CN109912612A - A kind of synthesis, preparation and its application based on Molecular Logic Gates identification Cys, GSH and HOCl fluorescence probe - Google Patents
A kind of synthesis, preparation and its application based on Molecular Logic Gates identification Cys, GSH and HOCl fluorescence probe Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title abstract description 8
- 230000015572 biosynthetic process Effects 0.000 title abstract description 6
- 238000003786 synthesis reaction Methods 0.000 title abstract description 6
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- 238000001514 detection method Methods 0.000 claims abstract description 26
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 13
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- 230000008859 change Effects 0.000 claims description 20
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Landscapes
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
A kind of synthesis, preparation and its application based on Molecular Logic Gates identification Cys, GSH and HOCl fluorescence probe, belongs to small-molecule fluorescent probe field, is related to molecular computer technical field, which has structural formula shown in formula (I):The formula (I) novel fluorescence probe of the invention based on Molecular Logic Gates identification Cys, GSH and HOCl is to report for the first time, there is detection to limit low, sensitive height, Visual retrieval may be implemented in the good advantage of selectivity；Probe raw material of the present invention is cheap and easy to get, high income and preparation cost are low, is suitable for industrialized production；Prepare liquid of the invention is 100% buffer solution, consistent with physiological environment buffer capacity in organism；The present invention is with Cys, GSH and HOCl are as input signal, maximum fluorescence emission peak fluorescence intensity at 514 nm and 412 nm constructs Molecular Logic Gates as output signal, it is to be reported for the first time based on Molecular Logic Gates identification Cys, the fluorescence probe of GSH and HOCl is able to solve limitation brought by single identification fluorescence probe.
Description
Technical field
The invention belongs to small-molecule fluorescent probe fields, are related to molecular computer technical field, and in particular to Yi Zhongduo
Identify the synthetic method of fluorescence probe and the construction method of Molecular Logic Gates and its application in detection Cys, GSH and HOCl.
Background technique
Traditional semiconductor silicon material is to realize logical operation by the variation of voltage；Supramolecular chemistry then passes through
The variation of object and the interaction process of main body and its corresponding spectral signal, with outputting and inputting during logic calculation
It is corresponding one by one.And the logical operation of fluorescence probe, be by probe molecule to two or more target molecule into
The change in fluorescence of row identification and the generation in identification process.These target molecules are as input signal, and fluorescent emission is as defeated
Signal out, the two construct Molecular Logic Gates jointly, and different points is in extremely rapid succession detected in same biological sample to realize
Object is analysed, can be used to detect certain diseases.Typical fluorescence probe is single identification.However, the single identification of fluorescence probe
With certain limitation.In biological applications, there may be certain connections between active material, and a kind of active matter
The variation of matter may cause the variation of related substances concentration, these variations cannot be tracked by single identification.Dual identification or
The fluorescence probe of person's Multiple recognition is able to solve limitation brought by single identification fluorescence probe.
Typical fluorescence probe is single identification.However, the single identification of fluorescence probe has certain limitation.?
In biological applications, there may be certain connections between active material, and a kind of variation of active material may cause phase
The variation of material concentration is closed, these variations cannot be tracked by single identification.The fluorescence of dual identification or Multiple recognition is visited
Needle is able to solve limitation brought by single identification fluorescence probe.
Hypochlorous acid (HOCl) is generated in vivo under myeloperoxidase enzymatic by hydrogen peroxide and chloride ion, is
One of typical active oxygen (ROS), has strong oxidation susceptibility and antibacterial activity in biology.But it is secondary in organism
The exception of chloric acid is related with a series of diseases, such as cardiovascular disease, human erythrocyte damage, injury of lungs, rheumatic disease and cancer
Disease.In addition, excessive HOCl can cause oxidative stress, very important effect is played in cell death signal conduction.
Glutathione (GSH) is the nonprotein mercaptan that content is most in normal cell.GSH is in redox active and base
Because playing a significant role in regulation.Once however, GSH horizontal abnormality, it is possible to lead to many diseases, such as malignant tumour, mental and physical efforts
Failure, the diseases such as kidney failure.In addition, intracellular GSH acts also as cysteine (Cys) storage.As one of biological thiol,
Cys participates in biological oxidation stable state, biocatalysis, the removing toxic substances of xenobiotic and other important physiology courses.But Cys
Shortage will lead to slow growth, liver damage, muscle and fat loss, lesion and other diseases.
Excessive HOCl can cause oxidative stress, and intracellular Cys and GSH are important small molecule antioxidant, can be direct
Consume excessive HOCl in organism.However, specificity and rapid detection method, HOCl and Cys/ highly sensitive due to lacking
GSH under oxidative stress really shear be not yet it is fully aware of.Therefore, it is developed based on the fluorescence probe of Molecular Logic Gates,
For detecting the problem of Cys, GSH and HOCl are those skilled in the art's urgent need to resolve.
Summary of the invention
For overcome the deficiencies in the prior art and defect, good, sensitivity that the object of the present invention is to provide a kind of selectivity
Novel fluorescence probe that is high and being capable of Visual retrieval Cys, GSH and HOCl, correspondingly provides the preparation of the novel fluorescence probe
Method, raw material is cheap and easy to get, and preparation cost is low.
It is another object of the present invention to construct Molecular Logic Gates identification Cys, GSH and HOCl, lacking for single identification is avoided
It falls into.
To achieve the goals above, technical scheme is as follows:
A kind of fluorescence probe based on Molecular Logic Gates identification Cys, GSH and HOCl, more identification fluorescent probe molecule formulas are
C33H29N3O4, probe compound is abbreviated as probe NPCC, with structural formula shown in formula (I):
Formula (I).
A kind of synthetic method of above-mentioned fluorescence probe, using following steps:
Step (1): by known compound 10- acetyl -9- hydroxyl -2,3,6,7- tetrahydro -1H- pyrans simultaneously [2,3-f] pyrido [3,
2,1-ij] quinoline -11(5H) -one and benzaldehyde be dissolved in ethyl alcohol, piperazine heavy stone used as an anchor be added, is heated to reflux, and obtaining red solid is to change
Object 1 is closed, structural formula is as follows:
；
Step (2): phenylhydrazine and glacial acetic acid are added in the ethanol solution containing compound 1, is heated to reflux, obtains yellow solid
As compound 2, structural formula is as follows:
；
Step (3): the dichloromethane solution of acryloyl chloride is added drop-wise to molten containing the mixing of the methylene chloride of compound 2 and triethylamine
In liquid, room temperature reaction, silica gel column chromatography chromatographs to obtain probe compound NPCC.
In step (1), the 10- acetyl -9- hydroxyl -2,3,6,7- tetrahydro -1H- pyrans simultaneously [2,3-f] pyrido [3,2,
1-ij] quinoline-11(5H) -one: the molar ratio of benzaldehyde is 1:(1-1.05).
In step (1), the additional amount of the piperazine heavy stone used as an anchor is 6-9 drops.
In step (1), the reaction time is preferably 12-16 hours.
In step (1), the purification procedures are as follows: reaction solution is cooled to room temperature, and has solid precipitation, is filtered, with frost
Ethanol washing filter cake, vacuum drying.
In step (2), the compound 1: the molar ratio of phenylhydrazine is 1:(1-3).
In step (2), the additional amount of the glacial acetic acid is 0.5 mL -1 mL.
In step (2), the reaction time is preferably 3-6 hours.
In step (2), the purification procedures are as follows: reaction solution is cooled to room temperature, and has solid precipitation, is filtered, with frost
Ethanol washing filter cake, vacuum drying.
In step (3), the compound 2: acryloyl chloride: the molar ratio of triethylamine is 1:(1-1.2): (1-1.4).
In step (3), the temperature when acryloyl chloride is added is 0-5 DEG C, and the time is 0.5 hour.
In step (3), the reaction temperature after the acryloyl chloride is added dropwise is room temperature, and reaction temperature is 25-28 DEG C, instead
Preferably 2-4 hours between seasonable.
In step (3), silica gel column chromatography chromatography washing and dehydrating integrated machine used is preferably petroleum ether and methylene chloride volume ratio
1:1 composition.
The synthetic route of probe of the present invention is as follows:
。
Compound N PCC is writing a Chinese character in simplified form for fluorescent probe compounds of the present invention.
Fluorescence probe prepared by the present invention using it first is that for detecting Cys, GSH and HOCl under water environment.
Fluorescence probe prepared by the present invention using it second is that for constructing Molecular Logic Gates.
Above-mentioned application, specifically, including:
Probe is dissolved in dimethyl sulfoxide (DMSO), prepares probe mother liquor (1 mM)；Probe solution is added in prepare liquid；To
Surveying liquid is preferably Tris-HCl(10 mM, 1 mM CTAB) buffer, pH of cushioning fluid 7.4.
Preferably, when in the prepare liquid without Cys GSH, maximum fluorescence emission peak fluorescence at 514 nm of solution
Intensity is without significant change；When having Cys GSH in the prepare liquid, maximum fluorescence emission peak at 514 nm of mixed solution
Fluorescence intensity dramatically increases, the fluoresced green under 365 nm fluorescent lamps.
Preferably, when Cys being added in the prepare liquid, the maximum fluorescence emission peak fluorescence intensity at 514 nm reaches response
It is 60 minutes the time required to platform, when GSH is added in the prepare liquid, the maximum fluorescence emission peak fluorescence intensity at 514 nm reaches
It is 15 minutes to response platform required time.
Preferably, the probe can distinguish Cys and GSH to the difference of the response time of Cys and GSH with dynamics.
Preferably, when Cys being added in the prepare liquid, HOCl is added after sixty minutes, the mixed solution is in 365 nm
Green fluorescence weakens under fluorescent lamp, and the mixed solution occurs without obvious blue fluorescence.
Preferably, when GSH being added in the prepare liquid, HOCl is added after 15 minutes, the mixed solution is in 365 nm
Green fluorescence disappears under fluorescent lamp, and the mixed solution issues blue-fluorescence.
Preferably, the difference of Cys and GSH is added in the prepare liquid, adds HOCl, the mixed solution is in 365 nm
The case where green fluorescence changes with blue-fluorescence under fluorescent lamp is different.
Preferably, the mixed solution is different the case where green fluorescence changes with blue-fluorescence under 365 nm fluorescent lamps,
It can be with Visual retrieval Cys, GSH and HOCl.
Preferably, it is 8.95 nM, GSH that the concentrations lower limit of described probe in detecting Cys, GSH and HOCl, which are respectively as follows: Cys,
It is 0.43 μM for 4.26 nM, HOCl.
The Molecular Logic Gates are acted on based on fluorescence probe and Cys, GSH and HOCl, the variation building of fluorescence intensity
Made of, using maximum fluorescence emission peak fluorescence intensity at 514 nm and 412 nm as judgment basis.
The Molecular Logic Gates include OR type logic gate and AND type logic gate.
The Molecular Logic Gates include that the construction method difference of OR type logic gate and AND type logic gate is as follows:
(1) taking fluorescent probe compounds NPCC solution is template, with Cys perhaps one of GSH for input signal Cys or
Interaction occurs by GSH and probe compound NPCC so that probe compound NPCC green fluorescence under 365 nm fluorescent lamps increases
By force, OR type logic gate is constructed using maximum fluorescence emission peak at 514 nm as output signal；
(2) take fluorescent probe compounds NPCC solution be template, using GSH and HOCl in succession exist as input signal, GSH and with
Probe compound NPCC occur in succession interaction so that probe compound NPCC under 365 nm fluorescent lamps blue-fluorescence enhance,
AND type logic gate is constructed using maximum fluorescence emission peak at 412 nm as output signal.
Compared with prior art, the beneficial effects of the present invention are embodied in following several respects:
1, the novel fluorescence probe of identification Cys, GSH and HOCl of the invention are to report for the first time, and there is detection to limit low, sensitive height,
The good advantage of selectivity, and there is the ability and extensive potential using value for resisting other small molecules interference in organism；
2, probe raw material of the present invention is cheap and easy to get, high income and preparation cost are low, is suitable for industrialized production；
3, prepare liquid of the invention is 100% buffer solution, consistent with the buffer capacity of physiological environment in organism；
4, fluorescence probe of the invention is used for the detection of Cys, GSH and HOCl, its visible color change of naked eye may be implemented visual
Change detection；
5, the present invention with Cys, GSH and HOCl be used as input signal, 514 nm and 412 nm place fluorescent emission as export believe
Number building Molecular Logic Gates, be report for the first time based on Molecular Logic Gates identification Cys, GSH and HOCl fluorescence probe.
Detailed description of the invention
Fig. 1 is addition ultra-violet absorption spectrum and fluorescence emission spectrum of 2 middle probe of embodiment with Cys, GSH and HOCl
Situation of change；After the Cys of 10 μm of ol/L is added, the HOCl of 300 μm of ol/L is added after sixty minutes；It is added 10 μm of ol/L's
After GSH, the HOCl of 300 μm of ol/L is added after 15 minutes.
Fig. 2 is situation of change of 3 middle probe of embodiment with the increase fluorescence spectrum of Cys additional amount；Fig. 2 illustration is 514
In 365 nm fluorescence after scattergram and identification Cys that maximum fluorescence emission peak fluorescence intensity at nm changes with Cys concentration
Fluoresced green under lamp.
Fig. 3 is situation of change of 4 middle probe of embodiment with the increase fluorescence spectrum of GSH additional amount；Fig. 3 illustration is 514
In 365 nm fluorescence after scattergram and identification GSH that maximum fluorescence emission peak fluorescence intensity at nm changes with GSH concentration
Fluoresced green under lamp.
Fig. 4 is that GSH is added in 5 middle probe mixed liquor of embodiment, the HOCl of various concentration is added after 15 minutes, probe is glimmering
The situation of change of light spectrum；Fig. 4 illustration is maximum fluorescence emission peak fluorescence intensity at 412 nm as HOCl concentration increases
Scattergram and identification HOCl after in 365 nm fluorescent lamp exhibits blue fluorescence.
Fig. 5 is that Cys, GSH and other disturbance substances are added in 6 middle probe mixed liquor of embodiment；In figure, other are different
Interference substance is Hcy, His, Glu, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg, Pro, Thy, Lys, Gly, Na+, K+, F–, Cl–, Br–, I–, CO3 2–, HCO3 –, Ac–, SO4 2–, PO4 3–, NO3 –, NO2 –, OH, O2•–, HOCl and H2O2。
Fig. 6 is that GSH is added in 7 middle probe mixed liquor of embodiment, and HOCl and other disturbance objects are added after 15 minutes
Matter；In figure, other different dry immunity substances are Na+, K+, F–, Cl–, Br–, I–, CO3 2–, HCO3 –, Ac–, SO4 2–, S2O8 2–, PO4 3–,
ClO4 –, HSO3 –, HS–, NO3 –, OH, NO2 –,1O2, O2•–And H2O2。
Fig. 7 a is the dynamic experiment that 8 middle probe of embodiment and Cys and GSH are responded；Fig. 7 b be 9 middle probe of embodiment with
The dynamic experiment of HOCl response.
Fig. 8 is the schematic diagram of AND Molecular Logic Gates in OR Molecular Logic Gates and embodiment 11 in embodiment 10.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, but this should not be interpreted as to the scope of the above subject matter of the present invention is limited to the following embodiments, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
The synthesis of fluorescent probe compounds NPCC.
(1) synthesis of compound 1
By known compound 10- acetyl -9- hydroxyl -2,3,6,7- tetrahydro -1H- pyrans simultaneously [2,3-f] pyrido [3,2,1-ij]
Quinoline -11(5H) -one (897 mg, 3 mmol) and benzaldehyde (334 mg, 3.15 mmol) be dissolved in the ethyl alcohol of 25 mL, stir
The piperazine heavy stone used as an anchor of 6-9 drops is added in the process, flows back 14 hours.It after fully reacting, is cooled to room temperature, filters, with the ethanol washing of frost
Filter cake, vacuum drying, obtains the compound 1(896.4 mg of red solid, yield 77.2%).
Nuclear magnetic resonance hydrogen spectruming determining:1H NMR (400 MHz, CDCl3, ppm):1.92-1.99 (m, 4H), 2.76-
2.85 (m, 4H), 3.29-3.32 (m, 4H),7.42 (m, 4H), 7.70 (m, 2H), 7.94 (d, 1 H, J =
15.8 Hz), 8.46 (d, 1 H, J = 15.8 Hz)。
Carbon-13 nmr spectra measurement:13C NMR (100 MHz, CDCl3, ppm): δ = 20.14, 20.25,
21.22, 27.48, 49.82, 50.25, 98.06, 103.11, 105.36, 118.70, 122.75, 124.07,
128.88, 128.96, 130.55, 135.27, 144.41, 149.38, 152.26, 161.51, 179.36,
191.56。
High resolution mass spectrum measurement: [M+H]+ calcd for [C24H21NO4+H ]+: 388.1543. Found:
388.1545。
(2) synthesis of compound 2
The acetic acid of phenylhydrazine (0.162 g, 1.5 mmol) and 0.5 mL is added to containing compound 2(194 mg, 0.5 mmol)
25mL ethanol solution in.Reaction mixture is flowed back 4 hours.After completion of the reaction, there is sediment precipitation, pass through filtering point
From being washed with frozen ethanol, and be dried under vacuum.Obtain yellow solid compound 3(233 mg, yield 97.7%).
Nuclear magnetic resonance hydrogen spectruming determining:1H NMR (400 MHz, CDCl3, ppm): 2.00 (m, 4H), 2.82-2.90
(m, 4H), 3.30-3.31 (m, 4H), 3.53-3.60 (m, 1H), 4.21-4.29 (m, 1H), 5.09-5.15
(m, 1H), 6.85 (t, 1H, J = 31.4 Hz), 6.93 (d, 2H, J = 17.4 Hz), 7.23 (t, 2 H,J = 21.4 Hz), 7.30 (s, 1 H), 7.36-7.42 (m, 5 H), 13.85 (s, 1 H)。
Carbon-13 nmr spectra measurement:13C NMR (100 MHz, CDCl3, ppm): δ = 20.38, 20.51,
21.48, 27.67, 46.89, 49.65, 50.09, 63.43, 91.85, 103.15, 105.84, 113.15,
118.51, 119.56, 121.05, 125.99, 127.59, 129.10, 129.47, 142.27, 144.65,
147.12, 150.76, 151.57, 162.10, 167.36。
High resolution mass spectrum measurement: HR-MS:m/z [M+H]+ calcd for [C30H21N3O3+H ]+: 478.2125.
Found: 478.2115。
(3) synthesis of fluorescent probe compounds NPCC
At 0-5 DEG C, methylene chloride (5 mL) solution of acryloyl chloride (90.5 mg, 0.225 mmol) is added drop-wise to containing change
Close object 2(83.5 mg, 0.175 mmol) and triethylamine (25 mg, 0.25 mmol) methylene chloride (10 mL) mixed solution
In.After being added dropwise in half an hour, it is to slowly warm up to room temperature.It reacts 2.5 hours at room temperature, after the reaction was completed, by mixture
It is washed and is dried over anhydrous sodium sulfate with salt water (10 mL × 3).Combined organic layer is filtered, solvent is evaporated under reduced pressure.Crude product is logical
Silica gel chromatography (eluant, eluent is petroleum ether: methylene chloride=1:1) is crossed, probe NPCC is obtained, is red brown solid (60
Mg, yield 65%).
Nuclear magnetic resonance hydrogen spectruming determining:1H NMR (400 MHz, CDCl3, ppm): 2.00 (m, 4H), 2.82-2.90
(m, 4H), 3.30-3.31 (m, 4H), 3.53-3.60 (m, 1H), 4.21-4.29 (m, 1H), 5.09-5.15
(m, 1H), 6.85 (t, 1H, J = 31.4 Hz), 6.93 (d, 2H, J = 17.4 Hz), 7.23 (t, 2 H,J = 21.4 Hz), 7.30 (s, 1 H), 7.36-7.42 (m, 5 H), 13.85 (s, 1 H)。
Carbon-13 nmr spectra measurement:13C NMR (100 MHz, CDCl3, ppm): δ = 20.38, 20.51,
21.48, 27.67, 46.89, 49.65, 50.09, 63.43, 91.85, 103.15, 105.84, 113.15,
118.51, 119.56, 121.05, 125.99, 127.59, 129.10, 129.47, 142.27, 144.65,
147.12, 150.76, 151.57, 162.10, 167.36。
High resolution mass spectrum measurement: HR-MS:m/z [M+H]+ calcd for [C33H29N3O4+H ]+: 532.2231.
Found: 532.2222。
Embodiment 2
Uv-vis spectra and fluorescence emission spectrum variation when probe and Cys of the present invention, GSH and HOCl are acted on.
Probe prepared by embodiment 1 is dissolved in DMSO, the probe mother liquor that preparation concentration and probe concentration is 1 mM.Prepare liquid is
Tris-HCl(10 mM, 1 mM CTAB) buffer, pH of cushioning fluid 7.4.30 μ L are taken to be added to 3 mL from probe mother liquor
Prepare liquid in probe mixed solution, mixed solution middle probe ultimate density be 10 μm of ol/L.With UV, visible light spectrophotometric
Uv-vis spectra and fluorescence spectrum when meter and sepectrophotofluorometer test probe and Cys, GSH and HOCl act on change feelings
Condition.From fig. 1, it can be seen that being added Cys(10 μM) or GSH(10 μM) after, the maximal ultraviolet absorption peak of probe is blue from 471 nm
441 nm are moved to, probe fluorescence intensity at 514 nm enhances, and in the process, solution colour is turned yellow by crocus,
There is green fluorescence appearance under 365 nm fluorescent lamps, realizes Visual retrieval Cys and GSH.Cys and GSH are separately added into above-mentioned
Solution in continuously add HOCl(30 μM) after, it can be seen that visibly different experimental phenomena: be separately added into GSH's and HOCl
Probe solution color becomes colourless by yellow, and in 365 nm fluorescent lamp exhibits blue fluorescence, maximal ultraviolet absorption peak is from 441 nm
Place is blue shifted to 380 nm, and maximum fluorescence emission peak is blue shifted to from 412 nm from 514 nm, and there is ultra-violet absorption spectrum and fluorescence to send out
Penetrate the feature of spectra ratios type detection HOCl；The probe solution color for being separately added into Cys and HOCl becomes light yellow by yellow,
Green fluorescence shoals under 365 nm fluorescent lamps, and no blue line fluorescence occurs, glimmering at the ultraviolet absorption peak and 514 nm at 441 nm
Light emitting peak does not completely disappear.
Embodiment 3
The fluorescence titration experiment of probe identification Cys of the present invention.
Prepared by embodiment 2 takes 30 μ L to be added in 3 mL prepare liquids in probe mother liquor, probe ultimate density is 10 μm of ol/
The Cys mother liquor of different equivalents (0-4 equivalent) is added in L, as shown in Fig. 2, with the increase of Cys concentration, maximum fluorescence at 514 nm
Emission peak fluorescence intensity gradually increases, and the detection of probe identification Cys is limited to 8.95 nM.
Embodiment 4
The fluorescence titration experiment of probe identification GSH of the present invention.
Prepared by embodiment 2 takes 30 μ L to be added in the prepare liquid of 3 mL in probe mother liquor, probe ultimate density is 10 μ
The GSH mother liquor of different equivalents (0-5 equivalent) is added in mol/L, as shown in figure 3, with the increase of GSH concentration, it is maximum at 514 nm
Fluorescence emission peak fluorescence intensity gradually increases, and the detection of probe identification GSH is limited to 4.26 nM.
Embodiment 5
The fluorescence titration experiment of probe identification HOCl of the present invention.
Prepared by embodiment 2 takes 30 μ L to be added in the prepare liquid of 3 mL in probe mother liquor, probe ultimate density is 10 μ
The GSH mother liquor of 10 equivalents is first added in mol/L, after 15 minutes, then difference is added into the prepare liquid of above-mentioned addition probe and GSH
The HOCl mother liquor of equivalent (0-30 equivalent), as shown in figure 4, fluorescence intensity gradually drops at 514 nm with the increase of HOCl concentration
Low, fluorescence intensity gradually increases at 412nm, and the feature with fluorescence emission spectrum Ratio-type detection HOCl, probe identifies HOCl
Detection be limited to 0.43 μM.
Embodiment 6
The present invention identifies the fluorescence selectivity experiment of Cys and GSH.
Prepared by embodiment 2 takes 30 μ L to be added in the prepare liquid of 3 mL in probe mother liquor, probe ultimate density is 10 μ
Mol/L, then it is separately added into the following disturbance substance of 10 equivalents: Hcy, His, Glu, Asp, Val, Phe, Tyr, Ala, Ser,
Leu, Arg, Pro, Thy, Lys, Gly, Na+, K+, F–, Cl–, Br–, I–, CO3 2–, HCO3 –, Ac–, SO4 2–, PO4 3–, NO3 –, NO2 –,
OH, O2•–, HOCl and H2O2.With the ultraviolet and fluorescence of ultraviolet-uisible spectrophotometer and sepectrophotofluorometer detection test fluid
Spectrum change.As can be seen from Figure 5, when a variety of different interfering substances are added in test fluid, 471 nm ultraviolet absorption peaks become without obvious
Change, fluorescence emission peak is without obviously increasing at 514 nm.Hcy is that acid content containing sulfhydryl amino is least in normal organism, so
Interference is unconspicuous caused by the addition of Hcy.However, after Cys or GSH is added, the maximal ultraviolet absorption peak of test fluid from
441 nm are blue shifted at 471 nm, fluorescence intensity enhances at 514 nm.Experimental result illustrates that probe NPCC has Cys and GSH
There is good selectivity.
Embodiment 7
The present invention identifies the fluorescence selectivity experiment of HOCl.
Prepared by embodiment 2 takes 30 μ L to be added in the prepare liquid of 3 mL in probe mother liquor, probe ultimate density is 10 μ
Mol/L, is first added the GSH mother liquor of 10 equivalents, after 15 min, then is separately added into the following disturbance substance of 30 equivalents: Na+, K+, F–, Cl–, Br–, I–, CO3 2–, HCO3 –, Ac–, SO4 2–, S2O8 2–, PO4 3–, ClO4 –, HSO3 –, HS–, NO3 –, OH, NO2 –,1O2,
O2•–And H2O2.With the ultraviolet and fluorescence spectrum variation of ultraviolet-uisible spectrophotometer and sepectrophotofluorometer detection test fluid.
As can be seen from Figure 6, when a variety of different interfering substances are added in test fluid, 441 nm ultraviolet absorption peaks are without significant change, 514 nm
Place's fluorescence emission peak is without being substantially reduced, and the nothing of fluorescence emission peak obviously increases at 412 nm.However, after HOCl is added, test fluid
Maximal ultraviolet absorption peak 380 nm are blue shifted to from 441 nm, at 514 nm fluorescence emission peak fluorescence intensity weaken, 412
Fluorescence emission peak fluorescence intensity increases at nm, realizes ultra-violet absorption spectrum and fluorescence emission spectrum Ratio-type detection HOCl.It is real
It tests result and illustrates that probe NPCC has good selectivity HOCl.
Embodiment 8
The dynamic experiment of probe of the present invention and Cys and GSH, that is, after Cys or GSH being added, the fluorescence intensity of probe and time
Relationship.
Prepared by embodiment 2 takes 30 μ L to be added in the prepare liquid of 3 mL in probe mother liquor, probe ultimate density is 10 μ
Mol/L, is separately added into Cys the and GSH mother liquor of 10 equivalents, and the concentration of Cys and GSH are 100 μm of ol/L.With the excitation of 440 nm
Wavelength excitation tests the fluorescence intensity of probe with the fluorescence spectra of the variation of time.As shown in Fig. 7 a, with the increasing of time
Add, the fluorescence intensity at 512 nm gradually increases, and the prepare liquid that probe and Cys is added reached stationary value at 60 minutes, is added and visits
The prepare liquid of needle and GSH reached stationary value in 15 minutes.Experimental result illustrate probe can by dynamics distinguish Cys and
GSH。
Embodiment 9
Probe of the present invention and HOCl dynamic experiment add HOCl, the fluorescence of probe after that is, addition GSH reaches response platform
The relationship of intensity and time.
Prepared by embodiment 2 takes 30 μ L to be added in the prepare liquid of 3 mL in probe mother liquor, probe ultimate density is 10 μ
Mol/L, is separately added into Cys the and GSH mother liquor of 10 equivalents, then being separately added into the concentration of HOCl, Cys and the GSH of 30 equivalents is 100
The concentration of μm ol/L, HOCl are 300 μm of ol/L.It is excited with the excitation wavelength of 380 nm, the place test 412 nm of probe and 514 nm
Fluorescence intensity ratio (I412/I514) with the time variation fluorescence spectra.As shown in Fig. 7 b, as time increases,
The Cys of 10 equivalents is first added, the rear HOCl, I that 30 equivalents are added412/I514Fluorescence intensity ratio variation it is unobvious；First it is added 10
The GSH of equivalent, the rear HOCl, I that 30 equivalents are added412/I514Fluorescence intensity ratio dramatically increase, reach stable in 15 minutes
Value.Experimental result illustrates that probe can effectively distinguish Cys, GSH and HOCl by dynamics.
Embodiment 10
The building of OR type logic gate.
The building of OR type logic gate, principle are existed as shown in figure 8, using Cys or GSH as output signal with probe NPCC
Maximum fluorescence emission peak fluorescence intensity at 514 nm is output signal, and the maximum fluorescence emission peak fluorescence intensity at 514 nm increases
By force, corresponding true value be 1,514 nm place maximum fluorescence emission peak without obvious fluorescence emission peak when, corresponding truth table be 0.
When input is (0,0), 30 μ L are taken to be added in the prepare liquid of 3 mL from probe mother liquor prepared by embodiment 2, probe is final
Concentration is 10 μm of ol/L, its fluorescence intensity is tested under the excitation of 441 nm, is turned since intramolecular charge occurs for probe NPCC
It moves, probe is without obvious fluorescence, so output valve is 0；When input is (1,0), 30 are taken from probe mother liquor prepared by embodiment 2
μ L is added in the prepare liquid of 3 mL, and probe ultimate density is 10 μm of ol/L, the Cys mother liquor of 10 equivalents is added, Cys's is final
Concentration is 100 μm of ol/L, its fluorescence intensity is tested under the excitation of 441 nm, due to probe NPCC Intramolecular electron transfer quilt
Inhibit, maximum fluorescence emission peak fluorescence intensity of the probe at 514 nm dramatically increases, so output valve is 1；When input for (0,
1) when, 30 μ L are taken to be added in the prepare liquid of 3 mL from probe mother liquor prepared by embodiment 2, probe ultimate density is 10 μ
The GSH mother liquor of 10 equivalents is added in mol/L, and the ultimate density of GSH is 100 μm of ol/L, and it is glimmering to test its under the excitation of 441 nm
Luminous intensity, since probe NPCC Intramolecular electron transfer is suppressed, maximum fluorescence emission peak fluorescence of the probe at 514 nm is strong
Degree dramatically increases, so output valve is 1；When input is (1,1), 30 μ L is taken to be added from probe mother liquor prepared by embodiment 2
Into the prepare liquid of 3 mL, probe ultimate density is 10 μm of ol/L, and the GSH of the Cys and 10 equivalents that are separately added into 10 equivalents are female
The ultimate density of liquid, Cys and GSH are 100 μm of ol/L, its fluorescence intensity are tested under the excitation of 441 nm, due to probe NPCC
Intramolecular electron transfer is suppressed, and maximum fluorescence emission peak fluorescence intensity of the probe at 514 nm dramatically increases, so output
Value is 1.
The building of OR type logic gate, varying input signal, corresponding output signal truth table are as shown in table 1.
OR type logic gate truth table
| Input 1(Cys) | Input 2(GSH) | It exports (fluorescence intensity at 514 nm) |
| 0 | 0 | 0 |
| 1 | 0 | 1 |
| 0 | 1 | 1 |
| 1 | 1 | 1 |
Embodiment 11
The building of AND type logic gate.
The building of AND type logic gate, principle are existed as shown in figure 8, using GSH and HOCl as output signal with probe NPCC
Maximum fluorescence emission peak fluorescence intensity at 412 nm is output signal, and the maximum fluorescence emission peak fluorescence intensity at 412 nm increases
By force, when corresponding true value is at 12 nm of Isosorbide-5-Nitrae without obvious fluorescence emission peak, corresponding truth table is 0.When input is (0,0)
When, take 30 μ L to be added in the prepare liquid of 3 mL from probe mother liquor prepared by embodiment 2, probe ultimate density is 10 μ
Mol/L tests its fluorescence intensity under the excitation of 380 nm, and probe is without obvious fluorescence, so output valve is 0；When input for (1,
0) when, 30 μ L are taken to be added in the prepare liquid of 3 mL from probe mother liquor prepared by embodiment 2, probe ultimate density is 10 μ
The GSH mother liquor of 10 equivalents is added in mol/L, and the ultimate density of GSH is 100 μm of ol/L, and it is glimmering to test its under the excitation of 380 nm
Luminous intensity, fluorescence intensity is without obviously increasing at 412 nm for probe, so output valve is 0；When input is (0,1), from implementation
30 μ L are taken to be added in the prepare liquid of 3 mL in probe mother liquor prepared by example 2, probe ultimate density is 10 μm of ol/L, is added 30
The ultimate density of the HOCl mother liquor of equivalent, HOCl is 300 μm of ol/L, its fluorescence intensity is tested under the excitation of 380 nm, due to
Probe NPCC cannot directly and HOCl, and fluorescence intensity is without obviously increasing at 412 nm for probe, so output valve is 0；Work as input
When for (1,1), 30 μ L are taken to be added in the prepare liquid of 3 mL from probe mother liquor prepared by embodiment 2, probe ultimate density is
The GSH of 10 equivalents is added in 10 μm of ol/L, and the GSH mother liquor of 30 equivalents is added after 15 minutes, and the ultimate density of GSH is 100 μ
The ultimate density of mol/L, HOCl are 300 μm of ol/L, its fluorescence intensity are tested under the excitation of 380 nm, probe is in 412 nm
The maximum fluorescence emission peak fluorescence intensity at place dramatically increases, so output valve is 1.
AND type logic gate truth table
| Input 1(GSH) | Input 2(HOCl) | It exports (fluorescence intensity at 412 nm) |
| 0 | 0 | 0 |
| 1 | 0 | 0 |
| 0 | 1 | 0 |
| 1 | 1 | 1 |
Claims (12)
1. one kind identifies that Cys, GSH and HOCl fluorescence probe, probe compound are abbreviated as probe NPCC based on Molecular Logic Gates,
It is characterized in that, the chemical structure of the probe is as shown in the formula (I):
；
Formula (I).
2. a kind of synthetic method of small-molecule fluorescent probe for detecting Cys, GSH and HOCl according to claim 1, special
Sign is, the specific steps of which are as follows:
(1) 10- acetyl -9- hydroxyl -2,3,6,7- tetrahydro -1H- pyrans simultaneously [2,3-f] pyrido [3,2,1-ij] quinoline -11
(5H) -one, benzaldehyde and piperazine heavy stone used as an anchor flow back in ethanol, and compound 1 can be obtained in product filtering:
；
(2) compound 1, phenylhydrazine and glacial acetic acid flow back in ethanol, and compound 2 can be obtained in product filtering:
；
(3) dichloromethane solution of acryloyl chloride is added drop-wise in the methylene chloride mixed solution containing compound 2 and triethylamine,
It is to slowly warm up to room temperature, is reacted at room temperature, product obtains fluorescence probe NPCC after isolating and purifying.
3. synthetic method according to claim 2, which is characterized in that in step (1), hydroxyl -2 10- acetyl -9-,
3,6,7- tetrahydro -1H- pyrans simultaneously [2,3-f] pyrido [3,2,1-ij] quinoline -11(5H) -one: the molar ratio of benzaldehyde is 1:
(1-1.05), the additional amount of the piperazine heavy stone used as an anchor are 6-9 drops；In step (2), the compound 1: the molar ratio of phenylhydrazine is 1:(1-3),
The additional amount of the glacial acetic acid is 0.5-1 mL；In step (3), the compound 2: acryloyl chloride: the molar ratio of triethylamine is
1:(1-1.2): (1-1.4).
4. synthetic method according to claim 2, which is characterized in that in step (1), the heating temperature is 80 DEG C, instead
It is 12-16 hours between seasonable；In step (2), the heating temperature is 80 DEG C, and the reaction time is 3-6 hours；In step (3), institute
Temperature is 0-5 DEG C when stating the addition of acryloyl chloride, and the reaction time is 0.5 hour, after the acryloyl chloride is added, is slowly risen
It warms to room temperature, reaction temperature is 25-28 DEG C, and the reaction time is 2-4 hours.
5. synthetic method according to claim 2, which is characterized in that in step (1), it is described be filtered into be cooled to room temperature after
Filtering, and with frost ethanol washing, be dried in vacuo；In step (2), it is described be filtered into be cooled to room temperature after filter, and
With the ethanol washing of frost, vacuum drying；In step (3), the purification procedures are crude product reaction solution salt water (10
ML × 3) it washs and is dried over anhydrous sodium sulfate, the organic layer of merging is filtered, solvent is evaporated under reduced pressure, crude product passes through silicagel column color
Spectrum purifying (eluant, eluent is petroleum ether: methylene chloride=1:1).
6. a kind of small-molecule fluorescent probe or claim 2-5 times of detection Cys, GSH and HOCl according to claim 1
The application of the small-molecule fluorescent probe of one described detection Cys, GSH and HOCl obtained, it is characterized in that: in Tris-HCl(10
MM, pH=7.4,1 mM CTAB) Cys, GSH and HOCl are detected in buffer, it is raw in the buffer and organism
It is consistent to manage environment buffer capacity；It is described in the mM of Tris-HCl(10 mM, pH=7.4,1 CTAB) in buffer to Cys, GSH
It carries out being detected as prepare liquid with HOCl, probe mother liquor, which is added in prepare liquid, obtains mixed solution, utilizes the change in fluorescence of mixed solution
To detect the presence or absence of Cys, GSH and HOCl.
7. application according to claim 6, which is characterized in that described mixed when in the prepare liquid without Cys, GSH and HOCl
Close the transmitting of solution unstressed configuration；When having Cys in the mixed solution, mixed solution green-emitting under 365 nm fluorescent lamps is glimmering
Light, maximum fluorescence emission peak fluorescence intensity significantly increases at 514 nm；When having GSH in the mixed solution, the mixed solution
The fluoresced green under 365 nm fluorescent lamps, maximum fluorescence emission peak fluorescence intensity significantly increases at 514 nm；The mixing is molten
When Cys being first added in liquid, HOCl, mixed solution green fluorescence under 365 nm fluorescent lamps are added after reaching response platform
Weaken, no blue-fluorescence occurs, and maximum fluorescence emission peak fluorescence intensity part is quenched at 514 nm, and maximum fluorescence is sent out at 412 nm
Penetrate peak fluorescence intensity reinforced partly；When GSH being first added in the mixed solution, HOCl is added after reaching response platform, it is described
Mixed solution redgreen fluorescence under 365 nm fluorescent lamps, there is blue-fluorescence appearance, and maximal ultraviolet absorption peak is blue from 441 nm
380 nm are moved to, maximum fluorescence emission peak is blue shifted to from 412 nm from 514 nm, and the probe has ultra-violet absorption spectrum and glimmering
The feature of optical emission spectroscopy Ratio-type detection HOCl.
8. application according to claim 7, which is characterized in that Cys or GSH is added in the mixed liquor, reaches response
The time of platform is different: being added the time required to Cys reaches response platform in the mixed liquor is 1 hour, is added in the mixed liquor
Entering the time required to GSH reaches response platform is 15 minutes, can distinguish Cys and GSH by dynamics；First add in the mixed liquor
Enter after Cys or GSH reaches response platform, adds HOCl, mixture solution color change under 365 nm fluorescent lamps
Difference, the maximum fluorescence emission peak at 514 nm and the maximum fluorescence emission peak fluorescence intensity at 412nm are different, and the solution exists
Color change difference and maximum fluorescence emission difference can effectively distinguish Cys, GSH and HOCl under 365 nm fluorescent lamps.
9. a kind of small-molecule fluorescent probe of detection Cys, GSH and HOCl according to claim 1, which is characterized in that institute
Quantitative detection of the fluorescence probe for Cys, GSH and HOCl is stated, the detection Cys detection is limited to 8.95 nM, the detection GSH
Detection be limited to 4.26 nM, the detection of the detection HOCl is limited to 0.43 μM.
10. a kind of change in fluorescence of the small-molecule fluorescent probe of detection Cys, GSH and HOCl according to claim 7 with plus
The difference for entering Cys, GSH and HOCl can construct Molecular Logic Gates model, which is characterized in that the Molecular Logic Gates are based on spy
Needle and Cys or GSH are acted on, and the principle of maximum fluorescence emission peak fluorescence enhancement is built-up at 514 nm, at 514 nm most
Big fluorescence emission peak fluorescence enhancement, exporting is that maximum fluorescence emission peak fluorescence intensity exports without significant change and is at 1,514 nm
0；When GSH is first added, HOCl is added after reaching response platform, maximum fluorescence emission peak fluorescence intensity significantly increases at 412 nm
Principle it is built-up, at 412 nm maximum fluorescence emission peak fluorescence intensity enhance, export as maximum fluorescence at 12 nm of Isosorbide-5-Nitrae
For emission peak fluorescence intensity without significant change, exporting is 0.
11. application according to claim 10, which is characterized in that the Molecular Logic Gates include OR type Molecular Logic Gates
With AND type Molecular Logic Gates, the construction method difference of the OR type Molecular Logic Gates and AND type Molecular Logic Gates is as follows:
(1) taking fluorescence probe mixed liquor described in claim 6 is template, is input signal, Cys with one of Cys or GSH
Or interaction occurs for GSH and fluorescence probe so that fluorescence probe mixed liquor green fluorescence under 365 nm fluorescent lamps increases
By force, maximum fluorescence emission peak fluorescence enhancement at 514 nm is believed using maximum fluorescence emission peak fluorescence intensity at 514 nm as output
Number building OR type Molecular Logic Gates；
(2) taking fluorescence probe mixed liquor described in claim 6 is template, is existed in succession using GSH and HOCl as input signal,
Interaction occurs in succession for GSH and HOCl and fluorescence probe so that the fluorescence probe mixed liquor is blue under 365 nm fluorescent lamps
Fluorescence enhancement, maximum fluorescence emission peak fluorescence enhancement at 412 nm, using maximum fluorescence emission peak fluorescence intensity at 412 nm as
Output signal constructs AND type logic gate.
12. the building of molecular logic according to claim 11 identifies Cys, GSH and HOCl based on Molecular Logic Gates more
Fluorescence probe solves limitation brought by single identification fluorescence probe, i.e., there may be certain between active material
System, and a kind of variation of active material may cause the variation of related substances concentration, and these variations cannot pass through single identification
To track.
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