Method for preparing a ratiometric fluorescent sensor for phycoerythrin based on a magnetic molecularly imprinted core-shell polymer

Abstract

A method for preparing a ratiometric fluorescent sensor for phycoerythrin based on a magnetic molecularly imprinted core-shell polymer is provided. With Fe.sub.3O.sub.4 magnetic nanoparticles as the core, blue fluorescence-emitting carbon quantum dots (B-CDs) are coupled on the surfaces of Fe.sub.3O.sub.4 magnetic nanoparticles, and SiO.sub.2 shells carrying template molecules (phycoerythrin) are grown on the surfaces of Fe.sub.3O.sub.4/B-CDs. Then, the molecularly imprinted polymer SiO.sub.2-MIPs are obtained by eluting the template molecules, that is, Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained. Fluorescence emission spectra of the dispersion of Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs in the presence of different concentrations of phycoerythrin are measured. By fitting the linear relationship between the ratios I.sub.phycoerythrin/I.sub.B-CDs of fluorescence emission peak intensities of phycoerythrin and B-CDs and the molar concentrations of phycoerythrin, the ratiometric fluorescent sensor for phycoerythrin is constructed.

Claims

1. A method for preparing a ratiometric fluorescent sensor for phycoerythrin based on a magnetic molecularly imprinted core-shell polymer, comprising the following steps: (1) preparation of aminated blue fluorescence-emitting carbon quantum dots (B-CDs) comprising: mixing 0.3 mL of 1,4-dioxane with 25 mL of catechol solution by ultrasound to obtain a first solution; transferring the first solution to a 50 mL high-pressure reactor with a polytetrafluoroethylene liner; heating the first solution in the high-pressure reactor for a first reaction at 180 C. for 12 hours to obtain a dark brown mixture; diluting the dark brown mixture with 20 mL of double-distilled water and centrifuging at 12,000 rpm to remove larger particles to obtain a supernatant; filtering the supernatant through a 0.4 m micro-filtration membrane to obtain a filtrate, and dialyzing the filtrate through a dialysis bag with a molecular weight cut-off of 1000 Da to remove unreacted experimental materials to obtain a second solution in the dialysis bag; collecting the second solution and subjecting to a rotary evaporation to remove 90% of water; and drying in a vacuum to obtain the B-CDs; and then dispersing the dried B-CDs in water to obtain a B-CDs aqueous dispersion; (2) preparation of carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles comprising: adding ferric chloride and ferrous chloride with a molar ratio of 2:1 into a 250 mL reaction flask to prepare a 100 mL mixed solution; adding 10 mL of ammonia water with a mass concentration of 25% to the mixed solution into the 250 mL reaction flask under N.sub.2 protection to obtain a third solution; stirring the third solution rapidly to cause a second reaction; adjusting pH of the third solution to alkaline with HCl after 10 minutes of the second reaction; adding 10 mL of trisodium citrate solution to the third solution to obtain a fourth solution; and then placing the 250 mL reaction flask containing the fourth solution in a water bath at 80 C. and continuously stirring the fourth solution for a third reaction for 30 minutes to obtain a first reaction production; obtaining the carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles by centrifugation, washing and drying of the first reaction product and dispersing the carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles in water to prepare a Fe.sub.3O.sub.4 aqueous dispersion; (3) preparation of magnetic molecularly imprinted core-shell polymers comprising: adding 2 mL of the B-CDs aqueous dispersion to 18 mL of aqueous dispersion containing 0.8 mL of the Fe.sub.3O.sub.4 aqueous dispersion to obtain a fifth solution; stirring the fifth solution for a period of 30 minutes, and then adding phycoerythrin and 20 L of 3-aminopropyltriethoxysilane to the fifth solution to obtain a sixth solution and allowing reaction of the sixth solution to proceed for 1 hour; then adding 40 L of ammonia water and 40 L of tetraethyl silicate to the sixth solution to obtain a seventh solution, and allowing reaction to proceed in the seventh solution away from light for 12 hours to obtain a second reaction product; centrifuging the second reaction product; and washing three times with a solution consisting of ethanol and acetonitrile at a volume ratio of 8:2 to remove the template molecules to obtain a product, and obtaining Fe.sub.3O.sub.4/B-CDs/SiO.sub.2 magnetic molecularly imprinted core-shell polymer (Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs) from the product by centrifugation, washing and drying; and then dispersing the Fe.sub.3O.sub.4/B-CDs/SO.sub.2-MIPs in water to provide a Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs aqueous dispersion; and (4) adding a plurality of molar concentrations of phycoerythrin to the Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs aqueous dispersion to form a plurality of homogeneous mixtures; incubating the plurality of homogeneous mixtures away from light for 5 minutes and measuring fluorescence emission spectra of the plurality of homogeneous mixtures; and by fitting a linear relationship between ratios I.sub.phycoerythrin/I.sub.B-CDs of fluorescence emission peak intensities of the phycoerythrin and the B-CDs and the plurality of molar concentrations of the phycoerythrin, the ratiometric fluorescent sensor for the phycoerythrin is constructed; wherein, in step (3), the mass concentration of the B-CDs is 1-10 mg/mL, the mass concentration of the carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles is 5-20 mg/mL, and the plurality of molar concentrations of the phycoerythrin in step (4) is 0.5-1 M.

2. The method for preparing the ratiometric fluorescent sensor for the phycoerythrin based on the magnetic molecularly imprinted core-shell polymer according to claim 1, wherein, in step (1), a size of each of the aminated B-CDs is 1-5 nm.

3. The method for preparing the ratiometric fluorescent sensor for the phycoerythrin based on the magnetic molecularly imprinted core-shell polymer according to claim 1, wherein, in step (2), a size of each of the carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles is 10-30 nm.

4. The method for preparing the ratiometric fluorescent sensor for the phycoerythrin based on the magnetic molecularly imprinted core-shell polymer according to claim 1, wherein, in step (4), a linear detection range of the molar concentrations of the phycoerythrin is 1-500 nM, and a detection limit of the phycoerythrin is 1-10 nmol/L.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic diagram showing the preparation for the ratiometric fluorescent sensor for phycoerythrin based on a magnetic molecularly imprinted core-shell polymer and the detection for phycoerythrin;

(2) FIG. 2 shows the fluorescence emission spectra of sensor system measured by the ratiometric fluorescent sensor of the present disclosure, corresponding to different molar concentrations of phycoerythrin;

(3) FIG. 3 shows the linear relationship between the ratios (I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak intensities of phycoerythrin and B-CDs and the different molar concentrations of phycoerythrin.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(4) The present disclosure will be further described below in conjunction with the drawings and specific embodiments.

Embodiment 1

(5) This embodiment relates to a method for preparing a ratiometric fluorescent sensor for phycoerythrin based on a magnetic molecularly imprinted core-shell polymer. A process of preparing the ratiometric fluorescent sensor and the principle of the ratiometric fluorescent detection of phycoerythrin are shown in FIG. 1, and the specific process steps are as follows.

(6) Preparation of aminated B-CDs: 0.3 mL of 1,4-dioxane and 25 mL of catechol solution are chosen to be mixed well by ultrasound, and transferred to a 50 mL high-pressure reactor with a polytetrafluoroethylene liner. Heating and reacting is performed at 180 C. for 12 hours to obtain a dark brown mixture. The prepared dark brown mixture is diluted with 20 mL of double-distilled water and centrifuged at 12,000 rpm to remove larger particles. The supernatant is collected and filtered through a 0.4 m micro-filtration membrane, and the filtrate is dialyzed through a dialysis bag with a molecular weight cut-off of 1000 Da to remove the unreacted experimental materials. The solution in the dialysis bag is poured out, subjected to rotary evaporation to remove 90% of the liquid, and then dried in a vacuum to obtain B-CDs. The B-CDs is stored at 4 C. from light or dispersed in solution to prepare dispersing B-CDs for subsequent experiments, wherein the average size of the B-CDs is 2 nm.

(7) Preparation of carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles: ferric chloride and ferrous chloride with a molar ratio of 2:1 are added into a 250 mL reaction flask to prepare a 100 mL mixed solution, 10 mL of ammonia water with a mass concentration of 25% is added into the reaction flask under N.sub.2 protection, stirring rapidly for reaction, the pH of the solution is adjusted to alkaline with HCl, after 10 min of reaction, 10 mL of trisodium citrate solution is added. The reaction flask is placed in a water bath at 80 C., and is continuously stirred for reaction for 30 min. The reaction product is centrifuged, washed and dried to obtain Fe.sub.3O.sub.4. The Fe.sub.3O.sub.4 is stored at 4 C. from light or dispersed in solution to prepare a dispersing Fe.sub.3O.sub.4 for subsequent experiments, wherein the average size of Fe.sub.3O.sub.4 is 15 nm.

(8) Preparation of magnetic molecularly imprinted core-shell polymers: 2 mL of B-CDs aqueous dispersion is added to 18 mL of aqueous dispersion containing 0.8 mL of Fe.sub.3O.sub.4, wherein the mass concentration of B-CDs is 2 mg/mL and the mass concentration of Fe.sub.3O.sub.4 magnetic nanoparticles is 10 mg/mL, after stirring and reacting for 30 min, template molecules (phycoerythrin) and 20 L of 3-aminopropyltriethoxysilane are added, wherein the dosage of phycoerythrin is 0.5 M, the reaction is continued for 1 hour, then 40 L of ammonia water and 40 L of tetraethyl silicate are added, and the reaction is carried out away from light for 12 hours. The reaction product is centrifuged and washed three times with a solution consisting of ethanol and acetonitrile at a volume ratio of 8:2 to remove the template molecules, and then Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained by centrifugation, washing and drying. The magnetic molecularly imprinted core-shell polymers are dispersed in solution to prepare a dispersion for use.

(9) At room temperature and stirring magnetically, a certain dosage of phycoerythrin is added to the polymer dispersion to form a homogeneous mixture and then incubated away from light for 5 minutes. Fluorescence emission spectra of the homogeneous mixture in the presence of different concentrations of phycoerythrin are measured. By fitting a linear relationship between the ratios (I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak intensities of phycoerythrin and B-CDs and the molar concentrations of phycoerythrin, the ratiometric fluorescent sensor for phycoerythrin is constructed. FIG. 2 shows the fluorescence emission spectra of sensor system measured by the ratiometric fluorescent sensor of the present disclosure, corresponding to different molar concentrations of phycoerythrin. As shown in FIG. 3, the linear detection range of molar concentration of phycoerythrin obtained from the ratiometric fluorescent sensor of the present disclosure, is 5-250 nM, and the detection limit is 2 nM.

(10) Embodiment 2: in this embodiment, the schematic diagram of the preparation process of the ratiometric fluorescent sensor and the principle of the ratiometric fluorescent detection of phycoerythrin are the same as embodiment 1, and the process steps for preparing aminated B-CDs and carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles are also the same as embodiment 1, wherein the average size of B-CDs is 3 nm and the average size of Fe.sub.3O.sub.4 is 20 nm. Other specific process steps are as follows.

(11) Preparation of magnetic molecularly imprinted core-shell polymers: 2 mL of B-CDs aqueous dispersion is added to 18 mL of aqueous dispersion containing 0.8 mL of Fe.sub.3O.sub.4, wherein the mass concentration of B-CDs is 5 mg/mL, and the mass concentration of Fe.sub.3O.sub.4 magnetic nanoparticles is 15 mg/mL, after stirring and reacting for 30 minutes, template molecules (phycoerythrin) and 20 L of 3-aminopropyltriethoxysilane are added, wherein the dosage of phycoerythrin is 0.8 M, the reaction is continued for 1 hour, then 40 L of ammonia water and 40 L of tetraethyl silicate are added, and the reaction is carried out away from light for 12 hours. The reaction product is centrifuged and washed three times with a solution consisting of ethanol and acetonitrile at a volume ratio of 8:2 to remove the template molecules, and then Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained by centrifugation, washing and drying. The magnetic molecularly imprinted core-shell polymers are dispersed in solution to prepare a dispersion for use.

(12) At room temperature and stirring magnetically, a certain dosage of phycoerythrin is added to the polymer dispersion to form a homogeneous mixture and then incubated away from light for 5 minutes. Fluorescence emission spectra of the homogeneous mixture in the presence of different concentrations of phycoerythrin are measured. By fitting a linear relationship between the ratios (I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak intensities of phycoerythrin and B-CDs and the molar concentrations of phycoerythrin, the ratiometric fluorescent sensor for phycoerythrin is constructed. The linear detection range of molar concentration of phycoerythrin is 5-500 nM, and the detection limit is 5 nM.

(13) Embodiment 3: in this embodiment, the schematic diagram of the preparation process of the ratiometric fluorescent sensor and the principle of the ratiometric fluorescent detection of phycoerythrin, and the process steps for preparing aminated B-CDs and carboxylated Fe.sub.3O.sub.4 magnetic nanoparticles are all the same as embodiment 1, wherein the average size of B-CDs is 5 nm and the average size of Fe.sub.3O.sub.4 is 25 nm. Other specific process steps are as follows.

(14) Preparation of magnetic molecularly imprinted core-shell polymers: 2 L of B-CDs aqueous dispersion is added to 18 mL of aqueous dispersion containing 0.8 mL of Fe.sub.3O.sub.4, wherein the mass concentration of B-CDs is 8 mg/mL, and the mass concentration of Fe.sub.3O.sub.4 magnetic nanoparticles is 20 mg/mL, after stirring and reacting for 30 min, template molecules (phycoerythrin) and 20 L of 3-aminopropyltriethoxysilane are added, wherein the dosage of phycoerythrin is 1 M, the reaction is continued for 1 hour, then 40 L of ammonia water and 40 L of tetraethyl silicate are added, and the reaction is carried out away from light for 12 hours. The reaction product is centrifuged and washed three times with a solution consisting of ethanol and acetonitrile at a volume ratio of 8:2 to remove the template molecules, and then Fe.sub.3O.sub.4/B-CDs/SiO.sub.2-MIPs are obtained by centrifugation, washing and drying. The magnetic molecularly imprinted core-shell polymers are dispersed in solution to prepare a dispersion for use.

(15) At room temperature and stirring magnetically, a certain dosage of phycoerythrin is added to the polymer dispersion to form a homogeneous mixture and then incubated away from light for 5 minutes. Fluorescence emission spectra of the homogeneous mixture in the presence of different concentrations of phycoerythrin are measured. By fitting a linear relationship between the ratios (I.sub.phycoerythrin/I.sub.B-CDs) of fluorescence emission peak intensities of phycoerythrin and B-CDs and the molar concentrations of phycoerythrin, the ratiometric fluorescent sensor for phycoerythrin is constructed. The linear detection range of molar concentration of phycoerythrin is 10-500 nM, and the detection limit is 8 nM.

(16) The above disclosures are only described as some preferred embodiments of the present invention. It should be noted that those skilled in the art can also make several improvements and modifications without departing from the principles of the present invention, and these improvements and modifications shall still fall within the protection scope of the present invention.