NaYF4: Yb, Er-coated NaYF4-coated SiO2-coated AgNPs as well as preparation method and application thereof in vitamin B12 detection

Abstract

The invention discloses NaYF4: Yb, Er (at) NaYF4 (at) SiO2 (at) AgNPs as well as a preparation method and application thereof in vitamin B12 detection. According to the method, a core NaYF4: Yb, Er and a core shell NaYF4: Yb, Er (at) NaYF4 are prepared through a high-temperature solvothermal reaction; and carrying out hydrophilic modification and fluorescence enhancement modification to obtain the nano probe. Compared with the prior art, the invention has the following advantages: (1) the NaYF4: Yb, Er (at) NaYF4 (at) SiO2 (at) Ag NPs can absorb exciting light at 980nm and convert the exciting light into visible fluorescence, and through an energy transfer mode, an up-conversion fluorescence emission peak and an ultraviolet absorption peak of vitamin B12 coincide, so that rapid detection is realized; (2) the nanoparticles and the application method have the advantages of high sensitivity, convenience and rapidness in operation, good specificity and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence detection, and relates to a fluorescence detection method for vitamin B 12 , specifically NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs, its preparation method and application in the detection of vitamin B 12 . Background Art

[0002] Vitamin B 12 is the most complex and largest B vitamin, and is a general term for all cobalamin substances with cyanocobalamin biological activity. Vitamin B 12 deficiency can lead to megaloblastic anemia or neuropathy. Clinically, B 12 deficiency is usually caused by insufficient intake or poor absorption. In the daily diet, the main sources of vitamin B 12 are animal products, such as milk, meat, eggs and fish. Vitamin B 12 can only be synthesized by certain bacteria or molds in soil, garbage, water, intestines and the omasum. No human or animal can synthesize vitamin B 12 by themselves. Vitamin B 12 has a low Recommended Dietary Intake (RDI). For adults, it is 2.4 mg per day, for pregnant women it is 2.6 mg per day, for lactating women it is 2.8 mg per day, and the intake for infants, children and adolescents should be slightly higher than the above standards. However, the RDI of vitamin B 12 for many infants, children and adolescents is even lower than the adult RDI of 2.4 mg. Therefore, the dairy industry, especially the industry involved in the production of infant formula, needs precise and highly sensitive vitamin B 12 quantitative analysis methods to accurately determine the content of vitamin B 12 in formula milk. However, due to the low content and complex physical and chemical properties of vitamin B 12 , it poses challenges to the compliance detection of conventional dairy products.

[0003] Upconversion nanoparticles have advantages such as anti-Stokes shift, near-infrared excitation, low interference background, narrow peak emission band, high photostability and penetration ability, and can achieve low background (under the excitation condition of 980 nm, there is almost no background fluorescence interference) and specificity. Summary of the Invention

[0004] Technical Problem to be Solved: In order to overcome the deficiencies of the prior art and obtain a detection method for vitamin B 12 with high sensitivity, high specificity, convenience and speed, the present invention provides NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs, its preparation method and application in vitamin B12 Application in detection.

[0005] Technical solution: A preparation method of NaYF4:Yb,Er@NaYF4@SiO2@AgNPs, the method comprising the following steps:

[0006] S1. Respectively add yttrium source, ytterbium source, and erbium source into a mixed solvent of oleic acid and 1-octadecene, degas at room temperature for 5 - 15 min, then heat to 140 - 160 °C under a nitrogen stream and strong stirring conditions, react for 30 - 45 min, and naturally cool to room temperature to form an oleic acid precursor; then add a methanol mixed solution of NH4F and NaOH, react at 50 °C for 30 - 45 min, and then react at 70 °C, 100 °C, and 120 °C for 30 - 45 min respectively at each stage; after the reaction, heat the solution to 290 - 310 °C in a nitrogen atmosphere, react for 1 - 2 h, and cool to room temperature; precipitate with ethanol, wash with a mixed solvent of ethanol and cyclohexane, and vacuum dry to obtain core nanoparticles NaYF4:Yb,Er;

[0007] S2. Add yttrium source into a mixed solvent of oleic acid and 1-octadecene, degas at room temperature for 5 - 15 min, then heat to 140 - 160 °C under a nitrogen stream and strong stirring conditions, react for 30 - 45 min, and naturally cool to room temperature to form an oleic acid precursor; then add a cyclohexane solution of the core nanoparticles NaYF4:Yb,Er obtained in S1, a methanol solution of NH4F and NaOH, react at 50 °C for 30 - 45 min, and then react at 70 °C, 100 °C, and 120 °C for 30 - 45 min respectively at each stage; after the reaction, heat the solution to 290 - 310 °C in a nitrogen atmosphere, react for 1 - 2 h, and cool to room temperature; precipitate with ethanol, wash with a mixed solvent of ethanol and cyclohexane, and vacuum dry to obtain core-shell nanoparticles NaYF4:Yb,Er@NaYF4;

[0008] S3. Ultrasonically disperse octanol and Triton X-100 in cyclohexane, at room temperature, add a cyclohexane solution of NaYF4:Yb,Er@NaYF4 and stir for 30 min, then add ammonium hydroxide solution to form an inverse microemulsion solution and stir for 1 h, add tetraethyl orthosilicate solution and stir and age for 14 h, centrifuge and wash to obtain SiO2-modified NaYF4:Yb,Er@NaYF4;

[0009] S4. Disperse the SiO₂-modified NaYF₄:Yb,Er@NaYF₄ obtained in S3 in an ethanol aqueous solution, stir with ultrasonic, add aminopropyltriethoxysilane thereto and stir magnetically, then add an AgNO₃ aqueous solution and continue stirring; then add a sodium citrate solution, heat to 100 °C and react for 20 - 30 min; after the reaction, cool to room temperature, centrifuge, separate and wash to obtain NaYF₄:Yb,Er@NaYF₄@SiO₂@Ag NPs.

[0010] Preferably, in S1, the dosage ratio of yttrium source, ytterbium source, erbium source, oleic acid, 1-octadecene, NH₄F, and NaOH is 0.2 - 0.3 g:0.07 - 0.08 g:0.007 - 0.008 g:6 - 8 mL:15 - 20 mL:0.14 - 0.15 g:0.1 - 0.2 g.

[0011] Preferably, in S2, the dosage ratio of yttrium source, oleic acid, 1-octadecene, NH₄F, NaOH and the core NaYF₄:Yb,Er is 0.3 - 0.4 g:6 - 8 mL:15 - 20 mL:0.14 - 0.15 g:0.1 - 0.2 g:50 mg.

[0012] Preferably, in S3, the dosage ratio of the core-shell NaYF₄:Yb,Er@NaYF₄, octanol, Triton X-100, cyclohexane, ammonium hydroxide solution and tetraethyl orthosilicate solution is 100 - 200 mg:6.7 - 8 mL:12 - 15 mL:10 - 15 mL:0.4 - 1 mL:50 - 100 μL.

[0013] Preferably, in S4, the dosage ratio of the APTES solution, AgNO 3、 and sodium citrate is 130 - 150 μL:7 - 80 mg:0.0174 g.

[0014] Preferably, the yttrium source, ytterbium source and erbium source are all chlorine compounds.

[0015] Preferably, the centrifugation speed in S1 and S2 is 6000 - 8000 rpm; the volume ratio of the washing liquids of ethanol and cyclohexane in S1 and S2 is 1:1.

[0016] NaYF₄:Yb,Er@NaYF₄@SiO₂@Ag NPs obtained by the method described in any one of the above.

[0017] The application of the above-mentioned NaYF₄:Yb,Er@NaYF₄@SiO₂@Ag NPs in detecting vitamin B in food or serum samples 12 , characterized in that the fluorescence excitation wavelength of the application is 980 nm, and the detection limit for vitamin B 12 is 0.01 nM.

[0018] Preferably, the standard curve of the application shows a good linear relationship within the ranges of 0.1 - 50 nM and 20 - 750 nM for VB 12 ; among which, the regression equations of VB 12 are y = -1461.02x + 40349.81 and y = -3890.75x + 43742.61 respectively.

[0019] The NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs of the present invention are used for detecting vitamin B 12 , and the principle lies in: performing simple pretreatment on the sample to be detected containing vitamin B 12 solution, adding the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe thereto. When vitamin B 12 is contained in the solution, due to the almost complete overlap between the emission spectrum of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe and the absorption spectrum of vitamin B 12 , a fluorescence quenching phenomenon occurs. Vitamin B 12 exhibits strong absorption characteristics within a specific wavelength range. When the excitation light passes through the solution, vitamin B 12 will absorb a large amount of excitation light energy, which significantly reduces the excitation light energy that can reach the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe, thereby causing a decrease in the fluorescence emission intensity of the fluorescence upconversion detection probe. When vitamin B 12 is not contained in the solution, since there is no substance that can significantly interact with the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe, at this time, the fluorescence upconversion detection probe will maintain a relatively stable fluorescence emission state under the action of the excitation light, and its fluorescence intensity will not show an obvious decrease or quenching phenomenon. Through the way of energy transfer, combined with the coincidence of the fluorescence emission peak of the upconversion nanoparticles and the ultraviolet absorption peak of vitamin B 12 , rapid, sensitive and specific detection of vitamin B 12 is achieved.

[0020] Advantages: (1) The NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs of the present invention can absorb the excitation light at 980 nm, convert it into visible fluorescence, and through the way of energy transfer, combined with the coincidence of the upconversion fluorescence emission peak and the ultraviolet absorption peak of vitamin B 12 , rapid detection is realized; (2) The nanoparticles and the application method have the advantages of high sensitivity, convenient operation, rapidity, good specificity, etc. Brief Description of the Drawings

[0021] Figure 1 Schematic diagram of the preparation process and detection strategy of the NaYF4:Yb,Er@NaYF4@SiO2@AgNPs fluorescence upconversion detection probe of the present invention.

[0022] Figure 2 Fluorescence and UV diagrams of the NaYF4:Yb,Er@NaYF4@SiO2@AgNPs fluorescence upconversion detection probe of the present invention: (a) Fluorescence spectra corresponding to optimizing the content of YbCl3·6H2O under the condition of a fixed doping amount of ErCl3·6H2O. The inset shows the fluorescence spectrum at 550 nm; (b) Fluorescence spectra corresponding to optimizing the content of ErCl3·6H2O under the condition of a fixed doping amount of YbCl3·6H2O. The inset shows the fluorescence spectrum at 550 nm; (c) Core-shell optimized fluorescence spectrum. The inset shows the fluorescence spectrum at 550 nm; (d) Fluorescence spectra of different tetraethyl orthosilicate solutions. The inset shows the fluorescence spectrum at 550 nm; (e) Fluorescence spectra modified with different contents of silver nanoparticles. The inset shows the fluorescence spectrum at 550 nm; (f) UV absorption spectra modified with different contents of silver nanoparticles.

[0023] Figure 3 Transmission electron microscopy images of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe of the present invention: (a) TEM image of the core NaYF4:Yb,Er; (b) TEM image of the core-shell NaYF4:Yb,Er@NaYF4; (c) TEM image of NaYF4:Yb,Er@NaYF4@SiO2; (d) TEM image of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs.

[0024] Figure 4 Infrared spectra of the NaYF4:Yb,Er, NaYF4:Yb,Er@NaYF4, NaYF4:Yb,Er@NaYF4@SiO2, Ag NO3, NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probes of the present invention.

[0025] Figure 5 XRD diagrams of the NaYF4:Yb,Er, NaYF4:Yb,Er@NaYF4, NaYF4:Yb,Er@NaYF4@SiO2, Ag NO3, NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probes of the present invention.

[0026] Figure 6 Fluorescence spectra (a) of the NaYF4:Yb,Er, NaYF4:Yb,Er@NaYF4, NaYF4:Yb,Er@NaYF4@SiO2, NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probes of the present invention and VB 12 Ultraviolet spectra (b) of NaYF4:Yb,Er, NaYF4:Yb,Er@NaYF4, NaYF4:Yb,Er@NaYF4@SiO2, AgNO3, AgNPs, NaYF4:Yb,Er@NaYF4@SiO2@AgNPs.

[0027] Figure 7 Fluorescence spectrum of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs of the present invention and ultraviolet spectrum of vitamin B 12

[0028] Figure 8 Mechanism of detecting VB by the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe of the present invention 12

[0029] Figure 9 Optimization diagram of detection conditions of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe of the present invention: (a) Influence of temperature on the fluorescence quenching efficiency of detecting VB 12 (concentration is 0.1 nM), (b) Influence of temperature on the fluorescence quenching efficiency of VB 12 (concentration is 50 nM), (c) Influence of reaction time on the detection of VB 12 (concentration is 0.1 nM), (d) Influence of reaction time on the detection of VB 12 (concentration is 50 nM).

[0030] Figure 10 Result diagram of specificity verification of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe of the present invention.

[0031] Figure 11 Fluorescence spectrum and linear fitting curve diagram of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe of the present invention, (a) Fluorescence spectrum of VB 12 with concentrations of 0.1 - 50 nM, (b) Linear fitting curve diagram of the emission peak area at 550 nm and VB 12 with concentrations of 0.1 - 50 nM, (c) Fluorescence spectrum of VB 12 with concentrations of 20 - 750 nM​Fluorescence spectrogram, (d) Linear fitting curve graph of the emission peak area at 550 nm and VB with a concentration of 20 - 750 nM 12 Detailed implementation manner

[0032] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all belong to the scope of the present invention. If not specifically specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0033] Example 1

[0034] Preparation method of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs, the method comprising the following steps:

[0035] S1. The NaYF4:Yb,Er in this example is NaYF4:20% Yb, 2% Er, and the contents of Yb and Er are atomic contents. In the embodiments of the present invention, the preparation method of the core NaYF4:20% Yb, 2% Er is specifically to add YCl3 (0.78 mmol), YbCl3 (0.2 mmol) and ErCl3 (0.02 mmol) into a 100 mL three-necked flask containing oleic acid (6 mL) and 1-octadecene (15 mL) respectively, degas at room temperature for 10 min, then heat to 160 °C for 30 min under a nitrogen stream and strong stirring to form an oleic acid precursor. After naturally cooling to room temperature, add 10 mL of a methanol solution containing NH4F (4.0 mmol) and NaOH (2.5 mmol), and stir at 50 °C for 40 min. Then, heat to 70 °C, 100 °C, 120 °C, each for 30 min. Then heat the solution to 300 °C under nitrogen for 1 h and cool to room temperature. The obtained core nanoparticles NaYF4:Yb,Er are precipitated with ethanol and subjected to configuration separation, washed 3 times with ethanol and cyclohexane (volume ratio 1:1), and dried in vacuum for later use.

[0036] ​S2. Add YCl3 (1 mmol) into a 100 mL three-necked flask containing oleic acid (6 mL) and 1-octadecene (15 mL), degas at room temperature for 10 min, then heat to 160 °C for 30 min under a nitrogen stream and vigorous stirring to form an oleic acid precursor. After naturally cooling to room temperature, add 10 mL of cyclohexane containing the core NaYF4:Yb,Er and a mixed solution of 10 mL of methanol containing NH4F (4.0 mmol) and NaOH (2.5 mmol), and stir in a 50 °C water bath for 30 min. Then, heat to 70 °C, 100 °C, and 120 °C, each for 30 min, and then heat to 300 °C under nitrogen for 1 h, and cool to room temperature. The obtained core-shell nanoparticles NaYF4:Yb,Er@NaYF4 are precipitated with ethanol, centrifuged, washed three times with ethanol and cyclohexane (volume ratio 1:1), and dried in vacuo for standby.

[0037] S3. Disperse 6.7 mL of octanol and 12 mL of Triton X-100 by ultrasonic stirring in 50 mL of cyclohexane, add 10 mL of cyclohexane containing 200 mg of core-shell nanoparticles NaYF4:Yb,Er@NaYF4 and stir for 30 min, add 0.4 mL of ammonium hydroxide solution to form an inverse microemulsion solution, stir for 1 h, add 50 μL of TEOS [tetraethyl orthosilicate] solution, age under stirring for 14 h, and finally centrifuge and wash three times with water and ethanol.

[0038] S4. Disperse the obtained modified core-shell upconversion nanoparticles into 10 mL of ethanol / water mixed solution, and stir ultrasonically for 30 min. Then add 0.13 mL of APTES [aminopropyltriethoxysilane], and stir magnetically for 10 min. Then weigh an appropriate amount of AgNO3 and disperse it into 10 mL of deionized water, add it to the magnetically stirred mixed solution, and continue stirring for 20 min. Then add 0.0174 g of sodium citrate, heat to 100 °C and hold for 20 min. After cooling the mixture to room temperature, centrifuge to obtain NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs nanocomposite particles, and wash three times with water and ethanol.

[0039] Result analysis:

[0040] Figure 2 are the fluorescence and ultraviolet diagrams of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe. In addition to the matrix selection and its crystallinity, the doping concentration of rare earths is also a decisive factor affecting its luminescence performance. In order to explore the influence of rare earth ion concentration on the 3+ ,Er 3+ fluorescence intensity of NaYF4:Yb, in this example, Yb 3+ with a doping concentration of 20% and Er3+ NaYF4: 20% Yb doped at a doping concentration of y% (y = 0.5, 1.0, 1.5, 2.0, 2.5) 3+ , y% Er 3+ upconversion fluorescence material. By Figure 2 (a) From the fluorescence spectrum, it can be clearly seen that when the Yb 3+ doping concentration is 20% and the Er 3+ doping concentration is 2%, the fluorescence intensity is the highest. Subsequently, with the Er 3+ doping concentration fixed at 2%, the doping molar concentration of Yb is adjusted. 3+ Among them, the doping molar concentrations of Yb are 10%, 20%, 40%, 60% and 80% respectively. By 3+ (b) From the fluorescence spectrum, it can be clearly seen that when the doping concentrations are 20% Yb Figure 2 and 2% Er 3+ , the fluorescence intensity is the highest. Therefore, the core adopts 20% Yb 3+ , 2% Er 3+ ion concentrations. The NaYF4 shell can provide a relatively uniform and stable crystal field environment for the rare earth ions on the surface of the NaYF4:Yb 3+ , Er 3+ core, optimize the energy transfer path, promote the energy transfer from the sensitizer ions to the activator ions, and facilitate the occurrence of the upconversion luminescence process, thereby enhancing the luminescence intensity. The thickness of the shell is changed by adjusting the ratio of OA to YCl3. The ratios of OA (mL) to YCl3 (mmol) used in this experiment are 1, 3, 6, 9 and 12 mL / 1 mmol respectively. By 3+ (c) From the fluorescence spectrum, it can be seen that when the ratio of OA (mL) to YCl3 (mmol) is 6 mL / 1 mmol, the fluorescence intensity is the highest. The hydrophilicity and hydrophobicity of the upconversion material itself do not meet the requirements for detecting VB Figure 2 . By coating the surface with an SiO2 shell and introducing hydrophilic groups, the originally hydrophobic upconversion material can be made hydrophilic, facilitating its dispersion and application in an aqueous environment. To explore the influence of different thicknesses of the silicon shell on the fluorescence of the modified fluorescent nanomaterials, different amounts of TEOS solution are added, and the volumes of the added solutions are 5 μL, 10 μL, 20 μL, 50 μL, 100 μL respectively. By 12 (d) From the fluorescence spectrum, it can be seen that when 10 μL of TEOS solution is added, the influence on the fluorescence intensity is the lowest. Silver nanoparticles have unique surface plasmon resonance (SPR) characteristics. Under the irradiation of light with a specific wavelength, the free electrons on their surface will undergo collective oscillation, generating a strong local electromagnetic field enhancement effect. When combined with the inner layer of NaYF4:Yb Figure 2 (d) From the fluorescence spectrum, it can be seen that when 10 μL of TEOS solution is added, the influence on the fluorescence intensity is the lowest. Silver nanoparticles have unique surface plasmon resonance (SPR) characteristics. Under the irradiation of light with a specific wavelength, the free electrons on their surface will undergo collective oscillation, generating a strong local electromagnetic field enhancement effect. When combined with the inner layer of NaYF4:Yb 3+, Er 3+ When combined, this local electromagnetic field can interact with the upconversion luminescence process of NaYF4:Yb 3+ , Er 3+ to enhance the fluorescence emission intensity of NaYF4:Yb 3+ , Er 3+ . By changing the peak shape, peak position, etc. of its emission spectrum, the optical properties of the entire material are optimized through the optical coupling between the two, making it have higher sensitivity in applications such as fluorescence detection and sensing. To explore the influence of different AgNO3 addition amounts on the fluorescence of NaYF4:Yb,Er@NaYF4@SiO2, different contents of AgNO3 were added, and the volumes of AgNO3 added were 0 μM, 0.045 μM, 0.09 μM, 0.225 μM, and 0.45 μM respectively. From Figure 2 (e) The fluorescence spectra, it can be seen that when 0.225 μM AgNO3 is added, the fluorescence intensity is maximally enhanced. This is attributed to the unique surface plasmon resonance (SPR) characteristics of Ag NPs. Figure 2 (f) shows the ultraviolet spectra of different AgNO3 addition amounts. When AgNO3 is added, Ag NPs are formed by high-temperature reduction of sodium citrate. Since the ultraviolet characteristic peak of Ag NPs is around 440 nm, it is confirmed that Ag NPs are successfully modified on the surface of NaYF4:Yb 3 + , Er 3+ @NaYF4@SiO2.

[0041] Figure 3 This is a transmission electron microscopy (TEM) image, from which the overall morphological structure of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs can be clearly seen. NaYF4:Yb,Er as the core part presents a relatively regular hexagonal nanocrystal morphology with a size of about 19 nm. On this basis, the shell structure wrapping NaYF4:Yb,Er increases the overall particle size to 21 nm. It closely adheres to the core NaYF4:Yb,Er and plays roles such as protection and performance regulation for the core. The SiO2 shell layer further wrapped on the outer layer of the core-shell structure expands the overall particle size to 25 nm. The SiO2 shell layer provides a stable isolation protection and a basis for function expansion for the material due to its chemical inertness and good biocompatibility. The silver nanoparticles (Ag NPs) modified on the outermost layer have a particle size of about 6 - 10 nm. These Ag NPs generate an optical synergy effect with the inner layer structure by virtue of their unique surface plasmon resonance characteristics, enhancing the overall fluorescence emission intensity of the material.

[0042] Figure 4is an infrared spectrum. In the materials of core, core-shell, SiO2-modified, and silver nanoparticle-modified, a broad absorption peak of O-H stretching vibration caused by intermolecular hydrogen bonding was observed at 3450 cm -1 The weak absorption peaks at 2926 cm- 1 , 2855 cm -1 correspond to asymmetric and symmetric stretching vibrations, confirming the residue of oleic acid on the surface of the core and core-shell nanoparticles. The absorption peaks of the long alkyl chain of oleic acid in the SiO2-modified and silver nanoparticle-modified materials decreased significantly, indicating the success of the water-repellency modification. Broad bands at approximately 1609 cm -1 and 1120 cm -1 have been assigned to asymmetric and symmetric C=O stretching vibrations of surface carboxylates, and only the intensity related to the carboxyl O-H bending vibration was observed at 1465 cm -1 . The infrared peaks of silica are mainly concentrated in the ranges of 1000 - 1200 cm -1 and 800 - 1000 cm -1 . The infrared peaks in the 1000 - 1200 cm - region are mainly caused by the stretching vibration of Si-H bonds. The infrared peaks in the 800 - 1000 cm -1 region are caused by the bending vibration of Si-O bonds. The infrared characteristic peak of silver nitrate is at 1385 cm -1 , and the infrared peak of silver nitrate appears in the upconversion material modified with silver nanoparticles, proving the successful modification of silver nanoparticles. The peak at 547 cm -1 may be the result of RE 3+ -F stretching vibration.

[0043] Figure 5 is the XRD pattern. It can be seen that the diffraction patterns of different samples show certain characteristics. Among them, the diffraction peaks of NaYF4:Yb,Er@NaYF4@SiO2, NaYF4:Yb,Er@NaYF4, and NaYF4:Yb,Er can correspond well to the data of the NaYF4:Yb,Er (JCPDS NO.28 - 1192) XRD standard card. This indicates the successful synthesis of NaYF4:Yb,Er and NaYF4:Yb,Er@NaYF4. Due to the small particle size of Ag nanoparticles, the full width at half maximum of all reflection peaks is relatively large. For the SiO2 part in the core-shell structure, no specific amorphous SiO2 peaks were observed in the XRD pattern. This is probably because the silica layer wrapped near the surface of NaYF4:Yb,Er@NaYF4 is very thin, so that no obvious characteristic peaks can be presented in the XRD pattern.

[0044] Figure 6Fluorescence image (a) and UV image (b) of the structural material to explore the effect of structural changes on its optical properties. For NaYF4:Yb,Er, its fluorescence intensity exhibits multiple characteristic peaks in the wavelength range of 500 - 600 nm. This luminescence mechanism is based on the energy transfer from Yb 3+ ions that absorb energy and transfer it to Er 3+ ions. In the fluorescence spectrum, these energy level transitions are manifested as fluorescence peaks at specific wavelengths. Due to the coating of the NaYF4 layer, surface defects and non-radiative transitions are reduced. This means that more electrons can return to the ground state through radiative transitions, thus enhancing the fluorescence intensity, which is manifested as an increase in fluorescence intensity in the fluorescence spectrum. The introduction of the SiO2 layer may change the crystal field environment around the Er 3+ ions. This change in the crystal field environment may affect the energy level structure of the Er 3+ ions, resulting in no increase or even a decrease in the radiative transition probability between some energy levels, thus leading to no enhancement of the fluorescence intensity. After loading Ag NPs, a significant change in fluorescence intensity occurs. This is due to the surface plasmon resonance (SPR) effect of Ag NPs. When rare earth ions are close to Ag NPs, they are in this enhanced electromagnetic field. According to the principles of quantum mechanics, the radiative transition rate is proportional to the intensity of the electromagnetic field. Therefore, the radiative transition probability of rare earth ions in the enhanced electromagnetic field increases, resulting in enhanced fluorescence intensity. In the UV image b, NaYF4:Yb / Er has certain absorption peaks in the UV region. These absorption peaks reflect the UV absorption transitions of Yb 3+ and Er 3+ ions. Although NaYF4:Yb / Er@NaYF4 is coated with a layer of NaYF4 on the basis of NaYF4:Yb / Er, in the UV image, their UV absorption peaks are almost the same. When a SiO2 layer is coated on the basis of NaYF4:Yb / Er@NaYF4, there is a relatively obvious characteristic absorption peak of SiO2 at about 230 nm. Ag NPs have a significant surface plasmon resonance (SPR) effect, and they have strong absorption characteristics in the UV-visible region. In this system, the SPR absorption peak of Ag NPs appears at about 400 - 450 nm. When Ag NPs are successfully loaded on the NaYF4:Yb / Er@NaYF4@SiO2 material, an absorption peak near 400 - 450 nm can be clearly observed in the UV image.

[0045] Example 2

[0046] The NaYF4:Yb,Er@NaYF4@SiO2@AgNPs fluorescence upconversion detection probe prepared in Example 1 is used for the detection of vitamin B 12 , and the specific detection steps are as follows:

[0047] Add 1 mL of the solution containing the NaYF4:Yb,Er@NaYF4@SiO2@AgNPs material into a test tube, and then add 50 μL of the test solution containing vitamin B 12 and shake well. React for 4 min at room temperature and place it into a fluorescence cuvette. Record all fluorescence spectra under 980 nm excitation. Using the blank reagent as a reference, with the fluorescence intensity as the vertical axis and the concentration as the horizontal axis, establish a standard curve.

[0048] Result analysis:

[0049] Figure 7 shows the fluorescence spectrum of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs and the ultraviolet spectrum of vitamin B 12 . After simple pretreatment, for the test sample of the solution containing VB 12 , add the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe. When VB 12 exists in the solution, due to the almost complete overlap between the emission spectrum of the NaYF4:Yb,Er@NaYF4@SiO2@AgNPs fluorescence upconversion detection probe and the absorption spectrum of VB 12 , fluorescence quenching occurs. VB 12 shows strong absorption characteristics in a specific wavelength range. When the excitation light passes through the solution, VB 12 will absorb a large amount of the excitation light energy, which significantly reduces the excitation light energy that can reach the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe, thereby causing the fluorescence emission intensity of the fluorescence upconversion detection probe to decrease.

[0050] Figure 8 shows the mechanism of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe for detecting VB 12 . By measuring the fluorescence decay curve to clarify the quenching mechanism, the fluorescence lifetime of the fluorophore remains constant in the inner filter effect (IFE). Experiments show that the fluorescence lifetime of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs hardly changes before and after adding VB 12 , confirming that the fluorescence quenching mechanism of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs in the presence of VB 12 is IFE.

[0051] Figure 9Optimization diagram of detection conditions for the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe. By optimizing the reaction ambient temperature of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe for detecting VB 12 (Figs. a - b), it was found that the temperature had little effect on the detection results. By optimizing the reaction time (Figs. c - d), it was found that as the reaction time gradually increased, the fluorescence intensity of the system showed different trends of change. In the initial stage of the reaction (about 1 - 3 minutes), the fluorescence intensity gradually decreased with the extension of time, indicating that as time passed, the interaction between the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe and VB 12 was continuously enhanced, and more VB 12 molecules interacted with the detection probe and affected its fluorescence characteristics. However, when the reaction time reached 4 minutes, the fluorescence intensity reached a relatively stable state. Further extending the reaction time (5 - 8 minutes), the fluorescence intensity did not show an obvious continuous decline. Therefore, 4 minutes of reaction was selected as the optimal reaction duration.

[0052] Figure 10 Verification result diagram of the specificity of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe. To evaluate the selectivity, interference molecules such as ions (Cu 2+ , Zn 2+ , Fe 3+ , Ni 2+ , Mn 2+ , Ca 2+ , Zr 2 + , NH4 + , K + , Pb 2+ , Ba 2+ , Al 3+ , Na + , Mg 2+ ) and vitamins (B1, B2, B3, B5, B6, B7, and C) were at a concentration (5 μM) 100 times higher than that of VB 12 (50 nM). All tests were performed in triplicate. Figure 10 As shown, the fluorescence signals corresponding to metal ions and other vitamins were negligible. When the target VB 12 was present, the fluorescence difference (F O - F) value increased significantly. These results indicate that this method has high selectivity for detecting the target VB 12 .

[0053] Figure 11Fluorescence spectra and linear fitting curves of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs fluorescence upconversion detection probe. The fluorescence intensity shows a good linear relationship in the ranges of 0.1 nM to 50 nM and 20 nM to 750 nM. 12 The linear curves are as follows. 12 The linear curve equations are y = -1461.02x + 40349.81 (R 2 = 0.991) and y = -3890.75x + 43742.61 (R 2 = 0.997). According to the principle that the LOD is three times the standard deviation of the blank signal, the LOD of 12 is calculated to be approximately 0.01 nM.

[0054] Example 3

[0055] The prepared NaYF4:Yb,Er@NaYF4:Yb,Er@SiO2@Ag NPs fluorescence upconversion detection probe was used to detect actual samples containing vitamin B 12 . The actual samples were functional beverages, multivitamin tablets, effervescent tablets, milk powder, and human serum. The functional beverages contained vitamin B 12 at concentrations of 0.2, 0.4, and 0.8 nM. The multivitamin tablets contained vitamin B 12 at concentrations of 4.5, 9, and 18 nM. The vitamin B 12 effervescent tablets contained vitamin B 12 at concentrations of 23, 46, and 92 nM. The milk powder contained vitamin B 12 at concentrations of 0.41, 0.83, and 1.66 nM. The human serum contained vitamin B 12The concentrations are 0.141, 0.128, 0.146, 0.133, 0.3, 0.29 nM. The detection process is as follows: For beverage samples, they can be directly diluted to an appropriate concentration with pure water before detection. For compound vitamin tablet samples, take one compound vitamin tablet and put it into a 50 mL centrifuge tube, add 20 mL of pure water, oscillate to dissolve, then centrifuge the solution at 6000 r / min for 15 min, discard the precipitate, collect the supernatant, filter it through a 0.22 μm filter membrane to obtain the stock solution of vitamin tablets, and dilute the filtrate to an appropriate concentration before detection. For effervescent tablet samples, take one effervescent tablet and put it into a 50 mL centrifuge tube, add 20 mL of pure water, oscillate to dissolve, filter it through a 0.22 μm filter membrane to obtain the stock solution of vitamin tablets, and dilute the filtrate to an appropriate concentration before detection. For milk powder samples, weigh 1.3 g of anhydrous disodium hydrogen phosphate, 1 g of anhydrous sodium metabisulfite, and 1.2 g of citric acid monohydrate, dissolve them in 100 mL of water; then weigh 2 g of milk powder, mix it with 10 mL of the above solution, add another 150 mL of water, hydrolyze at 121 °C for 10 min, adjust the pH to 4.5 after cooling, and then make up the volume to 250 mL with water, filter through a 0.22 μm filter membrane, pipette 5 mL of the filtrate, add 20 mL of water, adjust the pH to 6.8, and make up the volume to 100 mL with water. Add the sample solution containing vitamin B 12 to the solution of UCNPs@NaYF4@SiO2@Ag NPs materials, react at room temperature for 4 min, and put it into a fluorescence cuvette. Measure the fluorescence signal under 980 nm excitation, and calculate the content of vitamin B 12 in the actual sample through the standard curve.

[0056] To evaluate the detection effect of the established method on actual samples, this example selected functional beverages, compound vitamin tablets, effervescent tablets, milk powder, and human serum to detect the content of vitamin B 12 in them; the results are shown in Table 1 and Table 2 respectively, and good relative recoveries are obtained.

[0057] Table 1 Spike recoveries of VB 12 in samples

[0058]

[0059] Table 2 Spike recoveries of VB 12 in serum samples

[0060]

[0061] a Relative recovery rate = detected concentration / original concentration.

Claims

1. Preparation method of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs, characterized in that, The method comprises the following steps: S1. Respectively add yttrium source, ytterbium source, and erbium source into a mixed solvent of oleic acid and 1-octadecene, degas at room temperature for 5 - 15 min, then heat to 140 - 160 °C under a nitrogen stream and strong stirring conditions, react for 30 - 45 min, and naturally cool to room temperature to form an oleic acid precursor; then add a methanol mixed solution of NH4F and NaOH, react at 50 °C for 30 - 45 min, and then react at 70 °C, 100 °C, and 120 °C for 30 - 45 min respectively at each stage; after the reaction, heat the solution to 290 - 310 °C in a nitrogen atmosphere, react for 1 - 2 h, and cool to room temperature; precipitate with ethanol, wash with a mixed solvent of ethanol and cyclohexane, and vacuum dry to obtain core nanoparticles NaYF4:Yb,Er; S2. Add the yttrium source into a mixed solvent of oleic acid and 1-octadecene, degas at room temperature for 5 - 15 min, then heat to 140 - 160 °C under a nitrogen stream and strong stirring conditions, react for 30 - 45 min, and naturally cool to room temperature to form an oleic acid precursor; then add a cyclohexane solution of the core nanoparticles NaYF4:Yb,Er obtained in S1, a methanol solution of NH4F and NaOH, react at 50 °C for 30 - 45 min, and then react at 70 °C, 100 °C, and 120 °C for 30 - 45 min respectively at each stage; after the reaction, heat the solution to 290 - 310 °C in a nitrogen atmosphere, react for 1 - 2 h, and cool to room temperature; precipitate with ethanol, wash with a mixed solvent of ethanol and cyclohexane, and vacuum dry to obtain core-shell nanoparticles NaYF4:Yb,Er@NaYF4; S3. Ultrasonically disperse octanol and Triton X-100 in cyclohexane. At room temperature, add a cyclohexane solution of NaYF4:Yb,Er@NaYF4 and stir for 30 min, then add ammonium hydroxide solution to form a reverse microemulsion solution and stir for 1 h. Add tetraethyl orthosilicate solution and stir for aging for 14 h, then centrifuge and wash to obtain NaYF4:Yb,Er@NaYF4 modified with SiO2; S4. Disperse the NaYF4:Yb,Er@NaYF4 modified with SiO2 obtained in S3 in an ethanol aqueous solution, ultrasonically stir, add 3-aminopropyltriethoxysilane thereto and stir magnetically, then add an aqueous AgNO3 solution and continuously stir; then add a sodium citrate solution, heat to 100 °C and react for 20 - 30 min; after the reaction, cool to room temperature, centrifuge and separate, and wash to obtain NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs.

2. The preparation method of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs according to claim 1, characterized in that, In S1, the dosage ratio of the yttrium source, ytterbium source, erbium source, oleic acid, 1-octadecene, NH4F, and NaOH is 0.2 - 0.3 g:0.07 - 0.08 g:0.007 - 0.008 g:6 - 8 mL:15 - 20 mL:0.14 - 0.15 g:0.1 - 0.2 g.

3. The preparation method of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs according to claim 1, characterized in that, In S2, the dosage ratio of yttrium source, oleic acid, 1-octadecene, NH4F, NaOH to the core NaYF4:Yb,Er is 0.3 - 0.4 g:6 - 8 mL:15 - 20 mL:0.14 - 0.15 g:0.1 - 0.2 g:50 mg.

4. The preparation method of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs according to claim 1, characterized in that, In S3, the dosage ratio of core-shell NaYF4:Yb,Er@NaYF4, octanol, Triton X-100, cyclohexane, ammonium hydroxide solution and tetraethyl orthosilicate solution is 100 - 200 mg:6.7 - 8 mL:12 - 15 mL:10 - 15 mL:0.4 - 1 mL:50 - 100 μL.

5. The preparation method of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs according to claim 1, characterized in that, In S4, the dosage ratio of the APTES solution, AgNO 3、 sodium citrate is 130 - 150 μL: 7 - 80 mg: 0.0174 g.

6. The preparation method of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs according to claim 1, wherein, The yttrium source, ytterbium source and erbium source are all chlorine compounds.

7. The preparation method of NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs according to claim 1, characterized in that, The centrifugation speed in S1 and S2 is 6000 - 8000 rpm.

8. NaYF4:Yb,Er@NaYF4@SiO2@AgNPs prepared by the method according to any one of claims 1 - 7.

9. Use of the NaYF4:Yb,Er@NaYF4@SiO2@Ag NPs according to claim 8 in detecting vitamin B in food or serum samples 12 , characterized in that The fluorescence excitation wavelength of the application is 980 nm, and the detection limit for vitamin B 12 is 0.01 nM.

10. The application according to claim 9, wherein The standard curves of the applications show good linear relationships within the ranges of 0.1 - 50 nM and 20 - 750 nM for VB 12 respectively; among them, the regression equations of VB 12 are y = -1461.02x + 40349.81 and y = -3890.75x + 43742.61, respectively.

Images