CN105154066A - High-sensitivity fluorescent probe, method for preparing same and application of high-sensitivity fluorescent probe - Google Patents
High-sensitivity fluorescent probe, method for preparing same and application of high-sensitivity fluorescent probe Download PDFInfo
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- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title abstract description 21
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 99
- 229920000669 heparin Polymers 0.000 claims abstract description 99
- 229960002897 heparin Drugs 0.000 claims abstract description 99
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 32
- 229920001184 polypeptide Polymers 0.000 claims abstract description 31
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 31
- 239000000523 sample Substances 0.000 claims description 54
- 229920002567 Chondroitin Polymers 0.000 claims description 37
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 claims description 37
- 238000012360 testing method Methods 0.000 claims description 26
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 16
- JLZUZNKTTIRERF-UHFFFAOYSA-N tetraphenylethylene Chemical group C1=CC=CC=C1C(C=1C=CC=CC=1)=C(C=1C=CC=CC=1)C1=CC=CC=C1 JLZUZNKTTIRERF-UHFFFAOYSA-N 0.000 claims description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 108010022901 Heparin Lyase Proteins 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 11
- 230000015572 biosynthetic process Effects 0.000 claims description 9
- 238000003786 synthesis reaction Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- 238000013016 damping Methods 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 125000003368 amide group Chemical group 0.000 claims description 4
- 125000000539 amino acid group Chemical group 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 108010069848 AG 73 Proteins 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 abstract 1
- 239000000470 constituent Substances 0.000 abstract 1
- 229910052739 hydrogen Inorganic materials 0.000 abstract 1
- 239000001257 hydrogen Substances 0.000 abstract 1
- 239000003550 marker Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 60
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 20
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 11
- 238000001035 drying Methods 0.000 description 10
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 10
- 239000007790 solid phase Substances 0.000 description 7
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- ANUZKYYBDVLEEI-UHFFFAOYSA-N butane;hexane;lithium Chemical compound [Li]CCCC.CCCCCC ANUZKYYBDVLEEI-UHFFFAOYSA-N 0.000 description 4
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- UPJJNWCKFONBDW-UHFFFAOYSA-N 1-bromo-2-(1,2,2-triphenylethenyl)benzene Chemical group BrC1=CC=CC=C1C(C=1C=CC=CC=1)=C(C=1C=CC=CC=1)C1=CC=CC=C1 UPJJNWCKFONBDW-UHFFFAOYSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
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- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- 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 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 210000001557 animal structure Anatomy 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- RWCCWEUUXYIKHB-UHFFFAOYSA-N benzophenone Chemical compound C=1C=CC=CC=1C(=O)C1=CC=CC=C1 RWCCWEUUXYIKHB-UHFFFAOYSA-N 0.000 description 2
- 239000012965 benzophenone Substances 0.000 description 2
- GDTBXPJZTBHREO-UHFFFAOYSA-N bromine Substances BrBr GDTBXPJZTBHREO-UHFFFAOYSA-N 0.000 description 2
- 229910052794 bromium Inorganic materials 0.000 description 2
- 235000011089 carbon dioxide Nutrition 0.000 description 2
- 238000007819 clotting time assay Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 230000006837 decompression Effects 0.000 description 2
- CZZYITDELCSZES-UHFFFAOYSA-N diphenylmethane Chemical compound C=1C=CC=CC=1CC1=CC=CC=C1 CZZYITDELCSZES-UHFFFAOYSA-N 0.000 description 2
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
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- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
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- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- DSSYKIVIOFKYAU-XCBNKYQSSA-N (R)-camphor Chemical compound C1C[C@@]2(C)C(=O)C[C@@H]1C2(C)C DSSYKIVIOFKYAU-XCBNKYQSSA-N 0.000 description 1
- HCZMHWVFVZAHCR-UHFFFAOYSA-N 2-[2-(2-sulfanylethoxy)ethoxy]ethanethiol Chemical compound SCCOCCOCCS HCZMHWVFVZAHCR-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
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- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
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- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a high-sensitivity fluorescent probe TPE-1. The high-sensitivity fluorescent probe TPE-1 comprises a polypeptide AG73 with heparin recognition effects and AIE fluorogen. The AIE fluorogen is arranged at an end of the polypeptide AG73 and is used as a fluorescent signal radical marker. The invention further discloses a method for preparing the high-sensitivity fluorescent probe and application thereof. The high-sensitivity fluorescent probe, the method for detecting heparin and the application have the advantages that detection signals and heparin concentration have excellent linear relations; the heparin can be detected in diversified pH (potential of hydrogen) environments without being affected by the detection environments and complex constituents in detection systems; detection operation is easy and convenient and is high in speed, only samples need to be mixed with the fluorescent probe, and then the fluorescent intensity of the samples is instantly tested, so that corresponding heparin contents can be known; the contents of the heparin in the samples can be tested, and the quality of commercialized heparin can be evaluated.
Description
Technical field
The invention belongs to the quality test field of medicine, be specifically related to a kind of highly sensitive fluorescent probe TPE-1 and its preparation method and application.
Background technology
Heparin is the highly Sulfated mucopolysaccharide of one, is widely used in treatment and the prevention of various diseases clinically.In surgical operation, heparin is widely used as anti-coagulant.But the dosage of heparin must control in suitable scope clinically, otherwise side effect will be caused as profuse bleeding, thrombopenia, kaliemia and osteoporosis etc., even life-threatening under serious conditions.According to existing result of study, in surgical operation, the rational dosage range of heparin is 2-8U/mL, and during later stage rehabilitation medication, the rational dosage range of heparin is 0.2-1.2U/mL.Therefore, the concentration how detecting heparin is quickly and easily very important.Heparin concentration detection means conventional at present has activated clotting time assay method (activatedclottingtimeassay), APTT method (activatedpartialthromboplastintimeassay), electrochemical method, electrophoretic method and surface enhanced Raman spectroscopy method etc.But these methods all Shortcomings parts.Activated clotting time assay method and these two classical ways of APTT method use clinically, but but there is following obviously shortcoming: the concentration 1) being all indirect inspection heparin, therefore test result and truth can be caused to have deviation, the uncertain factor such as temperature humidity by environment affects comparatively large, and the data obtained is not very accurately and reliably; 2) test duration is long; 3) testing expense is high.And electrochemical method detects not high to the selectivity of heparin, be easily subject to the interference of other molecules and ion in system, therefore may provide spurious response.And Raman spectrometer instrument price is expensive, in addition, detect by surface enhanced Raman spectroscopy method and need first to prepare corresponding gold or SERS substrate, because gold and silver are precious metal, therefore the impact prepared by substrate of the higher and test result of the method cost is larger, cause the fluctuation of data, accuracy reliability is poor.
Therefore, extremely need to develop highly sensitive, the highly selective detection of a kind of novel detection technique for heparin.This technology must meet the following conditions: 1) optionally can detect heparin, the interference of other compositions in the not examined system of detection signal; 2) highly sensitive, detection sensitivity must lower than the lowest dose level concentration of heparin use clinically; 3) detection method is reliable, stable, detects not by the impact of microenvironment; 4) concentration of detection signal and heparin has good linear relationship, instead of the signal fluctuated up and down.
Summary of the invention
Goal of the invention: the present invention is directed to the present situation such as inaccurate, the consuming time length of data, testing expense height that current heparin detection method exists, by Development of Novel fluorescent probe technique, heparin is carried out to the detection of highly selective, high sensitivity.This technology is used for heparin detection and has the following advantages: 1) detection signal and heparin concentration present extraordinary linear relationship; 2) detect and can carry out under multiple pH environment, the impact of complicated ingredient in not examined environment and detection system; 3) detect speed easy and simple to handle fast, sample only need mix with fluorescent probe by the method, then tests its fluorescence intensity immediately and can learn corresponding heparin content; 4) both can test the content of heparin in sample, also can identify the quality of commercialization heparin.
Technical scheme: in order to solve the problems of the technologies described above, the invention provides a kind of highly sensitive fluorescent probe TPE-1, this fluorescent probe TPE-1 includes the polypeptide A G73 of recognition reaction and the AIE fluorophore being marked at one end of polypeptide A G73 as fluorescent signal group with heparin.
Wherein, the sequence of aforementioned polypeptides AG73 is RKRLQVQLSIRT.
Wherein, above-mentioned AIE fluorophore is tetraphenyl ethylene molecule.
A preparation method of highly sensitive fluorescent probe TPE-1, comprises the following steps:
1) synthesis of tetraphenyl ethylene molecule;
Be dissolved in by ditan (2.02g, 12mmol) in the tetrahydrofuran (THF) of 50mL drying, displacement nitrogen protection is placed in-78 DEG C of cooling baths.Inject the n-Butyl Lithium hexane solution that 5mL concentration is 2.5M, and keep this thermotonus 1 hour.4-bromine benzophenone (2.35g, 9mmol) is dissolved in the tetrahydrofuran (THF) of 10mL drying, slowly injects above-mentioned solution, and stirred overnight at room temperature.Add aqueous ammonium chloride solution cancellation reaction, dichloromethane extraction, is spin-dried for after organic phase anhydrous sodium sulfate drying.Gained crude product is dissolved in 80mL toluene, adds tosic acid (342mg, 1.8mmol), point water refluxes 5 hours.Question response liquid is cooled to room temperature, adds 10% sodium bicarbonate aqueous solution extraction, is spin-dried for after organic phase anhydrous sodium sulfate drying.Column chromatography is purified to obtain bromo tetraphenyl ethylene, productive rate about 40%.Bromo tetraphenyl ethylene (1.6g, 3.9mmol) is dissolved in the tetrahydrofuran solution of 30mL drying, nitrogen protection, is placed in-78 DEG C of cooling baths.Inject the n-Butyl Lithium hexane solution that 2mL concentration is 2.5M, and keep this thermotonus 2 hours.Add excess dry ice, and continue room temperature reaction and spend the night.Underpressure distillation is except desolventizing, and column chromatography is purified to obtain target carboxyl substituted tetraphenyl ethylene molecule.
1HNMR(CDCl
3,300MHz)δ:12.67(s,1H,-COOH),7.70(d,2H,Ph),7.14-6.97(m,17H,Ph).HRMScalculatedforC
27H
19O
2([M-H]
-):375.1391,observed375.1381.
2) synthesis of polypeptide A G73;
Polypeptide A G73 synthesizes according to solid phase polypeptide synthesis.Jin Sirui bio tech ltd synthesizes.
3) fluorescent mark of polypeptide A G73.
The fluorescent mark of polypeptide A G73 forms amido linkage by the amino of nitrogen end on the carboxyl on carboxyl substituted tetraphenyl ethylene molecule and polypeptide amino acid residue and obtains fluorescent probe molecule TPE-1.
HPLC99.7％；MScalculatedfor[M+3H]
+619.03,observed619.35.
Above-mentioned a kind of highly sensitive fluorescent probe TPE-1 is in the application of heparin context of detection.
Above-mentioned highly sensitive fluorescent probe TPE-1, to chondroitin polysulfate detection method of content in heparin sample, comprises the following steps:
1) probe molecule TPE-1 is prepared storing solution 1: get TPE-1, be dissolved in DMSO, compound concentration is the storing solution 1 of 10mM; Preparation pH be 7.4 10mMHEPES buffered soln 100mL stand-by; In cuvette, add 50 μ LDMSO, add 1 μ LTPE-1 storing solution 1, add 950 μ LHEPES damping fluids, obtain probe solution; Heparin sample containing chondroitin polysulfate is added in above-mentioned probe solution, tests its fluorescence intensity, be recorded as Out1;
2) in above-mentioned solution, add heparinase, 37 DEG C are reacted four hours, test fluorescence intensity, and are Out2 by gained records of values; Using the ratio of Out2/Out1 as signal, the content of chondroitin polysulfate in sample can be drawn.
The present invention is to the detection by quantitative principle of heparin: in the solution of TPE-1, add heparin it can be made to send strong fluorescence, and the concentration of fluorescence intensity and heparin presents linear relationship; In this mixing solutions, add heparinase if continue, then gained hyperfluorescenceZeng Yongminggaoyingguang is quenched, this is because heparinase has cut off heparin molecule, the combination of heparin and TPE-1 is broken, and fluorescence disappears.
Cleaning Principle to possibility impurity chondroitin polysulfate in heparin: chondroitin polysulfate can be combined with TPE-1, in conjunction with the fluorescence produced lower than the heparin of comparable sodium, the fluorescence intensity after the heparin sample that therefore there is chondroitin polysulfate adds TPE-1 is weaker than pure heparin sample; Chondroitin polysulfate in heparin can suppress the activity of heparinase, makes heparinase can not prohepar molecule, if therefore there is chondroitin polysulfate in heparin sample, then after heparinase process, fluorescence can not be quenched, i.e. Out
2be greater than pure heparin.To sum up, if there is chondroitin polysulfate in the sample of heparin, then the Out of larger numerical value is obtained
2/ Out
1, with this ratio for signal, the content of chondroitin polysulfate in heparin can be calculated.
Beneficial effect: fluorescent probe TPE-1 of the present invention can carry out quantitative detection to the content of heparin in solution to be measured.Detection behavior has good linear relationship, can carry out detecting under pH scope widely, detect and can not preserve by hyparinoids from animal organs or the interference of common biomolecular ions, highly sensitive, the probe stable performance of detection for a long time and use.In addition, TPE-1 can also be used for the quality test of heparin, measures the content of the impurity chondroitin polysulfate that it may contain.Detecting the chondroitin polysulfate that may exist in heparin is also realized by the enhancing of signal, and the content of detection signal and chondroitin polysulfate has good linear relationship, detection sensitivity high, is therefore of very high actual application value.Polypeptide also comprises the peptide sequence (including the sequence of D-amino acid, L-amino acid, D-amino acid and the amino acid whose mixed type of L-and alpha-non-natural amino acid) of other and heparin specific recognition.The probe of AIE, except TPE, also comprises other AIE probe.In addition, AIE effectively can be connected by modes such as amido linkage, disulfide linkage, click chemistries with polypeptide.
The present invention is directed to the present situation such as inaccurate, the consuming time length of data, testing expense height that current heparin detection method exists, by Development of Novel fluorescent probe technique, heparin is carried out to the detection of highly selective, high sensitivity.This technology is used for heparin detection and has the following advantages: 1) detection signal and heparin concentration present extraordinary linear relationship; 2) detect and can carry out under multiple pH environment, the impact of complicated ingredient in not examined environment and detection system; 3) detect speed easy and simple to handle fast, sample only need mix with fluorescent probe by the method, then tests its fluorescence intensity immediately and can learn corresponding heparin content; 4) both can test the content of heparin in sample, also can identify the quality of commercialization heparin.
Accompanying drawing explanation
The molecular structure of Fig. 1 fluorescent probe TPE-1;
Fig. 2 is the working curve that heparin detects;
Fig. 3 is the working curve that lower concentration heparin detects;
The selectivity that Fig. 4 fluorescent probe TPE-1 detects heparin;
The light stability that Fig. 5 fluorescent probe TPE-1 detects heparin and pH stability;
Fig. 6 fluorescent probe TPE-1 detects the working curve of chondroitin polysulfate;
Fig. 7 unknown concentration heparin causes the fluorescent signal of fluorescent probe TPE-1;
Fig. 8 causes the fluorescent signal of fluorescent probe TPE-1 to draw its concentration data according to unknown concentration heparin;
Fig. 9 fluorescent probe TPE-1 detects the linear relationship of chondroitin polysulfate;
Figure 10 fluorescent probe TPE-1 detects the principle of chondroitin polysulfate;
Figure 11 tetraphenyl ethylene fluorescence molecule
1hNMR collection of illustrative plates;
Figure 12 tetraphenyl ethylene fluorescence molecule high resolution mass spectrum figure;
The HPLC purity of Figure 13 fluorescent probe molecule TPE-1 characterizes;
The mass spectral characteristi of Figure 14 fluorescent probe molecule TPE-1.
Embodiment
Below technical solution of the present invention is described in detail, but protection scope of the present invention is not limited to described embodiment.
The preparation of the highly sensitive fluorescent probe TPE-1 of embodiment 1
1) synthesis of tetraphenyl ethylene molecule;
Be dissolved in by ditan (2.02g, 12mmol) in the tetrahydrofuran (THF) of 50mL drying, displacement nitrogen protection is placed in-78 DEG C of cooling baths.Inject the n-Butyl Lithium hexane solution that 5mL concentration is 2.5M, and keep this thermotonus 1 hour.4-bromine benzophenone (2.35g, 9mmol) is dissolved in the tetrahydrofuran (THF) of 10mL drying, slowly injects above-mentioned solution, and stirred overnight at room temperature.Add aqueous ammonium chloride solution cancellation reaction, dichloromethane extraction, is spin-dried for after organic phase anhydrous sodium sulfate drying.Gained crude product is dissolved in 80mL toluene, adds tosic acid (342mg, 1.8mmol), point water refluxes 5 hours.Question response liquid is cooled to room temperature, adds 10% sodium bicarbonate aqueous solution extraction, is spin-dried for after organic phase anhydrous sodium sulfate drying.Column chromatography is purified to obtain bromo tetraphenyl ethylene, productive rate about 40%.Bromo tetraphenyl ethylene (1.6g, 3.9mmol) is dissolved in the tetrahydrofuran solution of 30mL drying, nitrogen protection, is placed in-78 DEG C of cooling baths.Inject the n-Butyl Lithium hexane solution that 2mL concentration is 2.5M, and keep this thermotonus 2 hours.Add excess dry ice, and continue room temperature reaction and spend the night.Underpressure distillation is except desolventizing, and column chromatography is purified to obtain target carboxyl substituted tetraphenyl ethylene molecule.
1HNMR(CDCl
3,300MHz)δ:12.67(s,1H,-COOH),7.70(d,2H,Ph),7.14-6.97(m,17H,Ph).HRMScalculatedforC
27H
19O
2([M-H]
-):375.1391,observed375.1381.
2) synthesis of polypeptide A G73;
Polypeptide A G73 synthesizes according to solid phase polypeptide synthesis.Jin Sirui bio tech ltd synthesizes.
3) fluorescent mark of polypeptide A G73.
The fluorescent mark of polypeptide A G73 forms amido linkage by the amino of nitrogen end on the carboxyl on carboxyl substituted tetraphenyl ethylene molecule and polypeptide amino acid residue and obtains fluorescent probe molecule TPE-1.
Concrete steps are as follows:
Get a certain amount of chloromethyl polystyrene resin in Solid-phase Polypeptide reactor, add the anhydrous DMF of 5mL (DMF) and soak 2 hours, decompression pumps solvent.By the polypeptide A G73 (0.01mmol) of FmocN-end protection; O-benzotriazole-tetramethyl-urea hexafluorophosphate (0.009mmol) and DMAP (0.25nmol) are dissolved in 4mLN; in dinethylformamide; and add N; N-diisopropylethylamine (0.02mmol), adds solid phase reactor room temperature and sways reaction 10 hours, and decompression pumps solvent; with DMF washing resin repeatedly.Add diacetyl oxide/pyridine/DMF (2/1/3, v/v) mixing solutions 6mL, sway 30 minutes.Use DMF (4mL × 3) successively, methylene dichloride (4mL × 3) and DMF (4mL × 3) washing resin, send the de-process of pyridine/DMF with 20%.The tetraphenyl ethylene fluorescence molecule (0.02mmol) of band carboxyl functional group is dissolved in DMF and adds room temperature in solid phase reactor and sways 4 hours, carry out tracing detection with Kaiser ' s reagent.After having reacted, resin washed with dichloromethane, vacuum is drained.In this Solid-phase Polypeptide reactor of dress, slowly add the mixing solutions 5mL of trifluoroacetic acid/tri isopropyl silane/water (95/2.5/2.5, v/v), and keep reaction 3 hours.Filter, nitrogen blow-off's major part solvent, in raffinate, pour 20mL anhydrous diethyl ether into, occur white flock precipitate, 5 DEG C centrifugal 10 minutes, removes upper strata solvent, adds anhydrous diethyl ether 20mL, after vibration centrifugal 10 minutes again, remove most of impurity in precipitation.Dissolve with ionized water after precipitation vacuum-drying, lyophilize obtains white crude polypeptide, is dissolved in 20% acetonitrile/water solution (containing 1% trifluoroacetic acid) and carries out high performance liquid chromatography separation, obtain fluorescent probe molecule TPE-19.7mg.
HPLC99.7％；MScalculatedfor[M+3H]
+619.03,observed619.35.
Probe TPE-1 utilizes solid phase polypeptide synthesis to prepare.Amino acid protective group deprotection adopts the mixed solution stirred overnight at room temperature of trifluoroacetic acid, thioanisole, 3,6-dioxa-1,8-octanedithiol and water to carry out.Product is purified eventually through preparative high-performance liquid chromatographic, and gained productive rate is 33%, and its high purity 99.7% identified by high performance liquid chromatography.Mass spectral characteristi finds that its molecular ion peak is 619.35 [M+3H]
+, mate very much with calculated value 619.03, demonstrate the accuracy of product.
The application of the highly sensitive fluorescent probe TPE-1 of embodiment 2
Probe molecule TPE-1 is prepared storing solution 1.Get TPE-1 (1.85mg, 1 μm of ol), be dissolved in 100 μ LDMSO, storing solution 1 concentration obtaining TPE-1 is 10mM.Preparation pH be 7.4 10mMHEPES buffered soln 100mL stand-by.In cuvette, add 50 μ LDMSO, add 1 μ LTPE-1 storing solution 1, add 950 μ LHEPES damping fluids, obtain probe solution.By heparin sample obtain solution to be tested, get a certain amount of adding in above-mentioned probe solution, test its fluorescence intensity, the concentration of heparin in sample can be drawn.
First we establish the working curve that heparin detects according to the method described above.As seen from Figure 2, the fluorescence intensity of probe TPE-1 presents extraordinary linear relationship with the concentration adding heparin, and linearly dependent coefficient reaches 0.9995, and linearity range is 0.32-5.5 μ g/mL.The now detection of heparin is limited to 35ng/mL.
Use fluorescent probe TPE-1 can detect minimum concentration and reach 3.8ng/mL, working method is also very easy.TPE-1 storing solution 1 is diluted 10 times, and after must diluting, DMSO storing solution 2 concentration of TPE-1 is 1mM.In cuvette, add 50 μ LDMSO, add 1 μ LTPE-1 storing solution 2, add 950 μ LHEPES damping fluids, obtain probe solution.Now gained probe solution can detect the heparin of lower concentration, and has good linear relationship (Fig. 3).
The selective enumeration method to heparin of the highly sensitive fluorescent probe TPE-1 of embodiment 3
Fluorescent probe TPE-1 shows good selectivity to heparin, can avoid the interference of other common biomolecules or negatively charged ion in detection system.In the mixing solutions of 95%HEPES and 5%DMSO, fluorescent probe TPE-1 does not almost have fluorescence.When the test sample added is heparin (5.8 μ g/mL) time, solution just can send strong blue-green fluorescent, and its fluorescence intensity enhances about 31 times.If test sample be hyparinoids from animal organs as chondroitin sulfate, hyaluronic acid time, fluorescence only has very faint enhancing.Fluorescent probe TPE-1 and the common biomolecules of testing are as DNA, RNA, ATP, and glucose, protein (BSA) or negatively charged ion are as also very faint in the effect of phosphate radical etc., obviously can not strengthen the fluorescence (Fig. 4) of fluorescent probe.
The reliable stability of embodiment 4 highly sensitive fluorescent probe TPE-1
Probe TPE-1 molecule performance is very stable.The solution of TPE-1 is at room temperature placed and within two months, is not occurred significantly to decompose.In addition, all show good light stability after TPE-1 molecule or itself and Heparin-binding, the continuous ultraviolet lighting of 2 hours does not cause the change of fluorescence intensity, and (Fig. 5 a).These datas illustrate that TPE-1 stablizes, and preservation that can be long-term and use, meet the needs of commercial applications.In addition, TPE-1 normally can work under pH environment widely.The pH span of control of test system utilized TPE-1 to carry out heparin between 3-10 and detect discovery, within the scope of the pH of 3-10, the heparin adding comparable sodium obtains the fluorescent signal of same intensity (Fig. 5 b).Illustrate that TPE-1 can carry out completely to the detection of heparin in the scope of pH3-10.
The highly sensitive fluorescent probe TPE-1 of embodiment 5 detects chondroitin polysulfate content in heparin sample the quality test-TPE-1 of heparin sample
Probe TPE-1, except may be used for the detection of heparin concentration in sample, can also be used for very carrying out quality inspection containing other impurity (chondroitin polysulfate) in heparin sample.Chondroitin polysulfate is a kind of height sulfation mucopolysaccharide of synthetic, is once pretended to be heparin to sell by people for adding in heparin sample.This impure heparin medicine caused serious side reaction and death in 2008 in Clinical practice.Therefore, detect the chondroitin polysulfate that may exist in heparin and there is great practical value.
Use TPE-1 to detect chondroitin polysulfate content in heparin sample, carry out in two steps:
The first step:
Probe molecule TPE-1 is prepared storing solution 1.Get TPE-1 (1.85mg, 1 μm of ol), be dissolved in 100 μ LDMSO, storing solution 1 concentration obtaining TPE-1 is 10mM.Preparation pH be 7.4 10mMHEPES buffered soln 100mL stand-by.In cuvette, add 50 μ LDMSO, add 1 μ LTPE-1 storing solution 1, add 950 μ LHEPES damping fluids, obtain probe solution (10 μMs).Heparin sample containing chondroitin polysulfate is added in above-mentioned probe solution, tests its fluorescence intensity, be recorded as Out
1.
Second step:
In above-mentioned solution, add heparinase, 37 DEG C are reacted four hours, test fluorescence intensity, and are Out by gained records of values
2.By Out
2/ Out
1ratio as signal, the content of chondroitin polysulfate in sample can be drawn.
As shown in Figure 6, when the content of chondroitin polysulfate in heparin sample increases, Out
2/ Out
1ratio increase thereupon, and present extraordinary linear relationship when the content of chondroitin polysulfate is between 1%-70%, can Out be passed through
2/ Out
1ratio determination sample in the percentage composition of chondroitin polysulfate.The detectability of the method to chondroitin polysulfate content reaches 0.001%.
The highly sensitive fluorescent probe TPE-1 of embodiment 6 detects heparin concentration
Probe molecule TPE-1 is prepared storing solution 1.Get TPE-1 (1.85mg, 1 μm of ol), be dissolved in 100 μ LDMSO, storing solution 1 concentration obtaining TPE-1 is 10mM.Preparation pH be 7.4 10mMHEPES buffered soln 100mL stand-by.In cuvette, add 50 μ LDMSO, add 1 μ LTPE-1 storing solution 1, add 950 μ LHEPES damping fluids, obtain probe solution (10 μMs).
Take heparin 3.3mg, be dissolved in 10mL ultrapure water, obtaining heparin Stock concentrations is 0.33mg/mL.Get 10 these heparin solutions of μ L, add in above-mentioned 1mL probe solution, test its fluorescence intensity, obtain curve, see Fig. 7, this curve maximum emission wavelength place fluorescence intensity is 2395, contrasts the working curve of the fluorescent probe TPE-1 that we set up, heparin concentration corresponding to 2395 fluorescence intensities is about 2.9 μ g/mL, coincide with the actual concentration that adds.
The highly sensitive fluorescent probe TPE-1 of embodiment 7 is to chondroitin polysulfate content detection in heparin sample
Probe molecule TPE-1 is prepared storing solution 1.Get TPE-1 (1.85mg, 1 μm of ol), be dissolved in 100 μ LDMSO, storing solution 1 concentration obtaining TPE-1 is 10mM.Preparation pH be 7.4 10mMHEPES buffered soln 100mL stand-by.In cuvette, add 50 μ LDMSO, add 1 μ LTPE-1 storing solution 1, add 950 μ LHEPES damping fluids, obtain probe solution (10 μMs).
Take chondroitin polysulfate 3.3mg, be dissolved in 10mL ultrapure water, obtaining chondroitin polysulfate concentration is 0.33mg/mL, and dilutes 10 times, and obtaining chondroitin polysulfate Stock concentrations is 0.033mg/mL.Take heparin 3.3mg, be dissolved in 10mL ultrapure water, obtaining heparin Stock concentrations is 0.33mg/mL.Mixed by heparin storing solution 99 μ L and chondroitin polysulfate storing solution 10 μ L, then in mixture, the content of chondroitin polysulfate is 1%.Get this mixed solution 20 μ L, add in probe solution (10 μMs), test its fluorescence spectrum, obtaining fluorescence intensity is Out
1.
Preparation heparinase storing solution: first prepare Heparinase I, the PBS Stock concentrations of II and III is respectively 250U/mL, 50U/mL and 25U/mL, mixes and dilute 10 times with volume ratio 1:5:10, obtains heparinase storing solution (~ 4.7U/mL).Get this heparinase solution 2 μ L, add in above-mentioned probe solution, and keep 37 DEG C to react 4h, test its fluorescence spectrum, obtaining fluorescence intensity is Out
2.Out can be tried to achieve thus
2/ Out
1=0.67, chondroitin polysulfate content corresponding on working curve is 1%.
Claims (6)
1. a highly sensitive fluorescent probe TPE-1, is characterized in that, this fluorescent probe TPE-1 includes the polypeptide A G73 of recognition reaction and the AIE fluorophore being marked at one end of polypeptide A G73 as fluorescent signal group with heparin.
2. the highly sensitive fluorescent probe TPE-1 of one according to claim 1, is characterized in that, the sequence of described polypeptide A G73 is RKRLQVQLSIRT.
3. the highly sensitive fluorescent probe TPE-1 of one according to claim 1, is characterized in that, described AIE fluorophore is tetraphenyl ethylene molecule.
4. the preparation method of a kind of highly sensitive fluorescent probe TPE-1 according to claim 1, is characterized in that, comprise the following steps:
1) synthesis of tetraphenyl ethylene molecule;
2) synthesis of polypeptide A G73;
3) fluorescent mark of polypeptide A G73: the fluorescent mark of polypeptide A G73 forms amido linkage by the amino of nitrogen end on the carboxyl on carboxyl substituted tetraphenyl ethylene molecule and polypeptide amino acid residue and obtains fluorescent probe molecule TPE-1.
5. a kind of highly sensitive fluorescent probe TPE-1 according to claim 1 is in the application of heparin context of detection.
6. highly sensitive fluorescent probe TPE-1 according to claim 1 is to chondroitin polysulfate detection method of content in heparin sample, it is characterized in that, comprises the following steps:
1) probe molecule TPE-1 is prepared storing solution 1: get TPE-1, be dissolved in DMSO, compound concentration is the storing solution 1 of 10mM; Preparation pH be 7.4 10mMHEPES buffered soln 100mL stand-by; In cuvette, add 50 μ LDMSO, add 1 μ LTPE-1 storing solution 1, add 950 μ LHEPES damping fluids, obtain probe solution; Heparin sample containing chondroitin polysulfate is added in above-mentioned probe solution, tests its fluorescence intensity, be recorded as Out1;
2) in above-mentioned solution, add heparinase, 37 DEG C are reacted four hours, test fluorescence intensity, and are Out2 by gained records of values; Using the ratio of Out2/Out1 as signal, the content of chondroitin polysulfate in sample can be drawn.
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| CN108195808A (en) * | 2017-12-26 | 2018-06-22 | 中国石油大学(华东) | A kind of method for detecting oversulfated chondroitin sulfate in sodium heparin class impurity |
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| CN110963911A (en) * | 2019-11-19 | 2020-04-07 | 重庆师范大学 | AIE fluorescent probe for heparin detection and pH response, synthetic method and application |
| CN110963911B (en) * | 2019-11-19 | 2022-03-22 | 重庆师范大学 | AIE fluorescent probe for heparin detection and pH response, synthetic method and application |
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