Fluorescent Polarization Molecularly Imprinted Polymer and Its Application in the Tetection of Naringin

Background A uorescent magnetic surface molecular imprinting method was used to detect narigin by uorescence polarization technology. Method By using SiO 2 -coated magnetic particles as substrate and methlacrylic acid and acrylamide as monomers, a surface molecular imprinting polymer with both uorescence and magnetic characteristics was prepared and loaded with uorescein isothiocyanate. The binding ability of the prepared polymer was tested by uorescence polarization and ultraviolet (UV) spectrophotometry. Results Compared two other methods, the uorescence polarization method was more sensitive, and its limit of detection (LOD) was 0.1 mg/L. The recovery of the uorescence polarization method was higher than 81.3%. Conclusion It was shown that the uorescent magnetic surface molecular imprinting technique could be a new method to quickly and eciently detect naringin in food.


Abstract Background
A uorescent magnetic surface molecular imprinting method was used to detect narigin by uorescence polarization technology.

Method
By using SiO 2 -coated magnetic particles as substrate and methlacrylic acid and acrylamide as monomers, a surface molecular imprinting polymer with both uorescence and magnetic characteristics was prepared and loaded with uorescein isothiocyanate. The binding ability of the prepared polymer was tested by uorescence polarization and ultraviolet (UV) spectrophotometry.

Results
Compared two other methods, the uorescence polarization method was more sensitive, and its limit of detection (LOD) was 0.1 mg/L. The recovery of the uorescence polarization method was higher than 81.3%.

Conclusion
It was shown that the uorescent magnetic surface molecular imprinting technique could be a new method to quickly and e ciently detect naringin in food.

Background
Naringin, a kind of dihydro avonoid compound with multiple biological activities and pharmacological actions [1][2] , features activities of antivirus, anti-mutation, anti-allergy, anti-ulcer, anticancer, antiin ammation, analgesia and blood pressure reduction. It can be applied in the food industry as a avor modifying agent, natural pigment or bitterant for food and beverage production. However, it may poison human's genes [3] as it is one of the toughest substances for oxidation promotion among avonoid compounds that are able to resist [4][5][6] and promote oxidation according to research. Among the Chinese patent medicines, those against coughing contain a certain amount of avonoid compound, or naringin that is tested for more effective control over medicine quality via the currently-used methods of high performance liquid chromatography (HPLC) and spectrophotometry [7][8] . The latter is easy to present false-positive results due to its lower sample selectivity, while the former is costlier and time consuming in sample testing in spite of higher measuring precision and relatively thorough separation of structural analogues. Therefore, it is necessary to research how to detect naringin rapidly and effectively.
In recent years [9][10][11][12][13][14] , the molecular imprinting technique has been rapidly developing in elds of separation, catalyzing and sensor, owing to its speci city and practicality. Notably, the research on the surface molecular imprinting technique has solved the problems in the traditional molecular imprinting technique, such as di culties for elution and recombination of template molecules caused by highlycrosslinked net-structured polymers, and for e cient combination. The surface molecular imprinting technique is to compound molecularly imprinted polymers on the vehicle surface, forming a twodimension net structure which makes template molecules easy for adsorption and elution, and thus improves the capability of polymers to absorb and select [14][15][16] . In the meantime, the combination of the magnetic nano-materials and the surface molecular imprinting technique produces magnetic surface molecularly imprinted polymers, which can rapidly separate under external electric eld, with merits of active identi cation and fast separation [17][18] . The uorescent surface molecularly imprinted polymers produced by marking uorescent materials on the surface molecularly imprinted polymers have uorescence properties. Whether the template molecules have or have no uorescence properties, they can be detected by the uorescence method that greatly improves the sensitivity and speci city of the detection. The schematic diagram for this process is shown in Fig. 1. As a new popular method in uorescence detection, uorescence polarization, based on physical manifestation, is used to study the interaction among molecules in lives. It can simulate, to the most extent, the real life environment, while performing the real-time monitoring on the variation among molecules [19] . This experiment combines the magnetic and the uorescent surface molecular imprinting techniques to produce naringin uorescent magnetic surface molecularly imprinted polymers, and adopts a uorescence polarimeter to carry out real-time inspection on detection signals, thus achieving a new sensitive way to detect naringin rapidly and e ciently.

Magnetic inspection
As shown in Fig. 2, particles disperse in the water which appears to be black and turbid. By absorbing the particles in the water with a magnet, it can be clearly seen that all the particles gather at one side of the magnet, and thus the solution becomes clear again. Therefore, it can be demonstrated that the prepared

Infrared spectroscopy analysis
By inspecting between 4 000 400cm 1 with the infrared spectrometer, as shown in Fig. 3a, it can be seen that there are two characteristic peaks, at 561 cm 1 is an absorption peak called the characteristic vibration peak of Fe-O , while that at 1,400 cm 1 presents the vibration peak of hydroxide radical. So it is known that the preparation of Fe 3 O 4 magnetic nano-particles is correct. As shown in Fig. 3b, at 1,091 cm 1 is an absorption peak that is the characteristic peak of Si-O, demonstrating that SiO 2 is successfully covering the surface of Fe 3 O 4 particles.

Inspection by uorescence microscope
A uorescence microscope with a ten-time magni cation is used for uorescence detection on uorescent MIP-Fe 3 O 4 @SiO 2 and Fe 3 O 4 @SiO 2 , as shown in Fig. 4. By comparison of both two, it can be seen in Fig. 4a that uorescent circles clearly appear on the surface of silica gel, while nothing happens in Fig. 4b. This proves that uorescent molecules are successfully decorated on the uorescent MIP- Results by transmission electron microscope (TEM) A TEM with a 10,000-time magni cation is used for detection on Fe 3 O 4 , Fe 3 O 4 @SiO 2 and uorescent MIP- they combined with naringin in the solution to form a kind of macromolecular compound whose molecules slowed down the rotation, thus leading the light intensity of uorescence polarization to increase. The higher the concentration of naringin solution is, the more the naringin molecules that combine with uorescent MIP-Fe3O4@SiO2, the larger the volume of the large molecules, and the stronger the uorescence polarization integrity. Taking the deionized water as blank control, the uorescent surface molecularly imprinted polymers in the solution did not combine with naringin molecules at this moment, and the uorescence polarization value measured was 28.564. It increased with the increase of the mass concentration of naringin solution, due to the combination of the naringin molecules in the solution and the uorescent MIP-Fe 3 O 4 @SiO 2 . When the combination of both leading to a pretty small uorescence polarization value and an almost unchanged curse. By comparison of the two curses in Fig. 7, it can be known that the uorescent MIP-Fe 3 O 4 @SiO 2 has a speci c impact on the naringin detection. Meanwhile, the limit for uorescence detection on naringin is 0.1mg/L, with the linear detection range of 0.1 0.9mg/L (R2=0.995 8) and the linear equation of y=94.84x, which is obviously lower than that for ultraviolet spectrophotometry. Therefore, it indicates that the uorescence method used for detecting the capability of molecularly imprinted polymer to combine naringin is more sensitive.
Results of detecting the recovery rate of naringin in food As shown in

Discussion
This experiment has several steps: rst, introduce highly-active group -couple on the surface of silica gel with triethoxyvinylsilane as the coupling reagent through hydroxide radical -by taking Fe 3 O 4 @SiO 2 as the base and adopting the "grafting to" technology of using the interaction between Fe 3 O 4 @SiO 2 and hydroxide radical; second, add functional monomers (methacrylic acid and acrylamide) to form polymeric macromolecular chains and couple on the surface of the silica gel; third, add uorescent reagent for combining with the amide bond of acrylamide; last, add template and cross-linking agent to aggregate into a molecular imprinting layer. The uorescent MIP-Fe 3 O 4 @SiO 2 is obtained after the elution of template molecules. Magnetic nano-particles magnetize the surface molecularly imprinted polymers, and make them separate and aggregate rapidly under external electric eld, thus simplifying the operating steps. The nano-scale Fe 3 O 4 @SiO 2 formed by silica-gel-wrapped Fe 3 O 4 particles has a higher speci c area and surface activity, whose surface can couple with various coupling reagents, and will encounter swelling and resist corrosion in organic solvent due to the thermostability and rigidity of silica gel itself.
Therefore, the surface molecularly imprinted polymers made by Fe 3 O 4 @SiO 2 as the base have an excellent performance.
Meanwhile, this experiment proposed using the uorescence polarization method to evaluate the combining e ciency of uorescent surface molecularly imprinted polymers. Due to the fast rotation of the uorescence-marked nano-scale surface molecularly imprinted polymers in the solution and a small light intensity of uorescence polarization, the combination of such polymers and corresponding substrates generated new polymers with an increased volume, decreased spinning speed and increased light intensity of uorescence polarization. The difference of uorescence polarization values has some bearing on how many uorescent surface molecularly imprinted polymers in the solution combine with substrates. With the increase of the mass concentration of naringin solution, the combination of the naringin in naringin solution with different mass concentrations and the uorescent MIP-Fe3O4@SiO2 gradually peaked, while the light intensity of uorescence polarization increased to a stable value. The nonspeci c combination between uorescence NIP-Fe3O4@SiO2 and naringin molecules made such a combination ine cient, and thus the uorescence polarization strength had no obvious change with the increased mass concentration of the solution, with its change having nothing to do with the mass concentration of naringin solution (see Fig. 7). Therefore, it is feasible to use the uorescence polarization method to detect how fast the naringin uorescent MIP-Fe3O4@SiO2 can combine naringin. Comparing with the ultraviolet spectroscopy method in detection sensitivity, it is known that the lowest limit of detection for ultraviolet spectroscopy is 0.5mg/L, and that for uorescence polarization method is 0.1mg/L, with a linear detecting range of 0.1-0.9mg/L (R2=0.995 8). This can demonstrate that the new uorescence polarization method is more sensitive, e cient and convenient. The recovery rate of naringin in food can reach over 81.3% by using this method.

Conclusion
In conclusion, the prepared uorescent magnetic surface molecularly imprinted polymers have an excellent recovery rate, and the uorescence polarization has a higher speci city and sensitivity in detecting naringin in food. continuously into the ask 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 ask for reaction for 30min with the temperature rising to 80℃. Separate the product under external magnetic eld. Then wash it with deionized water six times to remove the unreacted substances before drying it for storage.

Preparation of Fe 3 O 4 @SiO 2
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 eld, and then use ultrapure water to wash it six times before drying it.
Preparation of uorescent Fe 3 O 4 @SiO 2 Activate 20g Fe 3 O 4 @SiO 2 in the methylsulphonicacid solution (50% of mass fraction). Place the activated Fe 3 O 4 @SiO 2 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-Fe 3 O 4 @SiO 2 . Put 3g vinyltriethoxysilane-Fe3O4@SiO2, 10g methacrylic acid, 1g acrylamide, 200mL distilled water and 0.018g ammonium persulfate into a 200mL round-bottom ask, to let them react for 7h at 70℃, then collect the product under external magnetic eld, and wash it with ethyl alcohol and distilled water for several times before drying it. Mix 1g product obtained from the above process, 15mg uorescein isothiocyanate, 5mL ethyl alcohol and 100mL distilled water, and keep shaking for 5h. Then collect the product under external magnetic eld, and wash it before drying it.

Preparation of uorescent MIP-Fe 3 O 4 @SiO 2
Dissolve the synthesized uorescent Fe 3 O 4 @SiO 2 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 crosslinking agent, and then keep stirring for 8h in 50℃ of water bath kettle. Collect the product under external magnetic eld, and then wash it before drying it.

Preparation of non-imprinted uorescent magnetic polymer (NIP-Fe 3 O 4 @SiO 2 )
Its preparation is the same as the above 2.3.1.4 section except for the step of adding naringin.
2.3.2 Inspection on the combining capability of naringin uorescent 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 uorescent MIP-Fe 3 O 4 @SiO 2 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): (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 uorescent MIP-Fe 3 O 4 @SiO 2 ; Q is the content (g/g) of naringin combining with MIP@Fe 3 O 4 of unit mass when being in equilibrium.
The method to detect how fast NIP-Fe 3 O 4 @SiO 2 can combine naringin is the same as above. Naringin NIP-Fe 3 O 4 @SiO 2 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 uorescence polarization Place equivalent uorescent MIP-Fe 3 O 4 @SiO 2 and NIP-Fe 3 O 4 @SiO 2 into the centrifuge tubes with naringin standard solution of different mass concentrations and deionized water, respectively, to make naringin molecules interact with uorescent MIP-Fe 3 O 4 @SiO 2 and NIP-Fe 3 O 4 @SiO 2 . Use a pipette to transfer a small amount of mixed solution in a 384 pore plate. Then place the pore plate in the uorescence polarimeter for detection.

Results of detection on the recovery rate of naringin in ketchup by uorescence polarization
Add the naringin of 0.12, 0.16, 0.25, 0.31 and 0.42mg to ve 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 uorescent MIP-Fe 3 O 4 @SiO 2 respectively, and then shake for 2h to make the uorescent MIP-Fe 3 O 4 @SiO 2 to combine fully with naringin molecules for achieving the adsorption equilibrium. Use the uorescence polarimeter to detect for uorescence intensity. As per the linear relation between the mass concentration of naringin solution and the uorescence intensity, the mass concentration of naringin in ve 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-Fe 3 O 4 @SiO 2 is used to perform uorescence polarization detection on naringin in the ketchup as the controlled trial. (2) where m' is the mass (mg) of the naringin added in the ketchup; m is the naringin mass (mg) detected by uorescence polarimeter; I is the recovery rate (%) of the naringin in the ketchup.

Declarations
Availability of data and material The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.