CN110261353A - A kind of zero background fluorescence detection method and its application of hydrazine - Google Patents
A kind of zero background fluorescence detection method and its application of hydrazine Download PDFInfo
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- CN110261353A CN110261353A CN201910406958.3A CN201910406958A CN110261353A CN 110261353 A CN110261353 A CN 110261353A CN 201910406958 A CN201910406958 A CN 201910406958A CN 110261353 A CN110261353 A CN 110261353A
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- hydrazine
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- background fluorescence
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- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 title claims abstract description 224
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000001917 fluorescence detection Methods 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 51
- 238000006482 condensation reaction Methods 0.000 claims abstract description 16
- 238000003384 imaging method Methods 0.000 claims abstract description 13
- 238000011065 in-situ storage Methods 0.000 claims abstract description 13
- 229960004194 lidocaine Drugs 0.000 claims abstract description 13
- HNJXPTMEWIVQQM-UHFFFAOYSA-M triethyl(hexadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](CC)(CC)CC HNJXPTMEWIVQQM-UHFFFAOYSA-M 0.000 claims abstract description 11
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 5
- 239000001257 hydrogen Substances 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 20
- 239000012491 analyte Substances 0.000 claims description 13
- 239000007853 buffer solution Substances 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 claims description 4
- 238000010189 synthetic method Methods 0.000 claims description 4
- 239000012429 reaction media Substances 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 2
- DCAYPVUWAIABOU-UHFFFAOYSA-N hexadecane Chemical compound CCCCCCCCCCCCCCCC DCAYPVUWAIABOU-UHFFFAOYSA-N 0.000 claims 2
- NRTLTGGGUQIRRT-UHFFFAOYSA-N triethylazanium;bromide Chemical compound [Br-].CC[NH+](CC)CC NRTLTGGGUQIRRT-UHFFFAOYSA-N 0.000 claims 1
- 239000000523 sample Substances 0.000 abstract description 39
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- 210000002966 serum Anatomy 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 34
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
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- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
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- VQLYBLABXAHUDN-UHFFFAOYSA-N bis(4-fluorophenyl)-methyl-(1,2,4-triazol-1-ylmethyl)silane;methyl n-(1h-benzimidazol-2-yl)carbamate Chemical compound C1=CC=C2NC(NC(=O)OC)=NC2=C1.C=1C=C(F)C=CC=1[Si](C=1C=CC(F)=CC=1)(C)CN1C=NC=N1 VQLYBLABXAHUDN-UHFFFAOYSA-N 0.000 description 1
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- 125000000031 ethylamino group Chemical group [H]C([H])([H])C([H])([H])N([H])[*] 0.000 description 1
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
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- 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"
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- 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"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G—PHYSICS
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- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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- 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"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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Abstract
The invention discloses a kind of zero background fluorescence detection method of hydrazine and its applications, the present invention promotes the condensation reaction between 4- lignocaine salicylide and hydrazine by cetyltriethylammonium bromide (CTAB) micella, indicator of the generated in-situ fluorescent material with intramolecular hydrogen bond as hydrazine proposes a kind of new method of hydrazine detection.This method constructs the detection pattern of zero background, and avoids the use of organic synthesis and organic solvent cumbersome during testing.Furthermore, in actual water sample, serum sample, living cells even mouse be imaged in it is extensive studies have shown that probe 4- lignocaine salicylide (DEASA) and original position sensing solutions are in sensitivity, anti-interference ability and imaging side face is with good stability and broad application prospect.
Description
Technical field
The invention belongs to hydrazine detection technique fields, are related to a kind of for detecting the methods and strategies of hydrazine.More particularly, to one
The zero background fluorescence detection method and its application of kind hydrazine.
Background technique
It is well known that hydrazine is a kind of widely used propellant, due to its intrinsic strong basicity and reproducibility, in work
Industry is widely used in field, mainly for the manufacture of chemical catalyst, medicine intermediate and agricultural chemicals.However, world health
Tissue is by the environmental contaminants with hypertoxicity and carcinogenicity that hydrazidines justice is in production and transportational process, and threshold limit is fixed
For 10ppb.In addition, the excellent aqueous solubility of hydrazine, is easily absorbed by oral cavity and skin contact, to kidney, lungs, liver even maincenter
The human organs such as nervous system cause serious damage.Therefore, for the detection of hydrazine, it is particularly significant for monitoring its trace concentration
, to meet highly sensitive in aqueous solution and there is the needs of good sensing capabilities.
Currently, the common analysis of the detection of hydrazine mainly has electrochemical analysis, HPLC/GS-MS method and capillary electricity
Swimming, but these methods usually require to carry out on high-cost laboratory apparatus, and sample preparation is cumbersome, and with fluorescence probe phase
Than the analysis not being suitable in biosystem.
Fluorescence probe can satisfy as the compound in a kind of sensing to object with special optical characteristic
The demand of growing solution environmental pollution and human health problems, and fluorescence probe takes in terms of sensitivity and selectivity
Obtained great progress.Developed so far it is a series of suitable for actual sample, living cells even organism fluorescence probe, these
Probe is simple and easy, can be used for label or bio-imaging, has them in terms of the early diagnosis of disease and environment pollution detection
There is great potential.
In addition, in order to improve the sensitivity for analysis of fluorescence probe and signal-to-noise ratio, although having had more in document report at present
Kind method designs and constructs molecular structure to improve response signal or reduce background interference.For example, the intramolecular electricity for passing through distortion
The photophysical process such as lotus transfer (TICT), fluorescence resonance energy transfer (FRET) and photo induced electron transfer (PET) design probe knot
Structure.But can completely avoid the interference of background fluorescence using the analysis method of probe and analyte in-situ preparation chromophore, and
And such probe usually has cleverly molecular structure, itself be it is non-luminous, can voluntarily be generated in conjunction with analyte luminous
Body realizes that the detection to analyte, and simple molecular structure can be effectively prevented from cumbersome synthesis and purification process, is
Relatively there is hydrazine detection method in the water of application value at present, hair is generated by the specific reaction between probe and target analytes
The analysis method of color group, to realize that zero background detection of high sensitivity of object provides a kind of new approaches.
Summary of the invention
In view of this, providing a kind of CTAB micella promotion the purpose of the present invention is aiming at the problems existing in the prior art
The condensation reaction of 4- lignocaine salicylide and hydrazine, novel sensing plan of the generated in-situ chromophore as hydrazine detection indicator
Slightly.
To achieve the goals above, technical scheme is as follows:
A kind of zero background fluorescence detection method of hydrazine promotes 4- bis- by cetyltriethylammonium bromide (CTAB) micella
Condensation reaction between ethylamino salicylide and analyte hydrazine, the generated in-situ new chromophore with intramolecular hydrogen bond is as hydrazine
The indicator of detection.
The present invention by 4- lignocaine salicylide (DEASA) in CTAB micella with the condensation reaction of hydrazine, in-situ preparation
The fluorescence indicator that is detected as hydrazine of new chromophore, realize zero background detection of hydrazine in water phase, avoid cumbersome organic
Synthesis and the use of organic solvent.
Specific detection method disclosed above, includes the following steps:
Step 1: PBS buffer solution is added in colorimetric cylinder as solvent；
Step 2: the CTAB of probe DEASA and certain concentration are added into the solvent of step 1.
Step 3: analyte hydrazine being added into the solution of step 2, and DEASA and hydrazine occur condensation reaction, generate shiner
Matter HDBM.
It should be noted that the reaction temperature of above-mentioned condensation reaction is room temperature, it is swift in response.
Preferably, the synthetic method of the new chromophore specifically comprises the following steps:
(1) using PBS buffer solution as reaction medium, 4- lignocaine salicylide and cetyltriethylammonium bromide is added
(CTAB)；
(2) analyte hydrazine is added in the solution that step (1) obtains, DEASA and hydrazine occur condensation reaction, ultimately generate this
New chromophore-luminescent substance HDBM of disclosure of the invention.
It should be noted that the present invention is by nuclear magnetic resonance spectroscopy to luminescent substance generated referring to Figure of description 1
HDBM carries out structural characterization, to show to be successfully generated luminescent substance HDBM by above-mentioned synthetic method.
Particularly, the concrete scheme disclosed by the invention for preparing luminescent substance HDBM is as follows:
The combination ratio that hydrazine and DEASA are measured by asmus method (Job method) is 1:2, by various concentration
CTAB in the presence of detect the spectrum of reaction solution, the CTAB concentration of optimization is 1 × 10-3mol·L-1, thus in 5mL colorimetric cylinder
The DMSO solution (5 × 10 of 20 μ L DEASA is added as solvent in the middle PBS buffer solution that 2mL is added thereto-3mol·L-1)
Afterwards, the aqueous solution (1molL of 2 μ L CTAB is then added-1), it adds after the hydrazine of 0.5 equivalent to get to luminescent substance HDBM.
Wherein, the structure of the luminescent substance HDBM generated in the present invention are as follows:
Preferably, the molar ratio of the analyte hydrazine and 4- lignocaine salicylide is 1:2.
Have when it is worth noting that the molar ratio of analyte hydrazine and 4- lignocaine salicylide being 1:2 in the present invention
Best illumination effect, therefore it is 1:2 that the molar ratio of the two is selected in fluorescence detection method disclosed in the present application.
Preferably, the concentration of the cetyltriethylammonium bromide (CTAB) is controlled 0.1~5 × 10-3mol·L-1
Between.
Wherein, the present invention controls the concentration of cetyltriethylammonium bromide (CTAB) 0.1~5 × 10-3mol·L-1It can promote the generation of luminescent substance HDBM in range, and currently preferred concentration is 1 × 10-3mol·L-1.And apply
It is 0.1~5 × 10 that people, which investigates range to the concentration of CTAB,-3mol·L-1, such as attached drawing 3 it is found that when the concentration of CTAB is 1 × 10- 3mol·L-1When, the fluorescence intensity highest of luminescent substance HDBM, so the concentration of the preferred CTAB of subsequent experimental is 1 × 10-3mol·
L-1。
It is a further aim of the invention providing a kind of zero background fluorescence detection method hydrazine in dicyandiamide solution of hydrazine
Application in Selective recognition and quantitative detection.
Preferably, the concentration of the cetyltriethylammonium bromide (CTAB) in the dicyandiamide solution is 1 × 10-3mol·
L-1。
It further include the detection method using hydrazine as answering in the detection of marker and imaging in application scenes
With.
It is worth noting that the invention discloses the condensation reactions between 4- diethylamino salicylide (DEASA) and hydrazine
The hydrazine sensing strategy of the chromophore of generation.Wherein, cetyl trimethylammonium bromide (CTAB) micella provides stable hydrophobic
Cavity is conducive to DEASA and hydrazine at micella interface and condensation reaction occurs.Meanwhile the luminescent substance of generation is in the guarantor of CTAB micella
Shield is lower to have better luminescent properties, so as to improve the sensitivity of detection, specific sensing principle such as 2 institute of Figure of description
Show.
It can be seen via above technical scheme that compared with prior art, the invention discloses a kind of zero background fluorescences of hydrazine
Detection method and its application, the present invention provides a kind of 4- lignocaine salicylides first generates in CTAB micella with hydrazine reaction
Novel sensing strategy of the new chromophore as hydrazine detection indicator, the zero background detection mode have hypersensitivity, are suitable for
The detection of trace hydrazine in actual sample and organism；
Secondly, detection scheme disclosed by the invention is established on the basis of the condensation reaction of non-fluorescence substance DEASA and hydrazine,
Suitable reaction site is provided for hydrophobicity probe and hydrophily hydrazine by introducing CTAB micella, reaction efficiency has obtained greatly
It is promoted, to contain the fluorescence-causing substance HDBM of intramolecular hydrogen bond as fluorescence signal indicator；
Then, the CTAB micella that the present invention uses makes in aqueous solution for the easy to detect feasible of hydrazine, without
It is adjusted by organic solvent；
Finally, sensing detection disclosed by the invention analysis successfully carries out the detection to hydrazine in actual sample, and
It is comparatively sensitive in living cells imaging, also further demonstrate that it not by the interference of complex environment and lasting the research of intracorporal hydrazine
The good performance of imaging effect.
In conclusion detection method disclosed by the invention can efficient selective identify hydrazine, and to hydrazine have very high spirit
Sensitivity.The present invention provides a kind of detection approach of new hydrazine, have many advantages, such as preparation simplicity and good biocompatibility, this
There is market application and promotional value for industry, life and biological field.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 is nuclear magnetic resonance spectroscopy of the HDBM of the present invention in deuterated DMSO.
Condensation reaction of the Fig. 2 between 4- diethylamino salicylide (DEASA) of the present invention and hydrazine generates the hydrazine of chromophore
Sensing principle figure.
Fig. 3 is DEASA of the present invention and fluorescence intensity figure of 0.5mM hydrazine when containing various concentration CTAB.
Fig. 4 is the fluorescence spectra in the DEASA solution of the invention containing CTAB with various concentration hydrazine.
Fig. 5 is the fluorescence spectra containing hydrazine He other analytes in DEASA and CTAB solution of the present invention.
Fig. 6 is the measurement table of hydrazine in actual sample of the present invention.
Fig. 7 is the confocal fluorescent image of HeLa cell and various concentration hydrazine after the present invention is handled with DEASA respectively.
Fig. 8 is the fluorescence imaging of hydrazine in Mice Body of the present invention.
Fig. 9 is the column diagram of HeLa cell survival rate of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The embodiment of the invention discloses one kind in CTAB micella 4- lignocaine salicylide and hydrazine reaction, generation newly add lustre to
Sensing strategy of the group as hydrazine indicator.
For a better understanding of the invention, further specific elaboration is made to the present invention below by following embodiment, but not
It can be regarded as limitation of the invention, it is some nonessential according to made by foregoing invention content for those skilled in the art
It improves and adjusts, be also considered as and be within the scope of the present invention.
The invention discloses a kind of zero background fluorescence detection methods of hydrazine, pass through cetyltriethylammonium bromide (CTAB)
Micella promotes the condensation reaction between 4- lignocaine salicylide and analyte hydrazine, generated in-situ new with intramolecular hydrogen bond
The indicator that chromophore is detected as hydrazine.
The new chromophore of the present invention-luminescent substance HDBM generation is that PBS buffer solution is added in colorimetric cylinder as molten
Then agent is added probe DEASA and CTAB, is eventually adding analyte hydrazine, condensation reaction occurs in CTAB micella, ultimately generates
Luminescent substance HDBM.
Specific solution is as follows:
The pure water solution of 2mL is added in 5mL colorimetric cylinder as solvent, the DMSO solution of 20 μ L DEASA is added thereto
(5×10-3mol·L-1) after, the aqueous solution (1 × 10 of CTAB is then added-3mol·L-1), add the hydrazine of 0.5 equivalent to get
To luminescent substance HDBM.
In order to further verify excellent effect of the invention, inventor has also carried out following experiment:
Experiment 1: the synthesis of luminescent substance HDBM and structural characterization
1, the synthesis of luminescent substance HDBM
The pure water solution of 2mL is added in 5mL colorimetric cylinder as solvent, the DMSO solution of 20 μ L DEASA is added thereto
(5×10-3mol·L-1) after, the aqueous solution (1 × 10 of CTAB is then added-3mol·L-1), add the hydrazine of 0.5 equivalent to get
To luminescent substance HDBM.
2, test analysis
Luminescent substance HDBM's1HNMR spectrum peak are as follows:
1H NMR (500MHz, DMSO-d6) δ (ppm) 11.47 (s, 2H), 8.59 (s, 2H), 7.26 (d, J=8.8Hz,
2H), 6.30-6.28 (m, 2H), 6.10 (d, J=1.7Hz, 2H), 3.36 (q, J=6.9Hz, 4H), 2.48 (t, J=7.0Hz,
4H)；
13CNMR spectrum peak are as follows:
13C NMR(126MHz,DMSO-d6)δ(ppm)δ161.08,133.44,106.85,104.49,97.50,44.29,
12.99.
The H spectrogram of luminescent substance is made for synthetic method disclosed by the invention and C spectrogram is learnt, the spectrum peak in attached drawing
It is corresponded with HDBM, i.e., provable luminescent substance HDBM is synthesized successfully.
Experiment 2: a kind of zero background fluorescence detection method based on hydrazine detects the concrete application of hydrazine in dicyandiamide solution
1, test experiments
In the aqueous solution containing 1.0mM CTAB and 50 μM of DEASA, hydrazine, K are successively separately added into every colorimetric cylinder+、Na+、Mg2+、Ca2+、F-、Cl-、NO3 -, Glu, the aqueous solution (1 × 10 of Cys, Gly, aniline-4mol·L-1), after mixing, test
Its fluorescence spectrum, referring to attached drawing 5 (A).
Comparison, in the aqueous solution containing 1.0mM CTAB, 100 μM of hydrazines and 50 μM of DEASA, successively to every colorimetric
Hydrazine, K are separately added into pipe+、Na+、Mg2+、Ca2+、F-、Cl-、NO3 -, Glu, the aqueous solution (1 × 10 of Cys, Gly, aniline-4mol·
L-1), after mixing, its fluorescence spectrum is tested, referring to attached drawing 5 (B).
2, test analysis
Probe DEASA has relative selectivity, a variety of potential interfering compounds involved in experiment, such as cation (K to hydrazine+, Na+, Mg2+And Ca2+), anion (F-, Cl-And NO3 -), amino acid (Glu, Cys and Gly) and even analog (aniline) are glimmering
Photoresponse can only be caused by hydrazine, and potentially species be interfered hardly to cause spectrum change.Further comparative test measurement
Show that coexisting for chaff interferent has little effect DEASA to the response of hydrazine.
The above results show that probe DEASA has very strong specificity to the detection of hydrazine, and suitable for physiological environment
Detection, this may be to be provided by the sensing strategy based on specific reaction, provide blank background and generated in-situ signal system
System.
To sum up, present invention can be implemented in Selective recognition hydrazines in aqueous solution.
Test 3: the probe DEASA measurement to the minimum detection limit of hydrazine quantitative detection
It is real to the titration containing 1.0mMCTAB and 50 μM of DEASA according to hydrazine using fluorescence emission spectrum at 25 DEG C
It tests, is calculated by 3 σ/k, obtain probe DEASA to the minimum detection limit of hydrazine up to 0.42nM, illustrate detection spirit of this method to hydrazine
Sensitivity is high, has potential application value in terms of showing probe DEASA in CTAB micella to hydrazine efficient detection.
Experiment 4: zero background fluorescence detection method of above-mentioned hydrazine is in practical water and the even mouse imaging of serum sample, living cells
The concrete application of middle detection hydrazine
(1) detection method detects the concrete application of hydrazine in actual water sample and serum sample
Firstly, the sensing analytical method proposed is applied to the detection in tap water, detects received from five places respectively
The seawater of collection, the hydrazine content in sewage and diluted human serum, test results are shown in figure 6.From the actual sample that Fig. 6 is shown
The measurement table of hydrazine learns, the rate of recovery and relative standard deviation (RSD) value are all within normal range (NR), other 2 high-caliber hydrazines
Content (respectively 2 μM and 10 μM) discloses the hydrazine residual in sewage.The original that the above results show probe DEASA and proposed
Position detection design is applicable to the quantitative analysis of actual sample.
(2) detection method detects the concrete application of hydrazine in living cells
HeLa cell and 50 μM of probe DEASA are incubated with 1 hour, after being washed with PBS buffer solution, various concentration is added
The hydrazine of (increasing to 150 μM from 10 μM) is further cultured for 2 hours, and the cell that unused hydrazine is further processed is as control sample, such as Fig. 7 institute
Show.Wherein Fig. 7 (a)~Fig. 7 (e) is followed successively by cell fluorescence when hydrazine concentration is respectively 0 μM, 10 μM, 50 μM, 100 μM, 150 μM
Imaging.
Learnt from Fig. 7, cell that unused hydrazine is further processed shows the fluorescence of complete darkness, illustrate probe DEASA together with
Reaction medium CTAB micella cannot trigger any fluorescence reaction.Zero background property of sensing determination has been affirmed in zero response, side by side
In addition to a possibility that different component interferes in living cells.In addition, can be captured after handling HeLa cell with 10 μM of hydrazines apparent
Green-fluorescent emission, this demonstrates the feasibility of bio-imaging sensed in situ.
(3) detection method detects the concrete application of hydrazine in organism
By probe DEASA (40 μ L, 50 μM of DMSO solution) through injection of skin into nude mice, and respectively in injection 0
Picture is imaged in shooting the first two after with 2 hours, as shown in Figure 8.Mouse itself does not shine, and fluorescent image in 2 hours still
It does not shine, it was demonstrated that relative stability of the DEASA in complex biological environment.After photo is opened in shooting second soon, identical
Other hydrazine (2 μ L, 0.1M) is injected at position, and timetable captures fluorescent image according to schedule, is then seen within 6 hours after injecting hydrazine
Fluorescence imaging is observed, the luminescent products HDBM generated during being derived from DEASA and hydrazine reaction illustrates proposed by the present invention
Sensing strategy in situ can also be applied to organism.
In addition, the emissive porwer obtained during 24 hours real-time detections can also normalizing quantization, and after 24 hours still
Hyperfluorescence can so be obtained without loss of strength, this shows that generated in-situ shiner has persistently effectively imaging in analysis
The advantages of.Therefore, good response feature and high stability that the program is shown, even if so that detection method disclosed by the invention
The detection of hydrazine can also be realized by condensation reaction in situ in biological level.
To sum up, detection method disclosed by the invention can be successfully applied to living cells imaging and Mice Body imaging, to say
Bright above-mentioned zero background fluorescence detection method has good biocompatibility.And for the biology for further proving detection method
Compatibility, applicant have also carried out phenetic analysis to HeLa cytotoxicity, and specific experiment result is as shown in experiment 5.
Experiment 5: the cytotoxicity experiment of zero background fluorescence detection method of above-mentioned hydrazine
By HeLa cell inoculation in 96 orifice plates, be incubated for for 24 hours, be added 50 μM of probe DEASA be incubated for 0 respectively, 1,2,4,8,
12, after 24 hours, cell is rinsed with phosphate buffer, and cytotoxicity is detected using CCK-8 method, wherein HeLa cell survival
The column diagram of rate is as shown in Figure 9.
From attached drawing 9 it is found that cell survival rate is still maintained at 90% or more after 50 μM of DEASA are incubated for 24 hours, it is able to
Prove that DEASA has relatively low cytotoxicity, this result shows bio-imaging of the probe DEASA to hydrazine in living cells to grind
Study carefully with good biocompatibility.
In conclusion there is the inspection without background interference the invention proposes a kind of multi-functional and direct hydrazine inspection policies
Brake is suitable for actual sample and biological vivo detection.CTAB micella makes detection feasible in aqueous solution, without logical
Organic solvent adjusting is crossed, and passes through in situ detection scheme, sensitivity and selectivity relatively high, nothing of the probe DEASA to hydrazine
Background property can also be used for that even bioanalysis is imaged into the cell.Good hydrazine fluorescence detection sensing solutions disclosed by the invention are
Realize that zero background detection of high sensitivity of object provides new detection approach, this is for industry, life and biological field
It is great to there is market to apply and promotional value.
To the above description of the disclosed embodiments and experimental example, professional and technical personnel in the field is made to can be realized or use
The present invention.Various modifications to these embodiments will be readily apparent to those skilled in the art, herein
Defined General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Cause
This, the present invention will not be limited to the embodiments shown herein, and is to fit to and principles disclosed herein and new
The consistent widest scope of clever feature.
Claims (7)
1. a kind of zero background fluorescence detection method of hydrazine, which is characterized in that promoted by cetyltriethylammonium bromide micella
Condensation reaction between 4- lignocaine salicylide and analyte hydrazine, the generated in-situ new chromophore with intramolecular hydrogen bond makees
For the indicator of hydrazine detection.
2. a kind of zero background fluorescence detection method of hydrazine according to claim 1, which is characterized in that the new chromophore
Synthetic method specifically comprises the following steps:
(1) using PBS buffer solution as reaction medium, 4- lignocaine salicylide and cetyltriethylammonium bromide is added；
(2) analyte hydrazine is added in the solution that step (1) obtains, DEASA and hydrazine occur condensation reaction, ultimately generate the present invention
Disclosed new chromophore-luminescent substance HDBM.
3. a kind of zero background fluorescence detection method of hydrazine according to claim 1 or 2, which is characterized in that the analyte
The molar ratio of hydrazine and 4- lignocaine salicylide is 1:2.
4. a kind of zero background fluorescence detection method of hydrazine according to claim 1 or 2, which is characterized in that the hexadecane
The concentration of base triethylammonium bromide is controlled 0.1~5 × 10-3mol·L-1Between.
5. a kind of zero background fluorescence detection method of hydrazine as described in claim 1 in dicyandiamide solution the Selective recognition of hydrazine and
Application in quantitative detection.
6. a kind of application of zero background fluorescence detection method of hydrazine according to claim 5, which is characterized in that further include institute
Detection method is stated using hydrazine as the application in the detection of marker and imaging.
7. a kind of application of zero background fluorescence detection method of hydrazine according to claim 5, which is characterized in that the solvent
The concentration of cetyltriethylammonium bromide in system is 1 × 10-3mol·L-1。
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