CN104694116A - A carbon-base fluorescence probe for a targeting living cell mitochondrion and preparation thereof - Google Patents

Abstract

Preparation of a novel living cell mitochondrion probe based on carbon dots (CDs) is disclosed. A product TPP-CDs is prepared by synthesizing the CDs surfaces of which are provided with amino by adopting citric acid and urea as raw materials through a hydrothermal method, grafting with a triphenylphosphine (TPP) small molecule the tail end of which is provided with carboxyl through an amidation reaction, and separating and purifying by utilization of an aqueous membrane and a glucan G-25 gel column. The mitochondrion probe is simple in preparation method and capable of large-scale production, has good water solubility, good light stability and wide excitation and emission spectrum ranges, and breaks the current situation that mitochondrion probes at present generally are poor in water solubility and liable to fluorescence quenching. In addition, the mitochondrion probe is low in cell toxicity and biological-friendly, and can be successfully used for mitochondrion imaging of living cells.

Description

A kind of mitochondrial carbon back fluorescent probe of target viable cell and preparation thereof

Technical field

The present invention relates to fluorescent nano material, specifically a kind of preparations and applicatio based on the Mitochondrially targeted probe of carbon point (Carbon Dots, CDs).

Background technology

CDs is as a kind of new fluorescent probe, except the exciting light spectrum width, the particle diameter that possess traditional quantum dot are little, outside good light stability, Stokes displacement is large, good biocompatibility, fluorescence lifetime are long advantageous property, especially there is hypotoxic feature, so be seen as strong substitute (Ruedas-Rama, the M.J. of quantum dot in biomarker, et al., Anal Chim Acta, 2012,751:1-23.).

Plastosome, as the energy plants of cell, participates in the significant process such as the oxidative phosphorylation of cell and lipid oxidation.As its dysfunction, then can have a strong impact on the state of cell, life entity shows the symptoms such as Fu Litelixishi ataxia, Parkinson's disease, diabetes and Heng Dingdun chorea.Therefore, plastosome imaging is for the diagnoses and treatment of subcellsular level significant (Sharma, A., et al., Biomacromolecules, 2012,13 (1): 239-52).

Current mitochondrial probe mainly contains Rhodamine 123 (Callahan J, K.J., Biomacromolecules, 2006, (7): 9), JC-1 (Wang, L., et al., Nano Lett, 2011.11 (2): p.772-80), Mitotracker (Minamikawa T, S.A., Journal of Cell Science1999 (112): 11) series etc.Although they are used widely all at present, but still there is poorly water-soluble, the easy cancellation of fluorescence, phototoxicity etc. not enough (Yang, Y., et al., Chem Commun, 2012.48 (3): 380-2).

Summary of the invention

For above problem, the invention provides a kind of preparation method of the water-soluble mitochondrial probe of carbon back of good light stability.

The technical solution used in the present invention is:

The mitochondrial carbon back fluorescent probe of a kind of target viable cell, it is the organoid probe for viable cell mitochondrial markers, take CDs as fluorophore, TPP is Mitochondrially targeted molecule, together with both being grafted on by amidate action between the carboxyl of TPP end of the amino on CDs surface.

The preparation method of described carbon back fluorescent probe: first with citric acid and urea for raw material hydrothermal method synthetic surface is with the CDs of amino, then by triphenylphosphine (TPP) small molecules (Marrache of end in amidate action grafting with carboxyl, S.and S Dhar, Proc Natl Acad Sci U S A, 2012.109 (40): 16288-93), product TPP-CDs is obtained with water system film and the separation and purification of dextran G-25 gel column.

Take citric acid as carbon source, urea is nitrogenous source, and hydrothermal method prepares the high quantum production rate CDs of N doping, by regulating and controlling the feed ratio of two kinds of raw materials, can obtain the CDs of a series of different carboxyl and amino ratio; 20ml hydrothermal reaction kettle is placed in retort furnace, and temperature of reaction is 180-200 DEG C, and the reaction times is 4-10h; The mol ratio of citric acid and urea is 6:1-0:1; Gained CDs solution is golden yellow liquid.

Because CDs is better water-soluble, and the TPP of terminal carboxyl(group) is soluble in ethanol, therefore choose the ethanol/water reaction system of 1:10-10:1 ratio, add the terminal carboxyl(group) that EDC/NHS activation is dissolved in the 40mg TPP of ethanol in advance, EDC, the molar ratio of NHS, TPP is 1:1.1:1.2, then adds 5ml and contains 20-120mg CDs aqueous solution stirring at room temperature reaction 12-48h; With dextran G-25 gel separator column separating-purifying after 0.22um water system membrane filtration, remove unreacted raw material, obtained aqueous solution is yellow liquid.

Mitochondrial probe tool prepared by the present invention has the following advantages:

(1) preparation method is simple, does not need loaded down with trivial details separation application;

(2) the Mitochondrially targeted fluorescent nano particles particle diameter synthesized is little, good water solubility;

(3) fluorescence quantum yield is high;

(4), there is not photobleaching phenomenon in good light stability;

(5) low, the good biocompatibility of cytotoxicity.

Accompanying drawing explanation

Fig. 1 is the transmission electron microscope photo of TPP-CDs;

Fig. 2 is the ultraviolet spectrogram of CDs, TPP and TPP-CDs;

Fig. 3 is the Zeta potential of CDs, TPP and TPP-CDs;

Fig. 4 is the infrared spectrogram of CDs, TPP and TPP-CDs;

Fig. 5 is the fluorescence spectrum figure of CDs and TPP-CDs;

Fig. 6 is the pH stability of CDs and TPP-CDs;

Fig. 7 is the anti-light Bleachability of CDs and TPP-CDs;

Fig. 8 is the cell mitochondrial fluorescence imaging picture of TPP-CDs;

Fig. 9 is the fluorescent co-location analysis of TPP-CDs and Mitotracker Deep Red in cell imaging.

Embodiment

Below by embodiment, the invention will be further described.

Embodiment

(1) preparation of CDs and separating-purifying:

Get citric acid 0.3mol respectively, urea 1.8mol is dissolved in 20ml deionized water, after stirring, solution is transferred in hydrothermal reaction kettle, retort furnace heats, temperature programming to 200 DEG C, reaction times is 10h, removes unreacted reactant, afterwards by for subsequent use for product lyophilize after gained golden solution freeze-drying method is concentrated after being down to room temperature with the separation and purification of dextran G-50 gel column.

(2) synthesis of TPP-CDs and separating-purifying:

Take 65mg CDs and 41mg TPP respectively, be dissolved in 5ml deionized water and ethanol respectively, first in the ethanolic soln of TPP, EDCHCl and NHS(mol ratio TPP:EDC:NHS=1:1.1:1.2 is added) under whipped state, after activated carboxyl 20min, CDs solution is slowly dropped in TPP solution, stirring at room temperature reaction 48h.Gained solution 50 DEG C revolves and boils off except ethanol, and after freeze-drying method is concentrated, G-50 sephadex column is separated and removes unreacted reactant, and products therefrom lyophilize is for subsequent use.(3) quantum yield measures (Yang, Y., et al., Chem Commun, 2012.48 (3): 380-2):

Choose Quinine Sulphate Di HC as standard substance, with 0.1M H ₂sO ₄make solvent, detect the fluorescent emission peak area under the ultraviolet absorptivity of fluorescent substance to be measured and standard substance and maximum fluorescence excitation wavelength respectively, select calculation formula to be:

In formula for quantum yield QY%, A are ultraviolet absorption value, I is fluorescence integrated emission intensity, for the refractive index of solvent, subscript S is reference, and subscript X is testing sample.

The quantum yield recording CDs and TPP-CDs is respectively 21%, and 15%.

(4) property representation of CDs and TPP-CDs

The surface tissue of (a) TPP-CDs and size dimension

Fig. 1 is the electron micrograph of TPP-CDs, and even by the TPP-CDs size that this method is obtained as seen from photo, grain size is about 25nm.

The spectral response curve of (b) CDs and TPP-CDs

Fig. 2 is the ultraviolet spectrogram of CDs and TPP-CDs.In figure there is charateristic avsorption band at 340nm place in visible CDs, the charateristic avsorption band of TPP is 261,267,274nm, and TPP-CDs has the charateristic avsorption band of CDs and TPP simultaneously, can according to this by reaction product purifies and separates during mixture after post reaction crossed by dextran G25 gel.

Fig. 3 is the infrared spectrogram of CDs and TPP-CDs.At 3411cm ^-1and 2971cm ^-1the stretching vibration absorption peak of place containing N-H and C-H, at 1750cm ^-1to 1650cm ^-1containing the stretching vibration absorption peak of C=O and the flexural vibration absorption peak of C=N and N-H in scope, at 1346cm ^-1there is the stretching vibration absorption peak of C-N at place, at 1200cm ^-1stretching vibration charateristic avsorption band containing C-O.As can be seen from the figure, existence-NH in CDs ₂with-CO-NH-, and in TPP-CDs ,-NH2 reduces, and proves part-NH ₂reaction is there occurs with-the COOH of TPP.

Fig. 4 is the Zeta potential of CDs, TPP and TPP-CDs.Can see that the Zeta potential of CDs is-18mV, illustrate that CDs surface does not only have-NH ₂, also have a large amount of-COOH.After reacting with the TPP of C-terminal, current potential increases to-7mV, has confirmed the existence of TPP group in reaction product further, and the raising of Zeta potential is conducive to cross-cell membrane transhipment (Marrache and Dhar2012) of nanoparticle.

Fig. 5 is the fluorescence spectrum figure of CDs and TPP-CDs, the increase as seen along with excitation wavelength in figure, and emmission spectrum all red shift occurs, and maximum excitation peak, at 340nm place, excites wide ranges, and when applying, the range of choice of excitation light source is wider.Maximum emission wavelength is at 420nm place, and peak width at half height is 80nm, and emission peak scope is from 410nm to 500nm, and it is multicolor luminous that this makes mitochondrial probe TPP-CDs to realize.

The fluorescent stability of (c) CDs and TPP-CDs

The RB buffered soln of preparation pH2-11, get the CDs solution that 2ml adds 200ul respectively, spectrophotofluorometer detects its fluorescence intensity.Fig. 6 is the changing conditions that the maximum fluorescence emission intensity pH value of CDs and TPP-CDs increases.Can see that raising the fluorescence intensity of CDs and TPP-CDs with pH value first raises rear in a slight decrease, and TPP-CDs comparatively its pH stability of CDs is better.

Fig. 7 is the anti-light bleaching situation of CDs and TPP-CDs.Be dissolved in by CDs and TPP-CDs respectively in substratum and be all incubated at copolymerization burnt special cell culture dish Hela cervical cancer cell with 2mg/ml concentration markers, the fluorescein-labelled Hela cell of same 20ug/ml in contrast.After hatching 24 hours altogether, PBS cleans three continuous light bleaching 110s under Olympus FV1000 laser co-focusing 488nm excitation light source 20% power, can see that the fluorescence-intensity decay of CDs and TPP-CDs is less than 10%, and under fluorescein kindred circumstances in contrast, decay reaches 57%.

Therefore can say that mitochondrial probe that we prepare possesses excellent pH stability and anti-light Bleachability.

(5) biologic applications of TPP-CDs

Fig. 8 is Hela cell mitochondrial mark photo.Because of TPP-CDs good water solubility, directly by its powder dissolution in substratum with 2mg/ml concentration markers Hela cervical cancer cell, and carry out common dye with business mitochondrial probe Mitotracker Deep Red.Olympus laser co-focusing FV1000 takes pictures.A, b are respectively TPP-CDs at 405nm (power 15%), 488nm(power 15%) fluorescence picture under exciting light, c is that Mitotracker Deep Red is at 635nm(power 2%) fluorescence picture under exciting light, d is the superposition picture of a, c.Can find out in figure, TPP-CDs all can obtain good imaging effect under 405nm and 488nm exciting light, visible TPP-CDs probe enters in tenuigenin, by the mark position of alternative line plastochondria commercialization probe Mitotracker Deep Red, TPP-CDs overlaps with business probe mark position substantially to the mark position of Hela cervical cancer cell, thinks pass flag cell mitochondrial.Fig. 9 carries out fluorescent co-location analysis to d further, and the Pearson's coefficient representing fluorescent co-location coincidence degree is respectively 0.76742.Show the mitochondrial probe successfully preparing special target.

Claims (4)

1. the mitochondrial carbon back fluorescent probe of target viable cell, is the organoid probe for viable cell mitochondrial markers, it is characterized in that:

Take CDs as fluorophore, TPP is Mitochondrially targeted molecule, together with both being grafted on by amidate action between the carboxyl of TPP end of the amino on CDs surface.

2. a preparation method for carbon back fluorescent probe described in claim 1, is characterized in that:

First with citric acid and urea for raw material hydrothermal method synthetic surface is with the CDs of amino, then pass through end in amidate action grafting and, with triphenylphosphine (TPP) molecule of carboxyl, obtain product TPP-CDs with water system film and the separation and purification of dextran G-25 gel column.

3., according to the preparation method of carbon back fluorescent probe described in claim 2, wherein the preparation of CDs is characterized in that:

Take citric acid as carbon source, urea is nitrogenous source, and hydrothermal method prepares the high quantum production rate CDs of N doping, by regulating and controlling the feed ratio of two kinds of raw materials, can obtain the CDs of a series of different carboxyl and amino ratio; 20ml hydrothermal reaction kettle is placed in retort furnace, and temperature of reaction is 180-200 DEG C, and the reaction times is 4-10h; The mol ratio of citric acid and urea is 6:1-0:1; Gained CDs solution is golden yellow liquid.

4., according to the preparation method of carbon back fluorescent probe described in claim 2, it is characterized in that:

Because CDs is better water-soluble, and the TPP of terminal carboxyl(group) is soluble in ethanol, therefore choose the ethanol/water reaction system of 1:10-10:1 ratio, add the terminal carboxyl(group) that EDC/NHS activation is dissolved in the 40mg TPP of ethanol in advance, EDC, the molar ratio of NHS, TPP is 1:1.1:1.2, then adds 5ml and contains 20-120mg CDs aqueous solution stirring at room temperature reaction 12-48h; With dextran G-25 gel separator column separating-purifying after 0.22um water system membrane filtration, remove unreacted raw material, obtained aqueous solution is yellow liquid.