CN105503768B - The preparation method of the fluorescence of alpha ketoglutaric acids/ultraviolet molecular probe and its application in biological specimen - Google Patents
The preparation method of the fluorescence of alpha ketoglutaric acids/ultraviolet molecular probe and its application in biological specimen Download PDFInfo
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- CN105503768B CN105503768B CN201610050322.6A CN201610050322A CN105503768B CN 105503768 B CN105503768 B CN 105503768B CN 201610050322 A CN201610050322 A CN 201610050322A CN 105503768 B CN105503768 B CN 105503768B
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- 239000003068 molecular probe Substances 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- KPGXRSRHYNQIFN-UHFFFAOYSA-N 2-oxoglutaric acid Chemical class OC(=O)CCC(=O)C(O)=O KPGXRSRHYNQIFN-UHFFFAOYSA-N 0.000 title claims abstract description 7
- 239000000523 sample Substances 0.000 claims abstract description 142
- 238000001514 detection method Methods 0.000 claims abstract description 65
- 210000002966 serum Anatomy 0.000 claims abstract description 65
- 239000000047 product Substances 0.000 claims abstract description 17
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000004090 dissolution Methods 0.000 claims abstract description 4
- 239000012065 filter cake Substances 0.000 claims abstract description 4
- GCWCHLULHAWMEH-UHFFFAOYSA-N hydrazine;methanol;hydrate Chemical compound O.OC.NN GCWCHLULHAWMEH-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000004451 qualitative analysis Methods 0.000 claims abstract description 4
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 4
- 239000002244 precipitate Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 65
- 238000000034 method Methods 0.000 claims description 64
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 49
- 238000012360 testing method Methods 0.000 claims description 28
- 210000004027 cell Anatomy 0.000 claims description 26
- 230000007246 mechanism Effects 0.000 claims description 22
- 238000003384 imaging method Methods 0.000 claims description 21
- 238000010521 absorption reaction Methods 0.000 claims description 20
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 18
- 230000008569 process Effects 0.000 claims description 18
- 238000012544 monitoring process Methods 0.000 claims description 14
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 12
- 230000008859 change Effects 0.000 claims description 12
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- 239000001888 Peptone Substances 0.000 claims description 7
- 150000001875 compounds Chemical class 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- IGHBXJSNZCFXNK-UHFFFAOYSA-N 4-chloro-7-nitrobenzofurazan Chemical compound [O-][N+](=O)C1=CC=C(Cl)C2=NON=C12 IGHBXJSNZCFXNK-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 6
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- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 5
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 claims description 5
- -1 filter Substances 0.000 claims description 5
- 238000000825 ultraviolet detection Methods 0.000 claims description 5
- 210000005253 yeast cell Anatomy 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 238000012937 correction Methods 0.000 claims description 4
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 4
- 229940066779 peptones Drugs 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 241000446313 Lamella Species 0.000 claims description 3
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- 238000010790 dilution Methods 0.000 claims description 3
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- 239000012086 standard solution Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 239000000460 chlorine Substances 0.000 claims description 2
- 229910052801 chlorine Inorganic materials 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 1
- HWXBTNAVRSUOJR-UHFFFAOYSA-N alpha-hydroxyglutaric acid Natural products OC(=O)C(O)CCC(O)=O HWXBTNAVRSUOJR-UHFFFAOYSA-N 0.000 claims 1
- 229940009533 alpha-ketoglutaric acid Drugs 0.000 claims 1
- 239000008121 dextrose Substances 0.000 claims 1
- 150000002825 nitriles Chemical class 0.000 claims 1
- 230000004044 response Effects 0.000 abstract description 22
- 238000004458 analytical method Methods 0.000 abstract description 12
- 238000011160 research Methods 0.000 abstract description 10
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 abstract description 6
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- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 16
- OJUGVDODNPJEEC-UHFFFAOYSA-N phenylglyoxal Chemical compound O=CC(=O)C1=CC=CC=C1 OJUGVDODNPJEEC-UHFFFAOYSA-N 0.000 description 12
- AIJULSRZWUXGPQ-UHFFFAOYSA-N Methylglyoxal Chemical compound CC(=O)C=O AIJULSRZWUXGPQ-UHFFFAOYSA-N 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000013461 design Methods 0.000 description 8
- 229940015043 glyoxal Drugs 0.000 description 8
- BTNMPGBKDVTSJY-UHFFFAOYSA-N keto-phenylpyruvic acid Chemical compound OC(=O)C(=O)CC1=CC=CC=C1 BTNMPGBKDVTSJY-UHFFFAOYSA-N 0.000 description 8
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- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
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- 239000001903 2-oxo-3-phenylpropanoic acid Substances 0.000 description 4
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 4
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 4
- DEDGUGJNLNLJSR-UHFFFAOYSA-N alpha-hydroxycinnamic acid Natural products OC(=O)C(O)=CC1=CC=CC=C1 DEDGUGJNLNLJSR-UHFFFAOYSA-N 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000004020 luminiscence type Methods 0.000 description 4
- 235000015277 pork Nutrition 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 230000000171 quenching effect Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
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- 125000000717 hydrazino group Chemical group [H]N([*])N([H])[H] 0.000 description 3
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- 239000011159 matrix material Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- 238000011896 sensitive detection Methods 0.000 description 3
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- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
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- 238000007689 inspection Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 229940107700 pyruvic acid Drugs 0.000 description 2
- 201000001275 rectum cancer Diseases 0.000 description 2
- 230000027756 respiratory electron transport chain Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- BWLBGMIXKSTLSX-UHFFFAOYSA-N 2-hydroxyisobutyric acid Chemical compound CC(C)(O)C(O)=O BWLBGMIXKSTLSX-UHFFFAOYSA-N 0.000 description 1
- OJPZNRMSJHFGTR-UHFFFAOYSA-N 2-oxidanylidene-2-phenyl-ethanal Chemical compound O=CC(=O)C1=CC=CC=C1.O=CC(=O)C1=CC=CC=C1 OJPZNRMSJHFGTR-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000007445 Chromatographic isolation Methods 0.000 description 1
- 238000004057 DFT-B3LYP calculation Methods 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 208000007976 Ketosis Diseases 0.000 description 1
- 101100491597 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) arg-6 gene Proteins 0.000 description 1
- 101100386053 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cys-3 gene Proteins 0.000 description 1
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 150000001323 aldoses Chemical class 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical class C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
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- 125000001309 chloro group Chemical group Cl* 0.000 description 1
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- OBISXEJSEGNNKL-UHFFFAOYSA-N dinitrogen-n-sulfide Chemical compound [N-]=[N+]=S OBISXEJSEGNNKL-UHFFFAOYSA-N 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- AGDZIJFMSISITG-UHFFFAOYSA-L disodium 2-oxopropanoate Chemical compound [Na+].[Na+].CC(=O)C([O-])=O.CC(=O)C([O-])=O AGDZIJFMSISITG-UHFFFAOYSA-L 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
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- 235000019253 formic acid Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004770 highest occupied molecular orbital Methods 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 150000002584 ketoses Chemical class 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
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- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000003077 quantum chemistry computational method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000004846 x-ray emission Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
- C07D271/12—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
-
- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
-
- 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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- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of preparation method of the fluorescence of alpha ketoglutaric acids/ultraviolet molecular probe and its application in biological specimen.Preparation method step is：NBD Cl are dissolved in chloroform, the g/mL of concentration of ordinary dissolution 0.002 ~ 0.012, then the hydrazine hydrate methanol solution that volumetric concentration is 0.2% ~ 1.2% is added, it is well mixed, brown precipitate is stirred to obtain at room temperature, is filtered, and filter cake washs through ethyl acetate, drying, obtain fluorescence/ultraviolet molecular probe of brown product alpha ketoglutaric acids.The qualitative and quantitative analysis of alpha ketoglutaric acids suitable for biological specimen, detection are sensitive, accurate, quick；Wherein biological specimen mainly includes serum, living cells, musculature etc., can be applied to analytical chemistry, life organic analysis, disease is examined in advance and the association areas such as clinical medicine detects.Have the advantages that good response, accuracys of data, reappearance, precision are high, equipment is convenient easy to operate, and exploitativeness is strong, is particularly suitable for the big data researchs such as high-volume sample combined sorting.
Description
Technical field
The invention belongs to analytical chemistry field, it is related to a kind of system of fluorescence of alpha-KG/ultraviolet molecular probe
Preparation Method and its application in biological specimen.
Background technology
At present, gas phase is mainly included for the analytical chemistry methods of alpha-KG（GC）, high performance liquid chromatography
And mass spectrum (HPLC)（MS）, UV-detector（UV-detector）Deng.And fluorescent molecular probe detection method quantity is very few, bag
Include and utilize nitro diazosulfide hydrazine（nitrobenzothiadiazole hydrazine DT）With naphthalimide hydrazine substituent
（hydrazino-substituted naphthalimide）Fluorescent molecular probe is monitored to alpha-KG.
Chromatographic detection method is applied in many fields, but is examined for the rapid batch of clinical medicine biology sample
It is not best selection for survey.Its reason is：First, technical step is complicated, and sample handling procedure is various, workload
Greatly, the operating time is long, efficiency is low etc..This is for medical biotechnology sample, particularly living cells, In vivo detection completely can not
It can realize.Secondly, sample requirements are big, and reagent consumption is big.The characteristics of biological specimen, is that sample size is small, and chromatogram joins
Close large-scale instrument detection method then generally require to pre-process large number of sample to be tested reach object purifying, enrichment,
The purpose of separation, therefore it is not particularly suited for trace sample analysis.Finally, large-scale instrument cost itself is high, and operates often step
It is careful, it is not easy to grasp and promotes, in the allegro modern society of high efficiency, the application of such method seems to have reached development
Bottleneck period.
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.However, for alpha-KG, such method is very rare, illustrates academia's mesh
Preceding research degree is also far from enough.From the point of view of the two methods reported at present, following lack be present and limit：
First, fluorescence response is poor, and sensitivity is relatively low, and principle of luminosity is unfavorable for detection of biological samples.From the point of view of existing report,
Existing probe is with after the reaction of determinand alpha-KG, about 7-9 times of Fluorescence Increasing, being difficult to realize highly sensitive detection.Although
Fluorescence Increasing is realized by the method for addition surfactant, but this method will destroy biotic environment while can bring larger
Error, thus be not suitable for the actually detected of biological specimen.Therefore, the Molecular Design of probe in itself has much room for improvement.
Next, detection accuracy, accurately the index such as close is to be improved.It is well known that relative to the higher letter of large-scale instrument
Number stability, bio-sensing often bring larger error.Although the signal self-correcting function of fluorescence probe can be effectively improved this
Shortcoming.But existing report is not implemented to improve to this shortcoming.And it is glimmering that existing probe molecule principle of luminosity is unfavorable for molecule
Light strongly increases.So the functional design and luminescence mechanism of probe have much room for improvement.
Again, the convenient degree of detection still has much room for improvement.At present, emerged in life organic analysis detection field many fast
Prompt convenient detection means, including colorimetric method, naked eyes monitoring etc..These methods are in fields such as cell analysis, monitored in vivo
It is widely used, but the colorimetric of alpha-KG/naked-eye observation technology is still in the blank of research.Therefore, will detect
Method further improves, and the convenient degree of experimental implementation is continued into the reality that raising is the exploitation of alpha-KG detection method
Demand.
Finally, the research to alpha-KG imaging technique is lacked.Cell/tissue imaging technique is that fluorescence probe is excellent
Unique embodiment of gesture.The technology can realize visualizing monitor and the treatment of disease, and the discovery for major diseases such as cancers is with examining
It is disconnected to have outstanding meaning.But this technology and application are not directed in the method that forefathers are reported.Thus, exploitation is suitable to cell
The molecular probe of imaging, the research mode that visualizing monitor platform will inherently change alpha-KG is established, is expected to
Realize the visual research to Cancerous disease caused by alpha-KG.Therefore, imaging technique and application are urgently carried out.
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 fluorescence of alpha-KG/ultraviolet
The preparation method of molecular probe, the molecular probe being prepared can be applied to detection of biological samples, have response good, data
The advantages that accuracy, reappearance, high precision, equipment is convenient easy to operate, and exploitativeness is strong, is particularly suitable for the combination of high-volume sample
The big data researchs such as screening.
Technical solution of the present invention is as follows：
A kind of preparation method of fluorescence of alpha-KG/ultraviolet molecular probe, step are：By NBD-Cl (7-
Chloro-4-nitrobenzo-2-oxa-1,3-diazole) it is dissolved in chloroform, the g/ of concentration of ordinary dissolution 0.002~0.012
ML, then add volumetric concentration be 0.2~1.2% hydrazine hydrate-methanol solution, be well mixed, stir at room temperature brown is sunk
Form sediment, filtering, filter cake washs through ethyl acetate, dries, and obtains fluorescence/ultraviolet molecular probe of brown product alpha-KG.
In above-mentioned preparation method, described NBD-Cl is dissolved in the preferred 0.006g/mL of concentration of ordinary dissolution in chloroform；Hydrazine hydrate-
Methanol solution volumetric concentration preferably 0.6%.
The fluorescence of described alpha-KG/ultraviolet molecular probe, Detection results index are as follows：
Detection sensitivity:0.9 μm of oL/L of test limit；Fluorescence Increasing multiple is 20~60 times；
Absorbing wavelength changes：Absorbed during detection to feux rouges and move 100 nm；
Color change：Color is changed into red from light green under fluorescent light；Color under uviol lamp is changed into green from colourless；
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 application of the fluorescence of above-mentioned alpha-KG/ultraviolet molecular probe：The alpha- ketone suitable for biological specimen
The qualitative and quantitative analysis of glutaric acid, detection are sensitive, accurate, quick；Wherein biological specimen mainly includes serum, living cells, muscle
Tissue etc., it can be applied to analytical chemistry, life organic analysis, disease is examined in advance and the association areas such as clinical medicine detects.
Alpha- ketone penta 2 in the fluorescence of alpha-KG of the present invention/ultraviolet molecular probe quantitative analysis biological specimen
When sour, the alpha-KG content suitable for detection serum.
The method of alpha-KG content, step include in fluorescence of the present invention/ultraviolet molecular probe detection serum：
1）Prepare solution
Probe HNBD storing solutions are prepared：Accurate fluorescence/ultraviolet the molecular probe for weighing alpha-KG is dissolved in anhydrous
Acetonitrile, compound concentration are 2 × 10-4M probe HNBD storing solutions；
Object alpha-KG to be measured（α-KA）Storing solution is prepared：Accurately weigh object alpha- ketone to be measured
Glutaric acid is dissolved in anhydrous acetonitrile, and compound concentration is 1 × 10-3M alpha-KG storing solution；
Serum storing solution is prepared：By serum and anhydrous acetonitrile by volume 5:1 mixing, the rpm of rotating speed 5000 centrifugation 20min,
Take supernatant to cross 0.2 μm of film process and obtain serum storing solution；
2）Establish the linear equation of serum/alpha-KG standard items
Take step 1）It is 2~900 μ that the object alpha-KG storing solution to be measured prepared, which dilutes to obtain gradient concentration,
M alpha-KG standard solution, 2~900 μM of alpha-KG standards after 200 μ L dilutions are then taken respectively
After product solution mixes with 100 μ L probe HNBD storing solutions and 650 μ L serum storing solutions, 50 μ L degree of addition be 10 mM,
PH7.4 PBS buffer solutions, shaking is well mixed, and 60min is placed at 37 DEG C, then through sepectrophotofluorometer and ultraviolet
Spectrometer detects, and establishes the linear equation or serum/α-KA concentration and UV absorption of serum/α-KA concentration and fluorescence signal intensity
The linear equation of signal intensity；
Alpha-KG content in fluorescence or UV process detection serum testing sample
a）Fluoroscopic examination testing sample：After 1000 μ L testing samples are injected into quartz colorimetric utensil, in entering in fluorescence detector
Row Scanning Detction, the intensity data for collecting fluorescent emission position substitute into the linear side of serum/α-KA concentration and fluorescence signal intensity
Journey, calculate to obtain alpha-KG content；
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 before and after reaction maximum absorption band volume efficiency and to substitute into serum/α-KA dense
Degree and UV absorption signal intensity, calculate to obtain alpha-KG content.
The application of alpha-KG content, optimal detection scope in above-mentioned fluorescence/ultraviolet molecular probe detection serum
For 3~900 μm of ol/L of fluorescence, ultraviolet 80~200 μm of ol/L.Determinand is entered in the method for fluoroscopic examination, ultraviolet detection respectively
The parallel repeated detection of row（n=7）, and calibrated with standard items, the optimal detection scope of fluorescence/ultraviolet detection is obtained, so as to root
The detection means optimized is selected to be quantified according to the concentration range of determinand contained by different samples.
Alpha- ketone penta 2 in the fluorescence of alpha-KG of the present invention/ultraviolet molecular probe qualitative analysis biological specimen
When sour, suitable for the naked eyes monitoring of alpha-KG blood serum sample, in living cells the monitoring of alpha-KG and
The monitoring of alpha-KG in musculature.
1st, the naked eyes of alpha-KG monitor in blood serum sample, and method is：By test serum sample and anhydrous acetonitrile
By volume 5:1 mixing, the rpm of rotating speed 5000 centrifugation 20min, takes supernatant to cross 0.2 μm of film process, takes gained supernatant 200 μ
L simultaneously sequentially adds 100 μ L probe HNBD storing solutions and 200 μ L alpha-KG storing solutions, then with pH 7.4 PBS
Buffer solution is settled to 1000 μ L, and resulting solution preserves 50~70min at 37 DEG C, is placed under fluorescence microscope and observes imaging, according to
Luminous situation judges whether contain alpha-KG in blood serum sample；
Color determination methods：If showing pale red under green or fluorescent lamp under uviol lamp, contain alpha-KG.
In probe HNBD storing solutions and alpha-KG the deposit liquid making method and above-mentioned detection serum
The method of alpha-KG content is identical.
2nd, in living cells alpha-KG monitoring, method is：Living cells to be measured is inserted 18 are cultivated in culture medium
~26h, living cells to be measured reach 2 × 10 in the medium 7~9×10 7During individual/mL, the spy of 3~5 times of 20 μM of volumes is added
Pin HNBD- acetonitrile solutions cultivate 10~12h altogether in 37 DEG C, with PBS washing three times, are placed under fluorescence microscope and observe
Imaging, judges whether contain alpha-KG in living cells to be measured according to luminous situation；
Determination methods：Show green under uviol lamp, then contain alpha-KG.
Described living cells preferred yeast bacterium cell；Described culture medium optimization protein peptone agar glucose.
3rd, in musculature alpha-KG monitoring, method is：Musculature to be measured is cut into 1~1.5 ㎜ of thickness
Lamella, the probe HNBD- acetonitrile solutions with 3~5 times of 20 μM of volumes, cultivate 1~2 h altogether in culture medium at 37 DEG C, so
Washed afterwards with PBS, be placed under fluorescence microscope and observe imaging, judged to treat whether contain in musculature according to luminous situation
There is alpha-KG；
Determination methods：Show green under uviol lamp, then contain alpha-KG.
Described culture medium is by the aqueous solution of 1wt% fat extract, 2wt% peptones and 2wt% glucose in 121 DEG C of cultures
20min is made.
The fluorescence of alpha-KG prepared by the present invention/ultraviolet molecular probe detection mechanism：
The design of the fluorescence of alpha-KG/ultraviolet molecular probe, principle are：By outstanding fluorescence female ring
NBD, utilize photo induced electron transfer mechanism（PET）Make its fluorescent quenching；The quenching group is to determinand alpha- ketone penta 2 simultaneously
Acid possesses good response characteristic, requires that the molecule advanced linear orbital energy of product has to change by a relatively large margin after reaction and causes PET mechanism
It is prevented from, so as to discharge hyperfluorescence signal and improve sensitivity；Other advanced linear orbital energy difference should reduce, so as to inhale
Red shift of wavelength is received, so as to show that visible light colors provide guarantee for naked-eye observation.
Fluorescence female ring NBD molecules have longwave absorption and transmitting, high quantum production rate, excellent Cell permeable, hypotoxicity
And the advantages that good availability.In alpha-KG molecular structure there is a carbonyl in carboxylic acid ortho position, and the group can
With electron donating group（Such as-amino）Reaction, change the electronics of group for effect.Therefore, parent Luminous Ring is made with NBD, by NBD-Cl
The elemental chlorine substitution of No. 7 positions of molecule turns into diazanyl, obtains fluorescence/ultraviolet molecular probe of alpha-KG of the present invention（I.e.
Probe HNBD）, then it is applied to detection of biological samples, mechanism is as shown in Figure 1（Wherein sent out in HNBD-alpha-KA fractions
Light group color is green）.
By Fig. 1 to the probe it can be seen from energy balane in excitation state in HNBD molecules, amino is as donor residues
Group（Donor 1）Its excited energy（-8.92eV）Far above the highest occupied molecular orbital energy of determinand alpha-KG
(- 11.34eV), thus the generation of PET electronic transfer process can be caused easily, so that probe is in fluorescent quenching shape in itself
State.And once combined with determinand, electron donating group is changed into the Donor2 shown in figure, its energy significantly reduce to
Under Acceptor energy, cause PET processes suppressed, so that fluorescence is released.The process will make probe anti-with object
Fluorescence intensity is substantially improved after answering, and significantly improves the sensitivity of detection signal.It is otherwise noted that in probe HNBD
After being combined into product HNBD-alpha-KA with object alpha-KG, the difference of its advanced linear orbital energy will diminish.Cause
This, from theoretic as can be seen that product absorption spectrum be possible to long-wavelength region move, and this point for solution before
Mention on realizing that naked-eye observation technical problem has great significance.Therefore, this organic molecule conduct of HNBD is built
The polyfunctional molecule probe of alpha-KG is practical.
Technical solution of the present invention has the beneficial effect that：
1）Sensitivity is higher：
Reported in literature congenic method detection at present is limited to 6 μM, and the detection limit is that surface-active is being added into system
Reached after the additional materials such as agent, and in the inventive method, inspection can be achieved under conditions of any surfactant is not added with
Survey is limited to 0.9 μM, and sensitivity greatly improves, while avoids the addition of additional materials, reduces error source.
2）Multi-channel detection, the degree of accuracy are higher：
Probe that the inventive method is developed while have fluorescent switch function and wavelength shift function concurrently, this function be with
Toward reporting with not available in fluorescence probe detection determinand α-KA method.The inventive method can with fluorescence by close to
Response enhancing is quantified caused by open mode, while is also quantified using volume efficiency caused by absorbing wavelength displacement.Its
In, ratio quantitative approach can effectively reduce ambient interferences, improve dosing accuracy, while UV absorption can quantitatively determine with fluorescence
Amount mutually correction, you can self-correcting function, so as to substantially increase the accuracy of testing result.
3）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 new
Grain husk.The probe that wherein PET processes make to be in cancellation state originally fluorescence after determinand is met significantly strengthens, and can bring highly sensitive sound
Should；Also, probe both with absorb red shift be using the nm of UV, visible light 380 wave number be starting point to red light district shift so that
The color of product is significantly changed, thus the probe of mechanism more single than other is more suitable for optical signal sensing analysis detection.And process
Middle fluorescence non-emissive displacement, thus without interference with colorimetric analysis.
4）A variety of biological sample imagings：
Successful implementation imaging of the determinand in living cells, musculature, this is also failed in forefathers' method
Realize.Very big impetus is played in further investigation of the realization of imaging for this biomarker of α-KA.
5）Naked eyes monitor, convenient and swift：
Probe of the present invention and detection method have filled up the blank of α-KA detection methods, monitor the naked eyes of determinand（Need not
Instrument, directly observe）It is possibly realized, detection process is more quick than average probe.
Brief description of the drawings
Fig. 1 is fluorescence/ultraviolet molecular probe detection and the principle of luminosity schematic diagram of alpha-KG of the present invention；
Fig. 2 is the probe HNBD and HNBD- α-KA of embodiment 1 high performance liquid chromatography-uv-spectrogram:(a) probe molecule
HNBD, (b) HNBD- α-KA, (c) probe HNBD, HNBD- α-KA；
Fig. 3 is the probe HNBD and HNBD- α-KA of embodiment 1 high phase liquid chromatogram-one-level（MS）And two level（MS/MS）Matter
Spectrogram：Probe HNBD (a: MS； b: MS/MS) ； HNBD-α-KA (c: MS；d: MS/MS)；
Fig. 4 is the linear equation of 2 serum of embodiment/α-KA concentration and fluorescence signal intensity；
Fig. 5 is the linear equation of 2 serum of embodiment/α-KA concentration and UV absorption signal intensity；
Fig. 6 is different determinand α-KA concentration in embodiment 2（1: 0 µM, 2: 5 µM, 3: 100 µM, 4: 300
µM, 5: 400 µM, 6: 600 µM, 7: 700 µM, 8: 900 µM, 9: 1000 µM,）Ultraviolet（On）And daylight
Lamp（Under）Photo (HNBD under irradiation: 20 µM; PBS buffer (pH 7.4)；
Fig. 7 is illuminated diagram of the blood serum sample under uviol lamp and fluorescent lamp, 1:Blank serum, 2:Blank+HNBD,
3:Blank+α-KA, 4:Blank+HNBD+ α-KA, wherein HNBD: 20 µM; α-KA: 200 µM, PBS buffer
(pH 7.4)；
Fig. 8 is the linear relationship of determinand α-KA and fluorescence signal in serum；1 be PBS solution data, 2 be in serum number
According to；
Fig. 9 is yeast cell imaging experiment：A (blank), b (+200 μM of α-KA of blank), c (+20 μM of blank
) and d (+20 μM of HNBD+200 μM of α-KA of blank) HNBD；
Figure 10 tests for fresh pork imaging of tissue：C (blank), d (+200 μM of α-KA of blank), g (blank+
20 μM of HNBD) and h (+20 μM of HNBD+200 μM of α-KA of blank)；
Figure 11 is various concentrations probe HNBD response intensity figures；
Figure 12 is fluorescence signal responses of the probe HNBD under with/without 200 μM of α-KA existence conditions of determinand：Up/down
Two Trendline are respectively fluorescence signal response of the probe molecule under with/without 200 μM of α-KA existence conditions of determinand；
Figure 13 is probe HNBD (20 μM) and α-KA (200 μM) in the fluorescence response that pH scopes are in 5.4 ~ 7.4；
Figure 14 temperature influences on the fluorescence signal of (200 μM) reactions of probe HNBD (20 μM) and α-KA；
Influence of Figure 15 ionic strengths to probe HNBD (20 μM) and α-KA (100 μM) reaction system；
Fluorescence response figures of Figure 16 probes HNBD to the object α-KA to be measured of various concentrations（α-KA concentration：0~900 µ
M）；
Figure 17 probes HNBD is to various concentrations pH value fluorescence response figure；
Figure 18 probes HNBD is to determinand α-KA and other material selectivity control experiment results.
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.
It is to connect Agilent 6460 using Agilent 1290 by efficient liquid phase-mass spectral analysis in the embodiment of the present invention
Triple quadrupole bar mass spectrometer system (Agilent, USA), and it is equipped with Agilent Jet Stream electrospray system high efficiency
Liquid chromatogram separation has been come by SB C18 posts (2.1 mm × 50 mm, 1.8 μm of i.d., Agilent, USA)
Into.Efficient liquid phase-ultra-violet analysis experiment is to use 1260 online vacuum suction devices of Agilent, carry out sample device automatically, be glimmering
Photodetector works together.Separation gradient is set to 0 minute: 70% A+30% B;10 minutes: 0% A+100% B; 15
Minute: 0% A+100% B;Wherein A and B is respectively the acetonitrile of 0.5% formic acid+5% and 100% acetonitrile solution.Fluorescence is examined
Survey is carried out using Hitachi's Hitachi F-7000 XRFs, and excitation wavelength is 475 nm, launch wavelength 540
Nm, it is 10.0 nm to excite with transmite slit width, voltage 400V, the nm/min of sweep speed 2400.Uv-vis spectra
It is to be carried out by the Bio ultraviolet-visual spectrometers of Cary 300, scanning range is 350-700 nanometers.Fluorescence imaging observation is logical
Cross Olympus, IX73-DP80 (Japan) inverted fluorescence microscopes are carried out.Isolating and purifying for compound is to use thin layer
Chromatography silica gel post is realized (filler 300-400 mesh).
Embodiment 1：Prepare fluorescence/ultraviolet molecular probe of alpha-KG
0.3g NBD-Cl are dissolved in 50mL chloroforms, the hydrazine hydrate-methanol for then adding 50mL volumetric concentrations 0.6% is molten
Liquid, it is well mixed, stirs 30min at room temperature, obtain brown precipitate, filter, filter cake washs through ethyl acetate, dries, and obtains 0.29g palm fibres
The fluorescence of color product, as alpha-KG/ultraviolet molecular probe（Probe HNBD）, yield 97%.
The fluorescence for the alpha-KG being prepared/ultraviolet molecular probe effect judge index is as follows：
Detection sensitivity:0.9 μm of ol/L of test limit；Fluorescence Increasing multiple is 20~60 times；
Absorbing wavelength changes：Absorbed during detection to feux rouges and move 100 nm；
Binary channels color change：Color is changed into red from light green under fluorescent light；Color under uviol lamp is by without discoloration
For green；
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.
Probe HNBD prepared by embodiment 1 and alpha-KG reaction feasibility checking：Take 0.1 gram of probe HNBD molten
In 80mL acetonitriles, add 2 times of equivalent alpha-KGs and 1h is stirred at room temperature thereto, obtain product HNBD- α-
KA, products therefrom prepare chromatographic isolation (chromatographic condition through half：Wates 600 controller；Detector：waters
2489；Elution requirement：Condition:5% A+95% B of 0-5 minutes 100% A, 5-7 minute;7-15 minutes：0% A+100%
B；15-18 minutes：0% A+100% B).
Characterized by high efficiency liquid chromatogram-ultraviolet, High Performance Liquid Chromatography/Mass Spectrometry, it is seen that product HNBD- α-KA are just
It is to be formed by the diazanyl of No. 7 positions of probe HNBD and the carbonyl synthesis of alpha-KG.Probe HNBD and product HNBD- α-
KA high efficiency liquid chromatogram-ultraviolet, High Performance Liquid Chromatography/Mass Spectrometry is shown in Fig. 2 and Fig. 3 respectively.
Embodiment 2：Detect alpha-KG content in serum（The alpha-KG prepared using embodiment 1
Fluorescence/ultraviolet molecular probe）
Take cancer patient's serum sample product（The cancer of the esophagus, right side breast cancer, right side colon, the carcinoma of the rectum）Add alpha- ketone penta
Alpha-KG serum model is made after diacid standard items（100 µM）As detection object：
Prepare solution
Probe HNBD storing solutions are prepared：Accurate fluorescence/ultraviolet the molecular probe for weighing alpha-KG is dissolved in anhydrous
Acetonitrile, compound concentration are 2 × 10-4M probe HNBD storing solutions；
Object alpha-KG to be measured（α-KA）Storing solution is prepared：Accurately weigh object alpha- ketone to be measured
Glutaric acid is dissolved in anhydrous acetonitrile, and compound concentration is 1 × 10-3M alpha-KG storing solution；
Serum storing solution is prepared：By serum and anhydrous acetonitrile by volume 5:1 mixing, the rpm of rotating speed 5000 centrifugation 20min,
Take supernatant to cross 0.2 μm of film process and obtain serum storing solution；
Establish the linear equation of serum/alpha-KG standard items
Take step 1）It is respectively 0 μ that the object alpha-KG storing solution to be measured prepared, which dilutes to obtain gradient concentration,
M, 5 μM, 100 μM, 300 μM, 400 μM, 600 μM, 700 μM, the alpha-KG mark of 900 μM and 1000 μM
Quasi- product solution, above-mentioned alpha-KG standard solution and 100 μ L probes HNBD storages after 200 μ L dilutions are then taken respectively
After standby liquid and 650 μ L serum storing solutions mixing, the PBS buffer solutions that 50 μ L degree are 10 mM, pH7.4, shaking mixing are added
Uniformly, 60min is placed at 37 DEG C, is then detected through sepectrophotofluorometer and ultraviolet spectrometer, it is dense to establish serum/α-KA
The linear equation of degree and fluorescence signal intensity（Fig. 4）Or the linear equation of serum/α-KA concentration and UV absorption signal intensity（Figure
5）；
Alpha-KG content in fluorescence or UV process detection serum testing sample
a）Fluoroscopic examination testing sample：After 1000 μ L test serums samples are injected into quartz colorimetric utensil, in fluorescence detector
Detection is inside scanned, the intensity data for collecting fluorescent emission position substitutes into the line of serum/α-KA concentration and fluorescence signal intensity
Property equation, calculate alpha-KG content be 99.07 ~ 99.34 μM；
b）Ultraviolet detection testing sample：After 1000 μ L test serums samples are injected into quartz colorimetric utensil, ultraviolet spectrometry is inserted
Photometer, collect the intensity of maximum absorption wavelength position, obtain before and after reaction maximum absorption band volume efficiency and substitute into serum/α-
KA concentration and UV absorption signal intensity, calculate alpha-KG content is 99.45 ~ 99.79 μM.
The present embodiment:Four kinds of serum model sample fluorescence（FLD）It is respectively with ultraviolet (UV) detection level：The cancer of the esophagus：
FLD: 99.07, UV： 99.45；Right side breast cancer：FLD: 99.20, UV： 99.58；Right side colon：FLD: 99.34,
UV： 99.48；The carcinoma of the rectum：FLD: 99.31, UV： 99.79；Error range：FLD：-0.93% ~ -0.56%； UV： -
0.55%~-0.21%.
As a result show, alpha-KG（α-KA）In 2~900 μM of concentration range（Than the 10 of document report-3
~10-5 The wider Ind. Eng. Chem. Res. 2015,54,2886 2893 of M）To being rung caused by probe HNBD of the present invention
Linear relationship is presented in induction signal（Fig. 4）, detect and be limited to 0.9 μm of ol/L（The 6 μM of Ind. Eng. reported better than document
Chem. Res. 2015, 54, 2886−2893 ）, and good coefficient correlation is shown in wider concentration range
（r2 = 0.9921.）.
Meanwhile the volume efficiency before and after the movement of UV absorption wavelength shows linear dependence to testing concentration（Fig. 5）,
This causes quantitative accuracy to greatly improve.Moreover, it is contemplated that the concentration to the determinand α-KA that may be present in biological sample
Rank, the range of linearity obtained by UV absorption ratio（80~190 μM）Fit into and quantified in biological sample, so as to improve
The biological applicability of method.The color of system is caused to be transformed into red by green in addition, significantly absorbing red shift, this acutely becomes
Change the naked eyes monitoring that ensure that trace determinand（Fig. 6）, this characteristic occurs first in fluorescence probe detection α-KA field,
The detection method that forefathers are reported is without this characteristic（Chem. Sci., 2014, 5, 4012）
1 is prepared respectively according to the solution manufacturing method of embodiment 2:Blank serum, 2:Blank+HNBD, 3:Blank+α-
KA, 4:Blank+HNBD+ α-KA, four kinds of solution to be measured, wherein HNBD: 20 µM; α-KA: 200 µM, PBS buffer
(pH 7.4), 60min is preserved under the conditions of 37 DEG C.It is visible by contrasting, blank+HNBD sample do not present absorb it is red
Move, while fluorescence is in cancellation state all the time under uviol lamp, this shows that serum matrix will not interfere to this probe.And add
After entering determinand α-KA, occur obvious green under uviol lamp, show that fluorescence is opened（PET is prohibited）Under fluorescent light simultaneously
There is orange red color (Fig. 6), show that the object α-KA progress color comparison of naked eye monitorings in blood serum sample can be realized（Figure
7）.
Embodiment 3：The naked eyes monitoring of alpha-KG in blood serum sample（Sample is the same as embodiment 2）
Method is：By test serum sample and anhydrous acetonitrile by volume 5:1 mixing, the rpm of rotating speed 5000 centrifugations
20min, take supernatant to cross 0.2 μm of film process, take gained the μ L of supernatant 200 and sequentially add 100 μ L probe HNBD storing solutions and
200 μ L alpha-KG storing solutions, are then settled to 1000 μ L, resulting solution is 37 with pH 7.4 PBS buffer solutions
DEG C 60min is preserved, be placed under fluorescence microscope and observe imaging, color is：It is green under uviol lamp, is light red, card under fluorescent lamp
It is bright to contain alpha-KG.
Probe HNBD storing solutions and alpha-KG deposit liquid making method are same as Example 2.
By Fig. 7 blank+HNBD sample, it can be seen that do not absorb red shift and occur, while fluorescence begins under uviol lamp
Cancellation state is in eventually, this shows that serum matrix will not interfere to this probe.And after adding determinand α-KA, in uviol lamp
It is lower obvious green occur, show that fluorescence is opened（PET is prohibited）Occurs orange red color under fluorescent light simultaneously, this is existing
Color comparison of naked eye observation is carried out to the object α-KA in blood serum sample showing to realize.Meanwhile by determinand α in serum-
KA and fluorescence signal linear relationship（Fig. 8）As can be seen that in human serum solution, it is glimmering in the range of 0~900 μM of determinand
Light number is that linear correlation is presented, while the relation established with UV absorption also complies with linear variability law.
Embodiment 4:The monitoring of alpha-KG in living cells（By taking saccharomycete as an example）
Method is：Yeast cell is inserted in Yeast protein peptone agar glucose and cultivates 24h, treats that yeast cell is being trained
Support in base and reach 2 × 107~9×10 7During individual/mL, the probe HNBD- acetonitrile solutions of 3 times of 20 μM of volumes are then added in 37
DEG C 12h is cultivated altogether, washed three times with PBS, insert and imaging is observed under fluorescence microscope, alpha- ketone in living cells to be measured
Glutaric acid color is：Green, and other control samples（A, b, c in Fig. 9）Without color, then provable contain is presented in green under the conditions of being somebody's turn to do
There is alpha-KG.
Comparison test analysis：In the case where ultraviolet wavelength excites, yeast cell（As shown in Figure 9）Respectively with blank, HNBD
(20 μM) and α-KA (200 μM) do not show fluorescent emission, wherein blank solution is in 37 DEG C after cultivating 24 h altogether
20 min are cultivated by cell in fat extract (1%), peptone (2%) and glucose (2%) mixture to be made.This table
Bright probe HNBD is that optics is immune for the various materials in cell, i.e., cellular matrix does not cause optical interference.And when addition
10 times amount determinand α-KA when, occur obvious launching effect under uviol lamp, the imaging of green fluorescence is smoothly observed
Arrive.In terms of this explanation probe HNBD has excellent membrane penetrating, while probe HNBD can apply to cell imaging.
Embodiment 5：The monitoring of alpha-KG in musculature（By taking fresh pork tissue as an example）
Method is：Fresh pork tissue is cut into 1 ㎜ of thickness lamella, it is molten with the probe HNBD- acetonitriles of 3 times of 20 μM of volumes
Liquid cultivates 1h at 37 DEG C,（The aqueous solution of 1wt% fat extract, 2wt% peptones and 2wt% glucose is in 121 DEG C of cultures
20min is made）, then washed with PBS, insert and imaging is observed under fluorescence microscopy device, alpha- in fresh pork tissue
Ketoglutaric acid color is：Green（Under uviol lamp）, it was demonstrated that contain alpha-KG.
Contrast test is verified：Experimental result（As shown in Figure 10）(37 DEG C, cultivate 24 h, c (blank), d (blank altogether
+ 200 μM of α-KA), g (+20 μM of HNBD of blank) and h (+20 μM of HNBD+200 μM of α-KA of blank), its is hollow
White liquor is that musculature cultivates 20min in the aqueous solution of 1wt% fat extract, 2wt% peptones and 2wt% glucose at 121 DEG C
It is made.Show in the case where being added without determinand, fluorescent emission is not almost presented after being cultivated altogether with probe for blank sample.With
After 10 times of equivalent determinand α-KA cultivate 60min altogether, fluorescent emission can be observed and significantly increase, show clearly histofluorescence
Image.These phenomenons confirm that the probe still has good penetrability for sturdy grain of meat tissue, thus are adapted to
It is imaged in musculature.
The experimental verification of the fluorescence of alpha-KG of the present invention/ultraviolet molecular probe all technical, specifically such as
Under：
Probe dosage optimization
Probe dosage is related to the important indicators such as sensitivity and the reagent consumption of detection, often as the primary of inspection optimization
Factor is investigated.According to the luminous characteristics of this probe, its sensitivity should belong to higher, therefore required when actually detected
Concentration and probe concentration should be relatively low.It is as higher in given concentration, reduce information strength by being likely to occur self-quenching.Therefore, it have selected 10 ~
50 μM of concentration range optimizes.As a result show, when testing concentration is 200 μM, concentration is 20 μM of probe
With most strong response.The concentration will be brought into follow-up Optimal Experimental process（Figure 11）.
Reaction time optimizes
Reaction time will influence probe molecule HNBD and determinand α-KA reaction efficiency and the extent of reaction, while also will certainly
Determine the stabilized soil pavement of final signal.Therefore, it is determined that after concentration and probe concentration, the time of reaction is optimized, temperature：37
DEG C, cushioning liquid PBS, pH 7.4.In figure 12 it can be seen that before the reaction in 60 minutes, fluorescence signal is in rising trend
Afterwards to plateau, so as to can determine that the reaction time is 60 minutes.By contrast, the probe solution without determinand does not occur then glimmering
The change of luminous intensity, this explanation signal response is caused by determinand.
The optimization of pH value of reaction system
PH values often have certain influence to the optical property of organic molecular probe, thus in the reaction often through buffering
Solution is controlled so as to meet to test needs.For application of the present invention（Serum, cell, tissue）Own characteristic, research
The pH value that is likely to be breached under animal body physiological environment（5.4~7.4）.From Figure 13（37℃, 60 min）In visible, physiology
In the range of pH value the fluorescence intensity response that is presented for the mixed system of object and probe of minor fluctuations influence it is very small.Cause
This, in common biosystem, i.e., when pH value is 7.4, organism acidity environment will not impact to the application of probe.
The optimization of reaction temperature
Influence of the temperature to chemical reaction is most important, biological specimen such as living cells, the tissue studied for the present invention
System is even more so.The temperature of usual animal body is generally 37 DEG C or so, and can probe have to object to be measured at this temperature
Good response will be related to the success or failure entirely tested.Such as Figure 14 (cushioning liquid：PBS buffer (pH 7.4), temperature：37
DEG C, the time：60 min) shown in, we are investigated fluorescence response caused by reaction of the temperature in 10 ~ 40 DEG C of scopes.
Therefrom it is seen that, this probe provides good fluorescence response at 37 DEG C with determinand reaction, thus particularly suitable for biology
The application and research of sample.
Influence of the ionic strength to fluorescence signal
Ionic strength, as distinctive factor in solution it is also possible to that fluorescence signal is caused necessarily to influence.In organism
In system, electrolyte is based on sodium chloride, therefore present invention determine that with the sodium chloride concentration under physiological condition（100 mM）To simulate
The electrolyte environment of actual sample system, so as to further prove feasibility of this method in practical systems.Figure 15（NaCl:
100 mM, cushioning liquid：PBS buffer (pH 7.4), temperature：37 DEG C, the time：60 min）In it is visible, sodium chloride is deposited
Almost do not impacted for fluorescence signal, this illustrates that this probe is entirely appropriate and treated in life system ionic strength atmosphere
Survey thing examinations.
Optical property and Mechanism Validation
A kind of it is contemplated that polyfunctional molecule probe for possessing novel luminescent mechanism of invention.Probe molecule HNBD hair
It is very faint to penetrate spectral intensity（As shown in figure 16）, show that the electronics row that fluorescence can be allowed effectively to be quenched occurs in the intramolecular
For.
To confirm that this behavior belongs to PET mechanism, the present invention has carried out mould with Quantum chemical calculation to probe excitation state
Intend（The general sea of density is theoretical, the horizontal B3LYP 6-31g (d, p) of base group1）.From the results of view, in probe molecule, the energy of amino
Amount is enough to cause effective PET mechanism to occur, so as to quenching fluorescence.And after HNBD combination determinands, its PET process is intended
System, then fluorescence release.In probe and the reacted mixed liquor of determinand, fluorescence intensity is obviously improved, and maximum can strengthen
60 times.This illustrates that rational PET Fluorescence Mechanisms design the handoff functionality for causing fluorescence probe to possess close/open.Not only such as
This, further to verify the mechanism, the present invention demonstrates different pH value to be influenceed on probe HNBD fluorescence.In view of HNBD institutes band
The alkalescence of diazanyl, devise with the experiment in hydrogen ion with amino electronics.From Figure 17, when pH value is in neutral acidity model
When enclosing, influenceed by PET, probe HNBD fluorescence activity is relatively low；When pH reduces, the hydrogen ion gradually increased causes by force to probe
Strong effect, the electronics on amino is prevented to move, so that fluorescence greatly enhances.This phenomenon has reconfirmed PET mechanism
In the presence of, while also illustrate that the reasonability of this probe design., whereas if be not PET mechanism, then HNBD probe absorption maximum
Spectrum is likely to occur blue shift, but this hypothesis substantially with experimental observation to phenomenon be not inconsistent.
In fact, the HNBD and reacted product HNBD- α-KA of object α-KA, which are shown, greatly absorbs red shift, this can
It can be due to the frontier orbit of the difference of HNBD- α-KA advanced linear orbital energy than probe HNBD molecules after product is converted to
The difference of energy has been reduced, and so as to result in the reduction for absorbing energy, that is, the red shift of absorption spectrum occurs.Meanwhile do not have
The Red Shift Phenomena of existing emission spectrum, this shows that this probe luminescence mechanism should belong to very novel fluorescent switch+absorption red shift mechanism.
Therefore, by these optical characteristics, the present invention can realize that sensitive detection response, naked eyes monitor, even contribution ratios school
Positive detection.This also indicates that the design of this probe is very successful simultaneously.
Probe molecule detection α-KA selectivity analysis
Following substances lay in liquid making method：Respectively with redistilled water Dissolved Amino Acids（Ala, Cys, His, Met,
Arg, Gln, Ile, Phe, Asn, Leu, Pro, Asp, Gly, Lys, Sar, Ser, Thr, Trp, Val,
Glu）, glucose（Glucose）, mannose（Galactose）, glyoxal（Glyoxal）, hydrogen peroxide (H2O2), Sodium Pyruvate
（sodium pyruvate (PAS)）, phenylglyoxal（phenylglyoxal）And pyroracemic aldehyde（methylglyoxal）, phenylpropyl alcohol
Ketone acid（phenylpyruvic acid）, the storing solution of above-mentioned substance is obtained, it is low dense needed for subsequent experimental for subsequent analysis
Degree solution dilutes on the basis of this storing solution to be made.
Based on the stronger fluorescence response of this probe, the present invention analyzes selectivity of the probe HNBD to α-KA, and it is more right
As including glucose（Glucose）, mannose（Galactose）, glyoxal（Glyoxal）, hydrogen peroxide (H2O2), pyruvic acid
Sodium（sodium pyruvate (PAS)）, phenylglyoxal（phenylglyoxal）, pyroracemic aldehyde（methylglyoxal）, benzene
Pyruvic acid（phenylpyruvic acid）To prepare.Probe HNBD is to determinand α-KA and other material selectivity control experiments
As a result as shown in figure 18, other materials include in figure：In a figures 100 it is micro- rub/liter amino acid：1: Ala 2: Cys 3: His
4: Met 5: Arg 6: Gln 7: Ile 8: Phe 9: Asn 10: α-KA 11: Leu 12: Pro 13: Asp
14: Gly 15: Lys 16: Sar 17: Ser 18: Thr 19: Trp 20: Val 21: Glu；5 mmoles in b figures/liter
α-KA, 200 it is micro- rub/liter galactolipin（Galactose(Gal)）, glyoxal Glyoxal (GO), hydrogen peroxide (H2O2), third
Ketone acid sodium sodium pyruvate (PAS), phenylglyoxal phenylglyoxal (PGO), pyroracemic aldehyde（methylglyoxal）
And phenylpyruvic acid（phenylpyruvic acid）.
First, the response of extreme difference is gone out to amino acids exhibit relative to determinand α-KA, HNBD, this should be due to amino acid
Have with α-KA in structure it is significantly different caused by.When addition excessive aldose, ketose, glyoxal, acetonate and phenylglyoxal
When, HNBD still maintains the selectivity outstanding to determinand α-KA.Although 200 μM of pyroracemic aldehyde causes the small of HNBD
Response, but this can't bring the interference of detection, and concentration that may be present is generally less than 2 μM to pyroracemic aldehyde in vivo after all.
Therefore, outstanding selectivity causes the entirely appropriate applications in biological specimen of HNBD.Meanwhile found by pH titration experiments,
In the interval range that pH value is 5.4 ~ 7.4, the intensity of fluorescence does not appear in significant changes, and this shows the probe in biotic environment i.e.
PH is completely applicable when being 7.4.In addition, temperature experiment also demonstrate that this probe is highly suitable for biological sample.
Claims (6)
1. a kind of method of fluorescence/ultraviolet molecular probe detection alpha-KG using alpha-KG, it is special
Sign is：Include the qualitative and quantitative analysis of alpha-KG in biological specimen；
The preparation method of the fluorescence of described alpha-KG/ultraviolet molecular probe, step are：NBD-Cl is dissolved in chlorine
In imitative, concentration of ordinary dissolution 0.006g/mL, hydrazine hydrate-methanol solution that volumetric concentration is 0.6% then added, is well mixed, room temperature
Under stir to obtain brown precipitate, filter, filter cake washs through ethyl acetate, dries, obtain the fluorescence of brown product alpha-KG/
Ultraviolet molecular probe；
The fluorescence of described alpha-KG/ultraviolet molecular probe, Detection results index are as follows：
Detection sensitivity:0.9 μm of oL/L of test limit；Fluorescence Increasing multiple is 20~60 times；
Absorbing wavelength changes：Absorbed during detection to feux rouges and move 100 nm；
Color change：Color is changed into red from light green under fluorescent light；Color under uviol lamp is changed into green from colourless；
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.
2. according to the method for claim 1, it is characterised in that the method for alpha-KG content in detection serum,
Step includes：
1）Prepare solution
Probe HNBD storing solutions are prepared：Accurate fluorescence/ultraviolet the molecular probe for weighing alpha-KG is dissolved in anhydrous second
Nitrile, compound concentration are 2 × 10-4M probe HNBD storing solutions；
Object alpha-KG to be measured（α-KA）Storing solution is prepared：Accurately weigh object alpha- ketone penta 2 to be measured
Acid is dissolved in anhydrous acetonitrile, and compound concentration is 1 × 10-3M alpha-KG storing solution；
Serum storing solution is prepared：By serum and anhydrous acetonitrile by volume 5:1 mixing, the rpm of rotating speed 5000 centrifugation 20min, takes
Clear liquid crosses 0.2 μm of film process and obtains serum storing solution；
2）Establish the linear equation of serum/alpha-KG standard items
Take step 1）It is 2~900 μM that the object alpha-KG storing solution to be measured prepared, which dilutes to obtain gradient concentration,
Alpha-KG standard solution, 2~900 μM of alpha-KG standard items after 200 μ L dilutions are then taken respectively
After solution mixes with 100 μ L probe HNBD storing solutions and 650 μ L serum storing solutions, it is 10 mM, pH7.4 to add 50 μ L degree
PBS buffer solutions, shaking is well mixed, 60min is placed at 37 DEG C, then through sepectrophotofluorometer and ultraviolet spectrometer
Detection, establish serum/α-KA concentration and fluorescence signal intensity linear equation or serum/α-KA concentration and UV absorption signal it is strong
The linear equation of degree；
3）Alpha-KG content in fluorescence or UV process detection serum testing sample
a）Fluoroscopic examination testing sample：After 1000 μ L testing samples are injected into quartz colorimetric utensil, in being swept in fluorescence detector
Detection is retouched, the intensity data for collecting fluorescent emission position substitutes into the linear equation of serum/α-KA concentration and fluorescence signal intensity, meter
Calculate to obtain alpha-KG content；
b）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 before and after reaction maximum absorption band volume efficiency and substitute into serum/α-KA concentration with
UV absorption signal intensity, calculate to obtain alpha-KG content.
3. according to the method for claim 2, it is characterised in that：Detection range is fluorescence 3~900 μm of ol/L, ultraviolet 80~
200µmol/L。
4. according to the method for claim 1, it is characterised in that the naked eyes monitoring of alpha-KG in blood serum sample,
Method is：By test serum sample and anhydrous acetonitrile by volume 5:1 mixing, the rpm of rotating speed 5000 centrifugation 20min, takes supernatant
Liquid crosses 0.2 μm of film process, takes the gained μ L of supernatant 200 and sequentially adds 100 μ L probe HNBD storing solutions and 200 μ L alpha-
Ketoglutaric acid storing solution, then it is settled to 1000 μ L with pH 7.4 PBS buffer solutions, resulting solution preserves 50 at 37 DEG C~
70min, it is placed under fluorescence microscope and observes imaging, judge whether contain alpha- ketone penta 2 in blood serum sample according to luminous situation
Acid；
Color determination methods：If showing pale red under green or fluorescent lamp under uviol lamp, contain alpha-KG.
5. according to the method for claim 1, it is characterised in that the monitoring of alpha-KG, method are in living cells：
Living cells to be measured is inserted into 18~26h of cultivation, living cells to be measured in culture medium and reaches 2 × 10 in the medium7~9 × 107Individual/
During mL, add 3~5 times of volumes, 20 μM of probe HNBD- acetonitrile solutions and cultivate 10~12h altogether in 37 DEG C, washed with PBS
Wash three times, be placed under fluorescence microscope and observe imaging, judge whether contain alpha- ketone in living cells to be measured according to luminous situation
Glutaric acid；
Determination methods：Show green under uviol lamp, then contain alpha-KG；
Described living cells is yeast cell；Described culture medium is peptone dextrose agar.
6. according to the method for claim 1, it is characterised in that the monitoring of alpha-KG, method in musculature
For：Musculature to be measured is cut into 1~1.5 ㎜ of thickness lamella, the probe HNBD- acetonitrile solutions with 3~5 times of 20 μM of volumes,
Cultivate 1~2 h altogether in culture medium at 37 DEG C, then washed with PBS, be placed under fluorescence microscope and observe imaging,
Judge treat whether contain alpha-KG in musculature according to luminous situation；
Determination methods：Show green under uviol lamp, then contain alpha-KG；
Described culture medium is by the aqueous solution of 1wt% fat extract, 2wt% peptones and 2wt% glucose in 121 DEG C of cultures
20min is made.
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