CN106281304B - A kind of fluorescence probe and preparation method thereof that can be used for malonaldehyde in living cells and be imaged - Google Patents
A kind of fluorescence probe and preparation method thereof that can be used for malonaldehyde in living cells and be imaged Download PDFInfo
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Abstract
The invention discloses a kind of fluorescence probes and preparation method thereof that can be used for malonaldehyde in living cells and be imaged, and belong to fluorimetric assay for biological materials technical field.The two positions are connected to obtain by fluorescence probe of the invention using 1,8-naphthalimide as Fluorescent reporter group with the effect partial that benzoyl hydrazine (containing substituted benzoyl hydrazine) is probe and malonaldehyde by certain linking group (such as imino group).The fluorescence probe can effectively detect malonaldehyde under physiological environment, and the bio-imaging of endogenous and exogenous malonaldehyde is successfully realized in living cells.The present invention realizes the malonaldehyde bio-imaging in living cells for the first time, currently preferred probe MDAP-1 high sensitivity (minimum detectability is down to 0.6 μM), fluorescence response is strong, the response time is short, responds to the selectivity that malonaldehyde has the advantage that unstressed configurations such as metal ion, oxidation activity product and active carbonyl compounds.
Description
Technical field
The present invention relates to fluorescent dyes used in fluorimetric assay for biological materials technical field, and in particular to one kind can be used for living cells
The fluorescence probe and preparation method thereof of interior malonaldehyde imaging.
Background technique
Fluorescence probe has many advantages, such as that spatial and temporal resolution is high, easy to operate and small to life process interference, is research life
The technical way of science, clinical treatment diagnosis and fluoroimmunoassay.In terms of the research of cell biology, fluorescence spectrum
Be usually used in situations such as tracking position and the variation of intracellular special component (target), thus disclose the specific physiology of biological target/
Pathologic function.There is the considerable commercial fluorescence probe for certain particular organisms target at present, such as can be used for cell
Dyestuff Hoechst 33342 of nuclear imaging etc..Since there are many intracellular biological target, visited for the fluorescence of particular target
The research of needle is current popular domain.
In terms of the growth and development of life entity and disease generation, oxidative stress (oxidative stress) plays one
Considerable role.Oxidative stress refers to that oxygen radical excessively generates and causes intracellular Antioxidative Defense System impaired, causes
Make oxygen radical and associated metabolites excessive aggregation, to generate the pathological state of a variety of toxicity to cell.Malonaldehyde
The important biomolecule marker (biomarker) of (Malondialdehyde, MDA) as oxidative stress, is intracellular lipid
The oxidation product of compound, the generation close relation of expression and a variety of diseases, it was reported that including leukaemia, glycosuria
The generation of a variety of diseases such as disease, tumour is all along with the raising of mda content, and therefore, the detection to intracellular malonaldehyde is right
In research biology related with oxidative stress and physiological action, the pathogenic mechanism of announcement related disease and the corresponding diagnosis and treatment side of offer
Method etc. has very important meaning.
Have a variety of detection methods for malonaldehyde at present, clinically using it is most wide be 2- thiobarbituricacidα- method,
This method is to be condensed the product progress ultraviolet detection of generation under strong acid hot environment to 2- thiobarbituricacidα- and malonaldehyde, excellent
Point is that detection limit is low, high sensitivity, the disadvantage is that cumbersome, poor specificity, poor repeatability (referring to Anal.Biochem.1979,
95,351-358 and Free Radical Biol.Med.1990,9,515-540).The shortcomings that overcome this detection method,
It is developed there are many detection method, including liquid chromatography, electrophoresis, Raman spectroscopy, mass spectrography etc., these methods
Defect is all the cumbersome pre-treatment of test sample, these pretreatment process generally require the reaction item of highly acid or/and heating
Part, thus detect object be substantially body fluid such as urine, serum etc., it is impossible to be used under physiological condition in living cells at
Picture.
Summary of the invention
It is an object of the invention to overcome the defect that can only currently detect malonaldehyde with external detection method, providing one kind can
The fluorescence probe being imaged for malonaldehyde in living cells.The object of the invention is also to provide the preparation methods of the fluorescence probe.
The purpose of the invention is achieved by the following technical solution:
It is a kind of to can be used for the fluorescence probe that malonaldehyde is imaged in living cells, general structure are as follows:
In general formula: X NH, CONH, NHCO, O, S, SO2、SO2NH or NHSO2；
R1For (CH2)nR5、(CH2)mOR6Or (CHR7CH2O)pR6, R2For (CH2)nR5Or (CH2)mOR6；
R3、R4It is H, Cl, Br, I, NO2、CN、CONHNH2Or COOR7；
R1、R2In: n, m, p are the integer in 0-18, R5For H or COOR6, R6For H or C1-18Alkyl (carbon atom number 1-
18 alkyl)；
R3、R4In: R7For H, C1-18Alkyl or M, M are metal ion such as Na+、K+Deng.
Preferably, in general formula: X NH, R1For CH2CH2CH3, R2For H, R3For CONHNH2Or H, R4For NO2、CONHNH2Or
H。
The preparation method that can be used for the fluorescence probe that malonaldehyde is imaged in living cells, includes the following steps: with ammonia
The methyl benzoate derivative that base replaces is starting material, passes through 1, the 8- naphthalene diformazan of Buchwald-Hartwig reaction and bromo
Acid imide coupling generates esters intermediary, then is condensed to yield fluorescence probe with hydrazine hydrate.
The fluorescence probe that malonaldehyde is imaged in living cells that can be used for is detecting the application in malonaldehyde: described is glimmering
Light probe can effectively detect malonaldehyde under physiological environment, and be successfully realized in living cells endogenous and exogenous the third two
The bio-imaging of aldehyde.
The present invention is the effect partial of probe and malonaldehyde with benzoyl hydrazine (containing substituted benzoyl hydrazine), with 1,8- naphthalene two
Carboximide is Fluorescent reporter group, and being connected the two positions by certain linking group (such as imino group) can be used for
The fluorescence probe that malonaldehyde is imaged in living cells.
The prior art is had the following advantages that the present invention and effect:
1, the present invention realizes the malonaldehyde bio-imaging in living cells for the first time, there is no similar report at present.
2, fluorescence probe MDAP-1 preferred for this invention, Stokes shift can reach~180nm, to keep away completely
The self-absorption for having exempted from dyestuff caused by small Stokes shift is serious, exciting light scattered color interference and thus bring detection spirit
The disadvantages of sensitivity declines is conducive to the biologic applications for playing dyestuff.
3, fluorescence probe of the invention has the ability of (pH=7.4) quantitative detection malonaldehyde under physiological environment, quantitative
Detection interval is 2-200 μM, and minimum detectability is down to 0.6 μM, sensitivity with higher.
4, fluorescence probe MDAP-1 fluorescence response of the invention is strong, and the response time is short, in the malonaldehyde effect with 1mM,
Enhance in 60 times of fluorescence enhancement in 5 minutes, in 10 minutes and reach 150 times in 110 times, 30 minutes, this big fluorescence enhancement and
Fluorescence quick response, it is high for reactivity and in the cell for the detection of the malonaldehyde of fast degradation, it is highly beneficial.
5, fluorescence probe MDAP-1 of the invention shows excellent selectivity.MDAP-1 is to each metal ion species, various
Oxidation activity product and various active carbonyls are all almost without fluorescence response.
6, fluorescence probe of the invention can be used for the imaging of endogenous and exogenous malonaldehyde in living cells.In hydrogen peroxide
The cellular oxidation induced stress during, the fluorescence of MDAP-1 along with hydrogen peroxide concentration increase also occur it is different degrees of
Enhancing, disclose the content of intracellular malonaldehyde along with the reinforcement of oxidative stress process raised bioprocess.Also,
Be added equivalent antioxidant --- after ascorbic acid and hydrogen peroxide, fluorescence does not change, this also confirms ascorbic acid from side
Oxidation resistance, illustrate the pharmic function of ascorbic acid.In terms of the cell imaging of exogenous malonaldehyde, MDAP-1's is glimmering
Light shows the phenomenon that gradually increasing along with the increase of additional concentration of malondialdehyde.
Detailed description of the invention
Fig. 1 is the fluorescence multiplication comparison diagram of probe MDAP-1, MDAP-2, MDAP-3 (10 μM) and MDA (1mM).
Fig. 2 is the fluorescence titration curve graph of MDAP-1 (10 μM) and MDA, MDA concentration is followed successively by 0 from the bottom to top, 2,5,10,
20、50、100、200、400、600、800、1000μM。
Fig. 3 is the fluorescence response relational graph of MDAP-1 Yu MDA concentration, and it is 0-200 μM that interior illustration, which is shown in MDA concentration ranges,
When, fluorescence intensity and MDA concentration are in a linear relationship.
Fig. 4 is fluorescence intensity-time chart of MDAP-1 (10 μM) and MDA (1mM) effect.
Fig. 5 is the fluorescence response figure of metal ion, ROS and RCS to MDAP-1, in figure, 1:MDAP-1,2:Mg2+, 3:Cd2 +, 4:Ca2+, 5:Mn2+, 6:Zn2+, 7:Hg2+, 8:Fe2+, 9:Cu2+, 10:H2O2, 11:ClO-；12:O2 -, 13:1O2, 14:OH,
15: acetone, 16: acetaldehyde, 17: glutaraldehyde, 18: acetylacetone,2,4-pentanedione, 19: formaldehyde, 20: glyoxal, 21: pyroracemic aldehyde, 22: malonaldehyde；
Black column and column with oblique line represent the fluorescence intensity that detectable substance concentration is respectively 0.1 and 1mM.
Specific embodiment
Further detailed description is done to the present invention below with reference to embodiment, but should not be construed as limiting the invention,
Without departing from the spirit and substance of the case in the present invention, modifications or substitutions made by the method for the present invention, step or condition are belonged to
In the scope of the present invention.Unless otherwise specified, technological means used in embodiment be well known to those skilled in the art it is normal
Rule means.
Embodiment 1
The synthetic route that can be used for malonaldehyde is imaged in living cells fluorescence probe MDAP-1, MDAP-2, MDAP-3 is as follows
Shown in reaction equation:
1. using Buchwald-Harwig amino coupled method synthesis intermediary (3a, 3b, 3c)
A. the synthesis of compound 3a
Weigh bromo- 2- propyl -1H- benzo [de] isoquinolin -1,3 (the 2H)-diketone of 300 milligrams of 6- (compound 1,
2.0mmol) (synthesis of compound 1 see J.Org.Chem.2013,78,3980-3988) is added in 50 milliliters of two mouth flasks,
Sequentially adding 370 milligrams of 5- amino -2- nitrobenzene methyls (compound 2a, 1.89mmol), (synthesis of compound 2a is shown in
Tetrahedron.Lett.2005,46,7477-7481), 42 milligrams of Pd2(dba)3(0.046mmol) and 66 milligrams (±) -2,
2'- is bis--(diphenyl phosphine) -1,1'- dinaphthalene (BINAP), it deaerates after being stirred, 18 milliliters of fresh distillations is added with syringe
Toluene is heated to boiling, and reaction overnight, is cooled to room temperature under reflux conditions.Chromatographic column is gone up after solvent is removed under reduced pressure, eluent is
Ethyl acetate/petroleum ether (4:1, v/v), obtains orange powder target product.Yield: 53.2%.
1H NMR(DMSO-d6, 400MHz) and δ 10.03 (s, 1H), 8.63 (d, J=8.0Hz, 1H), 8.54 (d, J=
8.0Hz, 1H), 8.43 (d, J=8.0Hz, 1H), 8.14 (d, J=12.0Hz, 1H), 7.86 (t, J1=J2=8.0Hz, 1H),
7.77 (d, J=8.0Hz, 1H), 7.48 (m, 1H), 7.44 (d, J=8.0Hz, 1H), 4.01 (t, J1=J2=8.0Hz, 2H),
3.85(s,3H),1.65(m,2H),0.93(t,J1=J2=8.0Hz, 3H)13C NMR(DMSO-d6,101MHz)δ166.57,
163.85,163.21,148.98,143.41,138.47,132.62,131.71,131.13,129.61,129.46,127.50,
126.91,124.97,122.79,118.30,116.73,116.07,103.92,53.55,41.45,21.26,
11.75.HRMS–ESI(m/z):[M+H+]calcd.for C23H20N3O6,434.1352；found,434.1388.
B. the synthesis of compound 3b
The similar compound 3a of the synthetic method of compound 3b, by 370 milligrams of 5- amino -2- nitrobenzene methyl (chemical combination
Object 2a, 1.89mmol) change 285 milligrams of 4-aminobenzoic acid methyl esters (compound 2b, 1.89mmol), the same compound of remaining step into
The synthesis of 3a.Yield: 45.6%.
1H NMR(DMSO-d6, 400MHz) δ 9.64 (s, 1H), 8.73 (d, J=8.0Hz, 1H), 8.51 (d, J=8.0Hz,
1H), 8.35 (d, J=8.0Hz, 1H), 7.96 (d, J=8.0Hz, 2H), 7.83 (t, J1=J2=8.0Hz, 1H), 7.60 (d, J
=8.0Hz, 1H), 7.44 (d, J=8.0Hz, 2H) .3.99 (t, J1=J2=8.0Hz, 2H) .3.84 (s, 3H), 1.65 (m,
2H),0.92(t,J1=J2=8.0Hz, 3H)13C NMR(DMSO-d6,101MHz)δ165.77,163.50,162.76,
146.15,145.16,132.66,131.08,130.83,129.16,129.15,125.66,123.09,122.73,122.11,
118.80,113.69,111.63,51.82,40.93,20.87,11.36.HRMS–ESI(m/z):[M+H+]calcd.for
C23H21N2O4,389.1501；found,389.1490.
C. the synthesis of compound 3c
The similar compound 3a of the synthetic method of compound 3c, by 370 milligrams of 5- amino -2- nitrobenzene methyl (chemical combination
Object 2a, 1.89mmol) change 285 milligrams of 3- Methyl anthranilates (compound 2c, 1.89mmol), the same compound of remaining step into
The synthesis of 3a.Yield: 63.5%.
1H NMR(DMSO-d6, 400MHz) δ 9.54 (s, 1H), 8.80 (d, J=8.0Hz, 1H), 8.52 (d, J=8.0Hz,
1H), 8.32 (d, J=8.0Hz, 1H), 7.96 (s, 2H), 7.83 (t, J1=J2=8.0Hz, 1H), 7.72 (d, J=8.0Hz,
1H), 7.69 (d, J=8.0Hz, 1H), 7.58 (t, J1=J2=8.0Hz, 1H), 7.37 (d, J=8.0Hz, 1H), 4.00 (t, J1
=J2=8.0Hz, 2H) .3.87 (s, 3H), 1.64 (m, 2H), 0.92 (t, J1=J2=8.0Hz, 3H)13CNMR(DMSO-d6,
101MHz)δ165.91,163.54,162.73,146.79,141.19,133.09,131.00,130.86,129.90,
129.30,128.88,125.98,125.30,123.99,122.10,122.01,122.00,112.06,108.46,52.25,
40.85,20.88,11.35.HRMS–ESI(m/z):[M+H+]calcd.for C23H21N2O4,389.1501；found,
389.1490.
2. the synthesis of fluorescence probe MDAP-1, MDAP-2, MDAP-3
The synthesis of A.MDAP-1
110 milligrams of compound 3a (0.26mmol) are dissolved in 8 ml methanols, 0.4 milliliter of hydrazine hydrate (8.25mmol) is added,
Heating stirring is refluxed overnight, it is cooling after decompression boil off solvent, crude oil through chromatographic column (eluent is ethyl acetate/methanol,
10:1) isolated orange target product MDAP-1.Yield: 55.0%.
1H NMR(DMSO-d6, 400MHz) and δ 10.03 (s, 1H), 8.63 (d, J=8.0Hz, 1H), 8.54 (d, J=
8.0Hz, 1H), 8.43 (d, J=8.0Hz, 1H), 8.14 (d, J=12.0Hz, 1H), 7.86 (t, J1=J2=8.0Hz, 1H),
7.77 (d, J=8.0Hz, 1H), 7.48 (m, 1H), 7.44 (d, J=8.0Hz, 1H), 4.01 (t, J1=J2=8.0Hz, 2H)
.3.85(s,3H),1.65(m,2H),0.93(t,J1=J2=8.0Hz, 3H)13C NMR(DMSO-d6,101MHz)δ166.57,
163.85,163.21,148.98,143.41,138.47,132.62,131.71,131.13,129.61,129.46,127.50,
126.91,124.97,122.79,118.30,116.73,116.07,103.92,53.55,41.45,21.26,
11.75.HRMS–ESI(m/z):[M+H+]calcd.for C23H20N3O6,434.1352；found,434.1388.
The synthesis of B.MDAP-2
The similar MDAP-1 of the synthetic method of MDAP-2 changes 110 milligrams of compound 3a (0.26mmol) into 100 milligrams of chemical combination
Object 3b (0.26mmol), remaining step obtain buff target product MDAP-2 with the synthesis of MDAP-1.Yield: 37.4%.
1H NMR(DMSO-d6, 400MHz) and δ 9.73 (s, 1H), 9.55 (s, 1H), 8.78 (d, J=12Hz, 1H), 8.51
(d, J=12Hz, 1H), 8.32 (d, J=8.0Hz, 1H), 7.89 (d, J=8.0Hz, 2H), 7.82 (t, J1=J2=8.0Hz,
1H), 7.48 (d, J=8.0Hz, 1H), 7.43 (d, J=8.0Hz), 4.52 (brs, 2H), 3.99 (t, J1=J2=8Hz, 2H),
1.64(m,2H),0.92(t,J1=J2=8.0Hz, 3H)13CNMR(DMSO-d6,101MHz)δ165.43,163.54,
162.76,146.21,143.64,132.98,131.03,129.28,129.15,128.40,127.47,125.37,122.41,
122.02,119.99,112.49,109.81,40.88,20.88,11.36.HRMS–ESI(m/z):[M+H+]calcd.for
C22H21N4O3,389.1614；found,389.1603.
The similar MDAP-1 of the synthetic method of C.MDAP-3 changes 110 milligrams of compound 3a (0.26mmol) into 100 milligrams of changes
It closes object 3c (0.26mml), remaining step obtains orange target product MDAP-3 with the synthesis of MDAP-1.Yield: 42.8%.
1H NMR(DMSO-d6, 400MHz) and δ 9.86 (s, 1H), 9.46 (s, 1H), 8.78 (d, J=8.0Hz, 1H), 8.46
(d, J=8.0Hz, 1H), 8.25 (d, J=8.0Hz, 1H), 7.86 (s, 1H), 7.77 (t, J1=J2=8.0Hz, 1H), 7.61
(m, 1H), 7.52 (m, 2H), 7.30 (d, J=8.0Hz, 1H), 4.57 (brs, 2H), 3.96 (t, J1=J2=8.0Hz, 2H),
1.62(m,2H),0.91(t,J1=J2=8.0Hz, 3H)13C NMR(DMSO-d6,101MHz)δ165.58,163.59,
162.78,147.16,140.68,134.55,133.23,129.56,129.26,128.85,125.20,124.61,122.10,
121.88,121.71,120.66,111.50,108.23,40.85,20.87,11.33.HRMS–ESI(m/z):[M+H+]
calcd.for C22H21N4O3,389.1614；found,389.1604.
The fluorescence response of embodiment 2 fluorescence probe MDAP-1, MDAP-2, MDAP-3 and malonaldehyde (MDA)
The stock solution (100mM) that fluorescence probe MDAP-1, MDAP-2 and MDAP-3 are prepared with DMSO takes a small amount of EP that is added to manage
In, it is diluted with PBS buffer solution (10mM, pH 7.4), a certain amount of MDA solution is added, then be diluted to probe end with DMSO and PBS
Concentration is 10 μM, the final concentration of 1mM of MDA (solvent is constituted at this time: DMSO:PBS=1:9, volume ratio, pH 7.4).EP is managed
Its fluorescence response situation is measured after incubating four hours at 37 DEG C.Taking maximum excitation wavelength is 370nm, the transmitting of three compounds
Wavelength is all in 550-553nm or so, and Stokes shift is all in 180nm or so, this Stokes shift is relative to common
Be for fluorescence probe it is sizable, can effectively avoid interference of the exciting light to transmitting, be highly beneficial for cell imaging；
Before the fluorescence intensity of three probes with MDA relative to reacting, 174,50 and 140 times (see Fig. 1) are enhanced respectively.Due to MDAP-1
Fluorescence response it is ideal, therefore select the probe is further to be studied.
The correlation of the amount of 3 fluorescence probe MDAP-1 of embodiment and malonaldehyde (MDA)
It takes the DMSO stock solution of MDAP-1 on a small quantity in EP pipe, is diluted with PBS buffer solution (10mM, pH 7.4), be added one
Quantitative MDA solution, then be diluted to final concentration of 10 μM of probe with PBS and DMSO, the final concentration of MDA is respectively 0,2,5,10,
20,50,100,200,400,600,800,1000μM.37 DEG C incubate 4 hours, detect solution fluorescence intensity (excitation wavelength:
370nm；Launch wavelength: 553nm.The fluorogram of MDAP-1-MDA titration is shown in Fig. 2；Its fluorescence intensity and the relational graph of MDA are shown in figure
3.Solvent is constituted: DMSO:PBS=1:9, volume ratio, pH 7.4).The results show that increase of the fluorescence intensity of MDAP-1 with concentration
And increase, and in 0-200 μM of concentration range, the concentration of fluorescence intensity and MDA show linear relationship, minimum detection limit warp
It is calculated as 0.6 μM.
Fluorescence-time correlation of 4 fluorescence probe MDAP-1 of embodiment and malonaldehyde (MDA)
It takes the DMSO stock solution of MDAP-1 on a small quantity in EP pipe, is diluted with PBS buffer solution (10mM, pH7.4), is added certain
The MDA solution of amount, then it is diluted to PBS and DMSO that probe is 10 μM final concentration of, final concentration of 1mM of MDA.It is incubated at 37 DEG C,
The solution of different time points is taken to detect its fluorescence response situation (excitation wavelength: 370nm；Launch wavelength: 553nm.Solvent is constituted:
DMSO:PBS=1:9, volume ratio, pH 7.4).MDAP-1 to the fluorescence response of MDA quickly, at 5 minutes 60 times of fluorescence enhancement,
110 times of fluorescence enhancement at 10 minutes reaches about 150 times (see Fig. 4) for 30 minutes.Since activity is high in the cell, is easy to drop by MDA
Solution, MDAP-1 are advantageous the imaging of MDA in the cell the good response speed of MDA.
Selectivity of the 5 fluorescence probe MDAP-1 of embodiment to the fluorescence response of MDA
The investigation object of the present embodiment is divided into three levels.
The MDAP-1 primarily looked at is to the fluorescence response situation of ion, by Mg2+、Cd2+、Ca2+、Mn2+、Zn2+、Hg2+、Fe2+
And Cu2+It is separately added into MADP-1 solution that (ion final concentration is respectively 0.1 and 1mM；Final concentration of 10 μM of MDAP-1；Solvent structure
At: DMSO:PBS=1:9, volume ratio, pH 7.4), the fluorescent emission situation of 553nm is detected under 370nm excitation, as a result sees figure
The probe MDAP-1 of 5, He Weijia ions compares, and probe MDAP-1 fluorescence is without significant change after adding ion.
Since MDA is the product of oxidative stress, and oxidative stress can also generate reactive oxygen species (Reactive Oxidative
Stress, ROS), therefore ROS has been investigated to the disturbed condition of probe, by H2O2、ClO-、O2 -、1O2And OH is added separately to
(ROS final concentration is respectively 0.1 and 1mM in MDAP-1 solution；Final concentration of 10 μM of MDAP-1；Solvent is constituted: DMSO:PBS=1:
9, volume ratio, pH 7.4), the fluorescent emission situation of 553nm is detected under 370nm excitation, as a result sees Fig. 5, after same addition ROS
Probe MDAP-1 fluorescence is without significantly changing.
Active carbonyl compound (the Reactive carbonyl being closer to MDA structure is finally investigated
Species, RCS), the acetone of 1mM, acetaldehyde, glutaraldehyde, acetylacetone,2,4-pentanedione, formaldehyde, glyoxal and pyroracemic aldehyde are added to MDAP-1
(RCS final concentration is respectively 0.1 and 1mM in solution；Final concentration of 10 μM of MDAP-1；Solvent is constituted: DMSO:PBS=1:9, volume
Than pH 7.4), the fluorescent emission situation of 553nm is detected under 370nm excitation, as a result sees Fig. 5, same probe after RCS is added
MDAP-1 fluorescence is without significantly changing.The above result shows that the fine selectivity to MDA of MDAP-1.
The cytotoxicity of 6 fluorescence probe MDAP-1 of embodiment
It is intracellular that the MDAP-1 (0,1,5,10 μM) of various concentration is added to the Hela being incubated for, it is small to continue incubation 24
When, using the proliferative conditions of mtt assay detection cell.It is bright that test result shows that MDAP-1 is not shown under 0-10 μM of concentration
Aobvious cytotoxicity.
Intracellular exogenous and endogenous MDA the imaging of embodiment 7
1. the cell imaging of exogenous MDA
5 μM of MDAP-1 is added in the Hela cell culture medium being incubated for, is separately added into after incubating 30 minutes different dense
The malonaldehyde (0,0.1,0.2,0.5mM) of degree continues to incubate 30 minutes, PBS buffer solution (pH 7.4) concussion washing is added 3 times (often
Secondary 5 minutes), cell culture medium is added, observes cellular morphology under laser confocal scanning microscope.It is using excitation light source
400nm.Collect the imaging picture of 500-650nm.The results show that MDAP-1 can make Hela cell have faint fluorescence,
Under the MDA effect of 0.1mM, fluorescence intensity enhances about 0.2 times；Under the MDA effect of 0.2mM, fluorescence intensity enhances about 1.0 times；
And under the MDA of 0.5mM effect, fluorescence intensity enhances about 2.7 times.The above results show that MDAP-1 can be used for exogenous MDA's
Cell imaging.
2. the cell imaging of endogenous MDA
Due to MDA be cellular oxidation stress product, the generation of endogenous MDA can pass through hydrogen peroxide inducing cell
Oxidative stress is realized.In this experiment, MDAP-1 of the HeLa cell first with 5 μM is incubated for 30 minutes, is using excitation light source
400nm is separately added into the hydrogen peroxide of 0.1 and 0.5mM at 0 moment, and the fluorescence intensity of 500-650nm was collected every 30 seconds.?
30 second moment fluorescence intensity has reached platform, and (total detection time is 6 minutes) fluorescence intensity is without significantly changing in the later time
Become.Under the hydrogen peroxide effect of 0.1mM, fluorescence intensity increases 2% with comparing for hydrogen peroxide is not added, and 0.5mM's is double
Oxygen water then increases 20%.
In order to prove MDA generation really due to oxidative stress generate, a kind of antioxidant --- ascorbic acid
(0.5mM) and hydrogen peroxide (0.5mM), which are simultaneously added to 5 μM of MDAP-1, to be made in used HeLa cell, and operating process is same as above,
The variation of fluorescence is had not seen in entire observation time.The above results illustrate the endogenous that MDAP-1 can be used for being induced by hydrogen peroxide
The cell imaging of MDA.
Claims (3)
1. a kind of can be used for the fluorescence probe that malonaldehyde is imaged in living cells, it is characterised in that general structure are as follows:
In general formula: X CONH, NHCO, S, SO2、SO2NH or NHSO2；
R1For (CH2)nR5、(CH2)mOR6Or (CHR7CH2O)pR6, R2For (CH2)nR5Or (CH2)mOR6；
R3、R4It is H, Cl, Br, I, NO2、CN、CONHNH2Or COOR7；
R1、R2In: n, m, p are the integer in 0-18, R5For H or COOR6, R6For H or C1-18Alkyl；
R3、R4In: R7For H, C1-18Alkyl or M, M are metal ion；
Or: X NH, R1For CH2CH2CH3, R2For H, R3For CONHNH2Or H, R4For NO2、CONHNH2Or H.
2. the preparation method of fluorescence probe described in claim 1, it is characterised in that include the following steps: the benzene replaced with amino
Methyl formate derivative is starting material, is coupled by Buchwald-Hartwig reaction and the 1,8-naphthalimide of bromo
Esters intermediary is generated, then is condensed to yield fluorescence probe with hydrazine hydrate.
3. application of the fluorescence probe described in claim 1 in detection malonaldehyde.
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