▎ 摘　　要

NOVELTY - Low-field nuclear magnetic resonance homogeneous immunoassay for detecting food-borne pathogens based on explosive amplification strategy of gadolinium quantum dots involves (a) coupling polyclonal antibody (Ab) of food-borne pathogen to magnetic graphene oxide (MGO) to obtain capture unit MGO@Ab, (b) mixing diethyltriaminepentaacetic acid, gadolinium chloride hexahydrate and water, heating, coupling L-cysteine to gadolinium-carbon quantum dots (CQDs), adding carboxy-modified polystyrene beads, EDC and NHS to ethanol, adding ethylenediamine-terminated polyethyleneimine and thiol-modified gadolinium-CQDs dispersion, coupling with polyclonal Ab of food-borne pathogen and (c) mixing PS@gadolinium-CQDs@Ab dispersion, MGO@Ab dispersion, and food-borne pathogen sample, analyzing and establishing quantitative relationship between longitudinal relaxation time difference of water protons and concentration of food-borne pathogens for determining concentration. USE - The low-field nuclear magnetic resonance homogeneous immunoassay is useful for detecting food-borne pathogens, preferably Vibrio parahaemolyticus, Vibrio vulnificus, Staphylococcus aureus, Escherichia coli and Salmonella (claimed). ADVANTAGE - The immunoassay has high sensitivity and accuracy and strong specificity and is simple and rapid. DETAILED DESCRIPTION - Low-field nuclear magnetic resonance homogeneous immunoassay for detecting food-borne pathogens based on explosive amplification strategy of gadolinium quantum dots for non-diagnosis or treatment purposes involves (a) (i) preparing magnetic graphene oxide (MGO) by a dual-solvent method and (ii) coupling the polyclonal antibody (Ab) of food-borne pathogen to the surface of MGO by 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) coupling reaction, blocking the non-specific binding site with bovine serum albumin and obtaining capture unit MGO@Ab, (b) (i) mixing 0.8-1.5 g diethyltriaminepentaacetic acid, 0.1-0.3 g gadolinium chloride hexahydrate and 10 ml water, performing ultrasonic dispersion for 10 minutes, heating the dispersion in a polytetrafluoroethylene reactor at 200℃ for 6-8 hours, dialyzing the product with water for 12 hours while changing the water once in every 3 hours and freeze-drying to obtain gadolinium-carbon quantum dots (CQDs), (ii) coupling L-cysteine to gadolinium-CQDs through EDC/NHS coupling reaction to obtain thiol-modified gadolinium-CQDs, (iii) adding 500-1000 μl 2.5 mg/ml carboxy-modified polystyrene beads, 12.2 mg EDC and 7.2 mg NHS to 10 ml ethanol, stirring at room temperature for 30 minutes, adding 200 μl ethylenediamine-terminated polyethyleneimine, reacting with EDC/NHS for 3 hours at room temperature, centrifuging the mixture at 3405 g for 5-10 minutes, washing, dispersing in 10 ml 0.1 M phosphate buffer saline (PBS) solution containing 10 wt.% N,N-dimethylformamide (DMF) and having pH of 7.5, adding 150-300 μl 0.01 M succinimide 3-(2-pyridyldithio)-propionate, reacting at room temperature for 1-3 hours, centrifuging, washing, redispersing in 10 ml 0.1 M PBS solution containing 10% DMF and having pH of 8, adding 1 ml thiol-modified gadolinium-CQDs dispersion, reacting at 4℃ for 8-10 hours, centrifuging, washing and dispersing the product in 10-20 ml water to obtain a spherical bush-like photosensitizer (PS)@gadolinium-CQDs complex dispersion and (iv) coupling the polyclonal Ab of food-borne pathogen to the spherical bush-like PS@gadolinium -CQDs complex through EDC/NHS coupling reaction to obtain biofunctionalized spherical bush-like PS@gadolinium-CQDs@Ab complex and (c) mixing 200 μl PS@gadolinium-CQDs@Ab dispersion, 50-200 μl MGO@Ab dispersion, and 1 ml food-borne pathogen sample to be tested in a sample bottle, shaking and incubating for 30-40 minutes, performing magnetic separation to obtain a capture unit-food-borne pathogen-signal unit sandwich structure, adding the capture unit-food-borne pathogen-signal unit sandwich structure to 800 μl 50mM dithiothreitol solution, reacting for 20 minutes, magnetically washing, adding 200 μl 0.01 M hydrochloric acid, reacting for 5 minutes, allowing to stand in a low-field nuclear magnetic resonance contrast agent relaxation analyzer, collecting T1 at 35℃, using IR pulse sequence measurement to determine the magnitude of the transverse relaxation time difference ΔT1 of water protons corresponding to a series of different concentrations of food-borne pathogen, establishing the quantitative relationship between the longitudinal relaxation time difference of water protons and the concentration of food-borne pathogens and determining the concentration of food-borne pathogens in unknown samples in accordance with the quantitative relationship.