CN110452250A - A kind of detection hydrazine fluorescence probe of fluorescein precursor structure - Google Patents
A kind of detection hydrazine fluorescence probe of fluorescein precursor structure Download PDFInfo
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- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
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- 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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Abstract
The present invention provides a kind of detection hydrazine fluorescence probe of fluorescein precursor structure, and in particular to fluorescence probe field, S1, after appropriate dichlorofluorescein and triethylamine are added in round-bottomed flask；S2, it is mixed after appropriate anhydrous dichloroethanes is added, the anhydrous dichloroethane solution in right amount dissolved with acryloyl chloride is added dropwise at leisure into round-bottomed flask, reacts, is monitored by TLC at room temperature；S3, to the end of reacting after, release vacuum state, be spin-dried for solvent, column chromatography obtains probe.With the strong properties of hydrazine and reacting for probe molecule substrate, the dichloro fluorogen parent that dissociates comes the present invention, and fluorescence from scratch, and then realizes that the detection to hydrazine is studied.
Description
Technical field
The invention belongs to fluorescence probe fields, and in particular to a kind of detection hydrazine fluorescence probe of fluorescein precursor structure.
Background technique
Fluorescein, also referred to as fluorescein, fluorescein, 3 ', 6 '-dihydroxy of chemical name spiral shell [isobenzofuran -1 (3H),
9 '-(9H)-ton -3- ketone] or 2- (6- hydroxyl -3- oxo -3H- xanthene -9- base) benzoic acid.Although the structure of fluorescein is similar
In the structure of rhodamine, but due to two phenyl ring by oxygen bridge connect in the same plane, the molecule is on rigid coplanar surface
There is strong yellow-green fluorescence with bigger conjugated structure, and due to the effect of exciting light.Fluorescein and its derivative
Have many advantages: excitation wavelength and launch wavelength are in visible-range, and fluorescence quantum yield is high in water, extinction coefficient
Height has no toxic side effect, low in cost etc..Therefore, it is typically used as biological stain, fluophotometer and redox instruction
Agent etc..
Summary of the invention
The object of the present invention is to provide a kind of detection hydrazine fluorescence probes of fluorescein precursor structure, derivative based on fluorescein
The design of object dichlorofluorescein has synthesized hydrazine detection fluorescence probe, and has carried out corresponding phenetic analysis to its molecular structure, transports
With the strong properties of hydrazine and reacting for probe molecule substrate, the dichloro fluorogen parent that dissociates comes, fluorescence from scratch, into
And it realizes the detection to hydrazine and studies.
The present invention provides the following technical solutions:
A kind of detection hydrazine fluorescence probe of fluorescein precursor structure, probe synthesis step are as follows:
S1, according to following formula,
After dichlorofluorescein (0.40g, 1.0mmol) and triethylamine (1.2mmol, 168 μ L) are added in round-bottomed flask,
S2, under 0 DEG C, nitrogen protection, be added the anhydrous dichloroethanes of 10mL after be mixed, slowly into round-bottomed flask
The anhydrous dichloroethane solution of 10mL dissolved with acryloyl chloride (1.5mmol, 123 μ L) is added dropwise in ground, reacts at room temperature, passes through TLC
Monitoring.
S3, to the end of reacting after, release vacuum state, be spin-dried for solvent, column chromatographs (petroleum ether: ethyl acetate=5:1
For leacheate), obtain probe.
Beneficial effects of the present invention:
With the strong properties of hydrazine and reacting for probe molecule substrate, the dichloro fluorogen parent that dissociates comes the present invention,
Fluorescence from scratch, and then realizes that the detection to hydrazine is studied.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.In the accompanying drawings:
Fig. 1 is that the detection of probe limits；
Fig. 2 is in the presence of 350 μM of N2H4, and at 25 DEG C, probe (10 μM) is in aqueous solution (DMSO/PBS=1:9, pH
=7.4) the time dependence change in fluorescence in；λ ex=504nm；
The absorption spectrum variation of probe (10 μM) in the case that Fig. 3 is the absence and presence hydrazine in buffer solution；
Fig. 4 be fluorescence spectrum variation (DMSO/PBS=1:9, v/v, 10mM, pH=7.4, at 25 DEG C, λ em/ex=
528/504nm)；
Fig. 5 is that probe (10 μM) are cationic in the spectrum when 504nm is excited to (350 μM) of N2H4；
Fig. 6 be various analytes (1000 μM) in buffer solution fluorescence response (DMSO/PBS=1:9, v/v,
10mM, pH=7.4；λ em/ex=530/504nm) spectrum of the anion when 504nm is excited at 25 DEG C；
Fig. 7 is spectrum of the amino-compound when 504nm is excited；
Fig. 8 is in the middle presence of DMSO/PBS (1:9, v/v) and there are when N2H4 (350 μM) at 25 DEG C, and pH is to probe (10
μM) 528nm at fluorescence intensity influence；
Fig. 9 is after being exposed to the hydrazine solution of various concentration under using 365nmUV light irradiation, to contain probe (0.5mM)
The fluorescence color of filter paper changes；
Figure 10 is the vitality test of the HeLa cell handled with (0-100 μM) of probe；
Figure 11 is fluorescence probe image (kex=504nm) (a, c) of Hela cell: only using probe (10 μM) at 37 DEG C
It is incubated for 1 hour cell；(b, d): with 10 μM probe pretreatment cell 1 hour, it is small then to pre-process 0.5 again with 330 μM of N2H4
When；(a, b): bright scene；(c, d): green channel (450-600nm)；
Figure 12 is the sensor mechanism of the 4- bromine bytyry probe of hydrazine；
Figure 13 is reaction mechanism figure when probe compound 1 detects hydrazine；
Figure 14 is the mass spectrogram of probe compound 1 and hydrazine effect front and back.
Specific embodiment
Unless otherwise indicated, all chemicals are obtained from commercial supplier, without further purification
Directly use.All measurements carry out in DMSO/PBS buffer (1:9, v/v, 10mM, pH=7.4), are dripped at room temperature
Fixed and selectivity experiment.Interior target BrukerDRX-400 and DRX- is being used as with TMS (tetramethylsilane, 0.00ppm)
Related compound is measured on 400/4 spectrometer1HNMR and13CNMR spectrum.1The data of HNMR are expressed as follows: chemical shift
(ppm), (s=is unimodal for multiplicity；D=is bimodal；T=triplet；Q=quartet, m=multiplet), integral, coupling constant
(Hz)。13CNMR spectrogram is with CDCl3(77.67ppm) is reference signal；Mass spectrum is recorded in E.I. mode.With pH-10C number
PH meter carries out all pH value measurements.All measurement experiments carry out at room temperature.
Using reagent: dichlorofluorescein, acryloyl chloride, methylene chloride, triethylamine, ethyl acetate, petroleum ether.
Using instrument: round-bottomed flask, silicagel column and other associated glass instruments of different size；F97pro fluorescence spectrophotometer
Photometer, Shanghai Asia honor RE-2000 Rotary Evaporators, 85-2 type constant temperature blender with magnetic force, DLSB-5/20 sub-cooled circulation
Pump, SHZ-DIII vacuum pump using circulatory water, WTH-203 type ultraviolet analysis instrument for three purposed, T-114 type electronic balance, 1800/1600 system
Column ultraviolet-uisible spectrophotometer, Japan Electronics JOEL400MHz Liquid NMR spectrometer etc..
Dichlorofluorescein (0.40g, 1.0mmol) and triethylamine (1.2mmol, 168 μ L) are added to according to above-mentioned formula
After in round-bottomed flask, under 0 DEG C, nitrogen protection, it is mixed after the anhydrous dichloroethanes of 10mL is added, it is slow into round-bottomed flask
The anhydrous dichloroethane solution of 10mL dissolved with acryloyl chloride (1.5mmol, 123 μ L) is slowly added dropwise, reacts, passes through at room temperature
TLC monitoring.After to the end of reacting, vacuum state is released, is spin-dried for solvent, (petroleum ether: ethyl acetate=5:1 is leaching to column chromatography
Washing lotion), obtain probe (193mg, yield 38%)；Compound 1:1HNMR(400MHz,CDCl3) δ ppm:8.01 (d, J=8.0Hz,
1H), 7.66 (m, 2H), 7.19 (s, 1H), 7.15 (d, J=8.0Hz, 2H), 6.82 (s, 2H), 6.64 (t, 2H), 6.60 (t, J
=4.0Hz, 2H), 6.28 (q, J=28.0Hz, 2H), 6.04 (d, J=12.0Hz, 2H)13CNMR (400MHz,DMSO-d6)δ
ppm:168.48,162.86,151.84,149.67,148.27,135.76,134.16,130.62,128.88,126.62,1
25.61,123.94,122.62,117.62,112.67.MS(ESI,m/z):C26H14Cl2O7,found[M]+509.
Experiment test:
1, the measurement of spectroscopic material and method and quantum yield
All aqueous solutions are prepared using deionized water.All measurement DMSO/PBS buffer (1:9, v/v, 10mM,
PH=7.4 it is carried out in).All pH value use pH-10C digital pH meter to carry out.Absorption spectrum uses VarianUV-
Cary5000 spectrophotometer record.Remembered using the HitachiF-7000 scanning sepectrophotofluorometer equipped with xenon flash lamp
Record fluorescence spectrum.For absorbing the quartz colorimetric utensil (3.5mL volume) for being included in 1cm × 1cm with the sample of fluorescence measurement
In.
The quantum yield of probe is determined according to following formula；
Wherein Φ is quantum yield；I is the integral area under revised emission spectrum；A is the extinction of excitation wave strong point
Degree；λ ex is excitation wavelength；η is the refractive index of solution；Subscript 1 and B correspond to unknown and standard.Select 0.1M rhodamine B
Ethanol solution is as standard, quantum yield 0.89.The quantum yield of probe is calculated as 0.91.
2, detectable limit calculates
N2H4Detectable limit (DL) calculated by linear function in Fig. 1 and following equation:
DL=3.3 σ/k
Wherein σ is (I525nm) probe blank solution fluorescence intensity standard derivative；K is linear calibration curve in Fig. 1
Slope；The concentration of probe is 9.8 × 10-8mol·L-1。
3, cell culture and fluorescence microscope imaging
In order to obtain the cell permeability of probe, Hela cell is maintained at 37 DEG C and contains 5%CO2Moist environment
In.(10 μM) of probe in cell and 1.0mL fresh culture are incubated for 1 hour, then by N2H4(330 μM) solution is added to
In addition it is incubated in 0.5 hour cell.Incubated cell is simultaneously flushed three times with PBS phosphate buffer solution, to remove before imaging
Remove free compound.The fluorescent image of Hela cell is carried out on inverted fluorescence microscope (DMI4000B).Control experiment is
In the case where other conditions are constant, Hela cell is only incubated with 1 with the probe (10 μM) in 1.0mL fresh culture
Hour, that is, be free of N2H4The fluorescent image of Hela cell is carried out in the case where (0 μM).
4, cell toxicity test
3- (4,5- dimethylthiazole) -2,5- diphenyl bromination tetrazolium (MTT) mensuration program of establishing criteria, which is tested, to be visited
Needle is tested for the cellular cytoxicity activity of Hela cell, to assess potential application of the probe in living cells imaging.By Hela
Cell is seeded in 96 hole assay plates 24 hours with the density (100 μ L total volumes/hole) of every 104 cells in hole.It is trained in serum-free
It supports in base and ready probe (5 μM, 10 μM, 25 μM, 50 μM, 100 μM) is added, then incubated together with cell 24 hours.It is logical
Cross the absorbance that microplate reader (German BertholdMithras2LB943) detects cell at 504nm.Control experiment passes through inspection
The growth medium without probe (0 μM) is surveyed to complete.
5, the time response of probe molecule
Fluorescence probe is the important parameter for measuring probe performance to the response speed of analyte.Fluorescence with quick response
Probe is expected to apply dynamic realtime during biomedical research to sense and monitor.
Probe (10 μM) is firstly evaluated in 50m Μ N2H4In the presence of, in 25 DEG C of aqueous solutions (DMSO/PBS=1:9, pH=
7.4) the time dependence change in fluorescence in.Fluorescence response shown in Fig. 2 is obtained by drawing the fluorescence intensity at 528nm
Curve.Add N2H4Afterwards, the fluorescence response of probe generates rapidly, and fluorescence intensity quicklys increase, and reached maximum value at 48 seconds, is
System reaction reaches balance, about 80 times of fluorescence intensity increase, responds significant.
6, the spectral characteristic of probe molecule
Under the encouragement of the tested results, had studied at 25 DEG C at aqueous solution (DMSO/PBS=1:9, pH=7.4)
Middle existence or non-existence N2H4Probe Absorption and emission spectra.
As shown in Figure 3 and Figure 4, the main absorption peak in the free probe ultraviolet-visible area in DMSO-PBS is located at
250nm.When by N2H4When (330 μM) are added in probe solution, the absorbance at 250nm is significantly reduced, and is occurred in 504nm new
Absorption peak, with pure 2', the absorption peak position consistency of 7'- dichlorofluorescein.In addition, the isoabsorptive point table at 403nm
Bright probe and N2H4Reaction be that one-to-one conversion forms noval chemical compound, become yellowish green from colourless with the colors of solution pigments
Color.Obviously, the open hole detection of hydrazine may be implemented.
Fluorescence analysis experiment shows in no N2H4In the case where, probe shows seldom fluorescence intensity.Use Luo Dan
Bright B is as standard, the fluorescence quantum yield Φ of probefIt is 0.91.As shown in the BB in Fig. 3 and Fig. 4, when the excitation at 504nm
When, the middle addition hydrazine into probe system, the new emission peak of appearance at 528nm, and with its intensity of the increase of hydrazine concentration by
It is cumulative to add.When the hydrazine amount of addition reaches 33 equivalent, fluorescence intensity increases up to platform, and about 1000 times of intensity increase, fluorescence face
Color becomes yellow green from colourless.These results indicate that probe has " OFF-ON " signal intensity to target detection molecule hydrazine, it is
Turn-on type probe.Meanwhile the fluorescence intensity at 528nm and hydrazine concentration show good linear relationship (R2=0.997),
Wherein N2H4Concentration in the range of 60-330 μM.N is obtained by calculating 3.3 σ/k based on the discussion for detecting limit above2H4
The probe in detecting of probe is limited to 9.8 × 10-8M (conversion is equivalent to 3.1ppb), the EPA lower than 10ppb allows threshold value, and is lower than
The fluorescence probe of most of reports.
7, the selectivity and competitiveness of probe molecule
High sensitivity, good selectivity, common-path interference are to determine the important references of probe molecule performance superiority and inferiority.In reality
In the environmental factor of border, Common Cations, the presence of anion and nitrogenous small molecule compound may identify the row of hydrazine to probe
To interfere, therefore the selectivity of probe is carried out, competitiveness test, to ensure detection knot when the actually detected hydrazine of probe
Fruit is true and reliable.
The selectivity of probe is assessed,
First by various interference analyte cations (Fe3+,Mg2+,Hg2+,Cu2+,Zn2+,Ba2+,Cd2+,Ni2+,Co2+,Mn2+,
Ag+), anion (SO3 2-,NO3 2-,HSO3-,F-,NO2-,SO4 2-,ClO-,HS-) and nitrogenous small molecule (NH3·H2O,Et3N,
DMF, DEA, OPD, Cys, Hcy and GSH etc.) (100 equivalent) dissolution with containing probe (10 μM) buffer solution in (DMSO/
PBS=1:9, pH=7.4).Measure fluorescence intensity of these determinands at 528nm respectively after 1 minute, and with not by
The blank sample of interference is compared.
As shown in Figures 5 to 7, the variation of the fluorescence intensity of interference analyte induction is very small, only N2H4In 528nm
Locate significant enhancing fluorescence intensity, along with color change green from colourless to bright orange under 365nmUV lamp.In order to study reality
The applicability of middle probe is applied on border, has carried out competitive experiment to the probe.From the figures it is clear that existing
In the case where various interference analytes, N is monitored2H4Interference very little.It should be pointed out that the same HS with nucleophilicity-
Not to identification, it is different from document of having registered.Therefore, probe can be used as N in complex environment2H4The sensing platform of detection.
8, the pH effect of probe molecule
Importance in practical applications is adapted in view of pH, has investigated influence of the pH to the fluorescence intensity of probe at 528nm.
As shown in figure 8, not observing apparent fluorescence enhancement (bottom black line institute in 4.0~10.0 wider pH range at 25 DEG C
Show).After hydrazine is added, since pH=4, response fluorescence enhances rapidly, after pH reaches 7 and 7, the response fluorescence intensity
Reach a platform.It is this as pH increases, fluorescence increases and reaches stable phenomenon reason after pH to neutrality and is:
(1) NH as alkalinity2NH2In acid condition, it is easy proton and is melted into salt；Existing NH in the form of salts2NH2's
Nucleophilicity is greatly lowered, and is hardly acted on probe；
It (2) is unprotonated residual free hydrazine with the hydrazine of probe effect, effective concentration is far below practical outer enriching
Degree.It is acid stronger, protonate more complete, free NH2NH2Concentration it is lower, naturally corresponding fluorescence intensity is lower；
(3) when pH is from acidity to neutral transition, protonation is greatly lowered, and dissociate NH2NH2Concentration with pH increase and
Increase, the corresponding fluorescence intensity that responds increases；
(4) after pH reaches neutrality or even alkaline, the protonation of hydrazine completely disappears, and the effective concentration of hydrazine is exactly in system
Additional concentration, the effect with probe are consistent, and naturally, response fluorescence intensity reaches extreme value in pH=7, and with pH
Increase and keep horizontal, is not further added by.
(5) in addition, as basicity increases, the fluorescence intensity of blank probe is increased slightly (right end shown in the black line of bottom), says
The OH of bright increase-Hydrolysis probes, dissociate a small amount of parent fluorogen, but well below hydrazine, there are situations, imply OH-It does not influence to examine
It surveys, probe pH has a wide range of application.
As shown in figure 8, existing in the DMSO/PBS (1:9, v/v) at 25 DEG C and there are when N2H4 (350 μM), pH pairs
The influence of fluorescence intensity at the 528nm of probe (10 μM), these are the result shows that probe steady is good, and at physiological ph
Detecting N2H4 has fabulous sensitivity.Therefore, which has very big potentiality, it is expected to for detecting in environmental sample
The tracer imaging of N2H4 in N2H4 and living cells.
9, the hydrazine in probe molecule detection gaseous state
In order to investigate concrete application of the probe in real life, detection of the probe to gaseous state hydrazine is further tested.For
Make to test easy to implement and practical application, using pH test paper as template, uses filter paper as absorbable substrate, by probe test paper
Change, is convenient for practical operation.
Before test, filter paper is immersed to DMSO solution (c=5 × 10 of probe-4mL-1) in and drying for standby.The bodies such as general
The hydrazine solution (0 μ l, 10 μ l, 100 μ l, 1mM, 10mM, 100mM, 1M) of product various concentration is packed into same specification closed glass with cover
In bottle (5ml, φ=1cm).Probe filter paper is cut to the circle similar with bottle cap internal diameter, and is close on the inside of bottle cap, on solution
Portion is not contacted with solution；Bottle cap is screwed again, " is smoked " in confined conditions 30 minutes, observation is then taken out, as a result such as Fig. 9 institute
Show.It can be found that corresponding test filter paper is observed under hand-held UV lamp 365nm excitation with the increase of hydrazine concentration
Fluorescence color is consistent with illustration in Fig. 8 from unstressed configuration → green fluorescence → yellow fluorescence significant change, fluorescence color this
The significant variation of kind is attributed to formation of the dichlorofluorescein as reaction product, it was demonstrated that still maintains same after probe test paper
Test performance.Therefore, probe can visually detect the N in actual sample2H4, there is biggish application potential.
10, the cell experiment of probe molecule
The cytotoxic activity (MTT) of probe molecule is tested:
In view of the above-mentioned excellent optical property of probe, the potentiality whether probe has biologic applications have further been probed into.
Firstly, 3- (4,5- dimethylthiazole -2- base) -2,5- diphenyltetrazolium bromide (MTT) will be used to visit to measure
For the cytotoxicity of HeLa Cells.Consider potential use of the probe in living cells imaging.Routinely test
Step, concentration and probe concentration be 5-100 μM it is 24 hours lower, estimation cell viability be greater than 92%, show the cytotoxicity of probe it is low,
As shown in Figure 10.This experimental results showed that, within the scope of a certain concentration, probe to HeLa cell almost without toxicity, therefore,
Probe has the potentiality of biologic applications.
Fluorescence imaging of the probe molecule in living cells:
Inverted fluorescence microscope image is shown in Figure 11.Hela cell and probe (10 μM) are incubated 1 hour at 37 DEG C
Afterwards, hypofluorescence cytoplasm (region Figure 11 c) is only detected in green channel.But hydrazine (330 μM) are added and incubate 0.5 hour
Afterwards, it observes under green channel, the significant increase (region Figure 11 d) of cell green fluorescence intensity, shows reacting simultaneously for probe and hydrazine
It confirms that probe has good penetrability to cell, and rapidly detects hydrazine in cell, there is good fluorescence imaging
Energy.
Recognition mechanism:
From above-mentioned experiment, it can be found that probe is the response type fluorescent probe molecule an of function admirable, the knowledge to hydrazine
Do not have that the response time is short, anti-interference, good selective.And the superiority of these performances and probe recognition mechanism point are not
It opens, probe is as follows to the recognition mechanism of hydrazine molecule:
(1) relative to the reaction recognition mechanism for passing through the free parent fluorogen out of hydrazinolysis ester bond in document, the skeleton of probe
On be also introduced into ester bond, still, introduced background acid is simplest α, and beta-unsaturated acid-acrylic acid exists in the molecule
Conjugate double bond system, it is easy to Isosorbide-5-Nitrae-Micheal addition reaction is carried out, to hide the foreshadowing with the addition reaction of nucleopilic reagent hydrazine；
(2) species, H are interfered relative to other nitrogenous or amino small molecule2NNH2Molecular volume is small, and nucleophilie nucleus ability is strong,
Space and electronic factor be all conducive to its easily with the identification division in substrate probe molecule, that is, α, β-are unsaturated conjugated
Isosorbide-5-Nitrae-Micheal addition reaction occurs for double bond, generates γ-H2The intermediate that NNH- replaces.Therefore, it is that the nucleophilic of probe adds
At reaction step, hydrazine has its precedence capability, improves its response speed and selectivity naturally.(the figure compared with documentation
12), H2NNH2When the probe molecule that nucleophilic attack 4- bromo-butyric acid ester group replaces, also there is end γ-H2The intermediate that NNH- replaces
Generation.Say that the two has a similar process from this point, but the difference is that, document hydrazine and 4- bromo-butyric acid ester group upper end base
Bromine is substitution reaction, and in this work is the nucleophilicity using hydrazine, with double bond nucleophilic addition.Due to probe identification division
For α, the unsaturated conjugated double bond of β-is said in energy point of view, it is easier to be generated Isosorbide-5-Nitrae-Micheal addition product with hydrazine, therefore, be visited
Needle relative to the probe molecule that recognition site is 4- bromo-butyric acid ester group comparatively, the former may have faster reaction rate,
Embody the shorter response time；
3. working as end group γ-H2After the intermediate that NNH- replaces generates, as the hydrazine molecule of parents' core, still there is one not participate in
The N of reaction, the lone pair electrons intramolecular attack ester carbonyl group on the N atom, leads to ester group cleavage, and dissociate the pure mother of probe
Body molecule, fluorescence restore.And another product is propionyl hydrazine in stable five-membered ring.Relative to scattershot intermolecular
The nucleophilic attack of nucleophilic attack, intramolecular is easier, and also shortens the response time.
Such as Figure 13 it can further be seen that acrylic is selected to be as the advantages of recognition group:
1. α is introduced on probe, the unsaturated conjugated double bond of β-, relative to the carbonyl of the direct attack short chain fatty acids of hydrazine, or
Hydrazine replaces end halogen atom, and the nucleophilic addition of hydrazine and the unsaturated conjugated double bond of α, β-is easier to realize；
2. the number of carbon is just right on acrylic acid, γ-H2After the intermediate that NNH- replaces generates, the following molecule
Interior necleophilic reaction, for involved reaction of atomic apart from upper suitable, subsequent products are the five-membered ring of ring strain very little.If selecting carbon
The more α of number, beta-unsaturated acid, when intramolecular nucleophilic attack ester carbonyl group, it is possible to create number of rings is greater than 6 heterocyclic compound, In
There is no five-membered ring to stablize in structure；
3. being used as simplest α, beta-unsaturated acid, structure is simple, and physical barrier is small, easily by hydrazine addition, and it is at low cost
It is honest and clean.
Exactly these advantages, in addition organic utilization of hydrazine parents' nuclearity, probe has hydrazine good selective and sensitive
Degree will be understood by, and some identification parameters or index, for example, detection limit, is better than document report, also illustrates that the probe is
The response type hydrazine fluorescence probe that one kind is had excellent performance, there is potential application value.
It is simultaneously the correctness of verifying mechanism, tests the mass spectrogram (Figure 14) of probe and hydrazine response front and back.The spy
Base peak of the needle before hydrazine is added is m/z=509, belongs to the peak M of probe.There is m/z=401 after hydrazine is added and sufficiently reacts
Peak, the peak is consistent with dichlorofluorescein molecular ion peak, and the relative molecular mass of dichlorofluorescein is 401.2g/mol, secretly
Show that this generates dichlorofluorescein really after the reaction was completed, further illustrate it is presumed that mechanism have its reasonability.
Advantages of the present invention:
1, it designs and is prepared for based on dichlorofluorescein being parent fluorogen, with simplest containing α, β-unsaturation is altogether
The acrylic acid of yoke double bond is as recognition site, one-step synthesis target-probe；
2, due to PET mechanism, probe is non-fluorescence active.The probe is reacted by hydrazinolysis, discharges parent fluorogen,
Restore fluorescence, can be used for quantitative determining N2H4(0-330μM).Further optical property is studies have shown that 1) probe is to N2H4's
Identification has high sensitivity, and detection limit is down to 3.1ppb (requirement lower than 10ppb)；2) selectivity of the fluorescence probe to hydrazine
Height, common yin, cationic and nitrogenous small molecule (ammonia, diethylamine and amino acid etc.) are small to identification Behavioral interference；3) probe pH
Tolerance is good, there is significant quick corresponding time (48 seconds) to hydrazine, and can be easily carried out naked eye to the colorimetric of hydrazine and glimmering
Photoresponse detection provides a kind of method of simple and quick visualization hydrazine.These results explanation, probe is that a performance is excellent
Different turn-on response type hydrazine identifies fluorescence probe；
3, probe has been carried out to the N in living cells2H4It is imaged, and shows that high intensity is fluorescence letter in green channel
Number.In addition, by after the probe test paper, can effective qualitative half-quantitative detection hydrazine steam, detection means is simple, and effect is fine.Cause
This, this fluorescence probe can be the N in biology and environmental system2H4Detection provides potential tool；
4, corresponding recognition mechanism is proposed on the basis of experiment, it is believed that containing α, the third of the unsaturated conjugated double bond of β-
Olefin(e) acid is bonded on fluorogen skeleton as recognition site, using parents' nuclearity of hydrazine, the two after Isosorbide-5-Nitrae-Micheal addition,
The processes such as intramolecular nucleophilic attack ester carbonyl group is broken ester bond, and dissociate parent fluorogen, and fluorescence signal expression restores.Wherein,
The introducing of the unsaturated conjugated double bond of α, β-improves the speed of response and selectivity of identification.
These are only the preferred embodiment of the present invention, is not intended to restrict the invention, although with reference to the foregoing embodiments
Invention is explained in detail, for those skilled in the art, still can be to foregoing embodiments institute
The technical solution of record is modified or equivalent replacement of some of the technical features.It is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (4)
1. a kind of detection hydrazine fluorescence probe of fluorescein precursor structure, it is characterised in that: probe synthesis step is as follows:
S1, according to following formula,
After appropriate dichlorofluorescein and triethylamine are added in round-bottomed flask；
S2, it is mixed after appropriate anhydrous dichloroethanes is added, is added dropwise at leisure in right amount into round-bottomed flask dissolved with acryloyl chloride
Anhydrous dichloroethane solution, react, monitored by TLC at room temperature.
S3, to the end of reacting after, release vacuum state, be spin-dried for solvent, column chromatography obtains probe.
2. a kind of detection hydrazine fluorescence probe of fluorescein precursor structure according to claim 1, it is characterised in that: S1 step
In rapid, after dichlorofluorescein (0.40g, 1.0mmol) and triethylamine (1.2mmol, 168 μ L) are added in round-bottomed flask.
3. a kind of detection hydrazine fluorescence probe of fluorescein precursor structure according to claim 1, it is characterised in that: S2 step
In rapid, under 0 DEG C, nitrogen protection, it is mixed, is added dropwise at leisure into round-bottomed flask molten after the anhydrous dichloroethanes of 10mL is added
There is the anhydrous dichloroethane solution of 10mL of acryloyl chloride (1.5mmol, 123 μ L), reacts, monitored by TLC at room temperature.
4. a kind of detection hydrazine fluorescence probe of fluorescein precursor structure according to claim 1, it is characterised in that: S3 step
In rapid, to the end of reacting after, release vacuum state, be spin-dried for solvent, (petroleum ether: ethyl acetate=5:1 is elution to column chromatography
Liquid), obtain probe.
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| CN113603712B (en) * | 2021-08-12 | 2023-07-14 | 兰州大学 | Preparation method of reactive fluorescent probe for rapid detection of hydrazine |
| CN113735872A (en) * | 2021-08-20 | 2021-12-03 | 兰州大学 | Fluorescent probe for rapidly detecting hydrazine compounds and synthesis and application thereof |
| CN113735872B (en) * | 2021-08-20 | 2022-06-28 | 兰州大学 | Fluorescent probe for rapidly detecting hydrazine compounds and synthesis and application thereof |
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