A dual-mode magnetic relaxation upconversion fluorescence biosensor and its application

Abstract

The invention relates to the technical field of food detection, and specifically discloses a dual-mode magnetic relaxation up-conversion fluorescent biosensor and its application, wherein the dual-mode magnetic relaxation up-conversion fluorescent biosensor comprises an antigen-antibody immune recognition unit, functional magnetic composite nanoparticles and functional up-conversion fluorescent nanoparticles, and the functional magnetic composite nanoparticles and functional up-conversion fluorescent nanoparticles are assembled through the antigen-antibody immune recognition unit to form a dual-mode magnetic relaxation up-conversion fluorescent biosensor for detecting aflatoxin B1 in food. The dual-mode magnetic relaxation up-conversion fluorescent biosensor described in the invention can detect aflatoxin B1 up to 0.16 ng / mL in MR mode and 0.038 ng / mL in FL mode, and the biosensor can ensure detection sensitivity and overcome the interference of colored samples in the detection of aflatoxin B1 in food, thereby effectively improving the accuracy of aflatoxin B1 detection in food.

Description

Technical Field

[0001] The present invention relates to the technical field of food detection, and in particular to a dual-mode magnetic relaxation up-conversion fluorescence biosensor and applications thereof. Background Art

[0002] Aflatoxin B1, chemical formula is C 17 H 12 O6, the pure product is colorless crystals with a relative molecular weight of 312.27. It is insoluble in water but soluble in oil and a variety of polar organic solvents, such as chloroform, methanol, ethanol, propanol, dimethylformamide, and insoluble in petroleum ether, ether and hexane. Aflatoxin B1 ranks first in carcinogenicity among known chemicals. In recent years, several methods for detecting aflatoxin B1 have been proposed, such as colorimetry, fluorescence sensors, plasmon resonance spectroscopy and surface enhanced Raman scattering. Among these methods, fluorescence sensors have high sensitivity and low detection limits. Fluorescence sensors based on upconversion nanoparticles (UCNPs) have excellent sensitivity, no photobleaching, and provide continuous luminescence, inducing only weak autofluorescence background and improving signal-to-noise ratio. However, there are complex food matrix interferences and relatively low sample recoveries, such as turbid and opaque matrices. In other words, sometimes the results obtained from single-mode-based fluorescence sensors may be inaccurate because single-mode signal transduction is subject to matrix interference.

[0003] Based on this, researching a dual-mode magnetic relaxation up-conversion fluorescence biosensor that can ensure detection sensitivity and has anti-interference detection capability and its application is a technical problem that needs to be solved by people in this technical field. Summary of the invention

[0004] The object of the present invention is to provide a dual-mode magnetic relaxation up-conversion fluorescence biosensor, comprising an antigen-antibody immune recognition unit, functional magnetic composite nanoparticles and functional up-conversion fluorescent nanoparticles. The functional magnetic composite nanoparticles and the functional up-conversion fluorescent nanoparticles are assembled through the antigen-antibody immune recognition unit to form a dual-mode magnetic relaxation up-conversion fluorescence biosensor for detecting aflatoxin B1 in food.

[0005] Preferably, the method for preparing the functional magnetic composite nanoparticles comprises the following steps:

[0006] S11, synthesis of aqueous UCNPs using solvothermal method;

[0007] S12. Aflatoxin B1-specific antibodies were immobilized on the surface of aqueous UCNPs based on the covalent coupling method to prepare functional UCNPs.

[0008] Preferably, the specific implementation of step S11 is:

[0009] S111, adding 12 mL OA, 30 mL 1-ODE and 1 mmol rare earth chloride complex into a three-necked flask for mixing, wherein Y:Yb:Er in the rare earth chloride complex is 78:20:2;

[0010] S112, heating the mixture from 123° C. to 160° C. in a nitrogen atmosphere and keeping the temperature until the mixture becomes a clear solution;

[0011] S113, add 30 mL of methanol solution containing 3.25 mmol NaOH and 3.5 mmol NH4F dropwise to the mixture, cool to 80°C and stir for 30 min;

[0012] S114, cooling to room temperature, adding ethanol and cyclohexane in a volume ratio of 1:1 to the mixture and centrifuging at 2000 g for 10 minutes;

[0013] S115, washing the mixture with ethanol and water to disperse the OA-UCNPs in chloroform, the washing times being 2-3 times;

[0014] S116. Take a certain amount of UCNPs-chloroform mixed solution and add it to a flask, then slowly add 20 ml of ethanol solution containing 300 mg PAA, and stir the solution at room temperature for 24 hours. After centrifugation, aqueous phase UCNPs can be obtained.

[0015] Preferably, the specific implementation of step S12 includes:

[0016] S121, disperse the aqueous UCNPs in PB buffer, add 200ul of EDC with a concentration of 10mg / mL and 120ul of NHS with a concentration of 10mg / mL to mix, and keep the reaction at room temperature for 30min, wherein the concentration of PB buffer is 0.01mM and the pH value is 7.4;

[0017] S122. The mixed reaction solution was rinsed with water and centrifuged at 2000g for 8 minutes, and then aflatoxin B1-specific antibody was added and mixed for 8 hours to obtain functional UCNPs, wherein the number of rinses was 2-3 times, the concentration of aflatoxin B1-specific antibody was 6ug / mL, PB was 0.01mM, and the pH value was 7.4.

[0018] Preferably, the method for preparing the functional magnetic composite nanoparticles comprises the following steps:

[0019] S21, synthetic aqueous Fe3O4;

[0020] S22, synthesis of Fe3O4@PCN-224;

[0021] S23. Based on the covalent coupling method, aflatoxin B1-specific antigen was mixed with Fe3O4@PCN-224 to prepare functional Fe3O4@PCN-224.

[0022] Preferably, the specific implementation of step S21 includes:

[0023] S211, 2 mmol Fe(acac) 3 was mixed with 10 mmol 1,2-hexadecanediol, 6 mmol oleic acid and 6 mmol oleylamine in 20 mL phenyl ether, and magnetically stirred under nitrogen flow;

[0024] S212, heating the mixture to 200° C. and maintaining for 30 minutes, then heating to 265° C. and refluxing for 30 minutes under a nitrogen flow;

[0025] S213, cooling the mixture to room temperature, precipitating a dark brown substance from the mixture using 40 mL of ethanol under air atmosphere, and centrifuging at 3500 g for 10 min;

[0026] S214, re-precipitating the mixture after the centrifugation with ethanol and re-dispersing it in hexane to generate oil-phase Fe3O4 nanoparticles;

[0027] S215, dissolving 10 mg of oil-phase Fe3O4 nanoparticles in 5 mL of toluene, then adding 5 mL of methanol containing 50 mg of DMSA and mixing, and leaving the mixture at room temperature for 24 h;

[0028] S216. Allow the mixture to stand at 30°C and vacuum dry the precipitate.

[0029] Preferably, the specific implementation of step S22 includes:

[0030] S221, 100 mg 0.13 mmol H2TCPP, 300 mg 0.93 mmol ZrOCl2·8H2O and 3.3 g 27 mmol benzoic acid were dissolved in 100 mL DMF, and the mixture was stirred at 95°C for 5 hours;

[0031] S222, centrifuging the stirred mixture at 9500 g for 15 min to collect PCN-224 nanoparticles, and washing the collected PCN-224 nanoparticles with DMF;

[0032] S223, ultrasonically disperse PCN-224 in 2.5 mL of ethylene glycol and preheat at 120 °C for 10 min, then add 3 mL of 15 mg / mL polyvinyl pyrrolidone and 5 mL of 1 mg / mL Fe3O4 aqueous solution dropwise and stir vigorously for 16 min;

[0033] S224. The mixture obtained after vigorous stirring was kept at 120° C. for 10 minutes and then cooled to room temperature. The cooled mixture was centrifuged and finally washed with water and ethanol several times to obtain Fe3O4@PCN-224.

[0034] Preferably, the specific implementation of step S23 includes:

[0035] S231, dispersing 5 mL of Fe3O4@PCN-224 with a concentration of 3.3 mg / mL in PB buffer, wherein the concentration of PB buffer is 0.01 mM and the pH value is 7.4;

[0036] S232, add 400 μL of 10 mg / mL EDC and 240 μL of 10 mg / mL NHS to the Fe3O mixture, and keep the reaction at room temperature for 30 min;

[0037] S233, washing the mixed solution after the reaction, and centrifuging it at 180° C. for 5 min, and the washing times are 2-3 times;

[0038] S234. Add 2 mL of 16 ug / mL aflatoxin B1-specific antigen to the mixed solution after centrifugation and mix for 8 hours to obtain functional Fe3O4@PCN-224, wherein the PB concentration of aflatoxin B1-specific antigen is 0.01 mM and the pH value is 7.4.

[0039] An application of a dual-mode magnetic relaxation up-conversion fluorescence biosensor, based on the dual-mode magnetic relaxation up-conversion fluorescence biosensor described above, is applied to the detection of aflatoxin B1 in food.

[0040] Compared with the prior art, the present invention provides a dual-mode magnetic relaxation upconversion fluorescence biosensor, which assembles functional upconversion nanoparticles and functional magnetic composite nanoparticles together through an antigen-antibody immune recognition unit, thereby constructing a dual-mode magnetic relaxation upconversion fluorescence biosensor for detecting aflatoxin B1 in food. The dual-mode magnetic relaxation upconversion fluorescence biosensor has a detection limit of 0.16 ng / mL for aflatoxin B1 in MR mode and a detection limit of 0.038 ng / mL for aflatoxin B1 in FL mode. Therefore, the biosensor not only ensures detection sensitivity but also overcomes the interference of colored samples, thereby improving the accuracy of aflatoxin B1 detection in food. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a schematic diagram of the assembly structure of a dual-mode magnetic relaxation upconversion fluorescence biosensor in the present invention,

[0042] Figure 2 This is a schematic diagram of the detection results of UCNPs and Fe3O4@PCN-224 in this embodiment.

[0043] Figure 3 : is a schematic diagram of the assembly detection results in this embodiment,

[0044] Figure 4 It is a schematic diagram of the detection results of aflatoxin B1 at different concentrations under the conditions of the presence of different metal ions in this embodiment. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings.

[0046] like Figure 1 As shown, Figure 1 The assembly structure of a dual-mode magnetic relaxation upconversion fluorescent biosensor is shown. The dual-mode magnetic relaxation upconversion fluorescent biosensor comprises an antigen-antibody immune recognition unit, functional magnetic composite nanoparticles and functional upconversion fluorescent nanoparticles, and the functional magnetic composite nanoparticles and functional upconversion fluorescent nanoparticles are assembled through the antigen-antibody immune recognition unit to form a dual-mode magnetic relaxation upconversion fluorescent biosensor for detecting aflatoxin B1 in food.

[0047] In this embodiment, the upconversion fluorescent nanoparticles are UCNPs, and the magnetic composite nanoparticles are Fe3O4@PCN-224. Since the fluorescence of the functional UCNPs is quenched by the PCN-224 in the functional Fe3O4@PCN-224, the self-diffusion coefficient of water protons near the Fe3O4 carrier is reduced, thereby obtaining excellent transverse relaxation performance; and on Fe3O4@PCN-224, the ΔT2 value in the assembled biosensor is significantly reduced, and aflatoxin B1 has an effect on the assembly process of the functional magnetic composite nanoparticles and the functional upconversion fluorescent nanoparticles, thereby changing the ΔT2 value and the fluorescence intensity of the anti-interference analysis of aflatoxin B1, so that the biosensor has both excellent sensitivity and anti-interference detection capability in slightly turbid and opaque samples.

[0048] In this embodiment, the hydrated particle size and morphology of the assembly were measured and evaluated using TEM (Transmission Electron Microscopy), DLS (Dynamic Light Scattering) and ELS (Electrophoretic Light Scattering). Figure 3As shown, 3a shows the TEM results of the assembly, 3b shows the DLS results of Ag-Fe3O4@PCN-224, Ab-UCNPs and the assembly, 3c shows the fluorescence change results of UCNPs, Ab-UCNPs, Fe3O4@PCN-224 and the assembly (the curves in 3c are ①②③④ from top to bottom), and 3d shows the transverse relaxation rate change results of Fe3O4@PCN-224, Ag-Fe3O4@PCN-224 and the assembly. Since UCNPs are coated with aflatoxin B1-forming Ab (Aflatoxin B1)-UCNPs-specific antibodies, Fe3O4@PCN-224 is coated with aflatoxin B1-specific antigens to form functional Fe3O4@PCN-224 (Ag- Fe3O4@PCN-224). The hydrated particle size of Ab-UCNPs increased from 212nm to 237nm, and their Zeta potential (Zeta potential refers to the potential of the shear plane, also called electrokinetic potential or electromotive potential, which is an important indicator to characterize the stability of colloidal dispersions) decreased from -18.01mV to -24.61mV. The hydrated particle size of Ag-Fe3O4@PCN-224 increased from 141nm to 180nm, and their Zeta potential decreased from 12.79mV to -18.01mV. The above results show that the surface modification of Ab-UCNPs and Ag-Fe3O4@PCN-224 was successful. The assembly of Ag-Fe3O4@PCN-224 and Ab-UCNPs induced by biorecognition events manifested as large polymer assemblies. DLS measurements further confirmed the formation of the assemblies, such as Figure 2 As shown, 2a and 2b both show the TEM image results of UCNPs, 2c shows the FTIR spectrum results of UCNPs, 2d shows the XRD results of Fe3O4@PCN-224, 2e shows the N2 adsorption isotherm results of Fe3O4@PCN-224, and 2f shows the lateral relaxation rate results of Fe3O4@PCN-224.

[0049] In this embodiment, the feasibility of the dual-mode sensor constructed by Ag-Fe3O4@PCN-224 and Ab-UCNPs was further evaluated, that is, the fluorescence intensity of UCNPs and the transverse relaxation rate of Fe3O4@PCN-224 were evaluated. Figure 2c shows the emission spectrum of UCNPs and the UV absorption spectrum of Fe3O4@PCN-224. As can be seen from the figure, there is a large overlap between the two spectra, which preliminarily indicates that a FRET process (fluorescence resonance energy transfer, which is a non-radiative energy transition process that transfers the energy of the donor excited state to the acceptor excited state through the electric dipole interaction between molecules) may occur between UCNPs and Fe3O4@PCN-224; further measurement of the fluorescence intensities of UCNPs, Ab-UCNPs, and the mixture of UCNPs and Fe3O4@PCN-224, which are 139.29, 131.5, and 126.36 respectively, at 543 nm. It can be seen that the fluorescence intensity of the assembled biosensor (43.71) is significantly lower than that of UCNPs, Ab-UCNPs, and their mixture, indicating that the FRET process of the assembled MR-FL biosensor is successfully achieved; at the same time, its r2 is measured on a 0.5 TLF-nuclear magnetic resonance instrument. From Figure 2 d, it can be seen that its r2 increases in the following order: Fe3O4@PCN-224 (42.12 mM-1 s-1) < Ag-Fe3O4@PCN-224 (43.47 mM-1 s-1) < assembly (102.10 mM-1 s-1), further confirming the feasibility of the assembled MR-FL biosensor.

[0050] Among them, the concentration of the antigen-antibody immune recognition unit is 2-12 μg / mL, the concentration of Fe3O4@PCN-224 is 1.5 mg / mL, and the assembly reaction time is 40 min.

[0051] In this embodiment, since detection conditions and materials such as the assembly time and the concentration of the surface recognition unit (antigen-antibody immune recognition unit) have a significant impact on the performance of the MR-FL biosensor. The influence of the concentration of the surface recognition unit on the fluorescence quenching efficiency can reflect the FRET efficiency and the sensitivity of the MR mode. Fluorescence quenching efficiency = E% = ([F0 - F] / F0) × 100%, where F0 and F represent the fluorescence intensities before and after the assembly of Ab-UCNPs respectively. Due to the gradual assembly of nanoparticles, as the concentration of antibody / antigen increases (2 to 12 μg / mL), the fluorescence quenching efficiency increases and ΔT2 decreases. A high fluorescence quenching efficiency reflects the excellent detection sensitivity of the FL mode. However, a low ΔT2 is not beneficial to the detection sensitivity of the sensor. In this embodiment, the concentration of the antigen-antibody immune recognition unit is preferably 6 μg / mL. As the reaction time and concentration of Ag-Fe3O4@PCN-224 increase, the fluorescence quenching efficiency increases and ΔT2 decreases.

[0052] Among them, the preparation method of the functional magnetic composite nanoparticles includes the following steps:

[0053] S11. Synthesize aqueous UCNPs using a solvothermal method, specifically:

[0054] S111, adding 12 mL OA (oleic acid), 30 mL 1-ODE (1-octadecylene) and 1 mmol rare earth chloride complex into a three-necked flask for mixing, wherein Y: Yb: Er in the rare earth chloride complex is 78:20:2;

[0055] S112, heating the mixture from 123° C. to 160° C. in a nitrogen atmosphere and keeping the temperature until the mixture becomes a clear solution;

[0056] S113, add 30 mL of methanol solution containing 3.25 mmol NaOH and 3.5 mmol NH4F dropwise to the mixture, cool to 80°C and stir for 30 min; in this step, in order to remove methanol, the mixed solution needs to be stirred at 100°C for 30 min and kept at a constant temperature for 1 h under a nitrogen flow;

[0057] S114, cooling to room temperature, adding ethanol and cyclohexane in a volume ratio of 1:1 to the mixture and centrifuging at 2000 g for 10 minutes;

[0058] S115, washing the mixture with ethanol and water to disperse the OA-UCNPs in chloroform, the washing times being 2-3 times;

[0059] S116, taking a certain amount of UCNPs-chloroform mixed solution and adding it to a flask, then slowly adding 20 ml of ethanol solution containing 300 mg PAA, and stirring the solution at room temperature for 24 hours, and then obtaining aqueous phase UCNPs after centrifugation;

[0060] S12, immobilizing aflatoxin B1-specific antibodies on the surface of aqueous UCNPs based on a covalent coupling method, thereby preparing functional UCNPs, specifically:

[0061] S121, disperse the aqueous UCNPs in PB (Phosphate Buffer) buffer, add 200ul of 10mg / mL EDC (N-(3-8-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride) and 120ul of 10mg / mL NHS (N-89 hydroxysuccinimide) to mix, and keep the reaction at room temperature for 30min, wherein the concentration of PB buffer is 0.01mM and the pH value is 7.4;

[0062] S122. The mixed reaction solution was rinsed with water and centrifuged at 2000g for 8 minutes, and then aflatoxin B1-specific antibody was added and mixed for 8 hours to obtain functional UCNPs, wherein the number of rinses was 2-3 times, the concentration of aflatoxin B1-specific antibody was 6ug / mL, PB was 0.01mM, and the pH value was 7.4.

[0063] In this embodiment, aqueous UCNPs were prepared based on the hot solvent method, and then aflatoxin B1-specific antibodies were fixed on the surface of aqueous UCNPs by a covalent coupling method to obtain functional UCNPs. The obtained functional UCNPs needed to be stored at 4°C for future use.

[0064] The method for preparing the functional magnetic composite nanoparticles comprises the following steps:

[0065] S21, synthesizing aqueous Fe3O4, specifically comprising:

[0066] S211, 2 mmol of Fe(acac)3 (iron (III) acetylacetonate) was mixed with 10 mmol of 1,2-hexadecanediol, 6 mmol of oleic acid and 6 mmol of oleylamine in 20 mL of phenyl ether, and the mixture was magnetically stirred under a nitrogen flow;

[0067] S212, heating the mixture to 200° C. and maintaining for 30 minutes, then heating to 265° C. and refluxing for 30 minutes under a nitrogen flow;

[0068] S213, cooling the mixture to room temperature, precipitating a dark brown substance from the mixture using 40 mL of ethanol under air atmosphere, and centrifuging at 3500 g for 10 min;

[0069] S214, re-precipitating the mixture after the centrifugation with ethanol and re-dispersing it in hexane to generate oil-phase Fe3O4 nanoparticles;

[0070] S215, dissolving 10 mg of oil-phase Fe3O4 nanoparticles in 5 mL of toluene, then adding 5 mL of methanol containing 50 mg of DMSA and mixing, and leaving the mixture at room temperature for 24 h;

[0071] S216, vacuum drying the precipitate of the mixture at 30°C;

[0072] S22, synthesis of Fe3O4@PCN-224, specifically including:

[0073] S221, 100 mg 0.13 mmol of H2TCPP (meso-tetrakis (4-carboxyphenyl) porphyrin), 300 mg 0.93 mmol of ZrOCl2·8H2O (zirconium oxychloride octahydrate) and 3.3 g 27 mmol of benzoic acid were dissolved in 100 mL of DMF (N, N-dimethylformamide), and the mixture was stirred at 95° C. for 5 hours;

[0074] S222, centrifuging the stirred mixture at 9500 g for 15 min to collect PCN-224 nanoparticles, and washing the collected PCN-224 nanoparticles with DMF;

[0075] S223, ultrasonically disperse PCN-224 in 2.5 mL of ethylene glycol and preheat at 120 °C for 10 min, then add 3 mL of 15 mg / mL polyvinyl pyrrolidone and 5 mL of 1 mg / mL Fe3O4 aqueous solution dropwise and stir vigorously for 16 min;

[0076] S224, the mixture obtained after vigorous stirring is kept at 120°C for 10 minutes and then cooled to room temperature, the cooled mixture is centrifuged, and finally washed with water and ethanol several times to obtain Fe3O4@PCN-224;

[0077] S23, mixing aflatoxin B1-specific antigen with Fe3O4@PCN-224 based on a covalent coupling method, thereby preparing functional Fe3O4@PCN-224, specifically comprising:

[0078] S231, dispersing 5 mL of Fe3O4@PCN-224 with a concentration of 3.3 mg / mL in PB buffer, wherein the concentration of PB buffer is 0.01 mM and the pH value is 7.4;

[0079] S232, add 400 μL of 10 mg / mL EDC and 240 μL of 10 mg / mL NHS to the Fe3O mixture, and keep the reaction at room temperature for 30 min;

[0080] S233, washing the mixed solution after the reaction, and centrifuging it at 180° C. for 5 min, and the washing times are 2-3 times;

[0081] S234. Add 2 mL of 16 ug / mL aflatoxin B1-specific antigen to the mixed solution after centrifugation and mix for 8 hours to obtain functional Fe3O4@PCN-224, wherein the PB concentration of aflatoxin B1-specific antigen is 0.01 mM and the pH value is 7.4.

[0082] The present invention also provides an application of a dual-mode magnetic relaxation up-conversion fluorescence biosensor, based on which the dual-mode magnetic relaxation up-conversion fluorescence biosensor described above is applied to the detection of aflatoxin B1 in food.

[0083] Based on the review of the above-mentioned dual-mode magnetic relaxation upconversion fluorescence biosensor, applying the dual-mode magnetic relaxation upconversion fluorescence biosensor to the detection of aflatoxin B1 in food should have the same technical effect, which will not be repeated here.

[0084] In order to further illustrate the working principle and technical effect of the present invention, a series of experiments are conducted to verify this.

[0085] In the following experiments, the final mixture was directly analyzed using NMI 20-CA (nuclear magnetic resonance imaging analyzer) and fluorescence spectrometer F96PRO (gain set to 10), where the Carr-Purcell-Meiboom-Gill sequence pulse sequence was used for T2 measurement, the nuclear magnetic resonance (NMR) frequency was 18 MHz, the repetition time was 7,000 ms, the echo time was 1.7 ms, the preamplifier gain was 3, the number of echoes was 2,000, and the sampling frequency was 100 KHz. The relationship between the concentration of aflatoxin B1 and ΔT2 (ΔT2 = Ttargets-T0, where Ttargets and T0 represent the time when transverse relaxation is present and absent of aflatoxin B1, respectively) and the FL (fluorescence) value are used for the quantitative analysis of aflatoxin B1.

[0086] 1. Detection of aflatoxin B1 using dual-mode MR-FL biosensor

[0087] 500 μL of Ab-UCNPs (concentration of 1.1 mg / mL), 500 μL of Ag-Fe3O4@PCN-224 (concentration of 3.3186 mg / mL) and 100 μL of aflatoxin B1 standard solution of different concentrations (concentrations are: 0.011, 0.022, 0.055, 0.11, 0.22, 0.55, 1.1, 2.2, 5.5, 11, 22, 55, 110, 220, 550, 1100, 2200 and 5500 ng / mL) were mixed and the mixture was incubated at room temperature for 40 min;

[0088] 2. Specificity analysis

[0089] All metal ions (aflatoxin B1, Mg2+, Cu2+, Pd2+, Fe2+, Ag2+, Ba2+, K2+, Zn2+, and Na+) were diluted to 220 ng / mL, mixed with 500 μL Ab-UCNPs (concentration of 1.1 mg / mL), 500 μL Ag-202 Fe3O4@PCN-224 (concentration of 3.3 mg / mL), and 100 μL standard solutions of different metal ions, and incubated at room temperature for 40 min. The T2 signal and fluorescence intensity were detected from the final mixture, as shown in Figure 2 . Figure 4 As shown, 4a shows the relaxation time of aflatoxin B1 at different concentrations, 4b shows the fluorescence change of aflatoxin B1 at different concentrations, 4c shows the standard curve of aflatoxin B1 at different concentrations in MR mode, and 4d shows the standard curve of aflatoxin B1 at different concentrations in FL mode.

[0090] 3. Detection of aflatoxin B1 in tap water, red wine, eggs and soy milk samples

[0091] The eggs and soy milk were pretreated using the existing analytical pretreatment method, namely: 2% trichloroacetic acid and raw eggs (or soy milk) (10 mL, 2:8 v / v) were mixed in a sample tube in a centrifuge and ultrasonicated for 10 minutes; then, the mixture was centrifuged at 5,000 g for 5 minutes to remove the fat layer, and the filtrate was stored at 4°C for further detection;

[0092] Aflatoxin B1 (concentrations: 22, 55, and 110 ng / mL, respectively) was diluted to 2, 5, and 10 ng / mL using tap water, red wine, pretreated eggs, or pretreated soy milk; 500 μL of Ab-UCNPs (concentration: 1.1 mg / mL), 500 μL of Ag-Fe3O4@PCN-214 (concentration: 3.3 mg / mL), and 100 μL of the above diluted food matrix solution containing aflatoxin B1 were mixed and incubated at room temperature for 40 minutes;

[0093] The results showed that both the T2 signal and the fluorescence intensity of the final mixed solution could be detected.

[0094] 4. Sample analysis

[0095] In order to evaluate the practical feasibility of the dual-mode MR-FL biosensor, after simple sample pretreatment, aflatoxin B1 was detected in turbid and opaque samples (eggs and soy milk) and transparent or colored samples (tap water and red wine; Table 1). As shown in Table 1, Table 1 shows the results of the dual-mode MR-FL biosensor for detecting aflatoxin B1 in various samples. The results show that in transparent or colored samples, the detection of aflatoxin B1 by upconversion fluorescence and magnetic relaxation sensing has excellent sample recovery, among which the sample recovery in FL mode is: 98.82% to 107.81%, RSD (relative standard deviation) <5.74%; the sample recovery in MR mode is: 100.76% to 109.78%, RSD <5.5%; due to the advantage of upconversion nanoparticles in resisting background color interference, the upconversion fluorescence mode does not affect the recovery of aflatoxin B1 in colored samples. However, in turbid and opaque samples, the sample recovery rate of FL mode is lower than that of MR mode, among which the sample recovery rate in FL mode is: 77.64% to 94.03%, RSD<8.6%; the sample recovery rate in MR mode is: 99.49% to 104.04%, RSD<8.29%. The fundamental reason is that the sample has only undergone simple pretreatment, and the protein and lipid present in eggs and soy milk have not been completely removed, resulting in a slightly viscous and opaque matrix, which verifies the advantages of the dual-mode method of the biosensor; that is, the sensitivity mode of FL allows the detection of trace aflatoxin B1 and overcomes the interference of colored samples, while the magnetic relaxation sensor overcomes the interference of turbid and opaque samples. Therefore, the MR-FL biosensor improves the sensitivity and accuracy of aflatoxin B1 detection.

[0096] Table 1 Results of the dual-mode MR-FL biosensor for the detection of aflatoxin B1 in various samples

[0097]

[0098] In summary, the dual-mode biosensor with MR and FL dual signals for detecting aflatoxin B1 based on Fe3O4@PCN-224 and UCNPs provided by the present invention has excellent sensitivity and effectively overcomes the interference of colored samples. Among them, the porous structure of Fe3O4@PCN-224 can reduce the self-diffusion coefficient of water protons, produce enhanced lateral relaxation phenomenon, and construct an anti-interference detection MR mode that can realize slightly turbid and opaque samples. The biosensor can be applied to the detection of aflatoxin B1 in colored, slightly turbid and opaque samples. The synergistic effect enables the dual-mode biosensor to show excellent anti-interference and excellent sensitivity in detection in complex food matrices, which will promote the development of rapid food safety detection technology in the future.

[0099] The above is a detailed introduction to a dual-mode magnetic relaxation up-conversion fluorescent biosensor and its application provided by the present invention. Specific examples are used in this article to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the core idea of ​​the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A dual-mode magnetic relaxation upconversion fluorescence biosensor, characterized in that: The invention comprises an antigen-antibody immune recognition unit, functional magnetic composite nanoparticles and functional up-conversion fluorescent nanoparticles, wherein the functional magnetic composite nanoparticles and the functional up-conversion fluorescent nanoparticles are assembled through the antigen-antibody immune recognition unit to form a dual-mode magnetic relaxation up-conversion fluorescent biosensor for detecting aflatoxin B1 in food, wherein: The method for preparing the functional up-conversion fluorescent nanoparticles comprises the following steps: S11, synthesis of aqueous UCNPs using solvothermal method; S12, immobilizing aflatoxin B1-specific antibodies on the surface of aqueous UCNPs based on a covalent coupling method to prepare functional UCNPs; The method for preparing the functional magnetic composite nanoparticles comprises the following steps: S21, synthetic aqueous Fe3O4; S22, synthesis of Fe3O4@PCN-224; S23. Based on the covalent coupling method, aflatoxin B1-specific antigen was mixed with Fe3O4@PCN-224 to prepare functional Fe3O4@PCN-224.

2. The dual-mode magnetic relaxation upconversion fluorescence biosensor according to claim 1, characterized in that: The specific implementation of step S11 is: S111, adding 12 mL OA, 30 mL 1-ODE and 1 mmol rare earth chloride complex into a three-necked flask and mixing, wherein Y:Yb:Er in the rare earth chloride complex is 78:20:2; S112, heating the mixture from 123° C. to 160° C. in a nitrogen atmosphere and keeping the temperature until the mixture becomes a clear solution; S113, add 30 mL of methanol solution containing 3.25 mmol NaOH and 3.5 mmol NH4F dropwise to the mixture, cool to 80°C and stir for 30 min; S114, cooling to room temperature, adding ethanol and cyclohexane in a volume ratio of 1:1 to the mixture and centrifuging at 2000 g for 10 minutes; S115, washing the mixture with ethanol and water to disperse the OA-UCNPs in chloroform, the washing times being 2-3 times; S116. Take a certain amount of UCNPs-chloroform mixed solution and add it to a flask, then slowly add 20 ml of ethanol solution containing 300 mg PAA, and stir the solution at room temperature for 24 hours. After centrifugation, aqueous phase UCNPs can be obtained.

3. The dual-mode magnetic relaxation upconversion fluorescence biosensor according to claim 2, characterized in that: The specific implementation of step S12 includes: S121, disperse the aqueous UCNPs in PB buffer, add 200ul of EDC with a concentration of 10mg / mL and 120ul of NHS with a concentration of 10mg / mL to mix, and keep the reaction at room temperature for 30min, wherein the concentration of PB buffer is 0.01mM and the pH value is 7.4; S122. The mixed reaction solution was rinsed with water and centrifuged at 2000g for 8 minutes, and then aflatoxin B1-specific antibody was added and mixed for 8 hours to obtain functional UCNPs, wherein the number of rinses was 2-3 times, the concentration of aflatoxin B1-specific antibody was 6ug / mL, PB was 0.01mM, and the pH value was 7.

4.

4. The dual-mode magnetic relaxation upconversion fluorescence biosensor according to claim 3, characterized in that: The specific implementation of step S21 includes: S211, 2 mmol Fe(acac) 3 was mixed with 10 mmol 1,2-hexadecanediol, 6 mmol oleic acid and 6 mmol oleylamine in 20 mL phenyl ether, and magnetically stirred under nitrogen flow; S212, heating the mixture to 200° C. and maintaining for 30 minutes, then heating to 265° C. and refluxing for 30 minutes under a nitrogen flow; S213, cooling the mixture to room temperature, precipitating a dark brown substance from the mixture using 40 mL of ethanol under air atmosphere, and centrifuging at 3500 g for 10 min; S214, re-precipitating the mixture after the centrifugation treatment with ethanol and re-dispersing it in hexane to generate oil-phase Fe3O4 nanoparticles; S215, dissolving 10 mg of oil-phase Fe3O4 nanoparticles in 5 mL of toluene, then adding 5 mL of methanol containing 50 mg of DMSA and mixing, and leaving the mixture at room temperature for 24 h; S216. Allow the mixture to stand at 30°C and vacuum dry the precipitate.

5. The dual-mode magnetic relaxation upconversion fluorescence biosensor according to claim 4, characterized in that: The specific implementation of step S22 includes: S221, 100 mg 0.13 mmol H2TCPP, 300 mg 0.93 mmol ZrOCl2·8H2O and 3.3 g 27 mmol benzoic acid were dissolved in 100 mL DMF, and the mixture was stirred at 95°C for 5 hours; S222, centrifuging the stirred mixture at 9500 g for 15 min to collect PCN-224 nanoparticles, and washing the collected PCN-224 nanoparticles with DMF; S223, ultrasonically disperse PCN-224 in 2.5 mL of ethylene glycol and preheat at 120 °C for 10 min, then add 3 mL of 15 mg / mL polyvinyl pyrrolidone and 5 mL of 1 mg / mL Fe3O4 aqueous solution dropwise and stir vigorously for 16 min; S224. The mixture obtained after vigorous stirring was kept at 120° C. for 10 minutes and then cooled to room temperature. The cooled mixture was centrifuged and finally washed with water and ethanol several times to obtain Fe3O4@PCN-224.

6. The dual-mode magnetic relaxation upconversion fluorescence biosensor according to claim 5, characterized in that: The specific implementation of step S23 includes: S231, dispersing 5 mL of Fe3O4@PCN-224 with a concentration of 3.3 mg / mL in PB buffer, wherein the concentration of PB buffer is 0.01 mM and the pH value is 7.4; S232, adding 400 μL of 10 mg / mL EDC and 240 μL of 10 mg / mL NHS to the Fe3O3 mixture, and keeping the reaction at room temperature for 30 min; S233, washing the mixed solution after the reaction, and centrifuging it at 180° C. for 5 min, and the washing times are 2-3 times; S234. Add 2 mL of 16 ug / mL aflatoxin B1-specific antigen to the mixed solution after centrifugation and mix for 8 hours to obtain functional Fe3O4@PCN-224, wherein the PB concentration of aflatoxin B1-specific antigen is 0.01 mM and the pH value is 7.

4.

7. An application of a dual-mode magnetic relaxation upconversion fluorescence biosensor, characterized in that: The dual-mode magnetic relaxation up-conversion fluorescence biosensor according to any one of claims 1 to 6 is applied to the detection of aflatoxin B1 in food.

Images