Detection kit for multiple fungaltoxins, and preparation method, detection method and application of detection kit

Abstract

The invention relates to the technical field of analysis and detection, and in particular, relates to a detection kit for multiple fungaltoxins, and a preparation method, a detection method and application of the detection kit. The kit comprises up-conversion nanoparticles respectively coupled with antigens of fungaltoxins to be detected, magnetic nanoparticles respectively coupled with antibodies of the fungaltoxins to be detected, and a standard substance solution of the fungaltoxins, and the fungaltoxins to be detected at least comprise zearalenone and fumonisin B1. The kit and the method have the advantages of high sensitivity, good specificity and the like, have important practical significance on ultra-sensitive detection of multiple fungaltoxins, and have good guiding significance on realization of a field rapid and ultra-sensitive detection technology.

Description

Technical field

[0001] The present invention relates to the field of analysis and detection, and in particular, to a detection kit of a variety of fungal toxins, a preparation method and a detection method and application thereof. Background technique

[0002] Food safety issues are increasingly received globally. In particular, the most common, the largest harm is the fungal toxin. The fungal toxin is a secondary metabolite and non-protein molecular compound produced by fungi under a certain temperature and humidity conditions. They are widely distributed, a wide variety, strong toxicity, difficult detection, and may have multiple toxins in the same contaminated food. Rodiatisin B1, FB1) and Corn Alkenone (Zeralnone, ZEN) are two fungal toxins that are generally existing in corn, oats, wheat and other cereals and dairy products. They tend to cause acute poisoning, chronic poisoning, teratogenicity and carcinogenesis, and inevitable, extensive, have a wide range of health. Theref...

Examples

preparation example Construction

[0057] The second aspect of the invention relates to the preparation method of the kit, specifically figure 1 In still shown in Fig. A, an aminocated upper-converted nanoparticle is obtained, and then the interaction of two miocaridin and biotinity of the two types of nanoparticles by the classic glutaraldehyde method is subsequently. -Fb1-BSA and Biotin-Zen-BSA two toxin artificial antigen coupling, forming two different upper conversion fluorescent probes of Biotin-FB1-BSA-UCNPS and Biotin-Zen-BSA-UCNPs; the magnetic nanoparticles are respectively The FB1 monoclonal antibody and zen monoclonal antibody were coupled to obtain two different capture probes of MCABS-FB1-MNPS and MCABS-ZEN-MNPs.

[0058] The present invention proposes a method of preparing an amino-modified UCNPS, synthesizing NAYF using solvent heat 4 Yb 3+ TM 3+ NAYF 4 Yb 3+ , Er 3+ UCNPS. At least the following steps:

[0059] 1 will 1mmol Reclin 3 · 6h 2 O (Y: YB: TM = 78: 20: 2), 8 ~ 10mL oleic acid and 12 ~ 20 ...

Embodiment 1

[0090] High sensitivity immunofluorescence sensors based on magnetic separation and two-color conversion were used to establish a method of simultaneously detectovoxin B1 and corn ionexone.

[0091] 1) Preparation of amino modified UCNPS:

[0092] Solvent thermal method to synthesize NAYF 4 Yb 3+ TM 3+ NAYF 4 Yb 3+ , Er 3+ UCNPS.

[0093] 11mmol Reclin 3 · 6h 2 O (Y: YB: TM = 78: 20: 2), 9 ml oleic acid and 15 ml 1-octadene were added to 100ml three bottles. In nitrogen or argon protective environments, the homogeneous solution was heated to 120 ° C and degassed.

[0094] 2 The mixed solution is continuously stirred for 30 minutes and then naturally cooled to room temperature. Will contain 2.5 mmol NaOH and 4mmolnh 4 The 10 ml of methanol solution was slowly added dropwise to the flask, and then stirred at 50 ° C for 30 minutes to consume all fluoride.

[0095] 3 The solution was heated to 120 ° C and maintained for a long time to ensure sufficient evaporation of methanol. The sol...

Embodiment 2

[0127] High sensitivity immunofluorescence sensors based on magnetic separation and two-color conversion are used to simultaneously detect the method of simultaneously detecting vodoxin B1 and corn nibricate in corn flour.

[0128]1) Weigh 20g pulverized corn flour sample, add 50 mL of a predetermined 70% methanol solution, and then stirred at a magnetic stirrer at high speed for 2 min, filtered with quantitative filter paper, and quantify 10 ml of filtrate, and use double steamed water. Dilution 10 times (in order to avoid the influence of fluorescence detection, the filtrate is not colored), filtered with 0.45 μm filter.

[0129] 2) Add FB1 and ZEN standards to the filtrate of the corn flour sample in three different concentrations, so that the final concentration of FB1 and ZEN is 0.1 ng / ml, 0.5 ng / ml and 2 ng / ml, respectively, resulting in three different concentrations. Cay ton sample to be tested.

[0130] 3) In two different upper conversion fluorescent probes contain...

Technical field

[0001] The present invention relates to the field of analysis and detection, and in particular, to a detection kit of a variety of fungal toxins, a preparation method and a detection method and application thereof. Background technique

[0002] Food safety issues are increasingly received globally. In particular, the most common, the largest harm is the fungal toxin. The fungal toxin is a secondary metabolite and non-protein molecular compound produced by fungi under a certain temperature and humidity conditions. They are widely distributed, a wide variety, strong toxicity, difficult detection, and may have multiple toxins in the same contaminated food. Rodiatisin B1, FB1) and Corn Alkenone (Zeralnone, ZEN) are two fungal toxins that are generally existing in corn, oats, wheat and other cereals and dairy products. They tend to cause acute poisoning, chronic poisoning, teratogenicity and carcinogenesis, and inevitable, extensive, have a wide range of health. Theref...

Examples

preparation example Construction

[0057] The second aspect of the invention relates to the preparation method of the kit, specifically figure 1 In still shown in Fig. A, an aminocated upper-converted nanoparticle is obtained, and then the interaction of two miocaridin and biotinity of the two types of nanoparticles by the classic glutaraldehyde method is subsequently. -Fb1-BSA and Biotin-Zen-BSA two toxin artificial antigen coupling, forming two different upper conversion fluorescent probes of Biotin-FB1-BSA-UCNPS and Biotin-Zen-BSA-UCNPs; the magnetic nanoparticles are respectively The FB1 monoclonal antibody and zen monoclonal antibody were coupled to obtain two different capture probes of MCABS-FB1-MNPS and MCABS-ZEN-MNPs.

[0058] The present invention proposes a method of preparing an amino-modified UCNPS, synthesizing NAYF using solvent heat 4 Yb 3+ TM 3+ NAYF 4 Yb 3+ , Er 3+ UCNPS. At least the following steps:

[0059] 1 will 1mmol Reclin 3 · 6h 2 O (Y: YB: TM = 78: 20: 2), 8 ~ 10mL oleic acid and 12 ~ 20 ...

Embodiment 1

[0090] High sensitivity immunofluorescence sensors based on magnetic separation and two-color conversion were used to establish a method of simultaneously detectovoxin B1 and corn ionexone.

[0091] 1) Preparation of amino modified UCNPS:

[0092] Solvent thermal method to synthesize NAYF 4 Yb 3+ TM 3+ NAYF 4 Yb 3+ , Er 3+ UCNPS.

[0093] 11mmol Reclin 3 · 6h 2 O (Y: YB: TM = 78: 20: 2), 9 ml oleic acid and 15 ml 1-octadene were added to 100ml three bottles. In nitrogen or argon protective environments, the homogeneous solution was heated to 120 ° C and degassed.

[0094] 2 The mixed solution is continuously stirred for 30 minutes and then naturally cooled to room temperature. Will contain 2.5 mmol NaOH and 4mmolnh 4 The 10 ml of methanol solution was slowly added dropwise to the flask, and then stirred at 50 ° C for 30 minutes to consume all fluoride.

[0095] 3 The solution was heated to 120 ° C and maintained for a long time to ensure sufficient evaporation of methanol. The sol...

Embodiment 2

[0127] High sensitivity immunofluorescence sensors based on magnetic separation and two-color conversion are used to simultaneously detect the method of simultaneously detecting vodoxin B1 and corn nibricate in corn flour.

[0128]1) Weigh 20g pulverized corn flour sample, add 50 mL of a predetermined 70% methanol solution, and then stirred at a magnetic stirrer at high speed for 2 min, filtered with quantitative filter paper, and quantify 10 ml of filtrate, and use double steamed water. Dilution 10 times (in order to avoid the influence of fluorescence detection, the filtrate is not colored), filtered with 0.45 μm filter.

[0129] 2) Add FB1 and ZEN standards to the filtrate of the corn flour sample in three different concentrations, so that the final concentration of FB1 and ZEN is 0.1 ng / ml, 0.5 ng / ml and 2 ng / ml, respectively, resulting in three different concentrations. Cay ton sample to be tested.

[0130] 3) In two different upper conversion fluorescent probes contain...

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