CN110470827B - Preparation method of resonance energy transfer nano structure based on ferritin - Google Patents
Preparation method of resonance energy transfer nano structure based on ferritin Download PDFInfo
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- CN110470827B CN110470827B CN201910787982.6A CN201910787982A CN110470827B CN 110470827 B CN110470827 B CN 110470827B CN 201910787982 A CN201910787982 A CN 201910787982A CN 110470827 B CN110470827 B CN 110470827B
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- 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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
- G01N21/763—Bioluminescence
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- 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
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- 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
- G01N33/531—Production of immunochemical test materials
Abstract
The invention relates to a preparation method of a resonance energy transfer nano structure based on ferritin, belonging to the field of novel nano materials; the invention utilizes the characteristic of controlling the dissociation/recombination of ferritin by pH and realizes the tri (2-phenylpyridine) iridium Ir (ppy) by adopting a three-step method for the first time 3 The molecule and the nano-gold Au NPs are uniformly embedded and assembled on the surface of the ferritin to prepare a composite material formed by Ir (ppy) 3 Ir (ppy) with resonance energy transfer characteristics for energy donor and Au NPs as energy acceptor 3 -ferritin-Au three-dimensional hybrid nanostructures; since Ir (ppy) 3 The ECL excitation spectrum has good spectrum overlap with the ultraviolet absorption spectrum of Au NPs, and the distance between the ECL excitation spectrum and the ultraviolet absorption spectrum on the surface of ferritin is less than 10 nm, so that efficient ECL-RET occurs between the ECL excitation spectrum and the Au NPs; the invention establishes an Ir (ppy) based method on the ferritin surface for the first time 3 A new research model of resonance energy transfer with Au NPs provides a new idea for the theoretical research of ECL-RET.
Description
Technical Field
The invention belongs to the field of novel nano materials.
Background
Electrochemiluminescence is a new sensing technology developed on the basis of chemiluminescence, is a product of mutual permeation of chemiluminescence and an electrochemical technology, and has the advantages of high sensitivity, low background noise, wide dynamic response range, strong controllability and the like. Electrochemiluminescence-resonance energy transfer (ECL-RET) refers to a process by which electron excitation energy is transferred from a donor to an acceptor in a non-radiative manner through dipole-dipole interactions when the ECL emission spectrum of the donor overlaps the absorption spectrum of the acceptor effectively. Compared with fluorescence resonance energy transfer, ECL-RET has the advantages of simple operation, low background signal interference, no need of excitation light source and the like. At present, ECL-RET has been widely used in the fields of nucleic acid hybridization analysis, immunoassay, cell analysis and detection, and the like. However, the establishment of the ECL-RET system does not leave open the proposition and application of new energy donor/acceptor pairs; the invention provides a new iridium complex Ir (ppy) prepared from tri (2-phenylpyridine) 3 The molecule is an energy donor, and the nano-Au NP is a novel ECL-RET energy donor/acceptor pair of an energy acceptor.
Ferritin is a soluble protein for storing iron, which widely exists in animals, plants and microorganisms, and the structure of ferritin comprises two parts: a protein moiety and an iron core moiety. Ferritin has excellent characteristics of pH-guided self-depolymerization and self-assembly, when the pH value is less than 3 or more than 10, the protein shell can be gradually dissociated into free fragments, however, when the pH value of the solution is adjusted to be restored to about 7.5, the free shell fragments can be self-assembled into a complete protein shell; the surface of the protein shell has a large number of functional groups such as amino, hydroxyl and the like, and a proper amount of nano gold particles and Ir (ppy) can be connected through gold-ammonia bonds or electrostatic adsorption 3 Molecule, therefore, when the external connection of nanogold and Ir (ppy) 3 The ferritin fragments of the molecules are uniformly mixed and the pH value of the ferritin fragments is adjusted to 7.5, so that a novel three-dimensional hybrid nanostructure with the outer surface combined with an energy supply/receptor can be obtained. Thus, ferritin is shown inFurther research and application in this field will further advance and develop the ECL-RET analysis.
Disclosure of Invention
The technical task of the invention is to make up the defects of the existing preparation method, and the preparation method is based on tris (2-phenylpyridine) iridium Ir (ppy) 3 The resonance energy transfer principle of nanogold is characterized in that ferritin is used as a carrier, a three-dimensional hybrid nanostructure integrating an energy donor and an energy acceptor is designed and synthesized on the surface of the ferritin, the distance between the energy donor and the energy acceptor is greatly shortened, the efficient transfer of the resonance energy on the surface of single ferritin is realized, and the method has obvious creativity.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
1. a preparation method of a resonance energy transfer nano-structure based on ferritin is characterized by comprising the following steps:
the first step is as follows: mixing 3-5 mL ferritin solution with concentration of 5 μ g/mL and 0.5-2.5 mL tris (2-phenylpyridine) iridium Ir (ppy) with concentration of 10 mmol/L 3 Mixing the solutions, adding 50-150 mu L of glutaraldehyde solution with the mass fraction of 50% as a cross-linking agent, continuously stirring the solution for 2 h, dialyzing, and purifying to remove redundant Ir (ppy) 3 Then, the resulting mixture was centrifuged and dispersed in 1 mL of a phosphate buffer solution having pH 7.4 to obtain ferritin-Ir (ppy) 3 Storing the solution at 4 ℃ for later use;
the second step: mixing 3-5 mL, a ferritin solution with the concentration of 5 mug/mL and 1.5-3.5 mL nano-gold Au NPs sol, adding 50-150 muL of glutaraldehyde solution with the mass fraction of 50% as a cross-linking agent, stirring 6 h away from light, dialyzing, purifying to remove redundant Au NPs, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain a ferritin-Au solution, and storing at 4 ℃ for later use;
the third step: mixing ferritin-Ir (ppy) 3 The solution is evenly stirred with ferritin-Au solution, and 0.1 is usedAdjusting the pH of the mixed solution to 2.5-4.0 by using mol/L HCl solution, and dissociating the protein shell of the ferritin into single fragments; continuously stirring 3 h until uniform, adjusting pH to 7.0-8.0 with 0.1 mol/L ammonia water solution, reassembling free ferritin coat segments into complete ferritin coat, and embedding Ir (ppy) on the surface of the coat segments 3 Uniformly assembling the nano-gold on the outer surface of single ferritin, dialyzing, and purifying to obtain Ir (ppy) 3 -three-dimensional hybrid nanostructure of ferritin-Au.
2. The method for preparing a ferritin-based resonance energy transfer nanostructure in accordance with claim 1, wherein the nanogold sol is prepared by the following steps:
measuring 4.12-6.12 mL of 1% HAuCl 4 Adding the solution and 100 mL ultrapure water into a clean and dry 250 mL three-neck round-bottom flask; slowly adding 10-30 mL sodium citrate solution dropwise under continuous stirring, heating and refluxing for 15-45 min, and stopping heating until the solution color finally becomes wine red; after cooling and filtration, the filtrate was finally obtained and stored at 4 ℃ until use.
Advantageous results of the invention
(1) The present invention utilizes a pH-directed protein depolymerization/recombination process based on Ir (ppy) 3 According to the resonance energy transfer principle of the nano-gold, the tris (2-phenylpyridine) iridium Ir (ppy) is embedded and assembled on the ferritin shell for the first time 3 The molecule and the nano-gold Au NPs obtain a novel three-dimensional hybrid nano-structure which is formed by Ir (ppy) 3 The nano-gold is used as an energy donor and an ECL energy acceptor, and is a nano-structure with high-efficiency resonance energy transfer efficiency.
(2) The invention provides a preparation method of a resonance energy transfer nanostructure, which is simple to operate and environment-friendly for the first time, solves the problems of complex operation, time and labor waste of the existing preparation method, has certain guiding significance for deep research of resonance energy transfer in ECL (electrochemiluminescence), and the prepared Ir (ppy) 3 ferritin-Au has obvious application value in electrochemiluminescence immunosensing analysis.
Detailed Description
The invention will now be further illustrated by reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.
Embodiment 1. A method for preparing a ferritin-based resonance energy transfer nanostructure, comprising the steps of:
the first step is as follows: mixing 3 mL ferritin solution with concentration of 5. Mu.g/mL and 0.5 mL iridium tris (2-phenylpyridine) Ir (ppy) with concentration of 10 mmol/L 3 Mixing the solutions, adding 50 μ L of 50% glutaraldehyde solution as crosslinking agent, stirring for 2 h, dialyzing, and purifying to remove excess Ir (ppy) 3 Then, the resulting mixture was centrifuged and dispersed in 1 mL of a phosphate buffer solution having pH 7.4 to obtain ferritin-Ir (ppy) 3 Storing the solution at 4 ℃ for later use;
the second step is that: mixing 3 mL, ferritin solution with concentration of 5 mug/mL and 1.5 mL nano-gold Au NPs sol, adding 50 muL glutaraldehyde solution with mass fraction of 50% as a cross-linking agent, stirring 6 h away from light, dialyzing, purifying to remove redundant Au NPs, centrifuging, dispersing into 1 mL phosphate buffer solution with pH of 7.4 to obtain ferritin-Au solution, and storing at 4 ℃ for later use;
the third step: mixing ferritin-Ir (ppy) 3 Uniformly stirring the solution and a ferritin-Au solution, adjusting the pH of the mixed solution to 2.5 by using 0.1 mol/L HCl solution, and dissociating the protein shell of ferritin into single fragments; stirring 3 h until uniform, adjusting pH to 7.0 with 0.1 mol/L ammonia water solution, wherein free ferritin coat fragments are reassembled into intact ferritin coat, and Ir (ppy) embedded on the surface of coat fragments 3 Can be uniformly assembled on the outer surface of single ferritin with nano gold, and Ir (ppy) can be obtained after dialysis and purification 3 -three-dimensional hybrid nanostructure of ferritin-Au.
Embodiment 2. A method for preparing a ferritin-based resonance energy transfer nanostructure, comprising the steps of:
the first step is as follows: 4 mL, 5 ug/mL in concentrationFerritin solution was mixed with 1.5 mL tris (2-phenylpyridine) iridium Ir (ppy) at a concentration of 10 mmol/L 3 Mixing the solutions, adding 100 μ L of 50% glutaraldehyde solution as crosslinking agent, stirring for 2 h, dialyzing, and purifying to remove excess Ir (ppy) 3 Then, the resulting mixture was centrifuged and dispersed in 1 mL of a phosphate buffer solution having pH 7.4 to obtain ferritin-Ir (ppy) 3 Storing the solution at 4 ℃ for later use;
the second step is that: mixing 4 mL, a ferritin solution with the concentration of 5 mug/mL and 2.5 mL nano-gold Au NPs sol, adding 100 muL of glutaraldehyde solution with the mass fraction of 50% as a cross-linking agent, stirring 6 h in a dark place, dialyzing, purifying to remove redundant Au NPs, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain a ferritin-Au solution, and storing at 4 ℃ for later use;
the third step: mixing ferritin-Ir (ppy) 3 Uniformly stirring the solution and a ferritin-Au solution, adjusting the pH of the mixed solution to 3.5 by using 0.1 mol/L HCl solution, and dissociating the protein shell of ferritin into single fragments; stirring 3 h until uniform, adjusting pH to 7.5 with 0.1 mol/L ammonia water solution, wherein free ferritin coat fragments are reassembled into intact ferritin coat, and Ir (ppy) embedded on the surface of coat fragments 3 Uniformly assembling the nano-gold on the outer surface of single ferritin, dialyzing, and purifying to obtain Ir (ppy) 3 -three-dimensional hybrid nanostructure of ferritin-Au.
Embodiment 3. A method for preparing a ferritin-based resonance energy transfer nanostructure, comprising the steps of:
the first step is as follows: 5 mL ferritin solution at a concentration of 5. Mu.g/mL was mixed with 2.5 mL tris (2-phenylpyridine) iridium Ir (ppy) at a concentration of 10 mmol/L 3 Mixing the solutions, adding 150 μ L of 50% glutaraldehyde solution as crosslinking agent, stirring for 2 h, dialyzing, and purifying to remove excess Ir (ppy) 3 Then, the mixture was centrifuged and dispersed in 1 mL of phosphate buffer pH 7.4 to obtain ferritin-Ir (ppy) 3 Storing the solution at 4 ℃ for later use;
the second step is that: mixing 5 mL, a ferritin solution with the concentration of 5 mug/mL and 3.5 mL nano-gold Au NPs sol, adding 150 muL of glutaraldehyde solution with the mass fraction of 50% as a cross-linking agent, stirring 6 h in a dark place, dialyzing, purifying to remove redundant Au NPs, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain a ferritin-Au solution, and storing at 4 ℃ for later use;
the third step: mixing ferritin-Ir (ppy) 3 Uniformly stirring the solution and a ferritin-Au solution, and adjusting the pH of the mixed solution to 4.0 by using 0.1 mol/L HCl solution, wherein the protein shell of ferritin is dissociated into single fragments; stirring 3 h to homogeneity, adjusting pH to 8.0 with 0.1 mol/L aqueous ammonia solution, wherein the free ferritin coat fragments can be reassembled into intact ferritin coat, and Ir (ppy) embedded on the surface of the coat fragments 3 Uniformly assembling the nano-gold on the outer surface of single ferritin, dialyzing, and purifying to obtain Ir (ppy) 3 -three-dimensional hybrid nanostructures of ferritin-Au.
Example 4. The nanogold sol was prepared according to the following steps:
4.12 mL of 1% HAuCl was measured 4 Adding the solution and 100 mL ultrapure water into a clean and dry 250 mL three-neck round-bottom flask; slowly adding 10 mL sodium citrate solution dropwise under stirring, heating and refluxing for 15 min, and stopping heating until the solution color is finally changed into wine red; after cooling and filtration, the filtrate was finally obtained and stored at 4 ℃ until use.
Example 5. The nanogold sol was prepared according to the following steps:
5.12 mL of 1% HAuCl was measured 4 Adding the solution and 100 mL ultrapure water into a clean and dry 250 mL three-neck round-bottom flask; slowly adding 20 mL sodium citrate solution dropwise under stirring, heating and refluxing for 30 min, and stopping heating until the solution color turns into wine red; after cooling and filtration, the filtrate was finally obtained and stored at 4 ℃ until use.
Example 6. The nanogold sol was prepared according to the following steps:
measured 12 mL of 1% HAuCl 4 The solution and 100 mL ultrapure water are added toClean dry 250 mL three neck round bottom flask; slowly adding 30 mL sodium citrate solution dropwise under stirring, heating and refluxing for 45 min, and stopping heating until the solution color is finally changed into wine red; after cooling and filtration, the filtrate was finally obtained and stored at 4 ℃ until use.
Claims (1)
1. A preparation method of a resonance energy transfer nano-structure based on ferritin is characterized by comprising the following steps:
the first step is as follows: mixing 3-5 mL ferritin solution with concentration of 5 μ g/mL and 0.5-2.5 mL tris (2-phenylpyridine) iridium Ir (ppy) with concentration of 10 mmol/L 3 Mixing the solutions, adding 50-150 mu L of glutaraldehyde solution with the mass fraction of 50% as a cross-linking agent, continuously stirring the solution for 2 h, dialyzing, and purifying to remove redundant Ir (ppy) 3 Then, the resulting mixture was centrifuged and dispersed in 1 mL of a phosphate buffer solution having pH 7.4 to obtain ferritin-Ir (ppy) 3 Storing the solution at 4 ℃ for later use;
the second step is that: mixing 3-5 mL, a ferritin solution with the concentration of 5 mug/mL and 1.5-3.5 mL nano-gold Au NPs sol, adding 50-150 muL of glutaraldehyde solution with the mass fraction of 50% as a cross-linking agent, stirring 6 h away from light, dialyzing, purifying to remove redundant Au NPs, centrifuging, dispersing into 1 mL of phosphate buffer solution with the pH of 7.4 to obtain a ferritin-Au solution, and storing at 4 ℃ for later use;
the third step: mixing ferritin-Ir (ppy) 3 Uniformly stirring the solution and a ferritin-Au solution, adjusting the pH of the mixed solution to 2.5-4.0 by using 0.1 mol/L HCl solution, and dissociating the ferritin protein shell into single fragments; continuously stirring 3 h until uniform, adjusting pH to 7.0-8.0 with 0.1 mol/L ammonia water solution, reassembling free ferritin coat segments into complete ferritin coat, and embedding Ir (ppy) on the surface of the coat segments 3 Uniformly assembling the nano-gold on the outer surface of single ferritin, dialyzing, and purifying to obtain Ir (ppy) 3 -three-dimensional hybrid nanostructures of ferritin-Au.
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