CN115575638A - Magnetic relaxation/fluorescence dual-mode sensor and preparation method and application thereof - Google Patents
Magnetic relaxation/fluorescence dual-mode sensor and preparation method and application thereof Download PDFInfo
- Publication number
- CN115575638A CN115575638A CN202211104310.9A CN202211104310A CN115575638A CN 115575638 A CN115575638 A CN 115575638A CN 202211104310 A CN202211104310 A CN 202211104310A CN 115575638 A CN115575638 A CN 115575638A
- Authority
- CN
- China
- Prior art keywords
- afb
- solution
- aflatoxin
- mabs
- pabs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Nanotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a magnetic relaxation/fluorescence dual-mode sensor and a preparation method and application thereof, belonging to the technical field of new functional materials and biosensing detection. The invention firstly synthesizes Gd 2 O 3 :Eu 3+ Nano-particles, amino-functionalization of which is carried out and then signal probe Gd is further synthesized 2 O 3 :Eu 3+ ‑AFB 1 -PAbs; secondly, a magnetic separation probe MB-AFB is synthesized 1 mAbs, synthesis of the two probes into a sandwich-structured dual-mode sensor, with target aflatoxin B in the middle 1 . The inventionThe prepared magnetic relaxation/fluorescence dual-mode immunosensor realizes the effect on aflatoxin B 1 Compared with other analysis technologies, the dual-mode immunosensor prepared by the invention has the advantages of higher accuracy, good sensitivity and double selection of signal output modes, and provides a new idea for detecting pollutants in food.
Description
Technical Field
The invention belongs to the technical field of new functional materials and biosensing detection, and particularly relates to a magnetic relaxation/fluorescence dual-mode sensor and a preparation method and application thereof.
Background
Aflatoxins (afflatoxins, afs) are a toxic class of secondary metabolites produced by aspergillus flavus, aspergillus knowae and aspergillus parasiticus, which fungi grow in various foods and cereals and pose a serious threat to food safety. Wherein, the aflatoxin B 1 （AFB 1 ) The most toxic, one of the most dangerous and deadly toxins. Numerous studies have shown that ingestion of AFB 1 Then, the human and the animal suffer from diseases such as liver cancer, kidney cancer and the like, so that the human and the animal are classified as carcinogenic substances by the international cancer research organization. AFB 1 The heat stability of the food is high, and the structure of the food cannot be damaged by normal processing means, so that the food is easy to enter a human body through a food chain and harm the health of human beings. AFB in cereals and products thereof, as defined in food safety standards 1 The maximum allowable limit of (2) is 20. Mu.g/kg. Therefore, it is necessary to develop a convenient and fast detection method with high sensitivity and accuracy.
At present, in AFB 1 The liquid chromatography-tandem mass spectrometry (LC MS/MS) based chromatography and the combined immunoadsorption test (ELISA) in the detection method are widely applied due to the advantages of higher accuracy, sensitivity and the like. However, LC MS/MS requires sophisticated instruments, complicated pre-treatment and skilled personnel, and ELISA methods using enzyme-based signal labels lack stability, thus preventing their application in rapid detection of actual samples. The fluorescence sensing method is a spectral analysis method developed in recent years, is widely applied to food safety detection due to the advantages of high sensitivity, simple experiment operation, high selectivity, low cost and the like, and is an ideal toxin quantitative detection method by utilizing a fluorescence immunoadsorption experiment method of a fluorescent material as a signal marker, so that the defects of the traditional enzyme-linked immunoassay method are overcome, and rapid development is achieved in recent years. Magnetic Relaxation Sensors (MRS) are based on the state of magnetic nanoparticles in aqueous solutionOr change in concentration, causing a longitudinal relaxation time (T) of the protons of the surrounding water 1 ) And transverse relaxation time (T) 2 ) The change occurs and can be used to identify and quantify various target analytes. Compared to fluorescence methods, MRS based assays are not affected by light scattering etc. and do not require laborious pre-treatment and purification, MRS methods do not affect the sensitivity of detection without pre-treatment since most biological and environmental samples have inherently lower magnetic field backgrounds. Although the two methods have the advantages of rapidness, sensitivity and simple operation on the detection of aflatoxin, a single detection mode is easily influenced by the test environment. In contrast, the dual-mode analysis strategy can reduce the influence of the detection environment and other interferences, thereby ensuring the accuracy of aflatoxin detection.
Xu and the like use functional MIL-88A to replace natural enzyme to catalyze a color development system and establish a sensitive detection system for aflatoxin B 1 The indirect competitive MOF-linked immunosorbent assay (MOFLISA) method of (1). Although the nano enzyme prepared by the method is more stable than natural enzyme, the nano enzyme has the defects of low catalytic efficiency and low detection sensitivity compared with the natural enzyme, and has the risk of false positive in low-concentration detection. Li establishes a quantum dot nanoparticle-based fluorescence immunoadsorption experiment (QBs-FLISA), cdSe/ZnS quantum dots are respectively coupled with two toxin antibodies to prepare fluorescent probes, and the fluorescent probes are successfully applied to aflatoxin B 1 (AFB 1 ) And Zearalenone (ZEN). Compared with the traditional enzyme-linked immunosorbent assay, the method greatly improves the detection sensitivity, but the method needs a complex pretreatment process and possibly has a fluorescence interference phenomenon to influence the detection accuracy. Chen et al are based on MB 250 And MB 30 The large difference in magnetic response suggests a new MRS sensing strategy in which MB is present 250 And MB 30 All capable of specifically recognizing a target to form an immune complex by MB 250 The magnetic response of (2) to separate the immune complexes and use of unreacted MB 30 Measured T 2 The values were read as signals and successfully applied to the detection of E.coli. But the methodBy using MB 30 As T 2 Signal output probe, which itself has weak magnetism, may possibly interact with MB 250 The phenomenon of nonspecific binding generated by agglomeration influences the accuracy of the detection result. The reported methods are all single detection modes and are easily influenced by the test environment, so that the detection accuracy is influenced. Therefore, it is important to construct a simple, fast, sensitive, and highly accurate dual-mode immunosensor for toxin detection.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention provides a magnetic relaxation/fluorescence dual-mode sensor, and a preparation method and an application thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a Gd-based material 2 O 3 :Eu 3+ The preparation method of the nanoparticle signal probe comprises the following steps:
1) Gd is synthesized by adopting a hydrothermal method 2 O 3 :Eu 3+ A nanoparticle;
2) For Gd 2 O 3 :Eu 3+ The nanoparticles are subjected to amino functionalization to obtain NH 2 -Gd 2 O 3 :Eu 3+ ；
3) Subjecting aflatoxin B 1 Polyclonal antibody AFB of (1) 1 Mixing the PAbs with PBS, adding PBS solution containing EDC and NHS and PBS solution containing NH 2 -Gd 2 O 3 :Eu 3+ And incubating to obtain the Gd-based solution 2 O 3 :Eu 3+ Nanoparticle signaling probe Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs。
Further, synthesizing Gd by a hydrothermal method in the step 1) 2 O 3 :Eu 3+ The specific process of the nano-particles is as follows: gd (NO) 3 ) 3 And Eu (NO) 3 ) 3 Adding the solution into a glycol solution; adding polyvinylpyrrolidone (PVP K30) into the solution in several times, heating while stirring until the polyvinylpyrrolidone is completely dissolved to obtain a mixed solution; adding dropwise a solution of ethanol containing thiourea to the mixtureStirring the mixed solution until the solution is light yellow, adding a NaOH solution, and stirring; transferring the obtained solution into a reaction kettle with a polytetrafluoroethylene lining, and heating at 200 ℃; after the reaction is finished, cooling the solution to room temperature, centrifuging to obtain precipitate, washing and drying the obtained precipitate, and calcining at 600 ℃ in air atmosphere to obtain the Gd 2 O 3 :Eu 3+ And (3) nanoparticles.
Further, gd is treated in the step 2) 2 O 3 :Eu 3+ The specific method for carrying out amino functionalization on the nano particles comprises the following steps: dissolving the nano particles in a mixed solution of water and ethanol, adding ammonia water while stirring, then adding TEOS (tetraethoxysilane) in batches, and washing after stirring; dissolving the obtained product in ethanol solution, adding ammonia water and APTES (aminopropyltriethoxysilane), stirring, washing to obtain amino-functionalized Gd 2 O 3 :Eu 3+ Nanoparticle NH 2 -Gd 2 O 3 :Eu 3+ 。
The polyvinylpyrrolidone (PVP K30) is added in batches because the PVP K30 is difficult to dissolve in the mixed solution, and the PVP K30 can be dissolved and uniformly mixed in the solution by adding in batches; the batch addition of TEOS is for better Gd 2 O 3 :Eu 3+ The surface is silanized to form a silicon dioxide layer.
Further, the mass-to-volume ratio of the nanoparticles to water and ethanol is as follows: 1 mg:2 mL:1 mL.
Further, the AFB in step 3) 1 PAbs, PBS containing EDC and NHS and PBS containing NH 2 -Gd 2 O 3 :Eu 3 + The volume ratio of the PBS solution of (1) is: 1:49:10:40.
The invention also provides a signal probe Gd prepared according to the preparation method of the signal probe 2 O 3 :Eu 3 + -AFB 1 -PAbs。
The invention also provides a magnetic separation probe MB-AFB 1 -a method of producing mAbs comprising the steps of: subjecting aflatoxin B 1 Monoclonal antibody AFB of 1 -mAbs were mixed with PBS followed by addition of a PBS solution containing EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide) and NHS (chemical substance of N-hydroxythiosuccinimide) and a PBS solution containing MB-NH 2 Incubating to obtain the magnetic separation probe MB-AFB 1 -mAbs。
Further, the AFB 1 mAbs, PBS containing EDC and NHS and PBS containing MB-NH 2 The volume ratio of the PBS solution of (1) is: 1:49:10:40.
The invention also provides the magnetic separation probe MB-AFB 1 Magnetic separation probe MB-AFB prepared by preparation method of-mAbs 1 -mAbs。
The invention also provides a Gd-based material 2 O 3 :Eu 3+ The magnetic relaxation/fluorescence dual-mode sensor of the nano particles is of a sandwich structure, and one end of the dual-mode sensor is provided with the magnetic separation probe MB-AFB prepared by the method 1 mAbs and the other end of the signal probe Gd prepared as described above 2 O 3 :Eu 3+ -AFB 1 PAbs with target aflatoxin B in the middle 1 。
The invention also provides the Gd-based 2 O 3 :Eu 3+ The preparation method of the magnetic relaxation/fluorescence dual-mode sensor of the nano particles comprises the following steps: a signal probe Gd 2 O 3 :Eu 3+ -AFB 1 PAbs and magnetic separation Probe MB-AFB 1 -mAbs mixing and then adding the target aflatoxin B 1 Incubating the solution to obtain the Gd-based peptide 2 O 3 :Eu 3+ Nanoparticle magnetic relaxation/fluorescence dual mode sensors.
The invention also provides the Gd-based 2 O 3 :Eu 3+ Magnetic relaxation/fluorescence dual-mode sensor of nanoparticles for detecting aflatoxin B 1 The use of (1).
Further, the dual-mode sensor is used for detecting aflatoxin B 1 The specific detection method in the application comprises the following steps:
taking out the Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs and MB-AFB 1 -mAbs mixing and then adding aflatoxin B at various concentrations 1 Incubating the solution, separating, taking out supernatant, and measuring fluorescence intensity and transverse relaxation time T of each supernatant 2 Obtaining the fluorescence intensity/T 2 Value and aflatoxin B 1 Linear relationship between solution concentrations;
taking Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs and MB-AFB 1 -mAbs are mixed and then aflatoxin B is added 1 Incubating the solution to be detected, separating, taking out supernatant, and detecting the fluorescence intensity and transverse relaxation time T in the supernatant 2 According to said fluorescence intensity/T 2 Value and aflatoxin B 1 Obtaining the linear relation between the solution concentrations to obtain the aflatoxin B in the solution to be detected 1 The concentration of (c).
Further, the Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs、MB-AFB 1 mAbs and varying concentrations of Aflatoxin B 1 The solution is mixed according to the volume ratio of 2; the Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs、MB-AFB 1 mAbs and Aflatoxin B 1 The solution to be tested is mixed according to a volume ratio of 2.
Further, the incubation temperature is 37 ℃; the separation method is magnetic separation.
Gadolinium oxide (Gd) 2 O 3 ) Is an easily obtained substrate material, and not only has excellent fluorescence performance, but also has magnetic performance. Gd (Gd) 2 O 3 As a high-quality fluorescent matrix material, the fluorescent material not only has a narrow emission band, but also has good chemical stability and light stability. And, gd 2 O 3 Middle Gd 3+ Radius of (1) and activator Eu 3+ 、Tb 3+ The equal radii are similar, and the rare earth luminescent ions are easily doped, so the rare earth doped Gd 2 O 3 The fluorescent material can improve the detection sensitivity. In addition, gd 2 O 3 Having paramagnetism, gd of the surface thereof 3+ Interact with water to produceA randomly changed local micro magnetic field for shortening transverse relaxation time T of hydrogen protons in water 2 Often as contrast agents for MR imaging.
Gd synthesized by hydrothermal method 2 O 3 :Eu 3+ Nanoparticles emitting red light under ultraviolet lamp irradiation at 260 nm wavelength and Gd in aqueous solution 2 O 3 :Eu 3+ The change in concentration can cause T 2 Change in value, thus synthesized Gd 2 O 3 :Eu 3+ The nanoparticles have dual signals of magnetic relaxation and fluorescence. The magnetic iron oxide nanoparticles (MB) are utilized to have high saturation magnetization and can be rapidly separated under an external magnetic field, and Gd 2 O 3 :Eu 3+ Not paramagnetic and not separated by the same magnetic field. Fixing the aflatoxin B on MB 1 The monoclonal antibodies (MAbs) of (1) as magnetic separation capture probes and Gd 2 O 3 :Eu 3+ Labeling aflatoxins B 1 The polyclonal antibodies (PAbs) of (1) are used as signal probes, and both the probes can specifically recognize the target AFB 1 Formation of a Sandwich-type immune Complex (MB-AFB) 1 -Gd 2 O 3 :Eu 3+ ）。
MB-AFB 1 -Gd 2 O 3 :Eu 3+ Can be removed rapidly by magnetic separation, using the remaining unreacted Gd 2 O 3 :Eu 3+ Probe to read T 2 Signal and fluorescence signal, T 2 The value and fluorescence intensity depend on unreacted Gd 2 O 3 :Eu 3+ The amount of (c). In the absence of the target, unreacted Gd is present due to the lack of an immune response 2 O 3 :Eu 3+ Excessive amount of T 2 The value is lower and the fluorescence intensity is higher. In contrast, introduction of the target initiates an immune response, resulting in Gd 2 O 3 :Eu 3+ Reduced amount, T 2 The value increases and the fluorescence intensity decreases.
Compared with the prior art, the invention has the following beneficial effects:
the invention is based on Gd 2 O 3 :Eu 3+ The nanometer particle prepares a magnetic relaxation/fluorescence double modeThe immunosensor has stronger specificity, sensitivity and accuracy, thereby realizing the aflatoxin B 1 High sensitivity detection of (2); compared with other analysis technologies, the dual-mode immunosensor prepared by the invention has the advantages of convenience in operation, higher accuracy, good sensitivity and dual selection of signal output modes; the invention provides a new idea for detecting the pollutants in the food, and has very important application value in the aspect of food safety detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is the Gd prepared in step (1) of example 1 2 O 3 :Eu 3+ SEM images of nanoparticles;
FIG. 2 is Gd in example 1 2 O 3 :Eu 3+ Fluorescence excitation and emission spectra of (a);
FIG. 3 is a graph showing the different concentrations of Gd in example 1 2 O 3 :Eu 3+ For solution T 2 The influence of the signal;
FIG. 4 is a diagram showing the Gd concentration in the fabrication process of the magnetic relaxation/fluorescence dual-mode immunosensor in example 2 2 O 3 :Eu 3+ -AFB 1 -effect of concentration of PAbs on detection sensitivity;
FIG. 5 is a graph showing the effect of incubation time on detection sensitivity during the preparation of the magnetic relaxation/fluorescence dual-mode immunosensor in example 3;
FIG. 6 is a graph showing fluorescence intensity measured in example 1 and aflatoxin B 1 Linear graph of relationship between solution concentration;
FIG. 7 shows T measured in example 1 2 Value and aflatoxin B 1 Linear graph of the relationship between solution concentrations.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
MB-NH described in the invention 2 、AFB 1 -PAbs and AFB 1 mAbs were purchased from Biotechnology, shanghai GmbH.
Example 1
(1) Synthesis of Gd 2 O 3 :Eu 3+ Nano-particles: while stirring, 1 mol/L Gd (NO) of 2.0 mL 3 ) 3 And 1.7 mL at 0.05 mol/L Eu (NO) 3 ) 3 The solution was slowly added to 25 mL in ethylene glycol, 2.0 g in polyvinylpyrrolidone (PVP K30) was slowly added to the above solution in 3 portions, stirred while heating until completely dissolved, an ethanol solution containing 0.11 g in thiourea was slowly added dropwise to the solution, stirred until the solution was pale yellow, 15 mL of 2 mol/L NaOH solution was slowly added, stirred for 30 min, the resulting solution was transferred to a 30 mL teflon lined reactor, and 2 h was heated at 200 ℃. After the reaction is finished, cooling the solution to room temperature, centrifuging to obtain a precipitate, washing with ethanol for 3 times, drying with a 60 ℃ oven, and calcining at 600 ℃ in an air atmosphere to obtain a final product Gd 2 h 2 O 3 :Eu 3+ The nanoparticles, SEM picture of FIG. 1, show Gd 2 O 3 :Eu 3+ The nanoparticles are in a columnar structure. 200 μ L of Gd dispersed in PBS at different concentrations (0.01 to 1 μ g/mL) 2 O 3 :Eu 3+ Measuring T of solution by NMR analyzer 2 Values (main instrument frequency: 19mhz, nech 15000, tw 20000 ms, ns 8), results are shown in fig. 3, different concentrations of Gd 2 O 3 :Eu 3+ Solutions with different T 2 The value is obtained.
(2) For Gd 2 O 3 :Eu 3+ Amino functionalization of the nanoparticles: gd prepared by the step (1) of 5 mg 2 O 3 :Eu 3+ Dissolving the nano particles in a mixed solution of 10 mL water and 5 mL ethanol, adding 800 mu L of ammonia water (25% w/w) under vigorous stirring, adding 200 mu L of TEOS every 20 min until the amount of the added TEOS is 600 mu L, stirring 2 h, washing with absolute ethanol for 3 times, dissolving the obtained product in 20 mL ethanol solution, adding 100 mu L of ammonia water (25% w/w), stirring for 10 min, adding 100 mu L of APTES, continuing stirring for 6 h, washing with absolute ethanol for 3 times after the reaction is finished to obtain amino functionalized Gd 2 O 3 :Eu 3+ Nanoparticle NH 2 -Gd 2 O 3 :Eu 3+ 。
(3) Signaling probe Gd 2 O 3 :Eu 3+ -AFB 1 Preparation of PAbs: 10.μ L (1 mg/mL) AFB 1 PAbs were mixed with 490. Mu.L PBS, 100. Mu.L PBS solution (0.01 mol/L, pH = 7.4) containing 50 mg/mL EDC and 25 mg/mL NHS was added, incubated at room temperature for 20 min with shaking to activate the carboxyl groups on the antibody, and then 400. Mu.L PBS solution containing 1mg NH 2 -Gd 2 O 3 :Eu 3+ The PBS of (3) was added to the mixed solution, and incubation with shaking was continued for 2 h, and after completion of the reaction, centrifugation was carried out at 7000 rpm for 5 min, followed by blocking of all non-specific binding sites 1 h with 1% BSA. After centrifugal washing, the product Gd 2 O 3 :Eu 3+ PAbs were redispersed in 1 mL PBS (0.01 mol/L, pH = 7.4) and stored at 4 ℃.
(4) Magnetic separation probe MB-AFB 1 Preparation of mAbs: 10 μ L (1 mg/mL) of AFB was taken 1 mAbs were mixed with 490. Mu.L PBS, 100. Mu.L PBS solution (0.01 mol/L, pH = 7.4) containing 50 mg/mL EDC and 25 mg/mL NHS was added, incubated at room temperature for 20 min with shaking to activate the carboxyl groups on the antibody, and then 400. Mu.L PBS solution containing 1mg MB-NH 2 Adding the PBS solution into the mixed solution, continuously oscillating and incubating the mixed solution for 2 h, and after the reaction is finished, magnetically separating and removing redundant AFB 1 -mAbs, followed by blocking with 1% BSA 1 h, magnetic separation washing the product MB-AFB 1 The mAbs were redispersed in 1 mL PBS (0.01 mol/L, pH = 7.4) and stored at 4 ℃.
(5) Collecting 200 μ L Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs（100 μg/mL），200 μL MB-AFB 1 mAbs (100. Mu.g/mL) were mixed and 100. Mu.L of 5 ng/mL AFB was added 1 The solution is incubated at 37 deg.C for 40 min, placed on a 0.42T magnetic separation rack for separation for 1 min, and the supernatant is taken out and placed in a new test tube for use.
(6) 200 μ L of the supernatant was taken in a microcuvette and the intensity of its emission peak at 609 nm, gd, was measured using a fluorescence spectrophotometer at an excitation wavelength of 260 nm 2 O 3 :Eu 3+ -AFB 1 Fluorescence excitation and emission spectra of PAbs are shown in FIG. 2. Taking 200 μ L of supernatant, and measuring T of the solution by NMR analyzer 2 Value (Main instrument frequency: 19MHz, NECH:15000, TW:20000 ms, NS: 8). By fluorescence intensity/T 2 Value and aflatoxin B 1 And (4) drawing a working curve according to the relation between the concentrations of the solutions. As shown in FIG. 6, the linear range of the fluorescence values was 0.02 to 5 ng/mL (y = -0.2159 lg (x) + 0.3926), and the lowest detection limit was 0.004 ng/mL. As in FIG. 7, by T 2 The linear range of the values was found to be 0.001 to 1 ng/mL (y =219.9456 lg (x) + 694.6999), with a minimum detection limit of 0.001 ng/mL.
Example 2
The steps (1) to (4) are the same as in example 1;
(5) For determining the Signaling Probe Gd 2 O 3 :Eu 3+ -AFB 1 Optimum concentration of PAbs, 200. Mu.L Gd concentration 50. Mu.g/mL, 100. Mu.g/mL, 500. Mu.g/mL, 1 mg/mL 2 O 3 :Eu 3+ -AFB 1 The PAbs solution was mixed with 200. Mu.L of 100. Mu.g/mL MB-AFB 1 mAbs were mixed and 100. Mu.L of AFB was added at different concentrations 1 (0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL), incubating at 37 ℃ for 40 min, placing on a 0.42T magnetic separation rack for separation for 1 min, taking out the supernatant, and placing in a new test tube for later use;
the experimental results are shown in FIG. 4 in the same step (6) as in example 1, and it can be seen from FIG. 4 that the signaling probe Gd is present in 2 O 3 :Eu 3+ -AFB 1 The optimal concentration of PAbs is 100. Mu.g/mL.
Example 3
The steps (1) to (4) are the same as in example 1;
(5) To determine the optimal incubation time, 200. Mu.L Gd was taken 2 O 3 :Eu 3+ -AFB 1 -PAbs（100 μg/mL），200 μL MB-AFB 1 mAbs (100. Mu.g/mL) were mixed and 100. Mu.L of different concentrations of AFB were added separately 1 The solution is incubated at 37 deg.C for 20 min, 40 min, 60 min and 120 min respectively.
The experiment result is shown in FIG. 5 in step (6) and example 1, and it can be understood from FIG. 5 that the optimal incubation time is 40 min.
Example 4
The steps (1) to (6) are the same as in example 1;
(7) Preparing aflatoxin B with concentration of 0.5 ng/mL 1 Respectively taking 200 mu L of the aflatoxin B, placing 200 mu L of the aflatoxin B in a microcuvette, measuring the emission peak intensity of the aflatoxin B at 609 nm by using a fluorescence spectrophotometer at an excitation wavelength of 260 nm to obtain the corresponding fluorescence intensity, substituting the obtained fluorescence intensity into the linear equation, namely y = -0.2159 lg (x) +0.3926 to obtain the aflatoxin B 1 The concentration value of (A) is 0.53 ng/mL; then 200 mul of the prepared aflatoxin B is taken respectively 1 Measuring the T of the solution by means of a NMR analyzer 2 Values (master instrument frequency: 19MHz, NECH 15000, TW 20000 ms, NS 2 The value is substituted into the linear equation y =219.9456 lg (x) +694.6999 to obtain the aflatoxin B 1 The concentration value of (A) is 0.52 ng/mL, and the results obtained by the two methods are close to the actual addition amount, and the two methods can be used for treating aflatoxin B 1 The content of (b) is detected.
The above description is only for the preferred embodiment of the present invention, and the protection scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention, the technical solution and the inventive concept of the present invention equivalent or change within the technical scope of the present invention.
Claims (10)
1. Gd-based 2 O 3 :Eu 3+ The preparation method of the signal probe of the nano-particles is characterized by comprising the following steps:
1) Gd is synthesized by adopting a hydrothermal method 2 O 3 :Eu 3+ A nanoparticle;
2) For Gd 2 O 3 :Eu 3+ The nanoparticles are subjected to amino functionalization to obtain NH 2 -Gd 2 O 3 :Eu 3+ ；
3) Subjecting aflatoxin B 1 Polyclonal antibody AFB of (1) 1 Mixing the PAbs with PBS, adding PBS solution containing EDC and NHS and PBS solution containing NH 2 -Gd 2 O 3 :Eu 3+ In PBSIncubating to obtain the Gd-based 2 O 3 :Eu 3+ Nanoparticle signaling probe Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs。
2. The Gd-based according to claim 1 2 O 3 :Eu 3+ The preparation method of the signal probe of the nano-particles is characterized in that Gd is synthesized by a hydrothermal method 2 O 3 :Eu 3+ The specific process of the nano-particles is as follows: gd (NO) 3 ) 3 And Eu (NO) 3 ) 3 Adding the solution into a glycol solution; then adding polyvinylpyrrolidone, heating and stirring until the polyvinylpyrrolidone is completely dissolved to obtain a mixed solution; dropwise adding an ethanol solution containing thiourea into the mixed solution, stirring until the solution is light yellow, adding a NaOH solution, and stirring; then heating, after the reaction is finished, cooling the solution to room temperature, centrifuging, washing and drying the obtained precipitate, and then calcining in air atmosphere to obtain the Gd 2 O 3 :Eu 3+ A nanoparticle;
the pair of Gd 2 O 3 :Eu 3+ The method for carrying out amino functionalization on the nano particles comprises the following steps: dissolving the nano particles in a mixed solution of water and ethanol, adding ammonia water under stirring, then adding TEOS, washing the obtained product after reaction, dissolving the product in the ethanol solution, adding ammonia water and APTES again, stirring, and washing again after the reaction is finished to obtain amino functionalized Gd 2 O 3 :Eu 3+ Nanoparticle NH 2 -Gd 2 O 3 :Eu 3+ 。
3. A Gd-based composition according to claim 1 or 2 2 O 3 :Eu 3+ Preparation method of nanoparticle signal probe Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs。
4. Magnetic separation probe MB-AFB 1 -a method for the production of mAbs, characterized in that it comprises the following steps: yellow wineAspergillus toxin B 1 Monoclonal antibody AFB of (1) 1 -mAbs are mixed with PBS and then PBS solution containing EDC and NHS and PBS solution containing MB-NH are added 2 Incubating to obtain the magnetic separation probe MB-AFB 1 -mAbs。
5. A magnetic separation probe MB-AFB as claimed in claim 4 1 Magnetic separation probe MB-AFB prepared by preparation method of-mAbs 1 -mAbs。
6. Gd-based 2 O 3 :Eu 3+ Dual mode sensor of magnetic relaxation/fluorescence of nanoparticles, characterized in that it is of sandwich structure with one end the magnetic separation probe MB-AFB of claim 5 1 -mAbs and at the other end the signaling probe Gd of claim 3 2 O 3 :Eu 3+ -AFB 1 -PAbs, intermediate target aflatoxin B 1 。
7. The Gd-based of claim 6 2 O 3 :Eu 3+ The preparation method of the magnetic relaxation/fluorescence dual-mode sensor of the nano particles is characterized by comprising the following steps: the signaling probe Gd of claim 3 2 O 3 :Eu 3+ -AFB 1 -PAbs and the magnetic separation probe MB-AFB of claim 5 1 -mAbs mixing and then adding the target aflatoxin B 1 Incubating the solution to obtain the Gd-based peptide 2 O 3 :Eu 3+ Nanoparticle magnetic relaxation/fluorescence dual mode sensors.
8. A Gd-based formulation according to claim 6 2 O 3 :Eu 3+ Magnetic relaxation/fluorescence dual-mode sensor of nanoparticles for detecting aflatoxin B 1 The use of (1).
9. Use according to claim 8, characterized in that the detection method comprises the following steps:
get theGd 2 O 3 :Eu 3+ -AFB 1 -PAbs and said MB-AFB 1 -mAbs mixing and then adding aflatoxin B at various concentrations 1 Incubating, separating, taking out supernatant, and measuring fluorescence intensity and transverse relaxation time T of each supernatant 2 Obtaining the fluorescence intensity/T 2 Value and aflatoxin B 1 Linear relationship between solution concentrations;
taking Gd 2 O 3 :Eu 3+ -AFB 1 -PAbs and MB-AFB 1 -mAbs are mixed and then aflatoxin B is added 1 Incubating the solution to be detected, separating, taking out supernatant, and detecting the fluorescence intensity and transverse relaxation time T in the supernatant 2 According to said fluorescence intensity/T 2 Value and aflatoxin B 1 Obtaining the linear relation between the solution concentrations to obtain the aflatoxin B in the solution to be detected 1 The concentration of (c).
10. The use according to claim 9, wherein said Gd is 2 O 3 :Eu 3+ -AFB 1 -PAbs and MB-AFB 1 -mAbs mixed in a volume ratio of 1:1; the incubation temperature is 37 ℃; the separation method is magnetic separation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211104310.9A CN115575638A (en) | 2022-09-09 | 2022-09-09 | Magnetic relaxation/fluorescence dual-mode sensor and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211104310.9A CN115575638A (en) | 2022-09-09 | 2022-09-09 | Magnetic relaxation/fluorescence dual-mode sensor and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115575638A true CN115575638A (en) | 2023-01-06 |
Family
ID=84580873
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211104310.9A Pending CN115575638A (en) | 2022-09-09 | 2022-09-09 | Magnetic relaxation/fluorescence dual-mode sensor and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115575638A (en) |
-
2022
- 2022-09-09 CN CN202211104310.9A patent/CN115575638A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Magnetic nanobead-based immunoassay for the simultaneous detection of aflatoxin B1 and ochratoxin A using upconversion nanoparticles as multicolor labels | |
| CN104280542B (en) | Double; two enhanced chemiluminescence immunoassays that and nanometer particle to mark luminous based on Reinforced by Metal amplifies | |
| Cui et al. | Homogeneous fluorescence-based immunoassay via inner filter effect of gold nanoparticles on fluorescence of CdTe quantum dots | |
| Liu et al. | Highly sensitive protein detection using enzyme-labeled gold nanoparticle probes | |
| Liao et al. | An ultrasensitive ELISA method for the detection of procalcitonin based on magnetic beads and enzyme-antibody labeled gold nanoparticles | |
| Wang et al. | Hue recognition competitive fluorescent lateral flow immunoassay for aflatoxin M1 detection with improved visual and quantitative performance | |
| Zhao et al. | Ultrasensitive dual-enhanced sandwich strategy for simultaneous detection of Escherichia coli and Staphylococcus aureus based on optimized aptamers-functionalized magnetic capture probes and graphene oxide-Au nanostars SERS tags | |
| Agoston et al. | Rapid isolation and detection of erythropoietin in blood plasma by magnetic core gold nanoparticles and portable Raman spectroscopy | |
| Xiong et al. | Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies | |
| KR20100107648A (en) | Combinational surface-enhanced raman scattering probe and method for detecting target substance by using the same | |
| CN114354591B (en) | Colorimetric/fluorescent double-mode biosensing detection method for rapidly detecting aflatoxin B1 | |
| Liu et al. | Upconversion nanoparticle as elemental tag for the determination of alpha-fetoprotein in human serum by inductively coupled plasma mass spectrometry | |
| Ding et al. | A SERS-based competitive immunoassay for highly sensitive and specific detection of ochratoxin A | |
| Tang et al. | Magnetic bead-based fluorescence immunoassay for aflatoxin B 1 in food using biofunctionalized rhodamine B-doped silica nanoparticles | |
| Fu et al. | Magnetic and fluorescent nanohybrids with surface imprinting silica as a dual-functional sensing platform for ratiometric fluorescence detection of phycoerythrin | |
| Hong et al. | A universal, portable, and ultra-sensitive pipet immunoassay platform for deoxynivalenol detection based on dopamine self-polymerization-mediated bioconjugation and signal amplification | |
| Li et al. | Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples | |
| Li et al. | Bioconjugated fluorescent zeolite L nanocrystals as labels in protein microarrays | |
| CN112014374A (en) | Surface-enhanced Raman immunoassay planar sensor and preparation method and application thereof | |
| Chen et al. | Cerium ions triggered dual-readout immunoassay based on aggregation induced emission effect and 3, 3′, 5, 5′-tetramethylbenzidine for fluorescent and colorimetric detection of ochratoxin A | |
| Hou et al. | Magnetic particle-based time-resolved fluoroimmunoassay for the simultaneous determination of α-fetoprotein and the free β-subunit of human chorionic gonadotropin | |
| Zhang et al. | A novel colorimetric immunoassay strategy using iron (iii) oxide magnetic nanoparticles as a label for signal generation and amplification | |
| Adeniyi et al. | Ultrasensitive detection of anti-p53 autoantibodies based on nanomagnetic capture and separation with fluorescent sensing nanobioprobe for signal amplification | |
| Li et al. | A highly sensitive immunofluorescence sensor based on bicolor upconversion and magnetic separation for simultaneous detection of fumonisin B1 and zearalenone | |
| Fan et al. | Precisely designed growth of dual-color quantum dots bilayer nanobeads for ratiometric fluorescent immunoassay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |