Summary of the invention
The objective of the invention is in order to solve existing detection enzyme activity method many is the negative signals that provide fluorescent weakening; And the sensitivity that causes detecting is low, poor stability, complex steps and the high problem of cost, and a kind of fluorescent small molecule probe that is used to detect enzymic activity is provided.
To achieve these goals, technical scheme of the present invention is following:
The present invention provides a kind of small molecules probe that is used to detect enzymic activity, and this small molecules probe is 3,4; 9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes (3,4; 9,10-tetra-(4-trimethylammoniobutyloxy-carbonyl)-perylene), its structural formula is:
The present invention provides a kind of preparation method who is used to detect the small molecules probe of enzymic activity, comprises the steps:
Step 1: the perylene acid anhydride is dissolved in the alkaline solution, filters, filtrating is regulated pH value to 8~9, adds four n-octyl bromination amine and 1; The 4-dibromobutane at 100 ℃~120 ℃ back flow reaction 1~3h, through extraction, distillation and purifying, obtains compound 3; 4,9,10-four-(4-brombutyl-ester group)-perylenes; The perylene acid anhydride of being stated, four n-octyl bromination amine and 1, the mol ratio of 4-dibromobutane are 1: 0.1: 10;
Step 2: with the compound that obtains 3,4,9 of step 1,10-four-(4-brombutyl-ester group)-perylenes are dissolved in the mixed solution of THF and water; In mixing solutions, add Trimethylamine 99,, after distillation, extraction and drying, obtain 3,4 at 60 ℃~70 ℃ 48~96h that reflux; 9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes, described 3; 4,9, the mol ratio of 10-four-(4-brombutyl-ester group)-perylenes and Trimethylamine 99 is 1: 20.
The present invention also provides a kind of application that is used to detect the small molecules probe of enzymic activity; This small molecules probe can be used for detecting the activity of enzyme; Its detection method is: small molecules probe and substrate molecule are configured to mixing solutions; Add the enzyme of different concns in the remix solution, the activity of enzyme is carried out fluoroscopic examination.
Preferably, described substrate molecule is ATP, polypeptide or nucleic acid molecule.
Preferably, described enzyme is Phosphoric acid esterase, proteolytic enzyme or nucleicacidase.
Preferably, described detection reaction system is: the total system of 400 μ L, and concentration is 8.2 Tris-HAc buffered soln for 5mM pH value, and concentration is 1-5 μ M small molecules probe, and concentration is 0.2-200 μ M substrate molecule, the enzyme of 0-5U/mL, surplus is a sterilized water.
Preferably, described detected temperatures is 25 ℃.
Preferably, described fluoroscopic examination condition is with the 442nm fluorescence excitation, the 450-650nm fluoroscopic examination.
The present invention also provides a kind of application that is used to detect the small molecules probe of enzymic activity; This small molecules probe can be used for detecting the activity of enzyme inhibitors; Its detection method is: small molecules probe and substrate molecule are configured to mixing solutions; Add the enzyme inhibitors of different concns in the remix solution, add enzyme again, the activity of enzyme inhibitors is carried out fluoroscopic examination.
Preferably, described enzyme inhibitors is Na
3VO
4, to Methyl benzenesulfonyl fluorine or trisodium phosphate.
Inventive principle
The present invention provides a kind of fluorescent small molecule probe that is used to detect enzymic activity, in the aqueous solution, exists with free monomeric form, can send tangible fluorescence; After adding substrate molecule in the solution; Substrate molecule has several negative charges because probe molecule has four positive charges, causes gathering of probe molecule because of the intensive electrostatic interaction between the two, thereby makes the fluorescent quenching of probe; The cancellation degree is deepened along with the increase of the amount of substrate molecule, reaches balance at last; After in system, adding enzyme, the electronegative group of substrate molecule is hydrolyzed and loses electric charge, and probe molecule becomes monomer freely again by gathering attitude; Thereby fluorescence recovers; The enzyme concn that adds is high more, and the resume speed of fluorescence is fast more, and it is also short more to reach the required time of balance; In above-mentioned system, add enzyme inhibitors, speed and degree that fluorescence recovers decrease the activity inhibited of enzyme.The concentration of suppressor factor is big more, and the inhibition degree of enzymic activity is also dark more.Therefore can be used to detect in real time enzymic activity and inhibitor activity thereof.
Beneficial effect of the present invention
The present invention provides a kind of fluorescent small molecule probe that is used to detect enzymic activity, and this small molecules probe is 3,4,9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes, this probe has four positive charges, and fabulous water-soluble (> 50mM is arranged).
The present invention also provides a kind of preparation method who is used to detect the fluorescent small molecule probe of enzymic activity; This method Shi Yi perylene acid anhydride is that raw material carries out alkylated reaction; In the terminal ionize of alkyl chain, its lotus that becomes positively charged is obtained then, the probe sample that makes is a red powder; Tangible green fluorescence is arranged in the aqueous solution, and compound method is simple.
The present invention also provides a kind of application that is used to detect the small molecules probe of enzymic activity; This small molecules probe can be used for detecting the activity of enzyme; Small molecules probe and substrate molecule are configured to mixing solutions, and then add the enzyme of different concns in the mixing solutions, the activity of enzyme is carried out fluoroscopic examination;
This small molecules probe also can be used for detecting the activity of enzyme inhibitors, and small molecules probe and substrate molecule are configured to mixing solutions, and then adds the enzyme inhibitors of different concns in the mixing solutions, adds enzyme again, and the activity of enzyme inhibitors is carried out fluoroscopic examination.The fluorescent signal that provides cancellation with prior art compares; The fluorescence enhanced signal that detection method of the present invention utilizes the controlled self-assembly of small molecules probe to bring; Can realize that, sensitive quick to enzymic activity and inhibitor activity detects; This detection method cost is low, and can directly in the big system of fluorescence ware, detect in real time, and is more stable.
Embodiment
The present invention provides a kind of small molecules probe that is used to detect enzymic activity, and this small molecules probe is 3,4,9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes, and its structural formula is:
This probe has four positive charges, and fabulous water-soluble (> 50mM is arranged).Tangible green fluorescence is arranged in the aqueous solution.
The present invention provides a kind of preparation method who is used to detect the small molecules probe of enzymic activity, comprises the steps:
Step 1: the perylene acid anhydride is dissolved in the alkaline solution, filters, filtrating is regulated pH value to 8~9, adds four n-octyl bromination amine and 1; The 4-dibromobutane at 100 ℃~120 ℃ back flow reaction 1~3h, through extraction, distillation and purifying, obtains compound 3; 4,9,10-four-(4-brombutyl-ester group)-perylenes; The perylene acid anhydride of being stated, four n-octyl bromination amine and 1, the mol ratio of 4-dibromobutane are 1: 0.1: 10;
Step 2: with the compound that obtains 3,4,9 of step 1,10-four-(4-brombutyl-ester group)-perylenes are dissolved in the mixed solution of THF and water; In mixing solutions, add Trimethylamine 99,, after distillation, extraction and drying, obtain 3,4 at 60 ℃~70 ℃ 48~96h that reflux; 9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes, described 3; 4,9, the mol ratio of 10-four-(4-brombutyl-ester group)-perylenes and Trimethylamine 99 is 1: 20.
Alkaline solution in the described step 1 is preferably the 5%KOH aqueous solution, for the not restriction of amount of alkaline solution, can get final product by Rong Xie perylene acid anhydride; The amount of substance of You Xuan De perylene acid anhydride and the volume ratio of alkaline solution are 1: 15, and the available Hydrogen chloride of filtrating is regulated pH value, and preferred temperature is 100 ℃; Extraction in the described step 1 is for to extract with chloroform, and the organic phase of telling is washed 3 times with the 15%NaCl aqueous solution, and distillation removes and desolvates; The crude product that obtains is crossed the chromatography column purifying, obtains compound 3,4; 9,10-four-(4-brombutyl-ester group)-perylenes.
In the described step 2 in the mixed solution of THF and water THF and water be to be 5: 1 blended by volume, for the not restriction of amount of mixed solution, can dissolve 3,4; 9,10-four-(4-brombutyl-ester group)-perylenes get final product, and are preferably 3; 4,9, the volume ratio that 10-four-(4-brombutyl-ester group)-perylenes are dissolved in THF amount of substance and mixed solution is 1: 15; Behind 60 ℃~70 ℃ backflow 48-96h, preferred temperature is 60 ℃, and distillation removes and desolvates; The crude product that obtains is soluble in water, and with chloroform extraction 3 times, the water distillation is also obtaining product 3 behind vacuum-drying 10h~20h under 50 ℃~100 ℃ conditions; 4,9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes.Synthetic route is following:
The present invention also provides a kind of application that is used to detect the small molecules probe of enzymic activity; This small molecules probe can be used for detecting the activity of enzyme; Its detection method is: small molecules probe and substrate molecule are configured to mixing solutions; And then add the enzyme of different concns in the mixing solutions, the activity of enzyme is carried out fluoroscopic examination.
Above-mentioned detection method is configured to small molecules probe and substrate molecule before the mixing solutions again; To the substrate molecule of different concns be joined respectively in the solution of small molecules probe; Carry out fluoroscopic examination behind the temperature-stable, along with the increase of the amount of substrate molecule, fluorescence intensity reduces gradually; Purpose is in order to detect fluorescence intensity when minimum, the concentration of substrate molecule.Described temperature is preferably 25 ℃, and the condition of fluoroscopic examination is the 442nm fluorescence excitation, the 450-650nm fluoroscopic examination.
Substrate molecule of the present invention should be the biomolecules with the probe oppositely charged, for enzyme to be detected selectivity is preferably arranged simultaneously, and after the enzyme effect, substrate molecule loses electric charge or electric density changes.Substrate molecule of the present invention is cheap, and preferably, described substrate molecule is ATP, polypeptide or nucleic acid molecule; ATP is as the substrate of SEAP; ATP is the intravital biological micromolecule of a kind of people of being present in, and an ATP molecule is formed by an adenosine molecule and three phosphate group condensations, has four negative charges; SEAP can generate uncharged adenosine at last with the phosphate group stepwise hydrolysis of ATP; Polypeptide is as the substrate of proteolytic enzyme, and nucleic acid molecule such as DNA or RNA can be used as the substrate of nucleicacidase.
Enzyme of the present invention is that lytic enzyme and substrate molecule are corresponding, can be Phosphoric acid esterase, proteolytic enzyme or nucleicacidase, is preferably SEAP, Proteinase K, trypsinase or nucleicacidase.
Preferably, described detection reaction system is: the total system of 400 μ L, and concentration is 8.2 Tris-HAc buffered soln for 5mM pH value, and concentration is 1-5 μ M small molecules probe, and concentration is 0.2-200 μ M substrate molecule, the enzyme of 0-5U/mL, surplus is a sterilized water.
More preferably; Described detection reaction system is: the total system of 400 μ L; 4 μ L concentration are 8.2 Tris-HAc buffered soln for 500mM pH value, and 20 μ L concentration are the small molecules probe solution of 20-100 μ M, and 80 μ L concentration are the substrate molecule solution of 1-1000 μ M; 1 μ L concentration is respectively the enzyme solution of 0-2000U/mL, and surplus is used the sterilized water polishing.
Preferably, described detected temperatures is 25 ℃.
Preferably, described fluoroscopic examination condition is with the 442nm fluorescence excitation, the 450-650nm fluoroscopic examination.
The present invention also provides a kind of application that is used to detect the small molecules probe of enzymic activity; This small molecules probe can be used for detecting the activity of enzyme inhibitors; Its detection method is: small molecules probe and substrate molecule are configured to mixing solutions; After adding the enzyme inhibitors of different concns in the remix solution, add enzyme again, the activity of enzyme inhibitors is carried out fluoroscopic examination.
Above-mentioned detection method is configured to small molecules probe and substrate molecule before the mixing solutions again; To the substrate molecule of different concns be joined respectively in the solution of small molecules probe; Carry out fluoroscopic examination behind the temperature-stable, along with the increase of the amount of substrate molecule, fluorescence intensity reduces gradually; Purpose is in order to detect fluorescence intensity when minimum, the concentration of substrate molecule.Described temperature is preferably 25 ℃, and the condition of fluoroscopic examination is the 488nm fluorescence excitation, the 450-650nm fluoroscopic examination.
In the above-mentioned detection method; For the selection of the selection of substrate molecule and enzyme with aforementioned consistent; Substrate molecule is preferably ATP, polypeptide or nucleic acid molecule, and enzyme is Phosphoric acid esterase, proteolytic enzyme or nucleicacidase, is preferably SEAP, Proteinase K, trypsinase or s1 nuclease.
Preferably; Described detection reaction system is: the total system of 400 μ L, and concentration is 8.2 Tris-HAc buffered soln for 5mM pH value, concentration is 1-5 μ M small molecules probe; Concentration is 0.2-200 μ M substrate molecule; Concentration is the enzyme inhibitors of 0-100 μ M, the enzyme of 0-5U/mL, and surplus is a sterilized water.
More preferably; Described detection reaction system is: the total system of 400 μ L; 4 μ L concentration are 8.2 Tris-HAc buffered soln for 500mM pH value, and 20 μ L concentration are that the small molecules probe solution 80 μ L concentration of 20-100 μ M are the substrate molecule solution of 1-1000 μ M, and 20 μ L concentration are the enzyme inhibitors solution of 0-2000 μ M; 1 μ L concentration is respectively the enzyme solution of 0-2000U/mL, and surplus is used the sterilized water polishing.
Preferably, described detected temperatures is 25 ℃.
Preferably, described fluoroscopic examination condition is with the 442nm fluorescence excitation, the 450-650nm fluoroscopic examination.
The present invention can select according to the kind of enzyme for enzyme inhibitors, does not have particular restriction, preferably Na
3VO
4, to Methyl benzenesulfonyl fluorine or trisodium phosphate.Na
3VO
4Can be used as the suppressor factor of SEAP, the suppressor factor of proteolytic enzyme is to the Methyl benzenesulfonyl fluorine, and the suppressor factor of nucleicacidase is a trisodium phosphate.
Below in conjunction with embodiment the present invention is done further detailed description.
Embodiment 13,4,9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes synthetic
1,2mmol perylene acid anhydride (PTCDA) is dissolved in the 30mL5%KOH aqueous solution, filters, filtrating is regulated pH value to 8~9 with Hydrogen chloride, 1 of the four n-octyl bromination amine of adding 0.02mmol and 20mmol; The 4-dibromobutane, 100 ℃ of back flow reaction 2 hours add chloroform extraction, and the organic phase of telling is washed 3 times with the 15%NaCl aqueous solution; Distillation removes and desolvates, and the crude product that obtains is crossed the chromatography column purifying, obtains pure compound compound 3; 4,9,10-four-(4-brombutyl-ester group)-perylenes;
2, with 2mmol compound 3,4,9,10-four-(4-brombutyl-ester group)-perylenes are dissolved in 30mL THF and the water mixed solution of (5:1 mixes by volume); The Trimethylamine 99 that adds 40mmol, 66 ℃ of backflow 72h, distillation removes and desolvates; The crude product that obtains is soluble in water, with chloroform extraction 3 times, obtain pure compound 3 after water distillation and the vacuum-drying; 4,9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes.
The nucleus magnetic hydrogen spectrum of probe and carbon spectrum spectrogram are as depicted in figs. 1 and 2:
1H-NMR (600MHz, D
2O): δ (ppm)=7.62-7.69 (m, 8H), 4.39-4.43 (t, 8H), 3.38-3.42 (t, 8H), 3.12 (s, 36H), 1.87-1.93 (m, 8H), 1.93-1.99 (m, 8H)
13C-NMR (150MHz, D
2O): δ (ppm)=169.1,130.9,129.7,127.7,126.6,126.5,120.7,65.6,65.0,52.6,24.5,19.1. can know that from these two spectrograms institute's synthetic probe structure is correct, and has higher purity.
Embodiment 23, and 4,9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes synthetic
1,4mmol perylene acid anhydride (PTCDA) is dissolved in the 60mL5%KOH aqueous solution, filters, filtrating is regulated pH value to 8~9 with Hydrogen chloride, 1 of the four n-octyl bromination amine of adding 0.04mmol and 40mmol; The 4-dibromobutane, 100 ℃ of back flow reaction 2 hours add chloroform extraction, and the organic phase of telling is washed 3 times with the 15%NaCl aqueous solution; Distillation removes and desolvates, and the crude product that obtains is crossed the chromatography column purifying, obtains pure compound compound 3; 4,9,10-four-(4-brombutyl-ester group)-perylenes;
2, with 4mmol compound 3,4,9,10-four-(4-brombutyl-ester group)-perylenes are dissolved in 60mL THF and the water mixed solution of (mixing in 5: 1 by volume); The Trimethylamine 99 that adds 80mmol, 66 ℃ of backflow 72h, distillation removes and desolvates; The crude product that obtains is soluble in water, with chloroform extraction 3 times, obtain pure compound 3 after water distillation and the vacuum-drying; 4,9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes.
The detection of embodiment 3 Phosphoric acid esterases
To Fig. 8 present embodiment is done detailed description in conjunction with Fig. 3
1, be that the ATP of 0 μ M, 5 μ M, 10 μ M, 20 μ M, 30 μ M, 40 μ M, 60 μ M, 100 μ M, 140 μ M and 200 μ M joins the small molecules probe 3,4,9 that 5 μ M embodiment 1 obtain respectively with concentration; In 10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylene solution, temperature-stable is after 25 ℃, with the 442nm fluorescence excitation; The fluorescence spectrum of scanning 450-650nm, Fig. 3 is the change curve of fluorescence intensity along with ATP concentration, as can be seen from the figure; Fluorescence intensity reduces along with the increase of ATP concentration gradually, when ATP concentration is 200 μ M, reduces to minimumly, and Fig. 4 is for adding the fluorescence photo before and after the ATP; Wherein scheming a is the fluorescence photo of the small molecules probe when not adding ATP; When figure b was 200 μ M for ATP concentration, the fluorescence photo of small molecules probe can be found out from figure a and figure b; After adding ATP, small molecules probe generation quencher.
2, the small molecules probe 3,4,9 that 5 μ M embodiment 1 is obtained, 10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes and 200 μ M ATP are configured to mixing solutions; Equilibrium temperature is at 25 ℃, and adding concentration in the remix solution respectively is 0U/mL, 0.1U/mL, 0.2U/mL; 0.5U/mL, 1.0U/mL, the SEAP of 2.5U/mL and 5.0U/mL is the fluorescent emission wavelength with 442nm; The 450-650nm fluoroscopic examination, the time is 30 minutes, detects to be limited to 0.02U/mL.
Described detection reaction system is: the system TV is 400 μ L; 4 μ L concentration are 8.2 Tris-HAc buffered soln for 500mM pH value; 20 μ L concentration are the small molecules probe solution of 100 μ M; 80 μ L concentration are the ATP solution of 1mM, and 1 μ L concentration is respectively the alkaline phosphatase enzyme solution of 0U/mL, 40U/mL, 80U/mL, 200U/mL, 400U/mL, 1000U/mL and 2000U/mL, and surplus is used the sterilized water polishing.
Fig. 5 is the fluorescence kinetics curve under the different alkaline phosphatase concentrations, and as can be seen from the figure, the amount of the enzyme of adding is many more, and fluorescence intensity is big more, and it is fast more that fluorescence recovers.Fig. 6 shows is the enzyme concn curve that fluorescence intensity changed along with the time when being 2.5U/mL, and as can be seen from the figure, along with the time is long more, fluorescence intensity is big more.Fig. 7 is for adding the fluorescence photo before and after the enzyme, and the fluorescence photo of figure a when not adding enzyme, figure b are the fluorescence photo of enzyme concn when being 2.5U/mL, from figure a with figure b contrast find out, add enzyme after, fluorescence intensity is recovered.
The selectivity experiment
Dispose the mixing solutions of 5 μ M small molecules probes and 200 μ M ATP; Equilibrium temperature is at 25 ℃; In mixing solutions, add 2.5U/mL SEAP (ALP) respectively (shown in Figure 8 a); Trypsin tyrpsin) 2.5U/mL (c shown in Figure 8), 2.5U/mL esterase (esterase) (b shown in Figure 8) and 2.5U/mL exonuclease I (ExoI) (d shown in Figure 8) place the fluorescence spectrum that scans 450-650nm after 30 minutes.As shown in Figure 8, the sample of other the three kinds of enzymes except that adding SEAP, fluorescence intensity has no significant change, and explains that this detection method has selectivity preferably to enzyme.
The detection of embodiment 4 proteolytic enzyme
1, be that the polyglutamic acid polypeptide of 0 μ M, 0.5 μ M, 1.0 μ M, 1.5 μ M, 2.0 μ M, 2.5 μ M, 3.0 μ M, 3.5 μ M, 4.0 μ M, 4.5 μ M, 5.0 μ M, 5.5 μ M, 6.0 μ M, 6.5 μ M, 7 μ M joins the small molecules probe 3 that 1 μ M embodiment 2 obtains respectively with concentration; 4; 9, in 10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylene solution, temperature-stable is after 25 ℃; With the 442nm fluorescence excitation, the fluorescence spectrum of scanning 450-650nm.Fig. 9 is the change curve of fluorescence intensity along with the polyglutamic acid peptide concentration, and as can be seen from the figure, fluorescence intensity reduces along with the increase of polyglutamic acid peptide concentration gradually, when the polyglutamic acid peptide concentration is 7 μ M, reduces to minimum.
2, the small molecules probe 3,4,9 that 1 μ M embodiment 2 is obtained; 10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes and 7 μ M polyglutamic acid polypeptide are mixed with mixing solutions, and equilibrium temperature is at 25 ℃, and in mixing solutions, adding concentration respectively is 0U/mL; 0.1U/mL, 0.2U/mL, the Proteinase K of 0.5U/mL and 1.0U/mL; With 442nm is fluorescence exciting wavelength, the 450-650nm fluoroscopic examination, and the time is 100 minutes.Detection is limited to 0.1mU/mL.
Described detection reaction system is: the system TV is 400 μ L; 4 μ L concentration are 8.2 Tris-HAc buffered soln for 500mM pH value; 20 μ L concentration are the small molecules probe solution of 20 μ M; 80 μ L concentration are the polyglutamic acid polypeptide solution of 35 μ M, and 1 μ L concentration is respectively the Proteinase K solution of 0U/mL, 40U/mL, 80U/mL, 200U/mL and 400U/mL, and surplus is used the sterilized water polishing.
Figure 10 shows is that Proteinase K concentration is the curve that fluorescence intensity changed along with the time during into 0.1U/mL, as can be seen from the figure, because the peptide bond that Proteinase K can hydrolysis polyglutamic acid polypeptide and it is degraded into the L-glutamic acid monomer; And make the negative charge density reduction of polypeptide originally, and probe molecule is disaggregation thereupon, thus fluorescence recovers; Proteinase K concentration is big more; Time is long more, and fluorescence intensity is big more, and reaches capacity gradually.
The detection of embodiment 5 nucleicacidases
1, be that the nucleic acid molecule of 0 μ M, 5 μ M, 10 μ M, 20 μ M, 30 μ M, 40 μ M, 60 μ M, 100 μ M, 140 μ M and 200 μ M joins the small molecules probe 3 that 1 μ M embodiment 2 obtains respectively with concentration; 4; 9, in 10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylene solution, temperature-stable is after 25 ℃; With the 442nm fluorescence excitation, the fluorescence spectrum of scanning 450-650nm.Experimental result shows: fluorescence intensity reduces along with the increase of nucleic acid molecule concentration gradually, reduces to during for 200nM minimum in nucleic acid molecule concentration.
2, the small molecules probe 3,4,9 that 1 μ M embodiment 2 is obtained; 10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes and 200nM nucleic acid molecule are mixed with mixing solutions, and equilibrium temperature is at 25 ℃, and in mixing solutions, adding concentration is 0U/mL; 1.0U/mL, 2.0U/mL, the s1 nuclease of 4.0U/mL and 10.0U/mL; With 442nm is fluorescence exciting wavelength, the 450-650nm fluoroscopic examination, and the time is 40 minutes.Detection is limited to 0.1U/mL.
Described detection reaction system is: the system TV is 400 μ L, and 4 μ L concentration are 8.2 Tris-HAc buffered soln for 500mM pH value, and 20 μ L concentration are the small molecules probe solution of 20 μ M; 80 μ L concentration are the nucleic acid molecule solution of 1 μ M; 1 μ L concentration is 0U/mL, 400U/mL, 800U/mL; The s1 nuclease solution of 1600U/mL and 4000U/mL, surplus is used the sterilized water polishing.
Experimental result shows: owing to s1 nuclease ability hydrolysed nucleic acid molecule it is degraded into single Nucleotide; And make the negative charge density reduction of nucleic acid chains originally, and probe molecule is disaggregation thereupon, thus fluorescence recovers; S1 nuclease concentration is big more; Time is long more, and fluorescence intensity is big more, and reaches capacity gradually.
Embodiment 6 alkaline phosphatase enzyme inhibitors Na
3VO
4Detection
1 is consistent with the step of implementing in 31;
2, the small molecules probe 3 that 5 μ M embodiment 1 is obtained; 4,9,10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes and 200 μ M ATP are configured to mixing solutions; Equilibrium temperature is at 25 ℃, and adding concentration in the remix solution respectively is the Na of 0 μ M, 25 μ M, 50 μ M, 75 μ M and 100 μ M
3VO
4, add the SEAP of 5.0U/mL again, be the fluorescent emission wavelength with 442nm, the 450-650nm fluoroscopic examination, the time is 30 minutes.
Described detection reaction system is: the system TV is 400 μ L; 4 μ L concentration are 8.2 Tris-HAc buffered soln for 500mM pH value; 20 μ L concentration are the small molecules probe solution of 100 μ M; 80 μ L concentration are the ATP solution of 1mM, and 20 μ L concentration are respectively the Na of 0 μ M, 500 μ M, 1000 μ M, 1500 μ M and 2000 μ M
3VO
4Solution, 1 μ L concentration is the SEAP of 2000U/mL, surplus is used the sterilized water polishing.
Figure 11 is the fluorescence kinetics curve under the different inhibitor concentration, and as can be seen from the figure, it is slack-off after adding suppressor factor that fluorescence probe recovers, the suppressor factor of adding recover slow more more, and promptly enzymic activity is suppressed more.
The suppressor factor of embodiment 7 proteolytic enzyme is to the detection of Methyl benzenesulfonyl fluorine
1 is consistent with the step of implementing in 41.
2, the small molecules probe 3,4,9 that 1 μ M embodiment 1 is obtained; 10-four-(4-Trimethylamine butyl oxygen-carbonyl)-perylenes and 7 μ M polyglutamic acid polypeptide are mixed with mixing solutions, and equilibrium temperature is at 25 ℃, add concentration in the remix solution respectively and be 0nM, 10nM, 20nM, 50nM, 75nM and 100nM to the Methyl benzenesulfonyl fluorine; The Proteinase K that adds 0.1U/mL again; With 442nm is the fluorescent emission wavelength, the 450-650nm fluoroscopic examination, and the time is 100 minutes.
Described detection reaction system is: the system TV is 400 μ L; 4 μ L concentration are that the 500mMpH value is 8.2 Tris-HAc buffered soln; 20 μ L concentration are the small molecules probe solution of 20 μ M, and 80 μ L concentration are the polyglutamic acid polypeptide solution of 35 μ M, 20 μ L concentration be respectively 0nM, 200nM, 400nM, 500nM, 1000nM, 1500nM and 2000nM to Methyl benzenesulfonyl fluorine solution; 1 μ L concentration is the Proteinase K of 40U/mL, and surplus is used the sterilized water polishing.
Experimental result shows: fluorescence probe recovers slack-off after adding suppressor factor, the suppressor factor of adding recover slow more more, and promptly enzymic activity is suppressed more.