CN111227799A - In-vivo detection method for brain tissue methylmercury - Google Patents
In-vivo detection method for brain tissue methylmercury Download PDFInfo
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- CN111227799A CN111227799A CN202010069573.5A CN202010069573A CN111227799A CN 111227799 A CN111227799 A CN 111227799A CN 202010069573 A CN202010069573 A CN 202010069573A CN 111227799 A CN111227799 A CN 111227799A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
- A61B5/0071—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence by measuring fluorescence emission
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/0033—Features or image-related aspects of imaging apparatus classified in A61B5/00, e.g. for MRI, optical tomography or impedance tomography apparatus; arrangements of imaging apparatus in a room
- A61B5/004—Features or image-related aspects of imaging apparatus classified in A61B5/00, e.g. for MRI, optical tomography or impedance tomography apparatus; arrangements of imaging apparatus in a room adapted for image acquisition of a particular organ or body part
- A61B5/0042—Features or image-related aspects of imaging apparatus classified in A61B5/00, e.g. for MRI, optical tomography or impedance tomography apparatus; arrangements of imaging apparatus in a room adapted for image acquisition of a particular organ or body part for the brain
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B2503/00—Evaluating a particular growth phase or type of persons or animals
- A61B2503/40—Animals
Abstract
The invention relates to a method for detecting methyl mercury in brain tissue in vivo, which comprises the following steps: (1) preparing a solution of the hexadecamer ferritin-gold nanocluster: (2) injecting 50-200 mu L of the hexadecamer ferritin-gold nanocluster solution prepared in the step (1) into a living body by adopting an intravenous injection, intraperitoneal injection or intragastric perfusion mode; (3) exciting and observing the fluorescence intensity of the brain of the living body by using a living body imager within 10 min-6 h, wherein the wavelength of the living body imager is 400-700 nm; if the living brain does not have fluorescence, the living brain tissue contains methylmercury; if the living brain has fluorescence, the living brain tissue does not contain methylmercury. The characteristic that the gold nanocluster can be used for biological imaging and the characteristic that the hexamer ferritin can cross a blood brain barrier are utilized, so that the gold nanocluster can enter brain tissues for biomedical imaging, and the gold nanocluster has high stability and specificity and does not need to use dyes; the hexadecamer ferritin-gold nanocluster can be applied to mercury detection of brain tissues in organisms with methyl mercury poisoning.
Description
Technical Field
The invention relates to a method for detecting brain tissue methylmercury in vivo, belonging to the preparation technology of biological probes and biological imaging markers.
Background
The fluorescent metal nanoclusters are an emerging fluorescent material with the size less than 2nm, have excellent photostability and sub-nanometer-sized biocompatibility, are easy to synthesize, and are receiving wide attention. By utilizing the characteristics, the fluorescent metal nanoclusters have participated in the fields of fluorescence biosensing, biological imaging and the like, and great development is achieved in the fields. The protein-gold nanocluster compound not only improves the fluorescence stability of the gold nanoclusters, but also expands the application range of the gold nanoclusters by means of the functional characteristics of the protein.
The detection of the protein-gold nanocluster on heavy metal elements such as mercury ions and lead ions is one of the most important properties, and in the existing research, the protein-gold nanocluster can detect mercury ions in an aqueous solution in vitro and detect mercury ions in organs such as the abdomen and the chest in vivo, but has no detection capability on mercury ions in brain tissues.
The protein-gold nanocluster can detect heavy metal elements such as mercury ions and lead ions, and is an important biological probe, but the protein-gold nanocluster does not have the capability of detecting the heavy metal elements such as mercury ions and lead ions in brain tissues at present.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a method for detecting the methyl mercury in the brain tissue in vivo, which utilizes the characteristic that a gold nano-cluster can form images in vivo, takes hexameric ferritin as a carrier, leads the hexadecameric ferritin to pass through a blood brain barrier by virtue of a brain nerve cell surface specific receptor and enter the brain tissue to detect inorganic mercury and methyl mercury, does not need to rely on dyes, and has the characteristics of high specificity, high sensitivity and tissue penetrating power.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for detecting methylmercury in brain tissue in vivo comprises the following steps:
(1) preparing a solution of the hexadecamer ferritin-gold nanocluster:
a) separating and purifying the hexadecyl-polymer ferritin, desalting by dialysis to prepare a hexadecyl-polymer ferritin solution with the concentration of 40 mg/mL-60 mg/mL, and preheating for 5-10 minutes at 35-40 ℃;
b) preparing a chloroauric acid solution with the concentration of 3-10 mM, adjusting the pH value to 6.5-8.0, and preheating for 5-10 minutes at 35-40 ℃;
c) preparing a sodium hydroxide solution and a hydrochloric acid solution with the concentration of 1M;
d) uniformly mixing the hexameric ferritin solution prepared in the step a and the chloroauric acid solution prepared in the step b, wherein the volume ratio of the hexameric ferritin solution to the chloroauric acid solution is 2: 1-1: 2, and adjusting the pH value of the mixed solution to be more than or equal to 10 by using the 1M sodium hydroxide solution prepared in the step c;
e) standing for reaction for 4-24 hours at 25-40 ℃, and adjusting the pH value of the mixed solution to be neutral by using the 1M hydrochloric acid solution prepared in the step c after the reaction is finished to obtain a solution of the hexadecamer ferritin-gold nanocluster with the concentration of 50-100 mu M;
(2) injecting 50-200 mu L of the hexadecamer ferritin-gold nanocluster solution prepared in the step 1 into a living body in an intravenous injection, intraperitoneal injection or intragastric perfusion mode;
(3) exciting and observing the fluorescence intensity of the brain of the living body by using a living body imager within 10 min-6 h, wherein the wavelength of the living body imager is 400-700 nm; if the living brain does not have fluorescence, the living brain tissue contains methylmercury; if the living brain has fluorescence, the living brain tissue does not contain methylmercury.
The solution of the hexadecamer ferritin-gold nanocluster prepared in the step 1 has pink fluorescence under the irradiation of an ultraviolet lamp.
Scanning the solution of the hexadecamer ferritin-gold nanocluster prepared in the step 1 by using a fluorescence spectrophotometer, exciting at 480-540nm, and having a maximum emission peak at 610-660 nm.
In step 3, the wavelength of the living body imager is 535 nm.
Compared with the prior art, the invention has the beneficial effects that:
the characteristic that the gold nanocluster can be used for biological imaging and the characteristic that the hexamer ferritin can cross a blood brain barrier are utilized, so that the gold nanocluster can enter brain tissues for biomedical imaging, and the gold nanocluster has high stability and specificity and does not need to use dyes. In addition, the hexadecamer ferritin-gold nanoclusters can be applied to the mercury detection of brain tissues in organisms (human bodies and mice) suffering from methyl mercury poisoning.
Detailed Description
The present invention will be further illustrated with reference to the following examples.
A method for detecting methylmercury in brain tissue in vivo comprises the following steps:
(1) preparing a solution of the hexadecamer ferritin-gold nanocluster:
a) separating and purifying the hexadecyl-polymer ferritin, desalting by dialysis to prepare a hexadecyl-polymer ferritin solution with the concentration of 40 mg/mL-60 mg/mL, and preheating for 5-10 minutes at 35-40 ℃;
b) preparing a chloroauric acid solution with the concentration of 3-10 mM, adjusting the pH value to 6.5-8.0, and preheating for 5-10 minutes at 35-40 ℃;
c) preparing a sodium hydroxide solution and a hydrochloric acid solution with the concentration of 1M;
d) uniformly mixing the hexameric ferritin solution prepared in the step a and the chloroauric acid solution prepared in the step b, wherein the volume ratio of the hexameric ferritin solution to the chloroauric acid solution is 2: 1-1: 2, and adjusting the pH value of the mixed solution to be more than or equal to 10 by using the 1M sodium hydroxide solution prepared in the step c;
e) and (c) standing for reaction for 4-24 hours at 25-40 ℃, and after the reaction is finished, adjusting the pH value of the mixed solution to be neutral by using the 1M hydrochloric acid solution prepared in the step (c), so as to obtain a solution of the hexadecamer ferritin-gold nanocluster with the concentration of 50-100 mu M.
(2) Injecting 50-200 mu L of the hexadecamer ferritin-gold nanocluster solution prepared in the step (1) into a living body by adopting an intravenous injection, intraperitoneal injection or intragastric perfusion mode;
(3) exciting and observing the fluorescence intensity of the brain of the living body by using a living body imager within 10 min-6 h, wherein the wavelength of the living body imager is 400-700 nm; if the living brain does not have fluorescence, the living brain tissue contains methylmercury; if the living brain has fluorescence, the living brain tissue does not contain methylmercury.
The solution of the hexadecamer ferritin-gold nanocluster prepared in the step 1 has pink fluorescence under the irradiation of an ultraviolet lamp. And a fluorescence spectrophotometer is adopted for scanning, excitation is carried out at 480-540nm, and the maximum emission peak is obtained at 610-660 nm.
Examples
Preparation of hexadecamer ferritin-gold nanocluster
Preparing a 40mg/ml solution of hexameric ferritin and pre-heating at 35 ℃ for 10 minutes, mixing it proportionally with a 5mM chloroauric acid solution having a pH of 7 and pre-heating at 40 ℃ for 5 minutes, stirring on a magnetic stirrer at 60r/min for 5 minutes to mix uniformly, and adjusting the pH to 11.5; then placing the mixture at 35 ℃ for reaction for 6h, and adjusting the pH value to be neutral; pink fluorescence was observed under an ultraviolet lamp.
Blood brain barrier in vitro cell model imaging
In vitro culture of neuroendothelial cells bEnd.3 with inoculum size of 3X 105And culturing for 24h at 37 ℃ in a 5% carbon dioxide incubator. When the coverage rate reaches 90%, starving the cells for 1h, adding the hexadecyl polymer ferritin-gold nanoclusters with the final concentration of 1-20 mu M into the culture solution, reacting for 1-4 h, discarding the culture solution, and washing with a phosphate buffer solution for three times. Cells were fixed with 4% paraformaldehyde for 20-60 minutes at room temperature. Observed with a confocal laser microscope. As a result, it was found that the cells fluoresced green when excited at 480-540 nm.
Detection of the Presence of methylmercury in Living brain tissue as Mercury ion Probe
The preparation method comprises the steps of injecting 10mg/kg of methylmercury into an abdominal cavity of a nude mouse aged 4 weeks, injecting a hexadecamer ferritin-gold nanocluster solution (50 mu M, 200 mu L) into the body of the mouse in a tail vein injection, abdominal cavity injection and intragastric administration mode after 0.5-4 hours, exciting and observing the brain fluorescence intensity of the mouse by using a mouse living body imager after 4 hours, and using the mouse injected with phosphate buffer solution into the abdominal cavity as a control. The results of the study found that the brain of the mice injected with methylmercury did not fluoresce as the blank mice, while the phosphate buffer group did.
Methyl mercury detection
Taking a mouse acute methyl mercury poisoning model as an example, injecting 50 mu M and 200 mu L of a hexadecameric ferritin-gold nanocluster solution into a mouse body in a tail vein injection manner, and observing after 4h, wherein the brain fluorescence of the mouse is quenched.
Claims (4)
1. A method for detecting methyl mercury in brain tissue in vivo is characterized in that: the method comprises the following steps:
(1) preparing a solution of the hexadecamer ferritin-gold nanocluster:
a) separating and purifying the hexadecyl-polymer ferritin, desalting by dialysis to prepare a hexadecyl-polymer ferritin solution with the concentration of 40 mg/mL-60 mg/mL, and preheating for 5-10 minutes at 35-40 ℃;
b) preparing a chloroauric acid solution with the concentration of 3-10 mM, adjusting the pH value to 6.5-8.0, and preheating for 5-10 minutes at 35-40 ℃;
c) preparing a sodium hydroxide solution and a hydrochloric acid solution with the concentration of 1M;
d) uniformly mixing the hexameric ferritin solution prepared in the step a and the chloroauric acid solution prepared in the step b, wherein the volume ratio of the hexameric ferritin solution to the chloroauric acid solution is 2: 1-1: 2, and adjusting the pH value of the mixed solution to be more than or equal to 10 by using the 1M sodium hydroxide solution prepared in the step c;
e) standing for reaction for 4-24 hours at 25-40 ℃, and adjusting the pH value of the mixed solution to be neutral by using the 1M hydrochloric acid solution prepared in the step c after the reaction is finished to obtain a solution of the hexadecamer ferritin-gold nanocluster with the concentration of 50-100 mu M;
(2) injecting 50-200 mu L of the hexadecamer ferritin-gold nanocluster solution prepared in the step 1 into a living body in an intravenous injection, intraperitoneal injection or intragastric perfusion mode;
(3) exciting and observing the fluorescence intensity of the brain of the living body by using a living body imager within 10 min-6 h, wherein the wavelength of the living body imager is 400-700 nm; if the living brain does not have fluorescence, the living brain tissue contains methylmercury; if the living brain has fluorescence, the living brain tissue does not contain methylmercury.
2. The in-vivo detection method of methylmercury in brain tissue according to claim 1, wherein: the solution of the hexadecamer ferritin-gold nanocluster prepared in the step 1 has pink fluorescence under the irradiation of an ultraviolet lamp.
3. The in-vivo detection method of methylmercury in brain tissue according to claim 1, wherein: scanning the solution of the hexadecamer ferritin-gold nanocluster prepared in the step 1 by using a fluorescence spectrophotometer, exciting at 480-540nm, and having a maximum emission peak at 610-660 nm.
4. The in-vivo detection method of methylmercury in brain tissue according to claim 1, wherein: in step 3, the wavelength of the living body imager is 535 nm.
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| CN202010069573.5A CN111227799B (en) | 2020-01-21 | 2020-01-21 | Method for manufacturing high-sensitivity biological probe for in-vivo detection of brain tissue methylmercury |
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Citations (6)
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| US20090069481A1 (en) * | 2003-12-29 | 2009-03-12 | Jingwu Zhang | External modification of composite organic inorganic nanoclusters |
| CN102150034A (en) * | 2008-08-05 | 2011-08-10 | 新加坡科技研究局 | Methods, compositions, and articles comprising stabilized gold nanoclusters |
| CN102735752A (en) * | 2012-06-11 | 2012-10-17 | 东南大学 | Tumor-targeting living body multimodality imaging method based on gold nano-clusters |
| CN103884701A (en) * | 2014-04-10 | 2014-06-25 | 中国烟草总公司郑州烟草研究院 | Mercury ion detection method |
| CN106583747A (en) * | 2016-12-06 | 2017-04-26 | 南华大学 | Preparation of protamine gold nanoclusters and application in analogue enzyme color comparison and fluorescence detection |
| CN106908427A (en) * | 2017-03-01 | 2017-06-30 | 哈尔滨师范大学 | Gold nanoclusters and carbon quantum dot composite fluorescence probe and its application |
-
2020
- 2020-01-21 CN CN202010069573.5A patent/CN111227799B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090069481A1 (en) * | 2003-12-29 | 2009-03-12 | Jingwu Zhang | External modification of composite organic inorganic nanoclusters |
| CN102150034A (en) * | 2008-08-05 | 2011-08-10 | 新加坡科技研究局 | Methods, compositions, and articles comprising stabilized gold nanoclusters |
| CN102735752A (en) * | 2012-06-11 | 2012-10-17 | 东南大学 | Tumor-targeting living body multimodality imaging method based on gold nano-clusters |
| CN103884701A (en) * | 2014-04-10 | 2014-06-25 | 中国烟草总公司郑州烟草研究院 | Mercury ion detection method |
| CN106583747A (en) * | 2016-12-06 | 2017-04-26 | 南华大学 | Preparation of protamine gold nanoclusters and application in analogue enzyme color comparison and fluorescence detection |
| CN106908427A (en) * | 2017-03-01 | 2017-06-30 | 哈尔滨师范大学 | Gold nanoclusters and carbon quantum dot composite fluorescence probe and its application |
Non-Patent Citations (1)
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| 张胜利: "铁储藏蛋白大小与形状的调控", 《中国博士学位论文全文数据库（工程科技Ⅰ辑）》 * |
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