CN110079311B

CN110079311B - Preparation method of nano fluorescent probe and application of nano fluorescent probe in detection of BV2 cell activation state

Info

Publication number: CN110079311B
Application number: CN201910382527.8A
Authority: CN
Inventors: 刘学; 毕秀丽; 刘禹霖; 陈扬
Original assignee: Liaoning University
Current assignee: Liaoning University
Priority date: 2019-05-09
Filing date: 2019-05-09
Publication date: 2022-06-14
Anticipated expiration: 2039-05-09
Also published as: CN110079311A

Abstract

The invention belongs to the technical field of fluorescent nano materials, and particularly provides a preparation method of a nano fluorescent probe, which comprises the following steps: completely dissolving a carbon source by using water, and adding a small molecular ligand into the solution for doping; ultrasonic dissolving and heating to obtain the target product. The carbon source is one or more of natural amino acids. The small molecular ligand is urea or ammonia water. In terms of molar ratio, carbon source: a small molecule ligand 1: 1-5. The nano fluorescent probe prepared by the invention is a spherical-like nano particle with a carbon atom as a skeleton structure and fluorescent property. The nanometer fluorescent probe can identify BV2 cells in different activation states, and has the characteristics of good biocompatibility, safety, no toxicity, stable action effect and the like. It mainly specifically recognizes BV2 cell membrane protein and cell factor; the preparation method has the advantages of simple process, easy operation, low preparation cost and easy popularization.

Description

Preparation method of nano fluorescent probe and application of nano fluorescent probe in detection of BV2 cell activation state

Technical Field

The invention belongs to the technical field of fluorescent nano materials, and particularly relates to a preparation method of a nano fluorescent probe and application of the nano fluorescent probe in detection of BV2 cell activation state.

Background

Microglia (BV2) are involved in the development of a range of neurodegenerative diseases, the activation and neuroinflammation of which are major features of neuropathology. It induces an endogenous immune response to central nervous system injury and disease, thereby exerting neuroprotective or neurotoxic effects. Any nervous system disorder, which usually results in inflammation and microglial activation with a concurrent increase in the number of glia cells, produces a change in phenotype, a phenomenon known as "reactive gliosis". In acute neurodegenerative diseases (stroke, cerebral hypoxia, cerebral trauma), microglial phenotype is changed and inflammatory mediators are released, wherein the released inflammatory mediators are mainly cytokines and chemokines. These acute inflammatory responses are generally beneficial for the survival of nerve cells, resulting in a reduction of secondary injury in the brain and repair of damaged tissues. Microglial-mediated chronic inflammation is involved in the pathological processes of various chronic neurodegenerative diseases, including: alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, etc. During chronic inflammation, microglia are activated for a long period of time and then continuously release a series of inflammatory mediators, resulting in oxidative stress. It is generally accepted that the chronic inflammatory response mediated by microglia is harmful to the body and can cause damage to nervous tissue.

In modern biomedicine, BV2 cell-related central nervous inflammation is found to damage cerebral cortex and hippocampal region, and is manifested by behaviors of obvious transient learning and memory capacity reduction and the like. This stress response is often used in immune response studies to mimic the challenge of a single or multiple clonal immunizing agents, thereby identifying the immune-challenged state of the body.

In addition, in the presence of BV2 cells, neurostimulators have a toxic effect on dopamine neurons. Therefore, the research on the state and degree of inducing BV2 cell activation has important significance for distinguishing the activation state of microglia and monitoring the state of the microglia in real time.

Disclosure of Invention

The invention aims to innovate the defects of the existing fluorescent probe, quickly and efficiently combine the fluorescent probe with BV2 cells, generate different fluorescent signals according to different numbers of target binding sites and well distinguish the fluorescent signals on an enzyme labeling instrument and a fluorescence spectrophotometer.

The technical scheme adopted by the invention is as follows: a preparation method of a nano fluorescent probe comprises the following steps:

1) completely dissolving a carbon source by using water, and adding a small molecular ligand into the solution for doping;

2) ultrasonic dissolving, and heating at 220 deg.C to obtain the target product.

Preferably, in the above method for preparing a nano fluorescent probe, the carbon source is one or more of natural amino acids.

Preferably, in the above method for preparing a nano fluorescent probe, the small molecule ligand is an ammonia small molecule.

Preferably, in the above method for preparing a nano fluorescent probe, the small molecule ligand is urea or ammonia water.

Preferably, in the above method for preparing a nano fluorescent probe, the molar ratio of carbon source: a small molecule ligand 1: 1-5.

The nano fluorescent probe prepared by the preparation method of the nano fluorescent probe is applied to detecting the BV2 cell activation state.

Preferably, the application as described above,

1) filtering, dialyzing and freeze-drying the nano fluorescent probe, dispersing the nano fluorescent probe in PBS buffer solution to obtain fluorescent nano probe solution, and storing the fluorescent nano probe solution at the normal temperature below 20 ℃ in a dark place;

2) adding the nano fluorescent probe solution into a 96-well plate containing BV2 cells, incubating for 5min, and detecting the fluorescence intensity.

Preferably, in the above application, the concentration of the fluorescent probe solution in the step 1) is 5-20 mg/L.

Preferably, in the above application, step 2), the volume of the cell suspension added to each well of the 96-well plate is 100 μ L, the density of the cell suspension is 100-10000/mL, and the volume of the nano fluorescent probe solution added to each well is 100 μ L.

Preferably, in the above application, the incubation temperature is 20-25 ℃.

The invention has the beneficial effects that:

1. the nanometer fluorescent probe is a sphere-like nanometer particle which takes carbon atoms as a framework structure, has the size of 1-10nm and has the fluorescent property. The nano fluorescent probe is a fluorescent probe which is not passivated or modified. The reason that the nano fluorescent probe can be used for distinguishing BV2 cells in different activation states is that the nano fluorescent probe can be combined with over-expressed membrane protein, and the nano fluorescent probe can be distinguished when the number of the cells is between 100 and 10000. In addition, the nano fluorescent probe has fluorescent property and can be used for cell imaging.

2. The fluorescent signal based on the nano fluorescent probe can not only identify cells in different activation states, but also has the characteristics of good biocompatibility, safety, no toxicity, stable action effect and the like. It mainly specifically recognizes membrane proteins and cytokines; the preparation method has the advantages of simple process, easy operation, low preparation cost and easy popularization.

3. The nano fluorescent probe prepared by the invention has the particle size of below 10nm, is spherical particles which are uniformly distributed, has no passivation or modification on the surface and has no toxicity, so the nano fluorescent probe can be used in the field of biomedicine.

Drawings

FIG. 1 is transmission electron microscopy imaging of a nanophosphor probe.

FIG. 2 shows the fluorescence spectrum of the nano fluorescent probe at an excitation wavelength of 330nm and an emission wavelength of 380 nm.

FIG. 3 is a graph of the intensity of fluorescence of activated and unactivated cells labeled under a fluorescent microplate reader.

Detailed Description

EXAMPLE 1 preparation of Nanofluorescent Probe

Weighing 2g (11.5mmoL) of arginine and 0.09g of alanine (1mmoL), dissolving in 20mL of ultrapure water, adding 1.5g (25mmoL) of urea, completely dissolving by ultrasonic, and heating at 200 ℃ of a reaction kettle for 10 hours to obtain the nano fluorescent probe.

The nano fluorescent probe prepared above is subjected to electron microscope scanning, and the result is shown in fig. 1, and as can be seen from fig. 1, the prepared nano fluorescent probe has an average particle size of 4nm, is uniformly distributed, and is formed into uniformly distributed spherical particles.

FIG. 2 is a fluorescence spectrum obtained by fluorescence detection of a sample of the nano fluorescent probe prepared in example 1 at an optimum excitation wavelength using a fluorescence spectrophotometer. As can be seen, the optimal excitation wavelength of the nano fluorescent probe is 330nm, and the optimal emission peak is located at 380 nm.

Example 2

After the nano fluorescent probe prepared in example 1 was filtered, dialyzed, and lyophilized, 0.004g of the obtained solution was dissolved in 200mL of PBS buffer solution to make the concentration of the nano fluorescent probe solution 0.02mg/mL, 100. mu.L of cell suspension was added to each well of a 96-well plate to make the density of the cell suspension 10000/mL, and 100. mu.L of the nano fluorescent probe solution was added to each well, and incubated at 20 ℃ for 5 min.

The fluorescence intensity values were measured on a fluorescent microplate reader, repeated 8 times, as shown in FIG. 3. The fluorescence intensity of the nano fluorescent probe under the enzyme-linked immunosorbent assay is 2.2 multiplied by 10⁷. Normal BV2 cells are not activated, the concentration of specific components of membrane proteins and cytokines is low, and according to the non-specific adsorption principle, the combination of the nano fluorescent probe and biological thiol, polypeptide and the like generates a slight fluorescence enhancement phenomenon. The activated BV2 cell has specific membrane protein exposed on the cell surface by the stimulation stress reaction and releases cell factors, and after being specifically combined with the nano fluorescent carbon needle, the fluorescence quenching phenomenon occurs. In conclusion, the nano fluorescent probe is combined with the BV2 cell induced by activation to generate fluorescence quenching, and generates a fluorescence enhancement phenomenon with the cell not activated, and the probe has excellent repeatability and better accuracy.

In conclusion, the method has the advantages of low raw material price, simplicity and feasibility, and no need of complex reaction conditions in the preparation process, and can well identify the activated and non-activated states of the BV2 cells in the medical application. The fluorescent probe not only can be used as a nano fluorescent probe, but also has the characteristics of good biocompatibility, specific selection and identification, long action time, good stability, low price, easy obtainment and the like. In view of these excellent properties, the novel nano fluorescent probe is expected to be applied and researched in the medical diagnosis and treatment field.

Claims

1. An application of a nano fluorescent probe in detecting the activation state of BV2 cells is characterized in that,

1) filtering, dialyzing, freeze-drying, dispersing in PBS buffer solution to obtain fluorescent nano probe solution with concentration of 5-20mg/L, and storing at 20 deg.C under normal temperature in dark place;

2) adding the nano fluorescent probe solution into a 96-well plate containing BV2 cells, wherein the volume of cell suspension in each well of the 96-well plate is 100 mu L, the density of the cell suspension is 100-10000/mL, the volume of the nano fluorescent probe solution in each well is 100 mu L, the incubation temperature is 20-25 ℃, incubating for 5min, and detecting the fluorescence intensity;

the preparation method of the nano fluorescent probe comprises the following steps: weighing 2g of arginine and 0.09g of alanine, dissolving in 20mL of ultrapure water, adding 1.5g of urea, completely dissolving by ultrasonic, and heating at 200 ℃ in a reaction kettle for 10 hours to obtain the nano fluorescent probe.