are respectively dissolved in 10mL of phosphate buffer containing 10mg of sucrose and 1mL of Tween 20(0.1mol/L, pH 7.4) to obtain a mixture containing 6 kinds of fluorescent antibody probes;
step 3, preparing an immunochromatographic test strip;
(1) preparing a bonding pad, a test line and a quality control line: uniformly spraying the 6 kinds of fluorescent antibody probe mixed liquid prepared in the step 2 on glass fiber to prepare a bonding pad, wherein the spraying amount is 10 mu L/cm; marking 6 test lines on the nitrocellulose membrane by using a pencil, marking as T1, T2, T3, T4, T5, T6 and 1 quality control line C1, wherein the line width is 2mm, and the interval between adjacent lines is 3 mm;
taking antigens of staphylococcus aureus enterotoxin A, B, C, D, E and F, respectively marking as SE1-BSA, SE2-BSA, SE3-BSA, SE4-BSA, SE5-BSA and SE6-BSA, then correspondingly spraying the antigens to test lines T1, T2, T3, T4, T5 and T6 in sequence to obtain test lines T1, T2, T3, T4, T5 and T6, wherein the spraying amount is 8 muL/cm; spraying goat anti-mouse IgG immunoglobulin to the position of a quality control line C1 to obtain a quality control line C1, wherein the spraying amount is 12 mu L/cm; drying and packaging the prepared combined pad and the nitrocellulose membrane at 37 ℃ for later use;
(2) assembling the test strip: sequentially adhering a sample pad, a combination pad, a cellulose membrane with a test line T1, a test line T2, a test line T3, a test line T4, a test line T5, a test line T6 and a quality control line C1 and an absorption pad on a PVC lining plate, wherein the length of an overlapped part between adjacent stickers is 4 mm; cutting into test strips with width of 3.5mm, sealing, and storing at 4 deg.C;
a method for detecting enterotoxin;
(1) preparing 9 mixed solutions containing A, B enterotoxins, C, D, E enterotoxins and F, wherein the concentration of each enterotoxin in each mixed solution is the same and is respectively 0, 0.05, 0.1, 0.3, 0.5, 0.8, 1, 2 and 3 ng/mL; dropwise adding the mixed solution onto a sample pad of an immunochromatography test strip, wherein one concentration corresponds to one test strip; and after 5min, placing the test strip in a portable fluorescence imager to observe the change conditions of the fluorescence color and the fluorescence intensity of the test line and the quality control line. If the test strip does not show fluorescence at the position of the quality control line C1, the test strip is invalid, and if the test strip shows fluorescence, the test strip is valid, and the detection result is valid. When no enterotoxin exists in the prepared mixed solution, the test line T1, the test line T2, the test line T3, the test line T4, the test line T5, the test line T6 and the quality control line C1 on the test strip have obvious fluorescence, and the fluorescence intensity of the test line T1, the test line T2, the test line T3, the test line T4, the test line T5 and the test line T6 in the test strip is gradually weakened along with the increase of the concentration of the enterotoxin. This is because the fluorescent antibody probe on the conjugate pad is bound to the enterotoxin in the mixed solution and then is not bound to the antigen in the test line, resulting in the decrease of fluorescence of the test line. Establishing a fluorescence color comparison card K1 for detecting the enterotoxin A according to a test line T1 arranged from low to high in the concentration of the enterotoxin A, obtaining a fluorescence color comparison card K2 for detecting the enterotoxin B, a fluorescence color comparison card K3 for detecting the enterotoxin C, a fluorescence color comparison card K4 for detecting the enterotoxin D, a fluorescence color comparison card K5 for detecting the enterotoxin E and a fluorescence color comparison card K6 for detecting the enterotoxin F in the same way. The prepared fluorescent colorimetric cards K1, K2, K3, K4, K5 and K6 can be used for realizing visual detection of enterotoxin, and can be used for qualitatively or semi-quantitatively detecting the enterotoxin A, B, C, D, E and F;
the fluorescence signal intensity of the test line and the quality control line is read by using a fluorescence immunochromatography reading instrument, and a standard curve y is established by utilizing the linear relation between the fluorescence signal ratio (T1/C1) of the test line T1 and the quality control line C1 and the concentration (the concentration range is 0-1 ng/mL) of the enterotoxin A 1 ＝0.9249-0.2847x 1 (correlation coefficient R) 2 ＝0.9943，y 1 The ratio of the fluorescence signals of T1/C1, x 1 As the concentration of enterotoxin a);
the linear relation between the fluorescence signal ratio (T2/C1) of the test line T2 and the quality control line C1 and the concentration of enterotoxin B is utilized to establish a standard curve y 2 ＝1.6419-0.5289x 2 (correlation coefficient R) 2 ＝0.9915，y 2 The ratio of the fluorescence signals of T2/C1, x 2 As the concentration of enterotoxin B);
the linear relation between the fluorescence signal ratio (T3/C1) of the T3 test line and the quality control line C1 and the concentration of enterotoxin C is used for establishing a standard curve y 3 ＝1.8657-0.6161x 3 (correlation coefficient R) 2 ＝0.9851，y 3 The ratio of the fluorescence signals of T3/C1, x 3 As the concentration of enterotoxin C);
fluorescence signal ratios (T4/C1) andestablishment of a Standard Curve y from the Linear relationship of the concentration of enterotoxin D 4 ＝2.5819-0.8129x 4 (correlation coefficient R) 2 ＝0.9916，y 4 The ratio of the fluorescence signals of T4/C1, x 4 As the concentration of enterotoxin D);
the linear relation between the fluorescence signal ratio (T5/C1) of the test line T5 and the quality control line C1 and the concentration of enterotoxin E is utilized to establish a standard curve y 5 ＝1.4357-0.4179x 5 (correlation coefficient R) 2 ＝0.9876，y 5 The ratio of the fluorescence signals of T5/C1, x 5 As the concentration of enterotoxin E);
the linear relation between the fluorescence signal ratio (T6/C1) of the T6 test line and the quality control line C1 and the concentration of enterotoxin F is used for establishing a standard curve y 6 ＝1.2536-0.4632x 6 (correlation coefficient R) 2 ＝0.9851，y 6 The ratio of the fluorescence signals of T6/C1, x 6 As the concentration of enterotoxin F);
therefore, the invention establishes a method for quantitatively detecting the enterotoxin A, B, C, D, E and F according to the fluorescence signal of the immunochromatographic test paper;
(2) simultaneously detecting 6 enterotoxins in the food sample;
the pretreatment of the rice cake sample refers to standard electrophoresis and immunoblotting method for detecting staphylococcus aureus enterotoxin A in import and export foods, and the pretreatment steps are as follows: a rice cake sample 1g was weighed, and 10mL of physiological saline was added thereto and homogenized to prepare a sample preparation solution. Detecting enterotoxin in the food sample according to the step 4, dropwise adding the preparation solution of the pretreated sample to the sample pad, and standing for reaction for 5 min; after the reaction is finished, putting the test strip into a portable fluorescence imager, firstly observing whether a quality control line C1 has fluorescence or not, if no fluorescence exists, indicating that the test strip is invalid, if the quality control line C1 has fluorescence, continuously observing the fluorescence colors of test lines T1, T2, T3, T4, T5 and T6 of the sample through naked eyes, and comparing the fluorescence colors with fluorescence colorimetric cards K1, K2, K3, K4, K5 and K6 respectively, and finding that the concentration of enterotoxin A is about 0.1ng/mL, the concentration of enterotoxin B is about 0.1-0.3 ng/mL, the concentration of enterotoxin C is about 0.3ng/mL, the concentration of enterotoxin D is about 0.5ng/mL, the concentration of enterotoxin E is about 0.5-0.8 ng/mL, and the concentration of enterotoxin E is about 0.8ng/mL, thereby realizing the visual and semi-quantitative detection of enterotoxin A, B, C, D, E and F;
reading fluorescence signal values of a test line T1, a test line T2, a test line T3, a test line T4, a test line T5, a test line T6 and a quality control line C1 by a fluorescence immunochromatographic reader, and respectively calculating the content of the enterotoxin according to the 6 quantitative standard curves established in the step 4; the method specifically comprises the following steps: substituting the fluorescence signal ratio T1s/C1s (0.8727) of enterotoxin A into the standard curve y of 0.9249-0.2847x, so that the concentration of the enterotoxin A is QA (0.9249-0.8727)/0.2847 of 0.183 ng/mL; similarly, the concentrations of enterotoxin B, C, D, E and F were found to be 0.237, 0.298, 0.5379, 0.6742 and 0.7854ng/mL, respectively, thereby enabling quantitative, rapid and accurate detection of enterotoxin A, B, C, D, E and F in the rice cake samples.
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations that do not depart from the spirit and scope of the invention are intended to be included within the scope of the appended claims.

Claims (9)

1. A preparation method of a fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins is characterized by comprising the following steps:
step 1, preparing carbon quantum dot fluorescent coding microspheres;
firstly, 1g of cane sugar and 10mL of phosphoric acid are mixed and stirred and then placed in a microwave oven with the power condition of 750W to be heated for 1min and 30s respectively, and 2 fluorescent carbon quanta are obtained and are marked as CQD1 and CQD 2; then mixing CQD1 and CQD2 in m different proportions to obtain a mixture of m carbon quantum dots, respectively dispersing the mixture in chloroform, respectively adding poly (maleic anhydride-alt-1-octadecene), polymethyl methacrylate and sodium dodecyl sulfate to perform first ultrasonic dispersion, finally adding sodium hydroxide to perform second ultrasonic dispersion, centrifuging and washing to obtain m carbon quantum dot fluorescence coding micro-dotsBall, denoted QB 1 、QB 2 、QB 3 、……、QB m ；
Step 2, taking m staphylococcus aureus enterotoxins, and respectively marking the m staphylococcus aureus enterotoxins as SE1, SE2, SE3, … … and SEm; the antibodies corresponding to the m staphylococcus aureus enterotoxins are respectively marked as Ab 1 、Ab 2 、Ab 3 、……、Ab m ；
Firstly, the fluorescence coding microsphere QB prepared in the step 1 is taken 1 Bovine serum albumin and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide were added to phosphate buffer to perform a first stirring reaction, followed by the addition of antibody Ab to SE1 1 And N-hydroxyl sulfo-succinimide, performing a second stirring reaction, and centrifuging after the reaction is finished to obtain a fluorescent coding microsphere labeled antibody probe QB 1 @Ab 1 ；
In the same way, QB is processed according to the steps 2 Ab antibody to SE2 2 Combining, QB 3 Ab antibody to SE3 3 In combination, … …, QB m Ab antibody to SEm m Combining to obtain QB 2 @Ab 2 、QB 3 @Ab 3 、……、QB m @Ab m A fluorescent antibody probe; namely, the m carbon quantum dot fluorescent coding microspheres obtained in the step 1 are used for correspondingly marking the enterotoxin antibodies one by one to obtain m fluorescent antibody probes; finally, dissolving m kinds of fluorescent antibody probes in equal mass in a phosphate buffer solution containing sucrose and Tween 20 to obtain a mixed solution containing m kinds of fluorescent antibody probes;
step 3, preparing the fluorescent coding microsphere test strip;
s1, uniformly spraying the fluorescent antibody probe mixed solution prepared in the step 2 onto a glass fiber membrane to serve as a bonding pad, and drying for later use; then marking m test lines T and 1 quality control line on the nitrocellulose membrane, wherein the test lines are respectively marked as T1, T2, T3, … … and Tm, the quality control lines are marked as C1, the lines of the test lines and the quality control lines keep a certain width, and a certain distance is kept between the lines;
taking m staphylococcus aureus enterotoxin antigens, and respectively marking as SE1-BSA, SE2-BSA, SE3-BSA, … … and SEm-BSA; then, spraying SE1-BSA to a test line T1, spraying SE2-BSA to a test line T2, and spraying … … and SEm-BSA to a test line Tm by using a film spraying machine to finish the preparation of the test line; spraying the goat anti-mouse IgG immunoglobulin to the position of a quality control line C1 to complete the preparation of the quality control line; drying the obtained nitrocellulose membrane for later use;
s2, taking a PVC lining plate as a substrate of the test strip, and respectively sticking a sample pad, a combination pad, a nitrocellulose membrane with m test lines T and 1 quality control line C1 and an absorption pad on the PVC lining plate substrate from left to right; the two adjacent stickers are mutually overlapped, the right end of the sample pad is overlapped with the left end of the combination pad, the right end of the combination pad is overlapped with the left end of the nitrocellulose membrane, and the right end of the nitrocellulose membrane is overlapped with the left end of the absorption pad; and after the bonding is finished, properly cutting to obtain the fluorescent coding microsphere test strip.
2. The method for preparing a fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins according to claim 1, wherein in the step 1, the mass ratio of the mixed CQD1 and CQD2 is 0-3: 0-2.5, and CQD1 and CQD2 are not 0 at the same time.
3. The method for preparing a fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins according to claim 1, wherein in step 1, the use amount ratio of the carbon quantum dot mixture, chloroform, poly (maleic anhydride-alt-1-octadecene), polymethyl methacrylate, sodium dodecyl sulfate and sodium hydroxide is 0.2-0.8 g: 5-10L: 1-2 g: 1-2 g: 0.1-0.5 g: 0.1-1 g; the time of the first ultrasonic dispersion is 5-20 min, and the time of the second ultrasonic dispersion is 15-30 min.
4. The method for preparing a fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins according to claim 1, wherein in the step 2, the first stirring reaction is carried out for 30-60 min; the time of the second stirring reaction is 60-120 min.
5. The method according to claim 1, wherein in step 2, the fluorescence-encoded microspheres QB are prepared by a method comprising the steps of 1 The dosage ratio of bovine serum albumin, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, phosphate buffer, antibody and N-hydroxy thiosuccinimide is 1-10 mu g: 10-100 mg: 1-2 mg: 10-20 mL: 10-20 μ g: 0.5-1 mg; the dosage ratio of the fluorescent antibody, the sucrose, the Tween 20 and the phosphate buffer solution is 3-30 mu g: 5-20 mg: 0.1-2 mL: 5-15 mL; the concentration of the phosphate buffer solution is 0.1mol/L, and the pH value is 7.4.
6. The method for preparing a fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins according to claim 1, wherein in step 3S 1, the test line and the quality control line have certain width of 2mm to 5mm and certain distance of 3mm to 8 mm; the drying temperature is 35-40 ℃; the spraying amount of the fluorescent antibody probe mixed solution sprayed on the glass fiber is 1-20 mu L/cm; the spraying amount of the staphylococcus aureus enterotoxin antigen sprayed to the test line is 5-20 mu L/cm; the spraying amount of the goat anti-mouse IgG immunoglobulin to the quality control line C1 is 5-20 mu L/cm.
7. The method for preparing a fluorescent coded microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins according to claim 1, wherein in step 3S 2, the width of the overlapping portion is 2-4 mm.
8. The use of the fluorescence-encoded microsphere test strip prepared by the method according to any one of claims 1 to 7 for detecting staphylococcus aureus enterotoxin is characterized by comprising the following steps:
(1) preparing an enterotoxin solution, dropwise adding the enterotoxin solution on a sample pad of a fluorescent coding microsphere test strip, standing for reacting for a period of time, placing the test strip in a portable fluorescent imager, and recording the change conditions of the fluorescence color and the fluorescence intensity of a test line and a quality control line;
correspondingly arranging different fluorescence pictures of the test lines according to the enterotoxin concentration from low to high; establishing a fluorescence colorimetric card for detecting enterotoxin SE1 by arranging fluorescence pictures of a test line T1, and recording the fluorescence colorimetric card as K1; arranging the fluorescence pictures of the test line T2, establishing a fluorescence colorimetric card for detecting enterotoxin SE2, and recording as K2; arranging the fluorescence pictures of the test line T3, establishing a fluorescence colorimetric card for detecting enterotoxin SE3, and recording as K3; … …, arranging fluorescence pictures of the test line Tm, and establishing a fluorescence colorimetric card for detecting the enterotoxin SEm, and recording the colorimetric card as Km;
further using a fluorescence immunochromatography reading instrument to read the fluorescence signal intensity of the test line and the quality control line C1, and establishing a standard curve by using the ratio of the fluorescence signal intensity of the test line and the quality control line C1 and the linear relation of the corresponding enterotoxin concentration: y = A + BX, wherein Y is the ratio of the fluorescence signal intensity of the test line to the quality control line C1, X is the concentration of enterotoxin, A is a constant term, and B is the coefficient of the equation;
(2) detection of enterotoxins in food samples: the pretreatment of the food sample refers to standard 'detection method of staphylococcus aureus enterotoxin A in import and export food electrophoresis and immunoblotting' to obtain a food sample solution;
the detection is the same as the step (1), the difference is that the enterotoxin solution is replaced by the food sample solution, the food sample solution is dripped on the sample pad, after standing reaction, whether the test strip quality control line C1 has fluorescence or not is firstly observed, if no fluorescence exists, the test strip is invalid, and the food sample solution needs to be re-detected by using a new test strip; if fluorescence exists, continuously observing the fluorescence colors of the sample test line T1, the test line T2, the test lines T3, … … and the test line Tm, respectively comparing the fluorescence colors with the fluorescence color comparison cards K1, K2, K3, … … and Km constructed in the step (1), and preliminarily judging the concentration range of the enterotoxin through the fluorescence colors, so that the visualization and semi-quantitative detection of the enterotoxin are realized;
obtaining fluorescence signal values of test lines T1, T2, T3, … … and Tm through a fluorescence immunochromatography reading instrument, and respectively recording the fluorescence signal values as T1s, T2s, T3s, … … and Tms; and (3) recording the fluorescence signal value of C1 as C1s, and calculating the ratio of the fluorescence signal intensity of the test line to the fluorescence signal intensity of the quality control line C1, substituting the ratio into the quantitative standard curve established in the step (1), and calculating the content of the enterotoxins, thereby realizing the rapid and accurate detection of the m enterotoxins in the food sample.
9. The use according to claim 8, wherein in step (1), the enterotoxin solution has a concentration of 0 to 1 mg/mL; the reaction time is 5-10 min; m in the steps (1) and (2) is less than or equal to 6 and is a positive integer.
CN201910799967.3A 2019-08-28 2019-08-28 Preparation method of fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins Active CN110618273B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910799967.3A CN110618273B (en) 2019-08-28 2019-08-28 Preparation method of fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910799967.3A CN110618273B (en) 2019-08-28 2019-08-28 Preparation method of fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins

Publications (2)

Publication Number Publication Date
CN110618273A CN110618273A (en) 2019-12-27
CN110618273B true CN110618273B (en) 2022-09-16

Family

ID=68922091

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910799967.3A Active CN110618273B (en) 2019-08-28 2019-08-28 Preparation method of fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins

Country Status (1)

Country Link
CN (1) CN110618273B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111929444A (en) * 2020-08-12 2020-11-13 四川大学华西医院 Effective immunoblotting PVDF membrane labeling method, primary anti-incubation method and elution and color development method

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101441212A (en) * 2008-12-04 2009-05-27 上海交通大学 Multiple-antigen synchronous detection method of quantum dot mark fluorescent immune
CN101762706A (en) * 2010-01-05 2010-06-30 中国农业大学 Method of detecting residue of small-molecule substance harmful to human body and a special kit
CN101812506A (en) * 2009-10-30 2010-08-25 中国人民解放军第三军医大学第一附属医院 Liquid chip encoded by multi-color quantum dot composite microspheres, as well as preparation method and detection method thereof
CN102053151A (en) * 2010-10-26 2011-05-11 华南理工大学 Fluorescent probe and method for rapidly detecting staphylococcus aureus by using same
CN102590506A (en) * 2012-02-16 2012-07-18 上海师范大学 Method for rapidly detecting and screening staphylococcus aureus
CN103482598A (en) * 2013-08-30 2014-01-01 吉林大学 Preparation method for monophosphate-adjuvant water-soluble fluorescent carbon quantum dot solid powder
CN104897906A (en) * 2015-06-25 2015-09-09 浙江大学 Dual quantitative protein toxin quantum dot fluorescence immunochromatographic strip and preparation method thereof
CN104914084A (en) * 2015-06-25 2015-09-16 浙江大学 Method for detecting proteins toxin by quantum dot cation exchange signal amplification technology
CN105353128A (en) * 2015-10-16 2016-02-24 江南大学 Colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins and preparation method thereof
CN105647527A (en) * 2016-01-22 2016-06-08 南京工业大学 Preparation method of yellow fluorescence carbon quantum dots
WO2018120855A1 (en) * 2016-12-28 2018-07-05 广州瑞博奥生物科技有限公司 Time-resolved fluorescent immunochromatographic test strip and kit for detecting myo, and preparation method therefor

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101441212A (en) * 2008-12-04 2009-05-27 上海交通大学 Multiple-antigen synchronous detection method of quantum dot mark fluorescent immune
CN101812506A (en) * 2009-10-30 2010-08-25 中国人民解放军第三军医大学第一附属医院 Liquid chip encoded by multi-color quantum dot composite microspheres, as well as preparation method and detection method thereof
CN101762706A (en) * 2010-01-05 2010-06-30 中国农业大学 Method of detecting residue of small-molecule substance harmful to human body and a special kit
CN102053151A (en) * 2010-10-26 2011-05-11 华南理工大学 Fluorescent probe and method for rapidly detecting staphylococcus aureus by using same
CN102590506A (en) * 2012-02-16 2012-07-18 上海师范大学 Method for rapidly detecting and screening staphylococcus aureus
CN103482598A (en) * 2013-08-30 2014-01-01 吉林大学 Preparation method for monophosphate-adjuvant water-soluble fluorescent carbon quantum dot solid powder
CN104897906A (en) * 2015-06-25 2015-09-09 浙江大学 Dual quantitative protein toxin quantum dot fluorescence immunochromatographic strip and preparation method thereof
CN104914084A (en) * 2015-06-25 2015-09-16 浙江大学 Method for detecting proteins toxin by quantum dot cation exchange signal amplification technology
CN105353128A (en) * 2015-10-16 2016-02-24 江南大学 Colloidal gold test strip for simultaneous detection of five staphylococcus aureus enterotoxins and preparation method thereof
CN105647527A (en) * 2016-01-22 2016-06-08 南京工业大学 Preparation method of yellow fluorescence carbon quantum dots
WO2018120855A1 (en) * 2016-12-28 2018-07-05 广州瑞博奥生物科技有限公司 Time-resolved fluorescent immunochromatographic test strip and kit for detecting myo, and preparation method therefor

Also Published As

Publication number Publication date
CN110618273A (en) 2019-12-27

Similar Documents

Publication Publication Date Title
Wang et al. Lanthanide-labeled fluorescent-nanoparticle immunochromatographic strips enable rapid and quantitative detection of Escherichia coli O157: H7 in food samples
CN106680496B (en) A kind of preparation of colorimetric fluorescence dual signal nanosphere and its application in immunochromatography quantitatively detects
CA1104928A (en) Oxidizing phenolic compounds in enzymatic immunoassay
WO2020199501A1 (en) Quick detection method for food allergens based on quantum dot fluorescence
CN111751527A (en) Novel coronavirus IgG and IgM antibody detection kit and preparation method and application thereof
CN110618273B (en) Preparation method of fluorescent coding microsphere test strip for simultaneously detecting multiple staphylococcus aureus enterotoxins
CN109239326A (en) Based on the micro-fluidic immuno-chip analysis method of magnetic particle nano enzyme and application
CN110988365A (en) Quantitative immune colloidal gold detection card and kit for cystatin C, microalbuminuria and urinary creatinine
CN201269879Y (en) Melamine detection test strip by color granule immunochromatography
CN107656048A (en) A kind of immuno-chromatographic test paper strip and its application of half-quantitative detection antigen or antibody
CN113917141A (en) Brucella latex microsphere antibody detection card and preparation method thereof
CN113912123A (en) Salmonella typhimurium multimode test strip based on magnetic molybdenum disulfide catalysis and photothermal effect, and detection method and application thereof
CN104931685A (en) Luminescence immune detection method based on recombinant antigen carrying His tag
JPH05223820A (en) Kit and method for detecting microorganism accompanied by periodontal disease using surfactant mixture as extraction composition
CN105510574B (en) Fluorescent nano probe and preparation method thereof and the method for the synchronous detection of Hazard factors a variety of in food
CN117233383A (en) Combined detection card for clostridium difficile antigen, toxin A and toxin B, preparation method thereof and kit
CN105785020A (en) Method for quickly detecting bacillus cereus
CN107727861B (en) A kind of pepsin assay kit and measuring method
CN109738638A (en) Direct immunization chromatographs detection method, test strips and the application for detecting Escherichia coli
CN106526166A (en) Rapid detection of lean meat powder in pork
US20040043510A1 (en) Particle-labeled protein and immuno-chromatograph using the same
CN111220802B (en) Clenbuterol hydrochloride small molecule hapten high sensitivity detection test paper based on nano antibody and preparation method thereof
CN113109325A (en) Pepsinogen I enzymatic chemiluminescence detection kit and preparation method and application thereof
CN113740543A (en) Kit for rapidly detecting proportion of glycosylated hemoglobin and preparation method thereof
Lu et al. A spatial color co-recognition immunochromatographic assay based on the hue-saturation-brightness color space to classify Salmonella serogroups

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant