CN107382900A - A kind of preparation method and applications of pH fluorescence probes - Google Patents
A kind of preparation method and applications of pH fluorescence probes Download PDFInfo
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Abstract
The present invention provides a kind of preparation method and applications of pH fluorescence probes, devise novel fluorescence probe CM BHNBD, it is derived from NBD (Zo of 4 nitrobenzene, 2 ethanedioic acid 13 two), it can be monitored to being acidified in intracellular mitochondrial/film of fluorogen, detected applied to pH in biological specimen body, have the advantages that good response, accuracys of data, reappearance, precision are high, exploitativeness is strong.
Description
Technical field
The invention belongs to analytical chemistry field, is related to a kind of preparation method of pH fluorescence probes and its in biological specimen
For the real-time detection method of pH value, acidifying monitoring specially in intracellular mitochondrial or mitochondrial membrane.
Background technology
At present, to H in intracellular mitochondrial/film+The analysis of monitoring, chemical method mainly include high performance liquid chromatography and matter
Spectrum, gas phase, UV-detector etc., fluorescent molecular probe detection method quantity are considerably less.Gas-chromatography detection method is very
It is multi-field to be applied, but be not best choosing for the quick detection of analysis field and clinical medicine biology sample
Select, first, gas-chromatography detection method technical step is complicated, and sample handling procedure is various, workload is big, the operating time is long, effect
Rate is low etc..This is completely not in the cards for medical biotechnology sample, particularly living cells, In vivo detection.Secondly, sample
Product demand is big, and reagent consumption is big.The characteristics of biological specimen, is that sample size is small, and chromatogram joint large-scale instrument detection
Method then generally requires to pre-process large number of sample to be tested to reach the purpose of object purifying, enrichment, separation, therefore simultaneously
It is not suitable for trace sample analysis.Finally, large-scale instrument cost itself is high, and it is careful to operate often step, be not easy to grasp and
Promote, in the allegro modern society of high efficiency, the application of such method seems the bottleneck period for having reached development.
Fluorescent molecular probe detection mainly carries out height selection detection to determinand by designing fluorescent molecular probe, so as to
The change of fluorescence response is produced, and highly sensitive detection is carried out to determinand using the difference property of optical signalling.Step is substantially
It is summarised as：MOLECULE DESIGN, sample detection etc..Detection and application based on fluorescent molecular probe are relatively new in analytical technology
Technology, its principle be by MOLECULE DESIGN thought, and luminophore is connected with reactive group to obtain molecular probe.During detection,
Probe identifies object by the specific reaction of reactive group, and the change of molecular structure will impact to luminophore,
Fluorescence signal is set to change, so as to realize detection or the bio-imaging to object.From the visible such method of its Cleaning Principle
Advantage is that selectivity is good, it is not necessary to which determinand separates, easy to operate, the batch quantity analysis suitable for sample.Institute is in this way in life
Life analysis and research field enjoys high praise.
At present, many fast and easily detection means, including colorimetric method, meat are emerged in life organic analysis detection field
Eye monitoring etc..These methods are widely used in fields such as cell analysis, monitored in vivo, but to the detection of pH in living cells
It is still in the blank of research.Therefore, detection method is further improved, it is living that the convenient degree of experimental implementation is continued into raising
The actual demand of internal pH detection method exploitation.
PH value plays the role of important physiology and pathology in cell autophagy and Apoptosis and stomach esophagus disease.
But newest evidence shows, intracellular acidification is the outstanding feature for embodying a concentrated reflection of intracellular lesion tissue.Although at present
More abnormal cell acidifying has been investigated, but has stilled need further to illustrate a variety of biological effects and multiple stimulation cells
Cell acidifying under interior conflict environment.In addition, medical science is non-to acidifying monitoring in intestinal mucosa and the gastrointestinal disease through drug screening at present
It is often urgent.
PH value is played an important role in terms of cell function, cell metabolism, is sustained life.Under normal circumstances, suitably
Acidity can adjust the activity of enzyme, promote the degraded of protein, so as to build favourable environment for metabolic cycles.However, pH value
Anomalous variation, such as it is acidified, it may occur that the damage of the cell dysfunction related to cell autophagy process, Apoptosis and organelle
Evil.For example, playing central role in mitochondria, cell differentiation and the growth of cell cycle are determined, and mitochondria is acidified implication
Mitochondrial autophagy, depolarising, and various diseases, including angiocardiopathy, DPN and cancer.Therefore, cell or tissue
Acidifying always means the generation of internal major injury.Therefore, cell or tissue acidifying always means internal major injury
Occur.
The content of the invention
The present invention is directed to the above-mentioned problems in the prior art, there is provided a kind of preparation method of pH fluorescence probes, this hair
Bright research and design novel fluorescence probe BHNBD and CM-BHNBD, they are derived from the NBD (3- of 4- nitrobenzene -2- ethanedioic acids -1
Two Zo), it can monitor to being acidified in intracellular mitochondrial/film of fluorogen, be detected applied to pH in biological specimen body, have and ring
The advantages that answering property is good, the accuracy of data, reappearance, high precision, exploitativeness are strong.
Meanwhile present invention also offers the real-time detection that above-mentioned pH fluorescence probes are applied to biological specimen inner cell pH value
Method.
Technical solution of the present invention is as follows：
A kind of preparation method of PH fluorescence probes, step include：
（1）By NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) according to concentration 0.001~-
0.008g/mL is dissolved in chloroform, then adds the solution of same volume chloroform, and the solution of chloroform is to contain volume integral in chloroform
The % of number 0.39~1 hydrazine hydrate-methanol solution, it is well mixed, stirs to obtain brown precipitate at room temperature, is separated out completely to precipitating, mistake
Filter, filter cake wash through ethyl acetate, dry, obtain NBD hydrazines；
（2）Pyridinium chloro-chromate is added in dichloromethane, adds 0.041~0.12g/mL of concentration, ultrasonic dissolution, Ran Houjia
Enter the g/mL of concentration 0.02~0.08 4-（Chloromethyl）Benzylated polyol, the h of reaction 3~4 is stirred at room temperature under nitrogen protection, has reacted
Into the absolute ether of rear addition 3~4 times of volumes of dichloromethane, stratification after 5min is stirred, removes supernatant, remove layer glue
Thing, then add the absolute ether of foregoing same volume, 20min is ground, it is molten with water, 5% sodium bicarbonate aqueous solution, saturated sodium-chloride successively
Liquid washs, and organic layer anhydrous sodium sulfate drying, revolving, obtains white crystal；
（3）By step（2）White crystal is dissolved in absolute ethyl alcohol, the g/mL of concentration of ordinary dissolution 0.002~0.006, is then added dense
0.002~0.004g/mL NBD hydrazines are spent, then the glacial acetic acid of absolute ethyl alcohol volume 1% is added dropwise, 3~4h of heating reflux reaction, reaction
Revolving removes solvent after end, then is purified through silicagel column, gradient elution, obtains Chinese red solid powder, i.e. PH fluorescence probes.
Further, step（1）In, in the hydrazine hydrate-methanol solution, hydrazine hydrate volumetric concentration is 0.2~1.2%.
Further, step（1）In, the NBD-Cl is dissolved in chloroform according to concentration 0.004g/mL.
Further, step（2）In, it is 0.083g/mL, Ran Houjia that pyridinium chloro-chromate, which is added to concentration in dichloromethane,
Enter 4-（Chloromethyl）The concentration of benzylated polyol is 0.05g/mL.
Further, step（2）In, the dichloromethane preferably newly steams anhydrous methylene chloride.
Further, step（2）In, reaction process is tracked using a plate, is terminal after consumption of raw materials is complete.
Further, step（2）In, the washing, be preferably washed with water successively three times, 5wt% sodium acid carbonates wash twice, it is full
Washed once with sodium chloride solution, each cleaning solution dosage is 0.5 times of absolute ether volume.
Further, step（3）In, step（2）White crystal concentration of ordinary dissolution in absolute ethyl alcohol is 0.004g/mL, so
The concentration for adding NBD hydrazines afterwards is 0.0032g/mL.
Further, step（3）In, the silicagel column purifying, preferably dichloromethane dissolves；The gradient elution, elution
Agent is dichloromethane：Methanol 500：1 to 200：1.
The PH fluorescence probes that the invention described above method is prepared（That is CM-BNBD probes）Effect judge index is as follows：
Absorbing wavelength changes：PH value, which is absorbed from acidity to neutrality to feux rouges, moves nearly 100 nm；
Detection range：Linear relationship in the range of pH1.98~7.00 be present.
The application of above-mentioned PH fluorescent molecular probes：Suitable for biological specimen, the pH detections point in intracellular mitochondrial/film
PH detection and analysis in analysis, tissue adherence, detection are sensitive, accurate, quick；Described biological specimen mainly include mucous membrane of esophagus,
Gastric mucosa, Hela cells etc., it can be applied to the correlations such as life organic analysis, analytical chemistry, disease are examined in advance and clinical medicine detects
Field.
A kind of real-time detection method for being used for pH value in intracellular mitochondrial/film using above-mentioned PH fluorescent molecular probes, step
Suddenly include：
1）Prepare solution
Prepare CM-BNBD probe storing solutions：The accurate PH fluorescent molecular probes that weigh are dissolved in anhydrous acetonitrile, and compound concentration is 1 × 10-4Mol/L, obtain CM-BNBD probe storing solutions；
Prepare object buffer solution to be measured（Britton-Robison buffer solutions）：With 0. 04 mol/L phosphoric acid, boric acid and acetic acid
It is formulated, is adjusted with the 0. 2 mol/L NaOH aqueous solution to required pH value during use；
2）Establish the linear equation of pH value and fluorescence signal intensity
Take step 1）The buffer standard product that the pH value that the object buffer solution to be measured prepared dilutes to obtain gradient concentration is 2~7 is molten
Liquid, respectively take 200 μ L respectively with 30 μ LCM-BNBD probes storing solutions and 770 μ L acetonitriles, shaking is well mixed, is placed at 37 DEG C
5min, then detected through sepectrophotofluorometer or ultraviolet spectrometer, establish the linear equation of pH value and fluorescence signal intensity；
Further, step 2）In, the buffer standard product solution PH of gradient concentration is followed successively by 2,3,4,5,6,7.
3）PH linear relationship is established, using a）Or b）Method determines sample P H values
a）Fluorescence detection：After 1000 μ L testing samples are injected into quartz colorimetric utensil, ultraviolet specrophotometer is inserted, is collected maximum
The intensity of absorbing wavelength position, obtain maximum absorption band volume efficiency before and after reacting and substitute into ultraviolet linear relationship, calculate
Obtain intracellular H+Levels, determine pH value；
b）Ultraviolet detection treats method：After 1000 μ L testing samples are injected into quartz colorimetric utensil, ultraviolet specrophotometer is inserted, is collected most
The intensity of big absorbing wavelength position, obtain maximum absorption band volume efficiency before and after reacting and substitute into ultraviolet linear relationship, count
Calculate to obtain intracellular H+Levels, determine pH value.
Above-mentioned detection method, respectively in the method for fluoroscopic examination or ultraviolet detection to intracellular H+Carry out parallel repeated detection
（n=7）, and calibrated with standard items, the optimal detection scope of fluorescence/ultraviolet detection is obtained, so as to according to contained by different samples
The concentration range of determinand come select optimize detection means quantified.
The Small-molecule probe detection mechanism of acidifying monitoring in intracellular mitochondrial/film prepared by the present invention（Such as Fig. 1）：In order to
Fluorescent switch mechanism is solved, the excited level rail of CM-BNBD fluorophors and reactive group is calculated in the method for quantum calculation
Road.As a result it is visible, in H+Under existence condition, CM-BNBD forms stable conjugated structure R-C=N+, relatively low energy is caused, so as to
Electronics is inhibited from R-C=N+Shifted to the electronics of NBD fluorogens, prevent PET, so as to which fluorescence is opened.Understand, the present invention
New H+The design of the fluorescent molecular probe of driving is rational.
Technical solution of the present invention has the beneficial effect that：
1）Sensitivity is higher：
The pH value of reported in literature congenic method detection at present in acid range wide scope it is seldom, and this probe is in acid range
Interior energy is established linear in pH out of 1.98~7.00.
2）Luminescence mechanism novel and unique, is more suitable for optical detection：
The luminescence mechanism of probe of the present invention is combination of the PET photo induced electron transfers mechanism with absorbing red shift, and principle is very novel, and
During fluorescence non-emissive displacement, thus without interference with colorimetric analysis.
3）A variety of biological sample imagings：
Imaging of the determinand in living cells and tissue is successfully realized, and has been carried out in cell and tissue a series of linear
Very big impetus is played in fluoroscopic examination, the further investigation of the realization of imaging for this mark of pH value.
4）Naked eyes monitor, convenient and swift：
Probe of the present invention and detection method add visually observed property, monitor the naked eyes of determinand（Without instrument, directly see
Survey）It is possibly realized, detection process is more quick than average probe.
Brief description of the drawings
Fig. 1 is PH fluorescent molecular probes and principle of luminosity；
Fig. 2 is PH fluorescent molecular probe H spectrograms；
Fig. 3 is PH fluorescent molecular probe C spectrograms；
Fig. 4 is PH fluorescent molecular probe mass spectrograms；
Fig. 5 is ultraviolet detection pH value；
Fig. 6 is the linear relationship of fluoroscopic examination pH value；
In vitro is established linear after Fig. 7 is addition reference probe MitoTracker Red；
Fig. 8 is that the fluorescence intensity of dynamic HELA cells (A ~ E) carries out time-tracking figure；
Fig. 9 is different pH value and the linear relationship of fluorescence signal in living cells；
Figure 10 is different pH value and the linear relationship of probe and the ratio of reference probe fluorescence intensity in living cells；
Figure 11 is the fluorogram in starved cells；
Figure 12 is that the pH value in starved cells is quantitatively schemed；
Figure 13 is the survival rate of Hela cells；
Figure 14 is that the fluorescence intensity of tissue (F ~ J) carries out time-tracking figure；
Figure 15 is the gastric mucosa under same time same concentration to fluorograms of the pH in different layer sections；
Figure 16 is the real-time detection of the pH in tissue；
Figure 17 optimizes for concentration and probe concentration, controls the μ L of buffer salt 200 (the Britton-Robinson buffer salts B- that pH is 1.98
R buffer salts：0.04mol/L phosphoric acid, acetic acid, boric acid+sodium hydroxide 0.2mol/L is prepared)；
Figure 18 optimizes for concentration and probe concentration, controls the μ L of buffer salt 200 (the Britton-Robinson buffer salts B- that pH is 1.98
R buffer salts：0.04mol/L phosphoric acid, acetic acid, boric acid+sodium hydroxide 0.2mol/L is prepared)；
Figure 19 is probe molecule to H+Selectivity analysis.
Embodiment
By describing the present invention in conjunction with specific embodiments, without departing from the idea case in the present invention described above, according to this
The various replacements or change that field ordinary technical knowledge and customary means are made, are included within the scope of the present invention.
In the embodiment of the present invention, efficient liquid phase-mass spectral analysis is to utilize the connections of Agilent 1290 Agilent 6,460 3
Weight quadrupole rod mass spectrometer system (Agilent, USA), and it is equipped with Agilent Jet Stream electrospray system high efficiency liquid
Phase chromatographic isolation is completed by SB C18 posts (2.1 mm × 50 mm, 1.8 μm of i.d., Agilent, USA).
Efficient liquid phase-ultra-violet analysis experiment is using 1260 online vacuum suction devices of Agilent, automatic progress sample device, fluorescence inspection
Survey device associated working.Separation gradient is set to 0 min: 70% A+30% B; 10 min: 0% A+100% B; 15 min: 0%
A+100% B;Wherein A and B is respectively the acetonitrile of 0.5% formic acid+5% and 100% acetonitrile solution.Fluoroscopic examination is to utilize Hitachi
Hitachi F-7000 XRFs are carried out, and excitation wavelength is 475 nm, launch wavelength is 540 nm, excites and launches
Slit width is 10.0 nm, voltage 400V, sweep speed 2400nm/min.Uv-vis spectra is by Cary 300
Bio ultraviolet-visual spectrometers are carried out, and scanning range is 350~700nm.Fluorescence imaging observation is by Olympus, IX73-
DP80 (Japan) inverted fluorescence microscopes are carried out.Isolating and purifying for compound is realized using thin-layer chromatography silicagel column
(filler 300-400 mesh).
In following embodiments, unless otherwise noted, room temperature is 20~30 DEG C.
Embodiment 1：Prepare PH fluorescence probes
A kind of preparation method of PH fluorescence probes, step include：
（1）By NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) according to concentration 0.004g/ml
It is dissolved in chloroform, then adds the solution of same volume chloroform, hydrazine hydrate-first containing volume fraction 1% in the solution of chloroform
Alcoholic solution（In hydrazine hydrate-methanol solution, hydrazine hydrate volumetric concentration is 1%）, it is well mixed, stirs 2h at room temperature and obtain brown precipitate,
Precipitation separates out completely, and filtering, filter cake washs through ethyl acetate, dries, obtains NBD hydrazines；
（2）Pyridinium chloro-chromate is added in new steaming anhydrous methylene chloride, the addition g/mL of concentration 0.083, ultrasonic dissolution, so
Concentration 0.05g/mL 4- is added afterwards（Chloromethyl）Benzylated polyol, reaction 3h is stirred at room temperature under nitrogen protection, after the completion of reaction（Adopt
It is terminal after consumption of raw materials is complete with a plate tracking reaction process）3.3 times of absolute ethers of dichloromethane are added, after stirring 5min
Stratification, supernatant is removed, remove a layer jelly, then add the absolute ether of foregoing same volume, ground 20min, use water successively
Wash three times, 5wt% sodium acid carbonates wash twice, saturated nacl aqueous solution wash once, each cleaning solution dosage is absolute ether volume
0.5 times, organic layer anhydrous sodium sulfate drying, revolving, obtain white crystal；
（3）By step（2）White crystal is dissolved in absolute ethyl alcohol, concentration of ordinary dissolution 0.004g/mL, then adds concentration
0.0032g/mL NBD hydrazines, then the glacial acetic acid of absolute ethyl alcohol volume 1% is added dropwise, heating reflux reaction 4h, reaction rotates after terminating
Solvent is removed, then is purified through silicagel column, gradient elution, obtains Chinese red solid powder, i.e. PH fluorescence probes.
The PH fluorescence probes that embodiment 1 is prepared, effect are as follows：
Detection sensitivity：Acid range interior energy is established linearly in pH out of 1.98~7.00, and can be used in acid range at present
Inner width range detection pH value it is seldom；
Absorbing wavelength changes：PH nearly 100nm of absorbing wavelength red shift out of 1.98~7.00；
Dual quantitative correction：Have fluorescent quantitation function concurrently and absorb peak ratio quantitative function；
Optical Mechanism index：Have PET fluorescent switch function concurrently and absorb red shift function.
The nuclear-magnetism of PH fluorescence probes and mass spectral characteristi such as Fig. 2~4.
Embodiment 2：Prepare PH fluorescence probes
A kind of preparation method of PH fluorescence probes, step include：
（1）By NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) according to concentration 0.008g/mL
It is dissolved in chloroform, then adds the solution of same volume chloroform, the hydrazine hydrate containing the % of volume fraction 1 in the solution of chloroform-
Methanol solution（In hydrazine hydrate-methanol solution, hydrazine hydrate volumetric concentration is 1.2%）, it is well mixed, stirs 2 h at room temperature and obtain brown
Precipitation, filtering, filter cake wash through ethyl acetate, dry, obtain NBD hydrazines；
（2）Pyridinium chloro-chromate is added in new steaming anhydrous methylene chloride, addition concentration 0.12g/ml, ultrasonic dissolution, then
Add the g/mL of concentration 0.08 4-（Chloromethyl）Benzylated polyol, 4 h of reaction are stirred at room temperature under nitrogen protection, add after the completion of reaction
Enter the absolute ether of 3 times of volumes of dichloromethane, stir stratification after 5min, remove supernatant, remove a layer jelly, then before adding
State the absolute ether of same volume, grind 20min, be washed with water successively three times, 5% sodium acid carbonate wash twice, saturated nacl aqueous solution washes
Once, each cleaning solution dosage is 0.5 times of absolute ether volume, organic layer anhydrous sodium sulfate drying, revolving, obtains white crystalline substance
Body；
（3）By step（2）White crystal is dissolved in absolute ethyl alcohol, the g/mL of concentration of ordinary dissolution 0.006, then adds concentration
0.004g/ml NBD hydrazines, then the glacial acetic acid of absolute ethyl alcohol volume 1% is added dropwise, heating reflux reaction 3h, reaction is rotated after terminating and removed
Solvent is removed, then is purified through silicagel column, gradient elution, obtains Chinese red solid powder, i.e. PH fluorescence probes.
Embodiment 3：Prepare PH fluorescence probes
A kind of preparation method of PH fluorescence probes, step include：
（1）By NBD-Cl (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) according to concentration 0.001g/mL
It is dissolved in chloroform, then adds the solution of same volume chloroform, the hydration containing the % of volume fraction 0.39 in the solution of chloroform
Hydrazine-methanol solution（In hydrazine hydrate-methanol solution, hydrazine hydrate volumetric concentration is 0.2%）, it is well mixed, stirs 2 h at room temperature and obtain
Brown precipitate, filtering, filter cake wash through ethyl acetate, dry, obtain NBD hydrazines；
（2）Pyridinium chloro-chromate is added in dichloromethane, concentration 0.041g/ml is added, ultrasonic dissolution, then adds concentration
0.02 g/mL 4-（Chloromethyl）Benzylated polyol, reaction 3h is stirred at room temperature under nitrogen protection, dichloromethane is added after the completion of reaction
The absolute ether of 4 times of volumes, stratification after 5min is stirred, remove supernatant, remove a layer jelly, then add foregoing same volume
Absolute ether, grind 20min, be washed with water successively three times, 5% sodium acid carbonate wash twice, saturated nacl aqueous solution wash once, every time
Cleaning solution dosage is 0.5 times of absolute ether volume, organic layer anhydrous sodium sulfate drying, revolving, obtains white crystal；
（3）By step（2）White crystal is dissolved in absolute ethyl alcohol, concentration of ordinary dissolution 0.002g/mL, then adds concentration
0.002g/mL NBD hydrazines, then the glacial acetic acid of absolute ethyl alcohol volume 1% is added dropwise, heating reflux reaction 4h, reaction is rotated after terminating and removed
Solvent is removed, then is purified through silicagel column, gradient elution, obtains Chinese red solid powder, i.e. PH fluorescence probes.
Embodiment 4：Using
It is used for the real-time detection method of pH value in biological specimen intracellular mitochondrial/film using PH fluorescence probes, step includes：
1）Prepare solution：
With manufacturing probe CM-BNBD storing solutions：The accurate hydrionic fluorescent molecular probe of detection that weighs is dissolved in anhydrous acetonitrile, prepares dense
Spend for 1 × 10-4Mol/L probe CM-BNBD storing solutions；
Prepare the buffer solution of the different pH value of object to be measured（Britton-Robison buffer solutions）：Cushioning liquid is with 0. 04
Mol/L phosphoric acid, boric acid and acetic acid are formulated, during use needed for the 0. 2 mol/L NaOH aqueous solution are adjusted on acidometer
PH value；
Liquid is matched somebody with somebody in the storage of the various ions and determinand that are equipped with needed for selectivity：Accurately weigh various determinand Li+;Na+; K+;
Mg2+; Ca2+;Ba2+;Sr2+;Fe2+;Fe3+;Co2+;Ni2+;Cu2+;Zn2+;Cr3+;Mn2+;Hg2+.F-;Cl-;Br-;I-;HCO3-;
NO2-;NO3-;N3 -;OAC-;PO4 3-;SO4 2-;S2-;
SO3 2-;H2O2;Hcy,GSH,Cys..Compound concentration is 1 × 10-3The storing solution of mol/L various determinands.
2）Establish the linear equation of pH value and fluorescence signal intensity
Take step 1）The pH value that the object storing solution to be measured prepared dilutes to obtain gradient concentration is followed successively by 2,3,4,5,6,7
Buffer standard product solution, after respectively taking 200 μ L to be mixed respectively with 30 μ L probe HNBD storing solutions and 770 μ L serum storing solutions, shaking
It is well mixed, 5min is placed at 37 DEG C, is then detected through sepectrophotofluorometer and ultraviolet spectrometer, establish pH value with it is glimmering
The linear equation of light signal strength；
3）PH linear relationship is established, determines sample P H values
a）Ultraviolet detection testing sample：After 1000 μ L testing samples are injected into quartz colorimetric utensil, ultraviolet specrophotometer is inserted, is received
Collect the intensity of maximum absorption wavelength position, obtain maximum absorption band volume efficiency before and after reacting and substitute into ultraviolet linear relationship
In, calculate to obtain H+Levels.
b）Ultraviolet detection testing sample：After 1000 μ L testing samples are injected into quartz colorimetric utensil, uv-spectrophotometric is inserted
Meter, the intensity of maximum absorption wavelength position is collected, obtain maximum absorption band volume efficiency before and after reaction and substitute into ultraviolet linear
In relation, H is calculated+Levels.
Interpretation of result：Fig. 5~7 show, CM-BNBD probes are 1.98~7.00 internal memories in linear relationship in acid range,
Practical application can be carried out.
4）The detection of intracellular photostability
After probe adds cell, Fig. 8 is obtained to the fluorescence intensity progress time-tracking of cell, it can be seen that the spy of this experiment
Performance of the photostability of pin inside cell is still better.Wherein concentration and probe concentration is 3 μm of ol/L.
5）PH linear relationships are established in intracellular detection
The fluorescence intensity of cell is detected in the culture environment of different pH value, and is linearly closed according to fluorescence intensity to establish fluorescence
System, obtain such as Fig. 9,10, the present invention and add reference probe MitoTracker Red to remove ambient interferences so that experimental data
It is more accurate.
6）Intracellular pH detection practical applications
Figure 11,12 are that cell is imaged when nutritional deficiency, it can be seen that the pH states at the cell of part are acid model
Enclose.（PH is in 5-6 or so）Using step 3）Detect some starved cells and data analysis, by its data point
Us are analysed it can be found that the pH value at intracellular some positions starts to tend to be acid.It is strong by gathering fluorescence to some of which part
Degree, bring into above it is resulting it is linear in pH be calculated have 6.7,4.7,6.5,3.9,4.9.Therefore also to proposed by the present invention
Nutritional deficiency also results in cell acidifying and verified.
Embodiment 5:Viable cell survival rate monitors（By taking Hela as an example）
The PH fluorescence that the cytotoxicity experiment of the present embodiment Hela cell survival rates is prepared mainly for the inspection embodiment of the present invention 1
Influence of the probe to cell survival.
Specific method is：Two groups of Hela cells are cultivated respectively, after cultivating 24h, calculate the number of cells in two groups respectively, so
PH fluorescence probes prepared by 3 μm of ol/L embodiment 1 are added in wherein one group afterwards, another set continues normal culture, simultaneously
Cultivate 6h.Then calculate respective number of cells percentage again, be added without probe for 1, add probe for 0.991.Therefore
It can be seen that the cytotoxicity very little of this probe, is adapted to organism（Such as Figure 13）.
Embodiment 6:The real-time detection of pH in tissue
1）Photostability and penetrability the detection experiment of tissue
After PH fluorescence probes prepared by embodiment 1 add in vitro gastric mucosa, the fluorescence intensity progress time-tracking of gastric mucosa is obtained
To Figure 14, it can be seen that performance of the photostability of the PH fluorescence probes of the present invention inside gastric mucosa is good.It is simultaneously right
The penetrability of probe is studied, and the fluorescence intensity pair of different layer sections is carried out to the gastric mucosa under same time same concentration
Than such as Figure 15, the membrane penetrating for going out this probe that can be seen can be relatively good.Wherein concentration and probe concentration is 3 μm of ol/L, reference probe
MitoTracker Red are 1 μm of ol/L.
2）The real-time detection of pH in tissue
Cultivate 3 groups of rabbits, Normal rabbits, the rabbit with reflux esophagitis and to reflux esophagitis feeding PPI medicines
Thing（Suppress and to the medicine for the treatment of inflammation）Rabbit, later experiments carry out when three groups of rabbits are put to death and dissected, take its stomach to glue
Film and mucous membrane of esophagus, and gastric mucosa to 3 groups of rabbits and mucous membrane of esophagus carry out fluoroscopic examination, it is by experiment it can be found that normal
The pH of the gastric environment of rabbit is in 2.3-3.5 or so, and the pH of oesophagus environment is in 5.2-6.4 or so；Rabbit with reflux esophagitis
The pH of sub- gastric environment is in 2.0-5.6 or so, and the pH of oesophagus environment is in 2.1-5.3 or so;To with reflux esophagitis feeding
PPI medicines（Suppress and to the medicine for the treatment of inflammation）The pH of rabbit gastric environment exist in 2.3-6.6 or so, the pH of oesophagus environment
2.5-6.5 left and right.A in Figure 16) gastric mucosa and the fluorogram B under mucous membrane of esophagus different conditions) gastric mucosa and mucous membrane of esophagus it is different
PH value under state.
The experimental verification of PH fluorescence probe all technicals prepared by the present invention, it is specific as follows：
First, probe dosage optimization
Probe dosage is related to the important indicators such as sensitivity and the reagent consumption of detection, often as the primary factor of inspection optimization
To be investigated.According to the luminous characteristics of this probe, its sensitivity should belong to higher, therefore the required probe when actually detected
Concentration should be relatively low.It is as higher in given concentration, reduce information strength by being likely to occur self-quenching.As a result show（Figure 17）Concentration
There is most strong response for 3 μM of probes.The concentration will be brought into follow-up Optimal Experimental process.
2nd, time dosage optimization
Reaction time will influence probe molecule and determinand H+Reaction efficiency and the extent of reaction, while will also determine final signal
Stabilized soil pavement.Therefore, it is determined that after concentration and probe concentration, the present invention optimizes to the time of reaction.Can be with from Figure 18
To find out, the response of probe of the invention is very sensitive, and signal can not obtain the reaction time in normal time range, from
And it can be seen that the sensitivity of probe is higher.
3rd, probe molecule is to H+Selectivity analysis
Following substances storing solution has respectively：A)1: blank;2,20mM Li+; 3: 20mM Na+; 4: 20mM K+; 5:
20mM Mg2+; 6: 20mM Ca2+; 7: 20mM Ba2+; 8: 20mM Sr2+; 9: 1mM Fe2+; 10: 0.1mM Fe3+;
11: 1mM Co2+; 12: 20mM Ni2+; 13: 1mM Cu2+; 14: 20mM Zn2+; 15: 0.1mM Cr3+; 16:
20mM Mn2+; 17.20mMHg2+.B) 1: 20mM F-; 2: 20mM Cl-; 3: 20mM Br-; 4: 20mM I-; 5:
20mM HCO3-; 6: 1mM NO2-; 7: 1mM NO3-; 8: 1mM N3 -; 9: 1mM OAC-; 10: 20mM PO4 3-;
11: 20mM SO4 2-; 12: 20mM S2-; 13: 20mM SO3 2-; 14: 0.1mM H2O2; 15: 1mMHcy, 16:
1mM GSH, 17:1mMCys. pH=1.98 and the μ L. of pH=7.00 Britton-Robinson buffer solutions 200.With
In subsequent analysis, low concentration solution needed for subsequent experimental dilutes on the basis of this storing solution to be made；
Based on the stronger fluorescence response of this probe, the present invention analyzes probe to H+Selectivity（Figure 19）：Relative to H+, probe
Other ions are shown with the response of extreme difference, this should be due to amino acid with α-KA have in structure it is significantly different caused by.When
When adding other materials such as phosphoric acid, acetic acid, boric acid+sodium hydroxide, probe is still maintained to determinand H+Outstanding selection
Property..Therefore, outstanding selectivity causes the entirely appropriate applications in biological specimen of probe CM-BNBD.In addition, temperature is tested
It also demonstrate that this probe is highly suitable for biological sample.
Claims (9)
1. a kind of preparation method of PH fluorescence probes, it is characterised in that step includes：
（1）NBD-Cl is dissolved in chloroform according to 0.001~-0.008g/mL of concentration, then adds the molten of same volume chloroform
Liquid, the solution of chloroform is hydrazine hydrate-methanol solution containing volume fraction 0.39~1% in chloroform, is well mixed, stirs at room temperature
Brown precipitate is mixed to obtain, is separated out completely to precipitating, filtering, filter cake washs through ethyl acetate, dries, obtains NBD hydrazines；
（2）Pyridinium chloro-chromate is added in dichloromethane, adds 0.041~0.12g/mL of concentration, ultrasonic dissolution, Ran Houjia
Enter the g/mL of concentration 0.02~0.08 4-（Chloromethyl）Benzylated polyol, the h of reaction 3~4 is stirred at room temperature under nitrogen protection, has reacted
Into the absolute ether of rear addition 3~4 times of volumes of dichloromethane, stratification after 5min is stirred, removes supernatant, remove layer glue
Thing, then add the absolute ether of foregoing same volume, 20min is ground, successively with water, 5wt% sodium bicarbonate aqueous solutions, saturated sodium-chloride
Solution washs, and organic layer anhydrous sodium sulfate drying, revolving, obtains white crystal；
（3）By step（2）White crystal is dissolved in absolute ethyl alcohol, the g/mL of concentration of ordinary dissolution 0.002~0.006, is then added dense
0.002~0.004g/mL NBD hydrazines are spent, then the glacial acetic acid of absolute ethyl alcohol volume 1% is added dropwise, 3~4h of heating reflux reaction, reaction
Revolving removes solvent after end, then is purified through silicagel column, gradient elution, obtains Chinese red solid powder, i.e. PH fluorescence probes.
2. preparation method according to claim 1, it is characterised in that：Step（1）In, the hydrazine hydrate-methanol solution
In, hydrazine hydrate volumetric concentration is 0.2~1.2%；The NBD-Cl is dissolved in chloroform according to concentration 0.004g/mL.
3. preparation method according to claim 1, it is characterised in that：Step（2）In, pyridinium chloro-chromate is added to two
Concentration is 0.083g/mL in chloromethanes, then adds 4-（Chloromethyl）The concentration of benzylated polyol is 0.05g/mL；The dichloromethane
Alkane steams anhydrous methylene chloride to be new；It is terminal after consumption of raw materials is complete using a plate tracking reaction process；The washing, preferably
Be washed with water successively three times, 5wt% sodium acid carbonates wash twice, saturated nacl aqueous solution wash once, each cleaning solution dosage is anhydrous second
0.5 times of ether volume.
4. preparation method according to claim 1, it is characterised in that：Step（3）In, step（2）White crystal is in nothing
Concentration of ordinary dissolution is 0.004g/mL in water-ethanol, and the concentration for then adding NBD hydrazines is 0.0032g/mL；The silicagel column purifying, choosing
Dissolved with dichloromethane；The gradient elution, eluant, eluent are dichloromethane：Methanol 500：1 to 200：1.
5. preparation method according to claim 1, it is characterised in that：The PH fluorescence probes effect being prepared judges to refer to
Mark is as follows：
Absorbing wavelength changes：PH value, which is absorbed from acidity to neutrality to feux rouges, moves nearly 100 nm；
Detection range：Linear relationship in the range of pH1.98~7.00 be present.
6. the application of PH fluorescent molecular probes is prepared in any one of Claims 1 to 5 preparation method, it is characterised in that：
Suitable for biological specimen, the pH detection and analysis in pH detection and analysis, tissue adherence in intracellular mitochondrial/film, detection spirit
It is quick, accurate, quick；Described biological specimen mainly includes mucous membrane of esophagus, gastric mucosa, Hela cells etc., and can be applied to life has
Machine analysis, analytical chemistry, disease is examined in advance and the association areas such as clinical medicine detects.
7. application according to claim 6, there is provided one kind using above-mentioned PH fluorescent molecular probes be used for intracellular mitochondrial/
The real-time detection method of pH value in film, it is characterised in that：Step includes：
1）Prepare solution
Prepare CM-BNBD probe storing solutions：The accurate PH fluorescent molecular probes that weigh are dissolved in anhydrous acetonitrile, and compound concentration is 1 × 10- 4Mol/L, obtain CM-BNBD probe storing solutions；
Prepare object buffer solution to be measured（Britton-Robison buffer solutions）：With 0. 04 mol/L phosphoric acid, boric acid and acetic acid
It is formulated, is adjusted with the 0. 2 mol/L NaOH aqueous solution to required pH value during use；
2）Establish the linear equation of pH value and fluorescence signal intensity
Take step 1）The buffer standard product that the pH value that the object buffer solution to be measured prepared dilutes to obtain gradient concentration is 2~7 is molten
Liquid, after respectively taking 200 μ L to be mixed respectively with 30 μ L CM-BNBD probes storing solutions and 770 μ L determinand storing solutions, shaking mixing is equal
It is even, 5min is placed at 37 DEG C, is then detected through sepectrophotofluorometer or ultraviolet spectrometer, establishes pH value and fluorescence signal
The linear equation of intensity；
3）PH linear relationship is established, using a）Or b）Method determines sample P H values
a）Fluorescence detection：After 1000 μ L testing samples are injected into quartz colorimetric utensil, ultraviolet specrophotometer is inserted, is collected maximum
The intensity of absorbing wavelength position, obtain maximum absorption band volume efficiency before and after reacting and substitute into ultraviolet linear relationship, calculate
Obtain intracellular H+Levels, determine pH value；
b）Ultraviolet detection treats method：After 1000 μ L testing samples are injected into quartz colorimetric utensil, ultraviolet specrophotometer is inserted, is collected most
The intensity of big absorbing wavelength position, obtain maximum absorption band volume efficiency before and after reacting and substitute into ultraviolet linear relationship, count
Calculate to obtain intracellular H+Levels, determine pH value.
8. application according to claim 7, it is characterised in that：Step 2）In, the buffer standard product solution PH of gradient concentration
It is followed successively by 2,3,4,5,6,7.
9. application according to claim 7, it is characterised in that：Respectively in the method for fluoroscopic examination or ultraviolet detection to cell
Interior H+Parallel repeated detection is carried out, and is calibrated with standard items, obtains the optimal detection scope of fluorescence/ultraviolet detection, so as to
The detection means optimized is selected to be quantified according to the concentration range of determinand contained by different samples.
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| CN113185966A (en) * | 2021-03-25 | 2021-07-30 | 湖南师范大学 | By using NH2Cr (VI) detection method using-CuMOFs as fluorescent probe |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108623578A (en) * | 2018-07-02 | 2018-10-09 | 湖南文理学院 | A kind of benzoxadiazole class pH fluorescence probes, preparation method and application |
| CN108623578B (en) * | 2018-07-02 | 2021-06-25 | 湖南文理学院 | Benzoxadiazole pH fluorescent probe, preparation method and application |
| CN112961146A (en) * | 2021-02-25 | 2021-06-15 | 安徽工业大学 | Hydrazone and amide compound containing nitrobenzo-2-oxa-1, 3-diazole and application thereof |
| CN112961146B (en) * | 2021-02-25 | 2023-07-21 | 安徽工业大学 | Hydrazone and amide compounds containing nitrobenzo-2-oxa-1, 3-diazole and application thereof |
| CN113185966A (en) * | 2021-03-25 | 2021-07-30 | 湖南师范大学 | By using NH2Cr (VI) detection method using-CuMOFs as fluorescent probe |
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