CN107677656A - A kind of ratio fluorescent nano probe and its application - Google Patents
A kind of ratio fluorescent nano probe and its application Download PDFInfo
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- CN107677656A CN107677656A CN201710890371.5A CN201710890371A CN107677656A CN 107677656 A CN107677656 A CN 107677656A CN 201710890371 A CN201710890371 A CN 201710890371A CN 107677656 A CN107677656 A CN 107677656A
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- G—PHYSICS
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- 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"
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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
- G01N21/62—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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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Abstract
The invention discloses a kind of ratio fluorescent nano probe and its application, and it is utilizedmCDs being capable of selectivity response ClO‑Characteristic, withmCDs is fluorescence probe, with [Ru (bpy)3]2+As reference reagent, structurem‑CDs@[Ru(bpy)3]2+Ratio fluorescent nano probe.As addition various concentrations ClO‑After being reacted, the fluorescence color of the ratio fluorescent probe gradually transits to red by blue-green, therefore can be used for ClO‑Quantitatively or semi-quantitatively detection;Meanwhile the ratio fluorescent nano probe can also be taken in cell, cell imaging is realized, therefore can be used for intracellular ClO‑The Visual retrieval of active oxygen radical.The present invention has the advantages that wide simple to operate, applicability, rapid reaction, the color change that can produce multicolour, can be to object progress multicolour, visualization, on-line quantitative analysis.
Description
Technical field
The present invention relates to a kind of ratio fluorescent nano probe and its application, belong to analytical chemistry and field of nanometer technology.
Background technology
Visual retrieval is because the signal of naked eyes identification can be provided, it is not necessary to which large-scale instrument aids in, suitable for real-time, live
Detection, makes it receive extensive concern.Fluorescence has spirit as one of a kind of classical visualization technique means in analytical chemistry
Sensitivity is high, high spatial and time resolution and simple to operation in terms of living cells and organism the advantages that, it is obtained extensively
Using.However, single-shot, which penetrates fluorescence probe generally presence, comes from the adverse effect such as instrument, environmental condition and probe molecule itself;
Meanwhile traditional fluorescence sense(Fluorescent quenching type and Fluorescence Increasing type)In the presence of change color it is single the problem of.On eliminating
State unfavorable factor and obtain accurate result, reference reagent structure Ratio-type can be introduced on the basis of single-shot penetrates fluorescence probe
Fluorescence probe, there is selectivity to respond to object and can produce gradual change and the multicolour of fluorescence color for it, and further improving can
Ability depending on changing sxemiquantitative, the quantitative analysis detection available for object in terms of chemical/biological.
Bio-imaging technology uses fluorophor, including inorganic material, as above conversion, quantum dot etc., organic material, such as green
Color fluorescin, red fluorescent protein or fluorescent dye etc. are marked.Fluorophor is set to reach higher point using exciting light
Sub- energy level, then launch the longer visible ray of wavelength, form vivo biodistribution light source and detected.In recent years, fluorescence skill
It is widely used in the research that art has been metabolized in some molecular biology and small molecule body.
Carbon point(CDs)Have become a kind of low cost, low toxicity, the photoluminescent with good biocompatibility
(PL)Inorganic material, it is widely used in chemical sensitisation and Enzyme assay, and bio-imaging.The present invention is special using carbon point
One property responds ClO-The characteristics of, by adding reference reagent, construct one kind and be applied to ClO-The ratio fluorescent nano of detection is visited
Pin, and cell imaging is applied it to, the ratio for being such as used for active oxygen radical is imaged, and has expanded and extend ratio fluorescent reagent
Realized in terms of cell imaging it is quantitative, real-time, non-invasively observe.
The content of the invention
The present invention penetrates the existing adverse effect of fluorescence probe for single-shot, it is proposed that a kind of ratio fluorescent nano probe, should
In ratio fluorescent nano probem- CDs can respond ClO in specific manner-, as addition various concentrations ClO-, can after being reacted
The fluorescence color of ratio fluorescent probe is set gradually to transit to red by blue-green, so as to realize to ClO-Sensitive, multicolour, can
Depending on changing quantitative detection.Meanwhile the ratio fluorescent nano probe applies also for cell imaging, the ClO of exogenous intake-Realize
Ratio is imaged.
To achieve the above object, the present invention adopts the following technical scheme that:
A kind of ratio fluorescent nano probe, its be withm- CDs is fluorescence probe, with [Ru (bpy)3]2+As reference reagent, structurem-CDs@[Ru(bpy)3]2+Ratio fluorescent nano probe.
It is describedm- CDs is using m-phenylene diamine (MPD) as raw material, and ethanol is solvent, at 180 DEG C after the h of hydro-thermal reaction 12, is passed through
Column chromatography chromatogram isolates and purifies what is obtained;Wherein, the mass volume ratio of m-phenylene diamine (MPD) and ethanol is 1:100 g/mL;Column chromatography color
Spectrum isolates and purifies the mixed solution that eluant, eluent used is dichloromethane and methanol, and both volume ratios are 15:1.
The construction method of the ratio fluorescent nano probe is:Willm- CDs solution and Ru (bpy)3Cl2Solution is by volume
6:1 mixing, it is made describedm-CDs@[Ru(bpy)3]2+Ratio fluorescent nano probe;Wherein,mThe concentration of-CDs solution is 20 μ
G/mL, Ru (bpy)3Cl2The concentration of solution is 40 μ g/mL.
A kind of ratio fluorescent nano probe is in quantitative detection ClO-In application, its application process includes following step
Suddenly:
1)The ClO of series concentration is separately added into the ratio fluorescent nano probe-, it is glimmering that its is determined after the min of hybrid reaction 5
Luminous intensity simultaneously draws fluorescence intensity with ClO-The standard curve of change in concentration, obtains regression equation;
2)Added into another ratio fluorescent nano probe and contain ClO-Testing sample, determine its fluorescence after the min of hybrid reaction 5
Intensity, recycle step 1)Gained regression equation calculation goes out ClO in sample-Content;
A kind of ratio fluorescent nano probe is in Visual retrieval ClO-In application, its application process comprises the following steps:
1)Agarose powder is added into the ratio fluorescent nano probe, is placed on the thick colloid of gained after heating stirring
The groove of lid is centrifuged, round pie is formed after its coolingm-CDs@[Ru(bpy)3]2+Agarose aquogel;
2)To gainedm-CDs@[Ru(bpy)3]2+The ClO of series concentration is separately added into agarose aquogel-, after reacting 5 min
The change of its fluorescence intensity and color is observed under 365 nm uviol lamps, and simultaneously using the fluorescence of digital camera shooting hydrogel
Imaging, to prepare standard color comparison card;
3)Added into the ratio fluorescent nano probe-agarose aquogel separately taken and contain ClO-Testing sample, react 5 min after
Observe its fluorescence intensity and color under 365 nm uviol lamps, and with step 2)Gained standard color comparison card is contrasted, visualization
ClO in judgement sample-Content;
Gainedm-CDs@[Ru(bpy)3]2+The content of agarose is 5% in agarose aquogel.
A kind of application of ratio fluorescent nano probe in cell imaging, including for intracellular ClO-Active oxygen is certainly
By the detection of base, it specifically includes following steps:
1)The ratio fluorescent nano probe is added in the culture dish equipped with cell, in 95% CO2/5% O2Atmosphere in train
After supporting 1 h, cell is washed 3-5 times with KRP buffer solutions, removes the ratio fluorescent nano probe for being introduced into cell, it is burnt using copolymerization
Microscope shoots in the case where excitation wavelength is two kinds of passages of 488nm and 543nm and is imaged;
2)To step 1)The middle cell for having ratio fluorescent nano probe of taking in adds 10 μM of ClO-, after further hatching 15 min
Cell is washed with KRP buffer solutions 3-5 times, removes the ClO for being introduced into cell-, use Laser Scanning Confocal Microscope in excitation wavelength for 488
Shooting cell imaging is carried out under two kinds of fluorescence channels of nm and 543 nm, is observedm-CDs@[Ru(bpy)3]2+Ratio fluorescent nano is visited
For ClO-Response, with realize to ClO-The measure of active oxygen radical.
The beneficial effects of the present invention are:
(1)[Ru(bpy)3]2+To pH and ClO-It is insensitive, therefore can be as the reference reagent in ratio fluorescent probe.
(2)Willm-CDs@[Ru(bpy)3]2+Agarose aquogel is made, is realizing ClO-While detection, combine solid
Body matrix is portable, easy storage, the characteristics of cost is low, nontoxic, suitable for the quick measure under varying environment.
(3)Ratio fluorescent probe response various concentrations ClO of the present invention-And fluorescence is showed by blue-green to red gradual mistake
Cross, multicolour, visual quantitative detection can be achieved, it is penetrated with respect to single-shot, and fluorescence sensitivity is higher, error is smaller, eliminates simultaneously
Come from the adverse effect such as instrument, environmental condition and probe molecule itself;Meanwhile it can respond ClO in specific manner-, it is
The on-line checking of active oxygen radical lays the foundation and example in cell and organism;
(4)Ratio fluorescent probe ClO suitable for solution of the present invention-Quantitative detection and intracellular ClO-Active oxygen radical
Ratio is imaged.
Brief description of the drawings
Fig. 1 is the present inventionm-CDs@[Ru(bpy)3]2+Ratio fluorescent nano probe response various concentrations ClO-Fluorescence it is strong
Spend variation diagram.
Fig. 2 is the present inventionm-CDs@[Ru(bpy)3]2+Agarose aquogel response various concentrations ClO-Color change figure.
Fig. 3 is the present inventionm-CDs@[Ru(bpy)3]2+The intracellular ClO of ratio fluorescent nano probe response-Ratio imaging.
Embodiment
In order that content of the present invention easily facilitates understanding, with reference to embodiment to of the present invention
Technical scheme is described further, but the present invention is not limited only to this.
Embodiment 1:m-CDs@[Ru(bpy)3]2+Structure and selectivity the response ClO of ratio fluorescent nano probe-
0.6 g m-phenylene diamine (MPD)s are dissolved in 60 mL absolute ethyl alcohol first, then solution is transitted to 100 mL polytetrafluoro
In ethene pyroreaction kettle, the h of heating response 12 at 180 DEG C, after question response kettle is cooled to room temperature, by products therefrom through post layer
Analysis chromatogram is isolated and purified(Eluant, eluent is dichloromethane:Methanol=15:1, v/v), obtained solution Rotary Evaporators will be molten
Agent is spin-dried for and is dried in vacuo, that is, obtains carbon pointm- CDs solid crystal.Again by volume 6:1 to 20 μ g/mL m-CDs
40 μ g/mL Ru (bpy) of middle addition3Cl2, it is madem-CDs@[Ru(bpy)3]2+Ratio fluorescent nano probe.
In gainedm-CDs@[Ru(bpy)3]2+The ClO of series concentration is separately added into ratio fluorescent nano probe-(ClO-'s
Concentration is followed successively by 1,2,3,4,5,6,7,8,9,10,15,20,25,30 μM), react 5 min and seen after under 365 nm uviol lamps
Examine the change of solution fluorescence intensity and color, it is seen that ratio fluorescent nano probe is gradually converted into red by blue-green(As Fig. 1 is
The ClO of various concentrations-Withm-CDs@[Ru(bpy)3]2+Reacted figure of fluorescence intensity changes).
Embodiment 2:m-CDs@[Ru(bpy)3]2+The preparation of agarose aquogel and selectivity response ClO-
Tom-CDs@[Ru(bpy)3]2+Middle addition agarose powder, will after heating stirringm-CDs@[Ru(bpy)3]2+Agarose glues
The colloid of thick shape is placed on the groove of centrifugation lid, and round pie is formed after its coolingm-CDs@[Ru(bpy)3]2+Agar
Syrup gel.
Tom-CDs@[Ru(bpy)3]2+The ClO of series concentration is separately added into agarose aquogel-(ClO-Concentration successively
For 0,10,20,30,40,50,100,200 μM), 5 min under 365 nm uviol lamps after observing its fluorescence intensity and face for reaction
Color change, while use the fluorescence imaging of digital camera shooting hydrogel(Such as Fig. 2).
Embodiment 3:m-CDs@[Ru(bpy)3]2+Ratio fluorescent nano probe is used for intracellular ClO-Ratio imaging
Willm-CDs@[Ru(bpy)3]2+Ratio fluorescent nano probe is added in the culture dish equipped with HeLa cells, in 95% CO2/
5% O2Atmosphere in culture 1 h after, wash cell 3-5 times with KRP buffer solutions, remove be introduced into HeLa cellsm-CDs@[Ru
(bpy)3]2+, cell in the case where excitation wavelength is two kinds of fluorescence channels of 488 nm and 543 nm shoot thin with Laser Scanning Confocal Microscope
Born of the same parents are imaged;Have again to intakem-CDs@[Ru(bpy)3]2+HeLa cells add 10 μM of ClO-, after further hatching 15 min,
Cell is washed with KRP buffer solutions 3-5 times, removes the ClO for being introduced into HeLa cells-, cell Laser Scanning Confocal Microscope is in excitation wave
Shooting cell imaging is carried out under a length of two kinds of passages of 488 nm and 543nm, is observedm-CDs@[Ru(bpy)3]2+Ratio fluorescent nano
Probe is to ClO-Response(Such as Fig. 3, wherein A be HeLa cells withmFluorescence after 1 h of-CDs cultures under 488nm excitation wavelengths
Co-focusing imaging figure;B is to have to intakemAfter 10 μM of ClO hatchings are added in-CDs HeLa cells, in 488nm excitation wavelengths
Under fluorescence co-focusing image;C be HeLa cells withm-CDs@[Ru(bpy)3]2+After cultivating 1 h, respectively in 488nm and
Fluorescence co-focusing image under 543nm excitation wavelengths;D is to have to intakem-CDs@[Ru(bpy)3]2+HeLa cells add
10 μM ClO-After hatching, the fluorescence co-focusing image under 488nm and 543nm excitation wavelengths respectively).
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
Claims (6)
- A kind of 1. ratio fluorescent nano probe, it is characterised in that:Withm- CDs is fluorescence probe, with [Ru (bpy)3]2+As reference Reagent, structurem-CDs@[Ru(bpy)3]2+Ratio fluorescent nano probe.
- 2. ratio fluorescent nano probe according to claim 1, it is characterised in that:The structure of the ratio fluorescent nano probe Construction method is:Willm- CDs solution and Ru (bpy)3Cl2Solution by volume 6:1 mixing, it is made describedm-CDs@[Ru(bpy)3]2+ Ratio fluorescent nano probe;Wherein,mThe concentration of-CDs solution is 20 μ g/mL, Ru (bpy)3Cl2The concentration of solution is 40 μ g/mL.
- 3. a kind of ratio fluorescent nano probe as claimed in claim 1 is in quantitative detection ClO-In application, it is characterised in that:Should Comprised the following steps with method:1)The ClO of series concentration is separately added into the ratio fluorescent nano probe-, its fluorescence is determined after the min of hybrid reaction 5 Intensity, and fluorescence intensity is drawn with ClO-The standard curve of change in concentration, draws regression equation;2)Added into another ratio fluorescent nano probe and contain ClO-Testing sample, it is strong to determine its fluorescence after the min of hybrid reaction 5 Degree, recycle step 1)Gained regression equation calculation goes out ClO in sample-Content.
- 4. a kind of ratio fluorescent nano probe as claimed in claim 1 is in Visual retrieval ClO-In application, it is characterised in that: Application process comprises the following steps:1)Agarose powder is added into the ratio fluorescent nano probe, is placed on the thick colloid of gained after heating stirring The groove of lid is centrifuged, round pie is formed after its coolingm-CDs@[Ru(bpy)3]2+Agarose aquogel;2)To gainedm-CDs@[Ru(bpy)3]2+The ClO of series concentration is separately added into agarose aquogel-, after reacting 5 min The change of its fluorescence intensity and color is observed under 365 nm uviol lamps, and simultaneously using the fluorescence of digital camera shooting hydrogel Imaging, to prepare standard color comparison card;3)To what is separately takenm-CDs@[Ru(bpy)3]2+Added in agarose aquogel and contain ClO-Testing sample, react 5 min after Observe its fluorescence intensity and color under 365 nm uviol lamps, and with step 2)Gained standard color comparison card is contrasted, visualization ClO in judgement sample-Content;Gainedm-CDs@[Ru(bpy)3]2+The content of agarose is 5% in agarose aquogel.
- A kind of 5. application of the ratio fluorescent nano probe in cell imaging as claimed in claim 1, it is characterised in that:For thin Intracellular ClO-The detection of active oxygen radical.
- 6. application of the ratio fluorescent nano probe in cell imaging according to claim 5, it is characterised in that:Its detection side Method comprises the following steps:1)The ratio fluorescent nano probe is added in the culture dish equipped with cell, in 95% CO2/5% O2Atmosphere in cultivate After 1 h, cell is washed 3-5 times with KRP buffer solutions, removes the ratio fluorescent nano probe for being introduced into cell, it is burnt aobvious using copolymerization Micro mirror shoots in the case where excitation wavelength is two kinds of passages of 488nm and 543nm and is imaged;2)To step 1)The middle cell for having ratio fluorescent nano probe of taking in adds 10 μM of ClO-, after further hatching 15 min Cell is washed with KRP buffer solutions 3-5 times, removes the ClO for being introduced into cell-, use Laser Scanning Confocal Microscope in excitation wavelength for 488 Shooting cell imaging is carried out under two kinds of fluorescence channels of nm and 543 nm, is observedm-CDs@[Ru(bpy)3]2+Ratio fluorescent nano is visited For ClO-Response, with realize to ClO-The measure of active oxygen radical.
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CN116396751A (en) * | 2023-04-03 | 2023-07-07 | 郑州中科生物医学工程技术研究院 | Fluorescent probe for double response to pH and GSSG, preparation method and application thereof |
CN116396751B (en) * | 2023-04-03 | 2024-02-27 | 郑州中科生物医学工程技术研究院 | Fluorescent probe for double response to pH and GSSG, preparation method and application thereof |
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