CN106749034B - Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker - Google Patents
Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker Download PDFInfo
- Publication number
- CN106749034B CN106749034B CN201611069757.1A CN201611069757A CN106749034B CN 106749034 B CN106749034 B CN 106749034B CN 201611069757 A CN201611069757 A CN 201611069757A CN 106749034 B CN106749034 B CN 106749034B
- Authority
- CN
- China
- Prior art keywords
- phenanthro
- fluorescent labeling
- ratio
- added
- double
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
- C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6447—Fluorescence; Phosphorescence by visual observation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5038—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving detection of metabolites per se
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
Abstract
The present invention relates to fluorescent labeling reagents, and in particular to a kind of pair of bisulfite and hypochlorite double-bang firecracker answer Ratio-type fluorescent labeling reagent and its synthetic method and application.The fluorescent labeling reagent is using phenanthro- imidazoles as fluorophor, and using the C=C double bond of activation as reaction active groups, chemical name is 2- (4- (- 2 base of 1- methyl-1 H phenanthro- [9,10d] imidazoles)-benzylidene) malononitrile.Its synthetic method is to obtain 4- benzimidazole benzaldehyde by phenanthrenequione, to two benzaldehydes, ammonium acetate and acetic acid back flow reaction, 4- methyl-benzoimidazole benzaldehyde is obtained again, it finally reacts to obtain 2- (4- (1- methyl-1 H phenanthro- [9,10d] -2 bases of imidazoles-benzylidene) malononitrile with malononitrile.The label of fluorescent labeling reagent of the invention is swift in response, selectivity is high, detection limits extremely low, simple synthetic method, has further pushed what biological micromolecule acted in life entity to probe into.
Description
Technical field
The present invention relates to fluorescent labeling reagents, and in particular to a kind of pair of bisulfite and hypochlorite double-bang firecracker answer Ratio-type
Fluorescent labeling reagent and its synthetic method and application.
Background technique
Nearly ten years, some biological micromolecules adjust organismic internal environment because of wide participation life vivo oxidation reduction reaction
It balances and is potentially applied to clinical medicine and is concerned.Among these, redox equilibrium active oxygen (ROS) and reduction
Sulfur species (RSS) play the part of pivotal player in adjusting bioprocess, such as cell Proliferation, differentiation and apoptosis, and it is wherein representative
Substance surely belongs to HSO3 -And ClO-.Although in recent years, people are in the response path of these biological micromolecules of extensive discussions, physiology function
The various performances such as energy, still, still there are many unknown aspects for the effect that these substances are played the part of in life system.Therefore, very
It is necessary to establish the instant detection method of one kind these biological micromolecules are completed to distinguish and be detected.
Up to the present, High Performance Liquid Chromatography/Mass Spectrometry, electrochemical analysis, capillary electrophoresis analysis and with organic probes
It is used to detect biological micromolecule with analytic approach such as fluorescence, Ramans based on nano material and is widely used.In these analysis sides
In method, fluorescence probe has super high sensitivity, detects and be applied to the unique advantages such as living organism system immediately.In addition, phase
Compare switching mode fluorescence organic probes, Ratiometric fluorescent probe greatly reduces by the shadow from external environment, instrument and equipment etc.
It rings.
Most of all, a kind of current Ratiometric fluorescent probe not yet can be used to detect HSO simultaneously3 -And ClO-, therefore,
Double-bang firecracker proposed by the invention answers fluorescent molecular probe for further appreciating that HSO3 -And ClO-Have in the intracorporal mechanism of action of machine
Far reaching significance.
Summary of the invention
The purpose of the present invention is a kind of labels to be swift in response, to HSO3 -And ClO-Selectivity is high, detection limits extremely low, synthesis side
Method simplicity answers Ratio-type fluorescent labeling reagent to bisulfite and hypochlorite double-bang firecracker；Present invention simultaneously provides its synthesis sides
Method and application.
It is of the present invention that Ratio-type fluorescent labeling reagent is answered to bisulfite and hypochlorite double-bang firecracker, it is with phenanthro- miaow
Azoles is fluorophor, and using the C=C double bond of activation as reaction active groups, chemical name is: 2- (4- (1- methyl-1 H phenanthro-
- 2 base of [9,10d] imidazoles)-benzylidene) malononitrile, chemical structural formula is:
The synthetic method for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent, including with
Lower step:
(1) it is added in flask by phenanthrenequione, to two benzaldehydes, adds ammonium acetate and acetic acid, be heated to reflux and reacted,
Reaction terminates, and is cooled to room temperature to solution, filters, and the solid washed with acetic acid, the filtrate filtered is added to ice water
In, yellow solid is stirred to get, yellow solid is merged with the solid after acetic acid washing, obtains intermediate product 4- benzimidazolyl
Benzaldehyde；
(2) the intermediate product 4- benzimidazole benzaldehyde that step (1) obtains is dissolved with anhydrous acetonitrile, potassium carbonate is added
It is heated to reflux, is cooled to room temperature with potassium hydroxide, the anhydrous acetonitrile of iodomethane, temperature rising reflux reaction, cooling, mistake is added
Filter obtains intermediate product 4- methyl-benzoimidazole benzaldehyde through rotary distillation, purifying after collecting filtrate；
(3) obtained intermediate product 4- methyl-benzoimidazole benzaldehyde is dissolved in anhydrous pyridine, heats up and stirs,
Malononitrile reaction is added, after the reaction was completed, cooling, filtering obtains red colored crystalline object, i.e. target product 2- (4- (1- methyl-1 H
- 2 bases of phenanthro- [9,10d] imidazoles-benzylidene) malononitrile (MPIBA).
Wherein:
In step (1), phenanthrenequione is 1:3:7:10 to the molar ratio of two benzaldehydes, ammonium acetate, acetic acid.
The time of back flow reaction is 50 minutes in step (1).
In step (2), the molar ratio of potassium carbonate, potassium hydroxide, iodomethane and intermediate product 4- benzimidazole benzaldehyde
For 1:1:1.2:1.
In step (2), potassium carbonate is added and potassium hydroxide is heated to reflux 30 minutes, is cooled to room temperature, iodomethane is added
Anhydrous acetonitrile, temperature rising reflux react 30 minutes.
In step (3), the molar ratio of malononitrile and intermediate product 4- methyl-benzoimidazole benzaldehyde is 1.5:1.
It in step (3), is warming up to 70 DEG C and stirs 10 minutes, malononitrile is added and reacts 1 hour.
The application for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent is thin for detecting
The exogenous HSO of born of the same parents3 -And ClO-Concentration, detection endogenous cellular HSO3 -And ClO-Concentration and fluorescence imaging.
Specifically includes the following steps:
One, the exogenous HSO of cell is detected3 -And ClO-Concentration:
(1) pH=7.40 is prepared, concentration is the PBS buffer salt solution of 10mM；Compound concentration is the HSO of 1mM3 -Acetonitrile mark
Quasi- solution, the ClO that concentration is 1mM-Acetonitrile standard solution and concentration be 0.05mM 2- (4- (1- methyl-1 H phenanthro- [9,
10d] -2 base of imidazoles)-benzylidene) and malononitrile acetonitrile solution；
(2) 0 μ L, 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, 15 μ L, 17.5 μ L, 20 μ L, 22.5 μ L, 25 μ L are taken respectively
Concentration is the HSO of 1mM3 -And ClO-Totally 22 parts of acetonitrile standard solution, be added separately in fluorescence cuvette, be separately added into 100 μ
L concentration is the PBS buffer salt solution of 10mM, and being then separately added into the 2- that 100 μ L concentration are 0.05mM again, ((1- methyl-1 H is luxuriant and rich with fragrance by 4-
And -2 base of [9,10d] imidazoles)-benzylidene) malononitrile acetonitrile solution, be finally separately added into acetonitrile constant volume to 1mL, mixing is equal
It is even；
Fluorescence intensity is tested by sepectrophotofluorometer, obtains fluorescence intensity ratio, detects ClO-Fluorescence intensity swashs
Hair wavelength is 440nm, detects HSO3 -The excitation wavelength of fluorescence intensity is respectively 330nm, 410nm；
(3) respectively with HSO3 -And ClO-Concentration be abscissa obtain using fluorescence intensity ratio as ordinate about cell
Exogenous HSO3 -And ClO-The linear equation of concentration, fluorescence intensity ratio；
(4) sample to be tested and acetonitrile after 1:4 is mixed by volume, are taken into 900mL, and 100 μ L concentration are added thereto and are
The acetonitrile solution of 2- (4- (- 2 base of 1- methyl-1 H phenanthro- [9,10d] imidazoles)-benzylidene) malononitrile of 0.05mM, according to outer
Source property HSO3 -And ClO-Concentration, fluorescence intensity ratio linear equation obtain HSO3 -And ClO-Concentration；
Two, for detecting living cells endogenous HSO3 -And ClO-Concentration:
(1) compound concentration is the HSO of 1mM3 -Acetonitrile standard solution, concentration be 1mM ClO-Acetonitrile standard solution, dense
Degree is the acetonitrile solution of 2- (4- (- 2 base of 1- methyl-1 H phenanthro- [9,10d] imidazoles)-benzylidene) malononitrile of 0.5mM, concentration
The SO for being 1mM for PMA solution that the LPS solution of 1 μ g/ml, concentration are 1 μ g/ml, concentration2Donor solution；
(2) living cells Hela cell is placed in culture medium and is cultivated, be divided into and do not cultivate 10 groups, inoculum concentration in every group of culture medium
It is 2 × 107~9 × 107A/mL cultivates 2- (4- (the 1- methyl-1 H phenanthro- for being first separately added into that 10 μ L concentration are 0.5mM for 24 hours
- 2 base of [9,10d] imidazoles)-benzylidene) malononitrile acetonitrile solution, then be separately added into 0 μ L, 7.5 μ L, 15 μ L, 22.5 μ L, 30
μ L concentration is the HSO of 1mM3 -Acetonitrile standard solution and ClO-Acetonitrile standard solution, cultivate 15min altogether in 37 DEG C, merging is altogether
Lower observation imaging is focused, the fluorescence intensity for collecting different colours optical channel obtains to carry out fluorescence intensity ratio about cell
Endogenous HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation；
(3) living cells Hela cell is placed in culture medium and is cultivated, be divided into and do not cultivate 4 groups, inoculum concentration in every group of culture medium
It is 2 × 107~9 × 107A/mL is cultivated for 24 hours；
It is used to the PMA solution that 100 μ L concentration are the LPS solution of 1 μ g/ml, 100 μ L concentration are 1 μ g/ml is added in first group
To stimulate cell to generate endogenic ClO-, add 2- (4- (the 1- methyl-1 H phenanthro- [9,10d] that 10 μ L concentration are 0.5mM
- 2 base of imidazoles)-benzylidene) acetonitrile solution of malononitrile cultivated, is imaged, and second group is not added LPS and PMA, only plus 10 μ L
Concentration is that the acetonitrile solution of 2- (4- (- 2 base of 1- methyl-1 H phenanthro- [9,10d] imidazoles)-benzylidene) malononitrile of 0.5mM is made
For control；
The SO that 100 μ L concentration are 1mM is added into third group2Donor is used to that cell is stimulated to generate endogenic HSO3 -, then
The second of 2- (4- (- 2 base of 1- methyl-1 H phenanthro- [9,10d] imidazoles)-benzylidene) malononitrile that 10 μ L concentration are 0.5mM is added
Nitrile solution is cultivated, and imaging, the 4th group is not added SO2Donor, only adding the 2- that 10 μ L concentration are 0.5mM, ((1- methyl-1 H is luxuriant and rich with fragrance by 4-
And -2 base of [9,10d] imidazoles)-benzylidene) malononitrile acetonitrile solution as control；
The burnt lower observation imaging of merging copolymerization, collects the fluorescence intensity of different colours optical channel, to carry out fluorescence intensity ratio
Value, according to endogenous cellular HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation, to obtain endogenous cellular
HSO3 -And ClO-Concentration.
Beneficial effects of the present invention are as follows:
(1) Ratio-type fluorescent labeling reagent of the invention is using phenanthro- imidazoles as parent ring, with the C=C double bond activated
For reaction site, make probe to HSO3 -And ClO-There is superior selectivity, and has apparent fluorescence signal and read.
(2) Ratio-type fluorescent labeling reagent of the invention response is sensitive, to ClO-Response time in a few seconds, to HSO3 -
Response within 40s.
(3) Ratio-type fluorescent labeling reagent of the invention detection limit is low, is compared to commercialized fluorescent labeling reagent, this
Invent proposed to HSO3 -And ClO-Detection limit be respectively 3.5nm/L and 7.5nm/L, well below both intracellular objects
Content existing for matter.
(4) Ratio-type fluorescent labeling reagent of the invention, the switching mode that compares fluorescent labeling reagent, with the ratio of fluorescence intensity
On the basis of value, rather than direct fluorescence intensity, the influence from external environment and instrument etc. can be greatly reduced.
(5) present invention is that the first is used to while distinguishing and detecting HSO so far3 -And ClO-Organic fluorescence probe.
(6) convenient and efficient for open hole detection due to the variation of ultra-violet colors after reaction.
(7) present invention is applied to the detection of living cells, the spy for further having pushed biological micromolecule to act in life entity
Study carefully.
Detailed description of the invention
Fig. 1 is the synthetic route chart of MPIBA；
Fig. 2 is the mass spectrogram in embodiment 1；
Wherein: the A, mass spectrogram of MPIBA；B, MPIBA and ClO-The mass spectrogram of reaction；C, MPIBA and HSO3 -The matter of reaction
Spectrogram；
Fig. 3 is the nuclear-magnetism H spectrum of MPIBA in embodiment 1；
Fig. 4 is the nuclear-magnetism C spectrum of MPIBA in embodiment 1；
Fig. 5 is MPIBA to HSO3 -Fluorescence intensity and Linear equations；
Fig. 6 is MPIBA to ClO-Fluorescence intensity and Linear equations；
Fig. 7 is the fluorogram of sample detection；
Wherein: A, detecting HSO in rainwater3 -The fluorogram of concentration；B, ClO in tap water is detected-The fluorogram of concentration；
Fig. 8 is MPIBA to HSO3 -Chromatic graph is researched and analysed and compared to the selectivity of molecule；
Wherein: A, HSO3 -To metal ion；B,HSO3 -To other reduction class sulfur species of anion；
Fig. 9 is MPIBA to ClO-Chromatic graph is researched and analysed and compared to the selectivity of molecule；
Wherein: A, ClO-To metal ion；B,ClO-To other active oxygens of anion and active nitrogen；
Figure 10 is to probe into pH value to the influence diagram of MPIBA and substance reaction effect；
Wherein: A, HSO3 -；B,ClO-；
Figure 11 is to probe into MPIBA to scheme the response time of substance；
Wherein: A, HSO3 -；B,ClO-；
Figure 12 is intracellular Fluorescence Linear equations；
Wherein: A, HSO3 -；B,ClO-；
Figure 13 is detection endogenous relevant cell figure；
Wherein: A, HSO3 -；B,ClO-；
Figure 14 is cell survival rate histogram.
Specific embodiment
The present invention is described further with reference to embodiments.
Embodiment 1
As shown in Figure 1, of the invention to HSO3 -And ClO-Double-bang firecracker answers Ratio-type fluorescent labeling reagent synthesis step to have 3 steps,
Using phenanthrenequione and to two benzaldehydes as raw material, specific synthetic operation is as follows:
(1) synthesis of intermediate 4- benzimidazole benzaldehyde:
600mg is added in the round-bottomed flask of 50ml to two benzaldehydes, the ammonium acetate of 315mg phenanthrenequione and 2.15g, then plus
The glacial acetic acid for entering 25ml is heated with stirring to reflux 50 minutes, is cooled to room temperature, and suction filtration obtains solid, washs to obtain with glacial acetic acid
Solid, the filtrate filtered, which is poured into ice water, stirs, and stirs to get yellow solid, filter and merge front washing after
Solid obtains intermediate 4- benzimidazole benzaldehyde 395.5mg, yield 96%.
(2) synthesis of intermediate 4- methyl-benzoimidazole benzaldehyde:
30ml anhydrous acetonitrile is added in 100ml round-bottomed flask, adds 336mg intermediate 4- benzimidazolyl benzene first
Aldehyde, 56mg KOH, 150mg K2CO3, be heated with stirring to reflux 30 minutes, be cooled to after room temperature be added with constant pressure funnel it is molten
There is 0.1ml CH3The acetonitrile solution of I, is added dropwise, and is warming up to reflux state and reacts 30 minutes, and cold filtration collects filtrate simultaneously
Revolving, crude product are further purified, and with (v n-hexane: v ethyl acetate=10:1) for eluant, eluent, obtain greenish yellow solid 4- methyl-
Benzimidazole benzaldehyde 280mg, yield 80.5%.
(3) target product 2- (4- (- 2 base of 1- methyl-1 H phenanthro- [9,10d] imidazoles)-benzylidene) malononitrile (MPIBA)
Synthesis:
It takes 176mg intermediate product 4- methyl-benzoimidazole benzaldehyde to be put into 25ml round-bottomed flask, it is anhydrous to be dissolved in 10ml
It in pyridine, is warming up to 70 DEG C and stirs 10 minutes, malononitrile 0.05ml is added and reacts again 1 hour, be put into refrigerator to slightly cooling, to
After solid is precipitated, red needle-like solid, as target product 2- (4- (1- methyl-1 H phenanthro- [9,10d] imidazoles-are directly filtered to obtain
2 bases-benzylidene) malononitrile, 158mg, yield 78.3%.
Intermediate 4- benzimidazole benzaldehyde is characterized as below:
1H NMR (DMSO-d6,500MHz), δ (ppm): 10.141 (s, 1H), 8.86 (d, J=8.5Hz, 2H), 8.62
(d, J=4.5Hz, 2H), 8.15 (q, J=8.5Hz, 4H), 7.79 (q, J=7.5Hz, 2H), 7.68 (d, J=7.0Hz,
2H),),2.953(s,N-H,1H).
13C NMR(DMSO-d6,500MHz),δ(ppm):(193.42,151.33,130.75,130.20,127.91,
127.71,126.97,126.22,125.98,124.94,124.10,122.35,121.97.MS m/z calcd for
322.37[M+H]+found 323.5.
Intermediate 4- methyl-benzoimidazole benzaldehyde is characterized as below:
1H NMR (DMSO-d6,500MHz), δ (ppm): 4.52 (s, 3H) 7.67 (m, 2H), 7.77 (m, 2H), 8.14 (d,
J=8.4Hz, 2H), 8.53 (d, J=8.4Hz, 2H), 8.57 (d, J=7.6Hz, 1H), 8.63 (d, J=7.6Hz, 1H), 8.86
(d, J=8.4Hz, 1H), 8.90 (d, J=8.4Hz, 1H), 10.10 (s, 1H)
13C NMR(DMSO-d6,500MHz),δ(ppm):(193.37,151.22,130.75,130.19,127.87,
127.68,126.16,125.92,124.98,124.14,123.53,122.35,122.01)MS m/z calcd for
336.1[M+H]+found 336.5.
Target product 2- (4- (- 2 bases of 1- methyl-1 H phenanthro- [9,10d] imidazoles-benzylidene) malononitrile is characterized as below:
1H NMR (DMSO-d6,500MHz), δ (ppm): 9.0 (d, J=8.5Hz, H), 8.89 (d, J=8.5Hz, 2H),
8.66 (d, J=7.5Hz, 2H), 8.62 (d, J=6.5Hz, 1H), 8.59 (d, J=5.5Hz, 2H), 7.80 (d, J=8.0Hz,
2H), 7.75 (d, J=3.0Hz, 2H), 7.68 (d, J=3.0Hz, H), 7.40 (d, J=3.0Hz, H), 4.37 (s, 3H, N-
CH3)
13C NMR(DMSO-d6,500MHz),δ(ppm):(161.01,150.98,137.47,131.24,,130.74,
127.90,127.72,126.24,125.00,122.36,122.07,113.74,82.53,36.90.MS m/z calcd for
384.1[M+H]+found 384.8.
MPIBA is subjected to Mass Spectrometer Method, is specifically shown in Fig. 2, wherein A is the mass spectrogram of MPIBA, and B is MPIBA and ClO-Instead
The mass spectrogram answered, C are MPIBA and HSO3 -The mass spectrogram of reaction.
MPIBA is subjected to magnetic resonance detection, is specifically shown in Fig. 3-4, wherein Fig. 3 is the nuclear-magnetism H spectrum of MPIBA, and Fig. 4 is
The nuclear-magnetism C of MPIBA is composed.
Embodiment 2
Detect the exogenous HSO of cell3 -And ClO-Concentration:
(1) pH=7.40 is prepared, concentration is the PBS buffer salt solution of 10mM；Compound concentration is the HSO of 1mM3 -Acetonitrile mark
Quasi- solution, the ClO that concentration is 1mM-Acetonitrile standard solution and concentration be 0.05mM 2- (4- (1- methyl-1 H phenanthro- [9,
10d] -2 base of imidazoles)-benzylidene) and malononitrile acetonitrile solution；
(2) 0 μ L, 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, 15 μ L, 17.5 μ L, 20 μ L, 22.5 μ L, 25 μ L are taken respectively
Concentration is the HSO of 1mM3 -And ClO-Totally 22 parts of acetonitrile standard solution, be added separately in fluorescence cuvette, be separately added into 100 μ
L concentration is the PBS buffer salt solution of 10mM, and being then separately added into the 2- that 100 μ L concentration are 0.05mM again, ((1- methyl-1 H is luxuriant and rich with fragrance by 4-
And -2 base of [9,10d] imidazoles)-benzylidene) malononitrile acetonitrile solution, be finally separately added into acetonitrile constant volume to 1mL, mixing is equal
It is even；
Fluorescence intensity is tested by sepectrophotofluorometer, obtains fluorescence intensity ratio, detects ClO-Fluorescence intensity swashs
Hair wavelength is 440nm, detects HSO3 -The excitation wavelength of fluorescence intensity is respectively 330nm, 410nm；
(3) respectively with HSO3 -And ClO-Concentration be abscissa obtain using fluorescence intensity ratio as ordinate about cell
Exogenous HSO3 -And ClO-The linear equation of concentration, fluorescence intensity ratio；Fig. 5 is MPIBA to HSO3 -Fluorescence intensity and linear
Graph of equation, wherein A, B are the HSO for being added 0-22.5 μM3 -Fluorogram, C is HSO3 -Fluorescence Linear equations.Fig. 6 is MPIBA
To ClO-Fluorescence intensity and Linear equations；Wherein, A is the ClO for being added 0-22.5 μM-Fluorogram, B is ClO-Photoluminescence line
Property graph of equation.
(4) sample to be tested and acetonitrile after 1:4 is mixed by volume, are taken into 900mL, and 100 μ L concentration are added thereto and are
The acetonitrile solution of 2- (4- (- 2 base of 1- methyl-1 H phenanthro- [9,10d] imidazoles)-benzylidene) malononitrile of 0.05mM, according to outer
Source property HSO3 -And ClO-Concentration, fluorescence intensity ratio linear equation obtain HSO3 -And ClO-Concentration.
Embodiment 3
Utilize the HSO in MPIBA detection rainwater3 -With the ClO in tap water-Content.
By rainwater sample to be measured and anhydrous acetonitrile, 1:4 is mixed by volume, takes gained liquid 900mL, and 100 are added thereto
μ L concentration is the MPIBA of 0.05mM, and acquired solution saves 1min at room temperature, rain is calculated according to 2 fluorescence intensity of embodiment
Contain HSO in water sample3 -Content, bring gained fluorescence intensity level (obtaining from Fig. 7 A) into photoluminescence line that Fig. 5 C is established
Property, institute's value is 1.9mM, i.e. HSO in rainwater3 -Content is 10.5mM.
By originally water sample to be measured and anhydrous acetonitrile, 1:4 is mixed by volume, takes gained liquid 900mL, and be added thereto
100 μ L concentration are the MPIBA of 0.05mM, and acquired solution saves 1min at room temperature, originally according to the judgement of 2 fluorescence intensity of embodiment
Whether contain ClO in water sample-, it is linear to bring gained fluorescence intensity level (obtaining from Fig. 7 B) into fluorescence that Fig. 6 B is established,
Institute's value is 2.3mM, i.e. ClO in tap water-Content is 12.5mM.
Fig. 7 is the fluorogram of sample detection.Wherein, A is HSO in detection rainwater3 -The fluorogram of concentration, B are to detect originally
ClO in water-The fluorogram of concentration.
Embodiment 4
To probe into MPIBA to HSO3 -Selectivity, some representative anion Cl-、Br-、I-、CH3COO-、ClO4 -、
SO4 2、H2PO4 -、S2O3 2-、SCN-, reduction sulfur species HS-, GSH, Cys, Hcy and metal ion Na+、K+、Mg2+、Fe3+、Cd2+、Co2 +、Ni2+、Hg2+、Al3+、Mn2+、Ag+、Cu2+、Zn2+Addition, which is given, to be probed into.As seen from Figure 8, several times HSO3 -The ion of concentration
Be added in MPIBA, caused by influence can be ignored.Under 365nm ultraviolet lamp, work as HSO3 -It is added it can be found that red glimmering
Light disappears, and blue-fluorescence occurs.In Fig. 8, A is that metal ion selectively influences MPIBA, and bottle is under 365nm ultraviolet lamp
The phenomenon that interference solion is added, B are some RSS, and Common Anions select Journal of Sex Research to probe, and bottle is corresponding is added
Phenomenon of the interfering ion solution under 365nm ultraviolet lamp.
Equally, in order to probe into MPIBA to ClO-Selectivity, in addition to same metal ion, active oxygen (ROS), active nitrogen
Including H (RNS),2O2、OH、TBHP、TBO-、KO2、ONOO-、NO2 -、NO3 -, NO is added in solution.From fig. 9, it can be seen that i.e.
Make to be several times as much as ClO-The interfering ion of concentration is added, caused by influence also to can be neglected.Equally under the ultraviolet lamp of 365nm,
Work as ClO-It is added it can be found that red fluorescence becomes green fluorescence.In Fig. 9, A is that metal ion selectively influences probe, bottle
The phenomenon that for interference solion is added under 365nm ultraviolet lamp, B is that some ROS, RNS select Journal of Sex Research to MPIBA, small
Bottle is the corresponding phenomenon that interfering ion solution is added under 365nm ultraviolet lamp.
In order to probe into influence of the pH value to ratio fluorescent, in the certain situation of the amount for the substance that probe and detectable substance is added
Under, it allows reaction to react in the section that pH is 4.0-10.0, probes into optimal reaction environment.The fluorescence as shown by Figure 10 A
Rate value maintains a higher level in the environment that pH is 6.0-10.0, this explanation is surveyed in neutral and weakly alkaline environment
Amount is more excellent environment, this and HSO3 -Pka be 7.20 to match.It is same as shown in Figure 10 B, in pH between 4.0-6.0, instead
Answer activity relatively low, when pH is in 6.0-10.0 environment, reactive ratio is significantly raised.
Figure 11 is to probe into MPIBA to scheme the response time of substance, and wherein A is that MPIBA adds HSO3 -Response time figure, B
ClO is added for MPIBA-Response time figure.
Embodiment 5
Utilize the HSO of MPIBA detection endogenous cellular3 -And ClO-Concentration:
(1) cell culture: this experimental selection Hela cell cultivates the cell recovered, and culture medium includes 10%
Ox embryo serum, 1% dual anti-, 89%DMEM, at 37 DEG C, 5%CO2Environment in cultivate the cell for 24 hours, to be grown fine and wait for
With, be divided into and do not cultivate 14 groups, in every group of culture medium inoculum concentration be 2 × 107~9 × 107A/mL；
(2) endogenous cellular HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation foundation:
1. compound concentration is the HSO of 1mM3 -Acetonitrile standard solution, concentration be 1mM ClO-Acetonitrile standard solution, dense
The acetonitrile solution for the MPIBA that degree is 0.5mM, concentration are the LPS solution of 1 μ g/ml, concentration is the PMA solution of 1 μ g/ml, concentration is
The SO of 1mM2Donor solution；
2. taking 10 groups of culture mediums, it is first separately added into 2- (4- (the 1- methyl-1 H phenanthro- [9,10d] that 10 μ L concentration are 0.5mM
- 2 base of imidazoles)-benzylidene) malononitrile acetonitrile solution, then be separately added into 0 μ L, 7.5 μ L, 15 μ L, 22.5 μ L, 30 μ L concentration
For the HSO of 1mM3 -Acetonitrile standard solution and ClO-Acetonitrile standard solution, 15min is cultivated altogether in 37 DEG C, under merging copolymerization is burnt
Observation imaging, the fluorescence intensity for collecting different colours optical channel obtain to carry out fluorescence intensity ratio about endogenous cellular
HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation.
Figure 12 A is intracellular HSO3 -The linear side of concentration and ratio fluorescent (blue-fluorescence intensity is than red fluorescence intensity)
Journey.Figure 12 B is intracellular ClO-The linear equation of concentration and fluorescence intensity ratio (green fluorescence intensity is than red fluorescence intensity).
(3) HSO that endogenous cellular generates3 -And ClO-Content detection:
4 groups of culture mediums are taken, 100 μ L concentration are the LPS solution of 1 μ g/ml, 100 μ L concentration are 1 μ g/ to being added in first group
The PMA solution of ml is used to that cell is stimulated to generate endogenic ClO-, add 10 μ L concentration be 0.5mM MPIBA acetonitrile it is molten
Liquid is cultivated, imaging, second group is not added LPS and PMA, only plus 10 μ L concentration be 0.5mM MPIBA acetonitrile solution as pair
According to；Figure 12 B is endogenous detection ClO-Relevant cell figure.
The SO that 100 μ L concentration are 1mM is added into third group2Donor is used to that cell is stimulated to generate endogenic HSO3 -, then
The acetonitrile solution that the MPIBA that 10 μ L concentration are 0.5mM is added is cultivated, and is imaged, the 4th group is not added SO2Donor only adds 10 μ L
The acetonitrile solution that concentration is the MPIBA of 0.5mM is as control；Figure 12 A is endogenous detection HSO3 -Relevant cell figure.
The burnt lower observation imaging of merging copolymerization, collects the fluorescence intensity of different colours optical channel, to carry out fluorescence intensity ratio
Value, according to endogenous cellular HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation, to obtain endogenous cellular
HSO3 -And ClO-Concentration.
Figure 13 A is endogenous HSO3 -Cytological map, be from left to right red channel in figure, blue channel, light field, overlapping,
Ratio, Figure 13 B are endogenous ClO-Cytological map, be from left to right green channel in figure, red channel, light field, overlapping, than
Rate.
Embodiment 6
Cell survival rate experiment:
Influence of the main verifying MPIBA toxicity of the experiment of cell survival rate to cell survival.It is added not in cell culture fluid
With the MPIBA probe (0M, 5M, 10M, 20M, 30M and 50M) of concentration, at 37 DEG C, 5%CO2Incubator in cultivate for 24 hours,
Then 4- methyl thiazolyl tetrazolium MTT (the 5mg mL of 25 μ L-1) 4 hours of culture in cell culture fluid are added to.As a result pass through
MTT cuvette method assesses cell survival rate.That group of cell survival of MPIBA is not added as 100%, various concentration MPIBA adds
The experimental group related data entered draws opposed cylinder Figure 14.
In the embodiment of the present invention efficient liquid phase-mass spectral analysis be using 1100 mass spectrometer system of Agilent (Agilent,
USA), and it is equipped with degasser, quaternary pump, autosampler, high performance liquid chromatography separation is by Hypersil GOLD C18
Column (2.1mm × 50mm, 1.8 μm of i.d., Agilent, USA) is completed.Fluorescence detection is to utilize Hitachi Hitachi F-4600
Fluorescence Spectrometer carries out, to ClO-Detection excitation wavelength is 420nm, to HSO3 -Detection excitation be respectively 330 and 440nm,
Excitation and transmite slit width are 10.0nm, voltage 400V, 2400 nm/min of scanning speed.Fluorescence imaging observation is to pass through
Olympus Fluo View FV1000 (Japan) is copolymerized coke to carry out, and is observed with 40 times of object lens.Isolating and purifying for compound be
It is realized using thin-layer chromatography silicagel column, wherein filler is 300-400 mesh.
Claims (8)
1. a kind of pair of bisulfite and hypochlorite double-bang firecracker answer Ratio-type fluorescent labeling reagent, it is characterised in that: with phenanthro- miaow
Azoles is fluorophor, and using the C=C double bond of activation as reaction active groups, chemical name is: 2- (4- (1- methyl-1 H phenanthro-
- 2 base of [9,10d] imidazoles)-benzylidene) malononitrile, chemical structural formula is:
2. a kind of synthesis described in claim 1 for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent
Method, it is characterised in that the following steps are included:
(1) it is added in flask by phenanthrenequione, to two benzaldehydes, adds ammonium acetate and acetic acid, be heated to reflux and reacted, reacted
Terminate, be cooled to room temperature to solution, filter, the solid washed with acetic acid, the filtrate filtered is added in ice water, stirs
It mixes to obtain yellow solid, yellow solid is merged with the solid after acetic acid washing, obtains intermediate product 4- phenanthro- imidazoles benzaldehyde；
(2) the intermediate product 4- phenanthro- imidazoles benzaldehyde that step (1) obtains is dissolved with anhydrous acetonitrile, potassium carbonate and hydrogen-oxygen is added
Change potassium to be heated to reflux, be cooled to room temperature, the anhydrous acetonitrile of iodomethane is added, temperature rising reflux reaction is cooling, and filtering will filter
Intermediate product 4- methyl-phenanthro- imidazolyl benzaldehyde is obtained through rotary distillation, purifying after liquid collection；
(3) obtained intermediate product 4- methyl-phenanthro- imidazolyl benzaldehyde is dissolved in anhydrous pyridine, heats up and stirs, be added
Malononitrile reaction, after the reaction was completed, cooling, filtering obtains red colored crystalline object, i.e. target product 2- (4- (1- methyl-1 H phenanthro-
- 2 bases of [9,10d] imidazoles-benzylidene) malononitrile.
3. the synthesis according to claim 2 for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent
Method, it is characterised in that: in step (1), phenanthrenequione is 1:3:7:10 to the molar ratio of two benzaldehydes, ammonium acetate, acetic acid.
4. the synthesis according to claim 2 for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent
Method, it is characterised in that: in step (1), the time of back flow reaction is 50 minutes.
5. the synthesis according to claim 2 for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent
Method, it is characterised in that: in step (2), potassium carbonate, potassium hydroxide, iodomethane and intermediate product 4- phenanthro- imidazoles benzaldehyde
Molar ratio is 1:1:1.2:1.
6. the synthesis according to claim 2 for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent
Method, it is characterised in that: in step (2), potassium carbonate is added and potassium hydroxide is heated to reflux 30 minutes, is cooled to room temperature, is added
The anhydrous acetonitrile of iodomethane, temperature rising reflux react 30 minutes.
7. the synthesis according to claim 2 for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent
Method, it is characterised in that: in step (3), malononitrile is with intermediate product 4- methyl-phenanthro- imidazolyl benzaldehyde molar ratio
1.5:1。
8. the synthesis according to claim 2 for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent
Method, it is characterised in that: in step (3), be warming up to 70 DEG C and stir 10 minutes, malononitrile is added and reacts 1 hour.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611069757.1A CN106749034B (en) | 2016-11-29 | 2016-11-29 | Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611069757.1A CN106749034B (en) | 2016-11-29 | 2016-11-29 | Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106749034A CN106749034A (en) | 2017-05-31 |
| CN106749034B true CN106749034B (en) | 2019-03-19 |
Family
ID=58905128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611069757.1A Active CN106749034B (en) | 2016-11-29 | 2016-11-29 | Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106749034B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760298A (en) * | 2017-09-28 | 2018-03-06 | 青岛科技大学 | A kind of fluorescence probe for detecting bisulfite and preparation method and application |
| CN108169189B (en) * | 2017-12-06 | 2020-07-07 | 济南大学 | Ratiometric fluorescent probe responding to sulfur dioxide/sulfite (hydrogen) salt |
| CN108484478A (en) * | 2018-05-10 | 2018-09-04 | 曲阜师范大学 | A kind of novel carbazoles fluorescence mercaptan labelled reagent and its synthetic method and application |
| CN111518066B (en) * | 2020-05-25 | 2022-03-25 | 河南省农业科学院农业质量标准与检测技术研究所 | Bifunctional fluorescent probe for identifying hypochlorite and bisulfite and preparation method and application thereof |
| CN113402468B (en) * | 2021-06-03 | 2022-07-19 | 山西大学 | Biphenylnitrile derivative with AIE and ESIPT characteristics and synthetic method and application thereof |
| CN113372282B (en) * | 2021-07-01 | 2023-03-24 | 齐齐哈尔大学 | Fluorescent probe for detecting bisulfite ions in real time and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104119856A (en) * | 2013-04-26 | 2014-10-29 | 中国科学院大连化学物理研究所 | Fluorescent probe for ratio detection of hypochloric acid and application thereof in biological systems |
| CN105038766A (en) * | 2015-06-25 | 2015-11-11 | 中国科学院合肥物质科学研究院 | Visible and reversible ratiometric fluorescent probe as well as preparation method and application thereof |
-
2016
- 2016-11-29 CN CN201611069757.1A patent/CN106749034B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104119856A (en) * | 2013-04-26 | 2014-10-29 | 中国科学院大连化学物理研究所 | Fluorescent probe for ratio detection of hypochloric acid and application thereof in biological systems |
| CN105038766A (en) * | 2015-06-25 | 2015-11-11 | 中国科学院合肥物质科学研究院 | Visible and reversible ratiometric fluorescent probe as well as preparation method and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| "Reactive"probe for hydrogen sulfite: Good ratiometric response and bioimaging application;Xiaohong Cheng，等;《Sensors and Actuators B: Chemical》;20130423;第184卷;第274-280页 |
| A Ratiometric Fluorescent Probe for Hypochlorite Based on a Deoximation Reaction;Weiying Lin，等;《Chemistry—A European Journal》;20090120;第15卷;第2305-2309页 |
| A ratiometric fluorescent probe for iron(III) and its application for detection of iron(III) in human blood serum;Lingliang Long，等;《Analytica Chimica Acta》;20131228;第812卷;第145-151页 |
| Differential sensing of fluoride and cyanide ions by using Dicyano substituted benzimidazole probe;Akul Sen Gupta，等;《Journal ofLuminescence》;20160113;第173卷;第165-170页 |
| Off-On-Off fluorescence behavior of an intramolecular charge transfer probe toward anions and CO2;Rashid Ali，等;《Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy》;20160531;第168卷;第21-28页 |
| 反应型化学传感器在阴离子检测中的应用;程晓红，等;《有机化学》;20160822;第36卷;第2822-2842页 |
| 菲并咪唑类双极发光材料的合成及其性能;管翔，等;《石油化工》;20131231;第42卷(第8期);第912-915页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106749034A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106749034B (en) | Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker | |
| Cheng et al. | A porphyrin-based near-infrared fluorescent sensor for sulfur ion detection and its application in living cells | |
| CN109081836B (en) | Mercury ion near-infrared fluorescent probe based on hemicyanine structure and preparation method and application thereof | |
| Chao et al. | A ratiometric fluorescence probe for monitoring cyanide ion in live cells | |
| CN107056769A (en) | A kind of L cysteines fluorescence probe and preparation method thereof | |
| Li et al. | A near-infrared fluorescent probe for Cu2+ in living cells based on coordination effect | |
| CN105712964B (en) | Preparation method and application of thiol fluorescent probe based on coumaroyl hydrazide | |
| Sun et al. | An efficient TP-FRET-based lysosome-targetable fluorescent probe for imaging peroxynitrite with two well-resolved emission channels in living cells, tissues and zebrafish | |
| CN108329302A (en) | A kind of half flower cyanines class near infrared fluorescent probe compound of sulfide specificly-response and its preparation method and application | |
| CN109535147A (en) | A kind of formaldehyde fluorescence probe of quick response and its preparation method and application | |
| Ai et al. | A dual-responsive fluorescent probe for detection of H2S and Cu2+ based on rhodamine-naphthalimide and cell imaging | |
| CN113999219A (en) | Double-site fluorescent probe and synthetic method and application thereof | |
| CN111072648A (en) | Fluorescent probe for detecting biological thiol in lysosome as well as preparation method and application thereof | |
| CN111807993A (en) | Near-infrared fluorescent compound for specifically detecting hydrazine and preparation method thereof | |
| Wang et al. | An ‘‘off–on–off’’sensor for sequential detection of Cu 2+ and hydrogen sulfide based on a naphthalimide–rhodamine B derivative and its application in dual-channel cell imaging | |
| CN114181204B (en) | Near infrared fluorescent probe for detecting viscosity and preparation and application thereof | |
| CN109180716B (en) | Multi-signal ratio type distinguishing detection H2O2And H2Design, synthesis and application of fluorescent probe of S | |
| CN107698557A (en) | Fluorescence probe based on pyridine connection pyrazoles acyl hydrazone derivative and its preparation method and application | |
| CN110643355A (en) | Fluorescent probe for detecting polarity of endoplasmic reticulum as well as preparation method and application thereof | |
| Li et al. | Water-soluble fluorescent probe for simultaneous detection of cyanide, hypochlorite and bisulfite at different emission wavelengths | |
| CN113637048B (en) | Two-photon fluorescent probe of gamma-glutamyl transpeptidase and preparation method and application thereof | |
| CN110357896B (en) | Compound, preparation and application thereof in detecting divalent copper ions and strong acid pH | |
| CN108373464B (en) | Formaldehyde fluorescent probe based on formaldehyde-induced catalysis of succinimide hydrolysis and preparation method and application thereof | |
| CN106867521B (en) | A kind of novel naphthalimide H2S fluorescence probe and the preparation method and application thereof | |
| CN106496239B (en) | The preparation and application of pH ratio fluorescents probe in a kind of lysosome |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211116 Address after: 257000 room b420, No. 59, Fuqian street, Dongying District, Dongying City, Shandong Province Patentee after: Dongying Guangli Lingang Industrial Park Co.,Ltd. Address before: 273165 No. 57, Jingxuan West Road, Qufu City, Jining City, Shandong Province Patentee before: QUFU NORMAL University |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221222 Address after: 257000 room b420, No. 59, Fuqian street, Dongying District, Dongying City, Shandong Province Patentee after: Dongying Guangli Lingang Industrial Park Co.,Ltd. Patentee after: Dongying Guangli Port Park Operation Co.,Ltd. Address before: 257000 room b420, No. 59, Fuqian street, Dongying District, Dongying City, Shandong Province Patentee before: Dongying Guangli Lingang Industrial Park Co.,Ltd. |