CN106518762A - Fluorescent probe for detecting formaldehyde in cell endoplasmic reticulums - Google Patents
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Abstract
The invention provides a fluorescent probe for detecting formaldehyde in cell endoplasmic reticulums. The chemical name of the fluorescent probe is 4-hydrazino-N-(2-aminoethyl-4-methyl benzenesulfonamido)naphthalimides, and the short name is Na-FA-ER. The invention further discloses properties and functions of the probe in the fields of fluorescence detection and bioimaging. According to the fluorescent probe, the fluorescence intensity of a probe solution is gradually strengthened along with the increase of formaldehyde concentration, so that the probe has relatively good application values in sensing detection of formaldehyde in in-vivo and in-vitro environments and has certain contribution for prevention and control work of formaldehyde.
Description
Technical field
Present invention is mainly applied to the detection of biological content of formaldehyde in vivo and in vitro, belongs to organic molecule fluorescence
Probe field.
Background technology
Formaldehyde also known as formaldehyde, of many uses in its daily productive life, which is in synthetic resin, surfactant, plastics, rubber
The industries such as glue, leather, papermaking, dyestuff, pharmacy, pesticide, photographic film, explosive, construction material are all widely used.However, first
Although aldehyde have the good reputation of " omnipotent industrial chemicals ", but and an out-and-out Health Killer.Many evidences all show, if
Mankind's Long Term Contact formaldehyde will cause a series of diseases, such as Alzheimer disease, cancer, apoplexy and miscarriage etc..It can be said that first
Aldehyde is a double-edged sword, if the formaldehyde in vivo and in environment can be detected in time, for human social development is by with important
Meaning.Therefore, formaldehyde fluorescent molecular probe field is all the focus of people's research all the time.This will to prevent and treat formaldehyde poisoning,
And the research of the physiological function of assessment cell, tissue and the generation of intravital formaldehyde, metabolic pathway and formaldehyde has important meaning
Justice.
Endoplasmic reticulum is the maximum organelle of intercellular membrane area, rich in abundant enzyme on the film surface that its height is folded,
To having great importance with the normal vital movement of organism.And under pathological condition, the membrane area and form of endoplasmic reticulum
Very big change can occur, so as to directly affect the generation of demethylation enzyme, and the content of formaldehyde in making endoplasmic reticulum changes.This
Invention is intended to invent a kind of probe of energy quick detection endoplasmic reticulum content of formaldehyde, and this also will produce formaldehydogenic machine to studying endoplasmic reticulum
Reason is significant.
At present, reported that moral Analysis Methods for Formaldehyde mainly has spectrophotography, electrochemical detection method, gas chromatogram both at home and abroad
Method, liquid chromatography, sensor method etc., but these detection methods often lay particular stress on field difference, and some are even cumbersome, should
With the destruction that can also carry out unrepairable in inconvenience, and test process to sample.Therefore, develop a kind of simple and fast, it is sensitive,
The method of efficient test content of formaldehyde is particularly important.Fluorescent probe there is popularity, high sensitivity and specificity and
Receive significant attention.
The content of the invention
Formaldehyde as mankind's daily life in a kind of common contaminant, with growth in the living standard, the mankind are also more next
More pay attention to impact of the formaldehyde to its daily life quality, therefore a kind of quick succinct detection method of invention has become everybody and grinds
The focus direction studied carefully.In order to tackle this demand, the problem to be solved in the present invention is to provide a kind of fluorescence of detection formaldehyde
Probe (Na-FA-ER).Probe of the present invention can be used to detect the formaldehyde in intracellular endoplasmic reticulum by Imaging-PAM.
The fluorescent probe of detection formaldehyde of the present invention, the fluorescent probe are named as 4-diazanyl-N-(2-amino
Ethyl-4-Methyl benzenesulfonyl amido) naphthalimide, abbreviation Na-FA-ER, its chemical structural formula such as formula () institute
Show：
（）
The preparation method of the fluorescent probe (Na-FA-ER) of above-mentioned detection formaldehyde is：
Prepare compound (3)：In the reaction vessel of suitable size, toluene is added thereto to, adds ethylenediamine (1), to first
Benzene sulfonyl chloride (2), is heated to reflux synthesizing compound N-this sulfonamide of (2-amino-ethyl)-4-methyl (3).
Prepare compound (5)：In the reaction vessel of suitable size, 1,8-4-bromonaphthalic anhydrides are added
(4), ethanol, N-this sulfonamide of (2-amino-ethyl)-4-methyl (3), be heated to reflux 4-bromo- N of generation-
(2 amino-ethyl-4-Methyl benzenesulfonyl amido) naphthalimide (5).
Prepare Na-FA-ER：In the reaction vessel of suitable size, 4-bromo- N-(2-amino second is added
Base-4-Methyl benzenesulfonyl amido) naphthalimide (5), add ethanol afterwards, under stirring add hydrazine hydrate (6), afterwards plus
Hot reflux generates compound 4-diazanyl-N-(2-amino-ethyl-4-Methyl benzenesulfonyl amido) naphthalimide
(7), referred to as：Na - FA - ER.
Above-mentioned Na-FA-ER to prepare reaction equation as follows：
。
Formaldehyde fluorescent probe Na-FA-ER detections direction of the present invention is environment and organism endoplasmic reticulum targeting formaldehyde
Detection.
As mentioned above fluorescent probe Na-FA-ER can sensitive single-minded identification formaldehyde, and by suppressing a-PET effects
Strengthen the mode of fluorescence realizing the identification of PARA FORMALDEHYDE PRILLS(91,95).
Fluorescent probe Na-FA-the ER of detection formaldehyde of the present invention itself with weaker yellow fluorescence, this be by
In diazanyl itself as an electron-donating group, its own two pairs of lone electron produces a-PET effects to whole fluorescence molecule group
Answer and the fluorescence of quencher molecule or so that probe molecule fluorescent weakening.After probe is acted on formaldehyde molecule, compound N a-
There is condensation reaction with formaldehyde in the diazanyl on FA-ER, be transformed into methylene hydrazine structure so that its a-PET effect is pressed down
System, causes fluorescence intensity substantially to increase.
Recognition mechanism is as follows：
。
Experiments prove that, after formaldehyde is added, the fluorescence intensity of probe solution is significantly increased, and the result also demonstrate that
Its potential value in the application of fluorescence imaging biology field.
Description of the drawings
Fig. 1：The nuclear magnetic spectrogram of probe Na-FA-ER（Hydrogen is composed and carbon is composed）；
Fig. 2：The ultra-violet absorption spectrum of probe Na-FA-ER；
Fig. 3：The selective enumeration method of probe Na-FA-ER PARA FORMALDEHYDE PRILLS(91,95)s；
Fig. 4：The titration experiments that probe Na-FA-ER are acted on formaldehyde；
Fig. 5：The dynamic experiment that probe Na-FA-ER are interacted with formaldehyde；
Fig. 6：The exogenous formaldehyde cell imaging experiment of probe Na-FA-ER；
Fig. 7：The endogenous formaldehyde cell imaging experiment of probe Na-FA-ER；
Fig. 8：The common location cell imaging experiment of probe Na-FA ER.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention will be further described, but the present invention is not limited by following embodiments
System, in embodiment, the number of compound corresponds to the number of compound in such scheme.
Embodiment 1
The synthesis of compound N-this sulfonamide of (2-amino-ethyl)-4-methyl (3)：
。
It is in 25 mL round-bottomed flasks, slow to (1) 1 mmol of ethylenediamine in toluene (5mL) under the conditions of 40 DEG C,
(2) 1.0 mmol of paratoluensulfonyl chloride is added in the case of being sufficiently stirred for, after being heated to reflux 3-4 h, room temperature is cooled to, to
The HCl/water solution of 5ml 1M, sucking filtration is added to remove filtering residue in reaction, filtrate is neutralized to a large amount of white solids of precipitation with NaOH and is
Only, reduce pressure sucking filtration, and solid vacuum drying obtains solid N-this sulfonamide of (2-amino-ethyl)-4-methyl (3).Yield：
72 %.Product directly carries out next step reaction without purification.
4-bromo- N of compound-(2-amino-ethyl-4-Methyl benzenesulfonyl amido) naphthalimide (5)
Synthesis：
。
In the round-bottomed flask of 25 mL, compound N-(2-amino-ethyl)-4-methyl benzenesulfonamide is added
(3) 0.5 mmol and 1, (4) 0.5 mmol of 8-4-bromonaphthalic anhydride mix, and are added thereto to 2 mL ethanol and flow back
Stirring, is cooled to room temperature, reaction system is placed in -5 DEG C of environment, has a large amount of solids to separate out, filtration under diminished pressure, filter cake ethanol
Washing 2-3 times, vacuum drying, obtains 4-bromo- N of compound-(- 4-methyl benzenesulfonamide of 2-amino-ethyl
Base) naphthalimide (5).Yield：75 %.Product directly carries out next step reaction without purification.
Compound 4-diazanyl-N-(2-amino-ethyl-4-Methyl benzenesulfonyl amido) naphthalimide (7)
Synthesis：
。
In 25 mL round-bottomed flasks, 4-bromo- N-(- 4-methyl benzenesulfonamide of 2-amino-ethyl is added
Base) (5) 0.3 mmol of naphthalimide, (2) 0.1 mL of hydrazine hydrate, 3 mL of ethanol is added, it is after being heated to reflux 2-3 h, cold
But to room temperature, filtration under diminished pressure, filter cake washing with alcohol 2-3 times, vacuum drying, Jing ethyl alcohol recrystallizations obtain faint yellow 4-hydrazine
Base-N-(2-amino-ethyl-4-Methyl benzenesulfonyl amido) naphthalimide (7).Yield：85 %.Probe Na-FA
The nuclear magnetic spectrogram of-ER is as shown in Figure 1.
1H NMR (400 MHz, DMSO) δ 9.15 (s, 1H), 8.61 (d, J = 8.2 Hz, 1H), 8.37
(d, J = 7.2 Hz, 1H), 8.25 (d, J = 8.6 Hz, 1H), 7.72 (t, J = 6.2 Hz, 1H),
7.66-7.56 (m, 3H), 7.24 (d, J = 8.5 Hz, 3H), 4.69 (s, 2H), 4.07 (t, J = 6.7
Hz, 2H), 3.03 (dd, J 1 = 13.2 Hz, J 2 = 6.5 Hz, 2H), 2.25 (d, J = 15.8 Hz, 3H).13C NMR (101 MHz, DMSO) δ 164.31, 163.39, 153.68, 142.91, 137.99, 134.67,
130.99, 129.93, 129.89, 128.70, 126.86, 124.51, 122.19, 118.87, 107.78,
104.40, 40.76, 40.59, 40.38, 40.17, 39.96, 39.75, 39.54, 39.33, 39.20, 21.36。
Embodiment 2
The uv absorption experiment detection of the formaldehyde fluorescent probe Na-FA-ER of series concentration gradient
PBS of the configuration containing 5% DMSO is used as detection mother solution, after uv absorption sub-ray spectrometer has been debugged, difference
The formaldehyde probe solution of variable concentrations is added, 2mL is settled to, after shaking up, is carried out UV absorbance detection（λ = 600 ~ 200
nm）, and establish the curve chart of uv absorption intensity and variable concentrations formaldehyde probe（See Fig. 2）.As seen from Figure 2, with
The increase of formaldehyde concentration and probe concentration, curve gradually increase in the absworption peak of λ=455 nm or so, and growth trend is than more uniform.
Embodiment 3
Endoplasmic reticulum formaldehyde fluorescent probe Na-FA-ER are tested to the selectivity of various active small moleculars
Solution prepares：It is 40 mM aldoketoneses, various conventional ions, aminoacid, active oxygen nitrogen class material to configure 10 mL concentration
PBS buffer solution；Configure the DMSO solution that 5 mL concentration are the 1 mM probes.
The PBS solution for adding 10 μ L probe mother solutions, 90 μ L DMSO and being separately added into all kinds of aldehyde ketones reaches its ultimate density
To 40 μM, the PBS buffer solution for being separately added into all kinds of conventional ions and aminoacid is that ultimate density reaches 5 mM, is separately added into
The PBS of all kinds of levels of reactive oxygen species makes its ultimate density reach 100 μM, is settled to 2 mL with PBS, shakes up
And fluorescence intensity detection (λ is carried out with spectrofluorophotometerex = 445 nm, λem=543 nm), then set up fluorescence strong
The curve chart from different particles (aldehyde ketone, aminoacid, conventional anion and cation and active oxygen nitrogen class) is spent, from all spectral lines
Fluorescence intensity data at middle selection λ=543 nm, and the formaldehyde probe is made to different ions (including first according to this data
Aldehyde) selectivity bar diagram（See Fig. 3）.Wherein, excitation wavelength is 445 nm；The concentration of probe：5 μM, test ion and ammonia
The concentration of base acid is 5 mM, and the concentration of active oxygen is 100 μM, and the concentration of Carbonyl compounds is 40 μM.The ion difference of addition
For：Probe, Biformyl, methyl-glyoxal, Sodium Pyruvate, hydroxy benzaldehyde, Chloral, acetaldehyde, paranitrobenzaldehyde, third
Ketone, sodium hypochlorite, hydrogen peroxide, tert-butyl peroxide, tertbutanol peroxide, N-acetyl-glycine, N-acetyl group-
L-cysteine, nitric oxide, D-homocysteine, magnesium chloride, sodium sulfate, sodium nitrite, sodium sulfite, sulfur hydrogenation
Sodium, L-arginine, calcium chloride, formaldehyde.
Embodiment 4
Fluorescence titration of the formaldehyde of variable concentrations to fluorescent probe Na-FA-ER is tested
Solution prepares：Configure 100 mL of aqueous solution of 100 mM formaldehyde；Configure 5 mL of the formaldehyde probe DMSO solution of 1 mM.
Preparation concentration and probe concentration is 5 μM of PBS buffer solution (5 % DMSO), is separately added into the formalin (0 of variable concentrations
- 500 μM) act on complete, then carry out fluorescence titration detection (λex = 445 nm, λem=543 nm), set up fluorescence drop
Determine spectrogram (see Fig. 4), find that, when concentration of formaldehyde reaches 500 μM, fluorescence intensity reaches saturation through detection.
Embodiment 5
The kinetic measurement that endoplasmic reticulum formaldehyde fluorescent probe Na-FA-ER are acted on formaldehyde
Solution prepares：Configure 100 mL of aqueous solution of 100 mM formaldehyde；Configure 5 mL of the formaldehyde probe DMSO solution of 1 mM.
10 μ L probe mother solutions, 90 μ L DMSO are added in 5 mL EP pipes, then different volumes are separately added into which
Formalin, make after the dilution of stand-by PBS buffer solution concentration of formaldehyde respectively reach 1 μM, 2 μM, 5 μM, 10 μM, 25 μM,
50 μM、100 μM、250 μM、500 μM、750 μM.Kinetic measurement (the λ of the probe is carried out subsequentlyex = 445 nm, λem
=543 nm), it is per minute test once, detect altogether 30 min, after end to be tested, set up the probe fluorescence intensity and when
Between kinetic curve (such as Fig. 5).With the increase of concentration of formaldehyde in detection system, saturation time is also reduced therewith, most fast full
It is about 10 min with the time.
Embodiment 6
The exogenous formaldehyde cell imaging test of endoplasmic reticulum formaldehyde fluorescent probe Na-FA-ER
It is 3 × 10 by density5The HeLa cells of individual/mL are inoculated in 35 mm culture dishs of imaging, in CO2Incubator（Temperature is
37 DEG C, 5 % CO2）Middle culture, after cell attachment, is divided into several groups of experimental grouies and is cultivated：(1) blank HeLa groups of cells；
(2) the additional 40 μM of formaldehyde of HeLa cells is incubated 30 min；(3) it is outer again after 30 min of the additional 40 μM of formaldehyde incubation of HeLa cells
Plus 5 μM of probes are incubated 30 min；After culture terminates, cell 2 ~ 3 times is rinsed with PBS, use Nikon fluorescence co-focusing
Microscope shoots the fluorescence photo of the HeLa cells cultivated under the conditions of this three groups respectively, it is found that (3rd) the HeLa cells organized are sent out
Go out intense fluorescence.(result is shown in Fig. 6).Wherein, excitation wavelength：488 nm, emission band：500 - 550 nm.Wherein：a )
The light field figure of HeLa cells；B) fluorogram of HeLa cells；C) it is the stacking chart of a and b；D) HeLa cells and formaldehyde
The light field figure being incubated altogether；E) the light field figure that HeLa cells are incubated altogether with formaldehyde；F) it is the stacking chart of d and e；g ) HeLa
The light field figure that cell is incubated altogether with probe and formaldehyde；H) fluorogram that HeLa cells are incubated altogether with probe and formaldehyde；I) it is g
With the stacking chart of h.Scale：20 μm.
Embodiment 7
The endogenous formaldehyde cell imaging test of endoplasmic reticulum formaldehyde fluorescent probe Na-FA-ER
It is 3 × 10 by density5The HeLa cell of individual/mL is inoculated in 35 mm culture dishs of imaging, in CO2Incubator（Temperature
Spend for 37 DEG C, 5 % CO2）Middle culture, after cell attachment, is divided into several groups of experimental grouies and is cultivated：(1) HeLa cells are additional
100 μM of sodium sulfitees are incubated 30 min；(2) it is outer again after 30 min of the additional 100 μM of sodium sulfitees incubation of HeLa cells
Plus 5 μM of probes are incubated 30 min；(3) the additional 5 μM of probes of HeLa cells are incubated 30 min；It is after culture terminates, slow with PBS
Rush liquid and rinse cell 2 ~ 3 times, shoot the HeLa cells cultivated under the conditions of this three groups with Nikon confocal microscope respectively
Fluorescence photo, it is found that (3rd) the HeLa cell organized can send obvious fluorescence signal.（As a result see Fig. 7）.Wherein,
Excitation wavelength：488 nm, emission band：500 - 550 nm.Wherein：A) the light field figure of HeLa cells；B) HeLa cells
Fluorogram；C) it is the stacking chart of a and b；D) HeLa cells and NaHSO3The light field figure being incubated altogether；E) HeLa cells
With NaHSO3The fluorogram being incubated altogether；F) it is the stacking chart of d and e；G) the light field figure that HeLa cells are incubated altogether with probe；h
) fluorogram that is incubated altogether of HeLa cells and probe；I) it is the stacking chart of g and h；J) HeLa cells and probe and NaHSO3Altogether
The light field figure of incubation；K) HeLa cells and probe and NaHSO3The fluorogram being incubated altogether；L) it is the stacking chart of j and k.Scale：
20 μm。
Embodiment 8
The common location cell imaging test of endoplasmic reticulum formaldehyde fluorescent probe Na-FA-ER
It is 3 × 10 by density5The cancer cell inoculation of individual/mL to imaging 35 mm culture dishs in, in CO2Incubator（Temperature is 37
DEG C, 5 % CO2）Middle culture, after cell attachment, is divided into two groups of experimental grouies and is cultivated：(1) cell and commerical grade endoplasmic reticulum
Red positioning dyestuff is incubated 30 min with 5 μM of probes after being incubated 15 min again；(2) cell and the red positioning of commerical grade endoplasmic reticulum
After dyestuff is incubated 15 min, be subsequently adding and 30 min be incubated with 40 μM of formaldehyde, then be incubated 30 min with 5 μM of probes；Wait to cultivate
After end, cell 2 ~ 3 times is rinsed with PBS, shot under the conditions of this two groups respectively with Nikon confocal microscope
The fluorescence photo of the cell of culture, can find the probe can altitude location matter is online in the cell, and be capable of identify which
In formaldehyde.Wherein, first group of experiment common location coefficient is 0.92, and second group of experiment common location coefficient is 0.95（As a result see figure
8）.Wherein, excitation wavelength：488 nm (green channel) and 561 nm (red channel), emission band：500-550 nm and
625 – 675 nm.Wherein：We can see that probe has good common location by the test of cell common location imaging experiment
Effect.A)-d) image is tested for endogenouss formaldehyde probe common location；E)-h) determined for exogenous property formaldehyde probe altogether
Position experiment image.Scale：20 μm.
Claims (3)
1. it is a kind of detection endocytoplasmic reticulum in formaldehyde fluorescent probe, it is characterised in that the fluorescent probe be named as 4-diazanyl-
N-(2-amino-ethyl-4-Methyl benzenesulfonyl amido) naphthalimide, abbreviation Na-FA-ER, its chemical structural formula is such as
Formula () shown in：
。
2. it is a kind of it is according to claim 1 detection endocytoplasmic reticulum in formaldehyde fluorescent probe preparation method, its feature
It is to comprise the steps：
1）In reaction vessel, add toluene, ethylenediamine, paratoluensulfonyl chloride, be heated to reflux synthesize compound N-(2-
Amino-ethyl)-4-methyl this sulfonamide；
2）In another reaction vessel, 1,8-4-bromonaphthalic anhydrides of addition, ethanol, N-(2-amino-ethyl)-
4-methyl this sulfonamide, is heated to reflux 4-bromo- N of generation-(2 amino-ethyl-4-Methyl benzenesulfonyl amido)
Naphthalimide；
3）Another reaction vessel is taken again, adds 4-bromo- N-(2-amino-ethyl-4-Methyl benzenesulfonyl amido) naphthalene
Acid imide, adds afterwards and hydrazine hydrate is added under ethanol, stirring, and post-heating backflow generates compound 4-diazanyl-N-(2
- amino-ethyl-4-Methyl benzenesulfonyl amido) naphthalimide, referred to as：Na - FA - ER.
3. application of the fluorescent probe described in claim 1 in formaldehyde in detection endocytoplasmic reticulum.
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