2.1 Materials and agents
Self-made ketchup by laboratory; FeCl2•4H2O, FeCl3•6H2O, ethyl alcohol, aqua ammonia, isopropanol, tetraethoxysilane(TEOS), methacrylic acid, acrylamide, acetone, ammonium persulfate, naringin and ethylene glycol dimethacrylate (EDMA) (all of them are analytical pure) from TCI Shanghai; methylsulphonicacid and fluorescein isothiocyanate (FITC) (both of them are analytical pure) from Sinopharm Chemical Reagent Co., Ltd.; vinyltriethoxysilane (analytical pure) from US Sigma-Alorich; chloroform (analytical pure) from Sinopharm Shanghai.
2.2 Instruments and devices
DF-101S Heat-collecting Thermostatic Magnetic Stirrer from Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd.; 101 Electrothermal Blowing Dry Box from Beijing Kewei Yongxing Instrument Co., Ltd.; JEM-2100F Transmission Electron Microscope from JEOL; RE5298A Rotary Evaporator from Shanghai Yarong Biochemical Instrument Co., Ltd.; IRprestige-21 Fourier Transform Infrared (FTIR) Spectrometer from Shimadzu Corporation; 2100UV Ultraviolet Spectrophotometer from Amersham Biosciences Corp.; BX40 Fluorescence Microscope from Olympus Corporation.
2.3.1 Preparation of fluorescent magnetic surface molecularly imprinted polymers
1) Preparation of Fe3O4 nano-particles
Add 1.72g FeCl2•4H2O, 4.72g FeCl3•6H2O and 80mL deionized water into a flask, release nitrogen continuously into the flask and then pour the mixture into the DF-101S heat-collecting thermostatic magnetic stirrer to make them dissolved by forceful stirring. Slowly instill 10mL aqua ammonia solution into the flask for reaction for 30min with the temperature rising to 80℃. Separate the product under external magnetic field. Then wash it with deionized water six times to remove the unreacted substances before drying it for storage.
- Preparation of Fe3O4@SiO2
Perform ultrasonic treatment to 300mg magnetic nano-particles, 50mL isopropanol and 4mL ultrapure water for 15min, and then successively add 5mL aqua ammonia and 2mL tetraethoxysilane. Keep stirring to let them react for 12h at room temperature. Collect the product under external magnetic field, and then use ultrapure water to wash it six times before drying it.
- Preparation of fluorescent Fe3O4@SiO2
Activate 20g Fe3O4@SiO2 in the methylsulphonicacid solution (50% of mass fraction). Place the activated Fe3O4@SiO2 and 30mL vinyltriethoxysilane into 400mL mixed solution of ethyl alcohol and water of 1:1 (V/V) to let them react for 31h at 50℃, and wash with ethyl alcohol and distilled water before drying, to get vinyltriethoxysilane-Fe3O4@SiO2. Put 3g [email protected], 10g methacrylic acid, 1g acrylamide, 200mL distilled water and 0.018g ammonium persulfate into a 200mL round-bottom flask, to let them react for 7h at 70℃, then collect the product under external magnetic field, and wash it with ethyl alcohol and distilled water for several times before drying it. Mix 1g product obtained from the above process, 15mg fluorescein isothiocyanate, 5mL ethyl alcohol and 100mL distilled water, and keep shaking for 5h. Then collect the product under external magnetic field, and wash it before drying it.
- Preparation of fluorescent MIP-Fe3O4@SiO2
Dissolve the synthesized fluorescent Fe3O4@SiO2 and 4mmol/L naringin into 100mL chloroform solution, keep releasing nitrogen into the solution for 5min, shake it for 6h before adding EDMA as the cross-linking agent, and then keep stirring for 8h in 50℃ of water bath kettle. Collect the product under external magnetic field, and then wash it before drying it.
- Preparation of non-imprinted fluorescent magnetic polymer (NIP-Fe3O4@SiO2)
Its preparation is the same as the above 22.214.171.124 section except for the step of adding naringin.
2.3.2 Inspection on the combining capability of naringin fluorescent magnetic surface molecularly imprinted polymers
1) Inspection on the combining capability by ultraviolet spectroscopy
First, prepare naringin solution with varied mass concentrations. Then place 0.05g fluorescent MIP-Fe3O4@SiO2 into such naringin solution for 1h. Then take away the clear liquid on the upper layer, to detect the UV absorption value of the solution after reaction via ultraviolet spectrophotometry. Thus the mass concentration of the solution after reaction can be calculated according to the standard curve. And the Q value is calculated by Equation (1):
where, c1 is the initial mass concentration (mg/L) of the naringin solution; c2 is the mass concentration (mg/L) of the naringin solution when being in equilibrium; v is the volume (L) of the naringin solution; m is the quality (g) of fluorescent MIP-Fe3O4@SiO2; Q is the content (g/g) of naringin combining with [email protected]3O4 of unit mass when being in equilibrium.
The method to detect how fast NIP-Fe3O4@SiO2can combine naringin is the same as above. Naringin NIP-Fe3O4@SiO2 is added into the standard solution to calculate the Q value, which will be compared with the detected results of imprinted polymers
- Inspection on the combining capability by fluorescence polarization
Place equivalent fluorescent MIP-Fe3O4@SiO2 and NIP-Fe3O4@SiO2 into the centrifuge tubes with naringin standard solution of different mass concentrations and deionized water, respectively, to make naringin molecules interact with fluorescent MIP-Fe3O4@SiO2and NIP-Fe3O4@SiO2. Use a pipette to transfer a small amount of mixed solution in a 384 pore plate. Then place the pore plate in the fluorescence polarimeter for detection.
2.3.3 Results of detection on the recovery rate of naringin in ketchup by fluorescence polarization
Add the naringin of 0.12, 0.16, 0.25, 0.31 and 0.42mg to five helpings of ketchup to make the ketchup have a total mass of 10mg respectively, then take 10mg ketchup without naringin to make solutions of 1,000mL, respectively. Take 5mL solution, respectively, add 0.05g synthesized fluorescent MIP-Fe3O4@SiO2 respectively, and then shake for 2h to make the fluorescent MIP-Fe3O4@SiO2 to combine fully with naringin molecules for achieving the adsorption equilibrium. Use the fluorescence polarimeter to detect for fluorescence intensity. As per the linear relation between the mass concentration of naringin solution and the fluorescence intensity, the mass concentration of naringin in five helpings of solution can be obtained, thus its mass can be calculated. The recovery rate I of naringin in the ketchup can be gained according to Equation (2). In the meantime, NIP-Fe3O4@SiO2 is used to perform fluorescence polarization detection on naringin in the ketchup as the controlled trial.
where m’ is the mass (mg) of the naringin added in the ketchup; m is the naringin mass (mg) detected by fluorescence polarimeter; I is the recovery rate (%) of the naringin in the ketchup.