CN109096384A - Green fluorescent protein base nanoparticle, preparation method and its application in terms of cell imaging and nucleus kernel dyeing - Google Patents
Green fluorescent protein base nanoparticle, preparation method and its application in terms of cell imaging and nucleus kernel dyeing Download PDFInfo
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- CN109096384A CN109096384A CN201810766889.2A CN201810766889A CN109096384A CN 109096384 A CN109096384 A CN 109096384A CN 201810766889 A CN201810766889 A CN 201810766889A CN 109096384 A CN109096384 A CN 109096384A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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Abstract
A kind of green fluorescent protein base nanoparticle, preparation method and its application in terms of cell imaging and nucleus kernel dyeing, belong to fluorescin based nano-material field.It is in deionized water, concentration thioflavin-T aqueous solution is added as molecule is constructed, adds bovine serum albumin(BSA) or human serum albumins aqueous solution, then heats the solution, finally solution is cooled to room temperature, to obtain green fluorescent protein base nanoparticle T-FPNs solution.In yeast cells dyestuff can be positioned as bio-imaging material and kernel with T-FPNs is shown in the experiment of Hela cell, the nanoparticle is suitble to be mass produced, because their synthetic method has the characteristics of inexpensive benefit, environmental protection, easy and biocompatibility；And the T-FPNs with excellent light stability can stain living cells and fixed cell kernel, realize quickly, be imaged in real time and without cleaning.
Description
Technical field
The invention belongs to fluorescin based nano-material fields, and in particular to a kind of green fluorescent protein base nanoparticle,
Preparation method and its application in terms of cell imaging and nucleus kernel dyeing.
Background technique
In recent years, the various nano materials with different function and property all achieved in design, manufacture it is very big into
Exhibition.Nano material (semiconductor-quantum-point, fluorescent carbon point, the cold light silicon nano, small organic molecule being largely developed
With organic fluorescence nanoparticle etc.) it is used for the application in relation to medical diagnosis, treatment aspect such as bio-imaging, drug delivery.But
Meanwhile the application of these materials receives different degrees of limitation, such as, lack crucial biocompatibility, to organism
Toxicity is big, complex synthetic route, it is with high costs, be not easy to popularize in an all-round way etc..
Nucleus kernel is the important component in cell, plays rRNA transcription, processing, assembles place
Role.The research that kernel and its associated biochemical are reacted in the visualization of kernel has great importance.Especially, in living cells
In the normal physiological characteristic of cell is maintained to the positioning of kernel, more facilitate the case where monitoring kernel in real time.However, mesh
Preceding most of kernel dyestuff does not have the function to the positioning of living cells kernel, is required to that the cells are fixed by complicated process
Poststaining.During fixation, it is likely that lose or change more Biont informations.Most commonly used kernel dyeing side
Method is needed using the antibody for being directed to a certain component of kernel.For example, introducing antifibrin element monoclonal antibody in cell, it is used to
Kernel is positioned.One advantage of this method is specificity of the antibody to its antigen, is capable of detecting when researcher
Targeted biomolecule or process.However, this process needs a specific antibody, this kind of antibody is usually valuableness
, it is frequently not commercially available, and there is still a need for another dyestuff is incorporated on antibody.So this method of the overall evaluation
It is complexity, it is expensive.It is unfavorable for the quick promotion of scientific experiment.
Summary of the invention
The object of the present invention is to provide a kind of green fluorescent protein base nanoparticle, preparation method and its in cell imaging and
The application of nucleus kernel dyeing aspect.The present invention is that green fluorescent protein base nanoparticle is prepared using one-step synthesis first
Then son carries out dyeing positioning to living cells imaging and kernel.
Green fluorescent protein base nanoparticle biocompatibility prepared by the present invention is fine, using the characteristics of its hypotoxicity with
Living cells is incubated for the purpose that the positioning of nucleus kernel may be implemented jointly, and photoluminescent property is stabilized ten hours or more.
The preparation method of green fluorescent protein base nanoparticle of the present invention and its to cell imaging and cell nucleus
The step of benevolence dyes is as follows:
1) thioflavin-T that concentration is 0.01~1mol/L (preferably 0.1~1mol/L) in deionized water, is added
(ThT) it is pure to add the ox blood that concentration is 0.01~1mol/L (preferably 0.1~1mol/L) as molecule is constructed for aqueous solution
The dosage mole of albumen (BSA) or human serum albumins aqueous solution, thioflavin-T and bovine serum albumin(BSA) or human serum albumins
It finally will be molten then by the solution in 50~100 DEG C (preferably 80~100 DEG C) of at a temperature of 5~20min of heating than for 1:1
Liquid is cooled to room temperature, to obtain green fluorescent protein base nano-particle solution of the present invention, green fluorescent protein Ji Na
The concentration of rice corpuscles is 0.64~64mg/mL.
2) by brewing yeast cell YPD (yeast extract powder peptone dextrose culture-medium) as culture medium, in 30 DEG C of constant temperature
In the lower incubation of oscillation until OD600 reaches 1.0 in oscillator；Take 1mL cell culture fluid that brewing yeast cell, gained is collected by centrifugation
Cell strain is washed 3 times with fresh YPD, and cell is then resuspended in 100 μ L, the base nanoparticle of green fluorescent protein containing 0.1mg/mL
In the YPD culture medium of son, places back in 30 DEG C of isothermal vibration devices and be incubated for 6 hours；Take 1mL cell culture fluid that wine is collected by centrifugation again
Brewer yeast cell, gained cell strain are resuspended 3~5 times with PBS buffer solution (pH 7.4), wash away culture medium and external as far as possible
Fluorescent nano particles, background interference when to avoid to co-focusing imaging；Drop finally is resuspended on glass slide in 2 μ L, as
The sample of cell imaging；
3) on the cover slip by HeLa cell inoculation, the culture in the Eagle culture medium (DMEM) of Dulbecco's improvement,
The culture solution of replacement in 3 days, control incubator environment are 37 DEG C and 5%CO2；Above-mentioned Hela cell inoculation after culture is being contained
Have and cultivated 1 hour on the coverslip of the DMEM culture medium of 1mg/mL T-FPNs, gently covers growth using new coverslip later
There is the coverslip of HeLa cell to be put into imaging chamber, wash 2 times with Hank's balanced salt solution (HBSS) and is maintained in HBSS；
It is finally imaged using ZEISS LSM700 confocal microscope, the confocal microscope is equipped with by Zen software control
100x oil-immersion objective (NA1.4), 488nm and 555 laser observe the effect dyed to nucleus kernel.
Thioflavine-T (ThT) is interspersed in bovine serum albumin(BSA) (BSA) skeleton, has synthesized new emission peak in green wavelength
Green fluorescent protein base nanoparticle T-FPNs, in yeast cells with show that T-FPNs can be done in the experiment of Hela cell
Dyestuff is positioned for biological image forming material and kernel.Green fluorescent protein base nanoparticle prepared by the present invention is suitble to extensive raw
It produces, because their synthetic method has the characteristics that inexpensive benefit, environmental protection, simplicity and biocompatibility.And have excellent
The T-FPNs of photostability can stain living cells and positioning living cells kernel, realize quickly, in real time and without cleaning be imaged.
Detailed description of the invention
Fig. 1: Fig. 1 (curve 2) is the fluorescent emission spectral curve of fluorescent nano particles prepared by embodiment 1；
The fluorescent nano particles of preparation are attached in cuvette, in Fluorescence Spectrometer, setting launch wavelength is 400nm,
Can detecte at 488nm has stronger emission peak.Fig. 1 (curve 3), Fig. 1 (curve 1) are needed for synthesizing nano-particle respectively
Constituent thioflavine-T and bovine serum albumin(BSA), they are under the excitation of phase co-wavelength without corresponding emission peak.
Fig. 2: Fig. 2 is colored graph of the fluorescent nano particles prepared by embodiment 1 to yeast cells.From the shooting to cell
As a result it sees, after addition fluorescent nano particles are incubated for jointly in yeast cells culture medium, green is presented in cell.Left figure is green
Fluorescence channel image, centre are combination pictures, and the right is that (confocal microscope can directly give three differences to bright field image
The image in channel is corresponding color channel image, combination picture and bright field image respectively).
Fig. 3: the imaging that fluorescent nano particles prepared by embodiment 1 position HeLa entoblast.In HeLa cell
After fluorescent nano particles are added, pass through the detection of confocal microscope, it is seen that by force green is presented in the position of intracellular kernel
Color fluorescence.The left side is green fluorescence channel image, and centre is combination picture, and the right is bright field image.
Specific embodiment
Embodiment 1:
In 0.8mL deionized water, the thioflavin-T aqueous solution 0.1mL that addition concentration is 1mol/L, which is used as, constructs molecule,
The Bovine Serum Albumin in Aqueous Solution 0.1mL, thioflavin-T of addition concentration 1mol/L and the dosage molar ratio of bovine serum albumin(BSA) are
1:1.Then by solution 100 DEG C at a temperature of heat 10min, finally solution is cooled to room temperature, obtains green fluorescent protein
Base nano-particle solution, the concentration of fluorescin base nanoparticle are 6.4mg/mL.
Embodiment 2:
Brewing yeast cell YPD (yeast extract powder peptone dextrose culture-medium) is vibrated at 30 DEG C and is incubated as culture medium
It educates until OD600 reaches 1.0；Take 1mL cell culture fluid that brewing yeast cell, the fresh YPD of gained cell strain is collected by centrifugation
Cell three times, is then resuspended in the 100 μ L that embodiment 1 obtains and contains 0.1mg/mL green fluorescent protein base nanoparticle by washing
YPD culture medium in, then place back in 30 DEG C of isothermal vibration device and be incubated for 6 hours；The centrifugation of 1mL cell culture fluid is taken to receive again
Collect brewing yeast cell, gained cell strain is resuspended 3~5 times with PBS buffer solution (pH 7.4), washes away culture medium and body as far as possible
Outer fluorescent nano particles, background interference when to avoid to co-focusing imaging；Drop finally is resuspended on glass slide in 2 μ L,
Cell imaging research is carried out using ZEISS LSM700 confocal microscope.
Embodiment 3:
On the cover slip by HeLa cell inoculation, the training in the Eagle culture medium (DMEM) of conventional Dulbecco's improvement
It supports, the culture solution of replacement in 3 days, control incubator environment is 37 DEG C and 5%CO2.Fluorescin base nanoparticle is to kernel
Staining procedure is as follows, and the Hela cell inoculation after above-mentioned culture is being contained 1mg mL-11 is cultivated on the DMEM coverslip of T-FPNs
Hour.Later, growth is gently covered using new coverslip has the coverslip of cell to be put into imaging chamber, balances salt with Hank's
Solution (HBSS) is washed twice and is maintained in HBSS.Finally it is imaged using ZEISS LSM700 confocal microscope, it is described
Confocal microscope is equipped with the 100x oil-immersion objective (NA1.4) by Zen software control, 488nm and 555 laser.For observing to core
The effect of benevolence dyeing.
Claims (3)
1. a kind of preparation method of green fluorescent protein base nanoparticle, it is characterised in that: in deionized water, concentration, which is added, is
Thioflavin-T the aqueous solution of 0.01~1mol/L adds the bovine serum albumin that concentration is 0.01~1mol/L as molecule is constructed
The dosage molar ratio of white or human serum albumins aqueous solution, thioflavin-T and bovine serum albumin(BSA) or human serum albumins is 1:1,
Then solution is finally cooled to room temperature, in 50~100 DEG C of at a temperature of 5~20min of heating to obtain green by the solution
Fluorescin base nano-particle solution, the concentration of green fluorescent protein base nanoparticle are 0.64~64mg/mL.
2. a kind of green fluorescent protein base nanoparticle, it is characterised in that: be prepared by method described in claim 1.
3. green fluorescent protein base nanoparticle described in claim 1 answering in terms of cell imaging or nucleus kernel dyeing
With.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112461807A (en) * | 2020-11-26 | 2021-03-09 | 山西大学 | Application of carbon quantum dots in targeted nucleolus wash-free imaging |
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| CN112461807A (en) * | 2020-11-26 | 2021-03-09 | 山西大学 | Application of carbon quantum dots in targeted nucleolus wash-free imaging |
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