CN103630518A - Novel method for detecting activity of hydrogen sulfide synthetase by using hydrogen sulfide fluorescence probe and application of method - Google Patents
Novel method for detecting activity of hydrogen sulfide synthetase by using hydrogen sulfide fluorescence probe and application of method Download PDFInfo
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Abstract
The invention belongs to the technical field of biological detection and relates to a novel method for detecting the activity of hydrogen sulfide synthetase by using a hydrogen sulfide fluorescence probe as well as an application approach of the method. The method is suitable for detection of the activity of hydrogen sulfide synthetase such as CBS, CSE and CL, and comprises the following steps: shearing a tissue sample into small blocks, grinding, performing cell lysis in a tissue cell lysis solution, performing protein quantification on a lysis product, as the drawings, adding a reaction system (comprising the tissue cell lysis product, L-cysteine and 5-pyridoxal phosphate) into a reaction pool on an outer ring of a reaction bottle, and adding an absorption system (comprising operating fluid of the H2S fluorescence probe containing a reaction adjuvant cetrimonium bromide) into an absorption pool on an inner ring of the reaction bottle, wherein H2S gas molecules generated in the reaction system are combined with the fluorescence probe in the absorption system, the system can be used for fluorescence assay and fluorescence imaging, the H2S content is quantitatively detected, and the activity of the H2S synthetase is intuitively reflected. The invention can provide a good method for research of H2S-related signal paths and significance and diagnosis of H2S in disease processes.
Description
One, technical field
The invention belongs to technical field of biological, be specifically related to new method and application thereof that a kind of fluorescence probe with sulfuretted hydrogen detects sulfuretted hydrogen synthase activity.This fluorescence probe detects enzymatic activity method and can be the mechanism in some important zymetology processes in research organization's cell, illustrates some important signal path in biosystem, for early diagnosis and the prevention of some major disease provides good supplementary means.
Two, background technology
For a long time, sulfuretted hydrogen (hydrogen sulfide, H
2s) be considered to a kind of poisonous gas, yet, along with to H
2going deep into of S research, finds that some histocytes in body can generate H
2s, it is nitrogen monoxide (nitric oxide, NO) and carbon monoxide (carbon monoxide, CO) found the third endogenous gaseous signal molecule afterwards that continues.Increasing experiment shows, H
2s is not only a kind of endogenic gaseous signal molecule, and has various kinds of cell protective effect, participates in multiple pathophysiological process in body.In mammal, take halfcystine or cysteine derivative as substrate catalysis generation H
2the endogenous enzymes of S has relative tissue specificity, cystathionie-γ-lyases (cystathionine-γ-lyase, CGL) mainly in cardiovascular system, express, cystathionie-beta-synthetase (cystathionine-β-synthase, CBS) is mainly expressed in central nervous system, liver and pancreatic tissue.Mercaptopyruvate transsulphurase (3-mercaptopyruvate sulfurtransferase, 3-MST) and cysteine lyase (cysteine lyase, CL) are also responsible for catalyzing endogenous property H
2the generation of S.Along with H
2the vital role of S in living things system is little by little familiar with, and differentiates and detection of biological endogenous H
2the method of S receives publicity day by day, H in detection of biological sample
2the accuracy of S concentration and reliability are extremely important for understanding its function, but due to H
2s is volatilizable gas molecule, is subject in vivo the impact of various complicated factors, causes its detection difficulty high, and testing result is unstable.And sulfuretted hydrogen synthetase activity performance in detection of biological tissue or cell better reflects that this histocyte produces H
2the ability of S, with respect to the H in direct-detection biosome
2s content, the method is more reliable and stable.So, directly perceived, special, the activity of sulfuretted hydrogen synzyme seems particularly important in detection of biological tissue or cell quantitatively.
Traditional detection H
2s method all needs complicated sample preparation as colourimetry, electrochemical methods, vapor-phase chromatography and sulphide precipitation, fluorescence detection method is due to easy, sensitivity is high, favorable repeatability, and can realize visual detection, therefore, novel luciferase assay reagent and detection method become the focus of research.Sulfuretted hydrogen fluorescence probe is highly sensitive, can be used as biological substance research indication mechanism, and can be used for H
2the indirect determination of sulfuretted hydrogen synthase activity in the quantitative detection of S and histocyte, in the fields such as diagnosis of research Microenvironment Properties and disease, be also widely used in addition, fluorescence detection method a kind of directly perceived, special, easy detection means of can yet be regarded as, is expected to replace traditional detection method.
The method of the traditional detection sulfuretted hydrogen synthase activity bottle-methylene blue laws that responds, but multigelation method cracking histocyte step in the method seems particularly loaded down with trivial details, the H in its detection reaction bottle absorption system
2s content is also be not as directly perceived, easy and sensitive as fluorescence imaging method of the present invention.The fluorescence probe adopting in method of the present invention is novel phenylpropyl alcohol two mercaptan compounds, it is the lactone conformation of a sealing, good stability, can long preservation use (20 ℃), without the absorption characteristic of visible region, the faint (fluorescence quantum yield: Φ=0.003), only fluorescence strengthens (fluorescence quantum yield: Φ=0.392) after reaction of probe autofluorescence, during for fluorescence imaging, signal to noise ratio (S/N ratio) is high, and can be used for the quantitative measurement of fluorescence; The detection of this fluorescence probe in phosphate buffer solution is limited to micromole (μ mol/L), 10 micromolar H in blood plasma and cell can be detected
2s concentration, detects effect stability.Fluorescence probe and H
2before and after S reaction, change rapidly, quantum yield is higher, under 37 ℃ of conditions and H
2s reaction 1h can reach high fluorescent, is applicable to H in reaction system
2the Fast Measurement of S; It is easy and simple to handle, and testing conditions is easily realized, and favorable repeatability is visual; And this fluorescence probe selectivity is high, can be specifically and H
2s reaction, is not disturbed as halfcystine, glutathione by other biological mercaptan.Therefore, the method is hopeful to be applied as a kind of novel sulfuretted hydrogen synthase activity detection technique.
Three, summary of the invention
The problem that the present invention need to solve is:
1. provide a kind of based on H
2the fluorescence probe of S, can be used for detecting the new method of sulfuretted hydrogen synthase activity.This fluorescence probe detects enzymatic activity method and can be the mechanism in some important zymetology processes in research organization's cell, illustrates some important signal path in biosystem, for some major disease early diagnosis and prevention good supplementary means is provided.
2. H is provided
2s fluorescence probe detects the application approach of sulfuretted hydrogen synthase activity.
Technical scheme of the present invention is:
1. the fluorescence probe with sulfuretted hydrogen detects the new method of sulfuretted hydrogen synthase activity:
(1) fluorescence probe adopting in method of the present invention is novel phenylpropyl alcohol two mercaptan compounds, it is the lactone conformation of a sealing, absorption characteristic without visible region, faint (the fluorescence quantum yield: Φ=0.003) of probe autofluorescence, only after reaction, fluorescence strengthens (fluorescence quantum yield: Φ=0.392), MW=591.65, its structural formula is:
(2) fluorescence probe and the H that in method of the present invention, adopt
2principle of luminosity and the specificity thereof of S reaction are original:
This fluorescence probe is the lactone conformation of a sealing, without the absorption characteristic of visible region, and probe (Nonfluorescent) and H
2s is reaction in phosphate buffered saline(PBS) (PBS, pH value 7.4), generates fluorophore (Fluorescent) and 3H-1,2-benzene dimercaptopropane-3-ketone.
H
2s is a kind of active nucleopilic reagent, in biosystem, can participate in nucleophilic displacement of fluorine, detects H
2s selectivity, key is to distinguish with other biological nucleopilic reagent, especially biological thiol is such as halfcystine and glutathione.Theoretically, H
2s can think a mercaptan of not replacing, and can carry out twice necleophilic reaction, and other mercaptan is if halfcystine is an alkyl nomercaptan, can only carry out necleophilic reaction one time.H
2the free sulfhydryl groups of S (SH) reacts and generates intermediate product A1 with electrophilic part A in fluorescence probe structure, if another electrophilic body in place, as the ester group in A1, SH group can experience the process of a spontaneous cyclisation, discharges fluorescence and product B.This method not only can stably be caught H
2thereby S generates degradable product B, also can measure quantitatively fluorescence intensity, the easy H that reflects intuitively again
2s content.A also can, with biological thiol as halfcystine etc. reacts, can not spontaneous cyclisation discharge fluorophore but generate product A 2.That therefore, has reported is novel by H
2the phenylpropyl alcohol mercaptan structure of S mediation, it produces fluorescence signal to H
2s has higher selectivity, not disturbed as halfcystine, glutathione by other biological mercaptan.
(3) reaction bulb absorption system of the present invention is formulated as follows:
A. prepare 100mmol/L phosphate buffer (PBS, PH=7.4)
A liquid: 1mol/L K
2hPO
43H
2o (MW=228.22) gets 9.13g+40ml ddH
2o;
B liquid: 1mol/L KH
2pO
4(MW=136.09) get 1.36g+10ml ddH
2o;
Get A liquid 40.1ml+B liquid 9.9ml, after mixing, use ddH
2o is settled to 500ml;
B. preparation detects H
2the fluorescence probe liquid of S: 4mg fluorescence probe (MW=591.65) is dissolved in to 13.5ml acetonitrile, the fluorescence probe mother liquor of compound concentration 50mmol/L,-20 ℃ of long preservation, during use, first get 100mmol/L phosphate buffer (PBS, PH=7.4) 500 μ l, then add acetonitrile 500 μ l, after mixing, get fluorescence probe mother liquor 20 μ l and add wherein mixing with it, the fluorescence probe working fluid of compound concentration 20 μ mmol/L;
C. prepare Cetrimide (CTAB): get 364mg CTAB (MW=364.45) and be dissolved in the Cetrimide mother liquor that is made into 10mmol/L in 100ml ethanol, normal temperature is preserved, during use, press 10mmol/L phosphate buffer (PBS for 1:1000, PH=7.4) dilute, be made into the Cetrimide working fluid of concentration 10 μ mol/L;
D. prepare reaction bulb absorption system: the sulfuretted hydrogen probe working fluid 100 μ l that get concentration 20 μ mmol/L, add on this basis reaction adjuvant Cetrimide mother liquor (CTAB, 10mmol/L) 10 μ l, and be settled to 10ml with phosphate buffer (PBS), be that CTAB final concentration is 10 μ mol/L, get the reaction system 1ml preparing and add reaction bulb absorption cell, matching while using.
(4) reaction bulb reaction system of the present invention is formulated as follows:
A. prepare 100mmol/L phosphate buffer (PBS, PH=7.4)
A liquid: 1mol/L K
2hPO
43H
2o (MW=228.22) gets 9.13g+40ml ddH
2o;
B liquid: 1mol/L KH
2pO
4(MW=136.09) get 1.36g+10ml ddH
2o;
Get A liquid 40.1ml+B liquid 9.9ml, after mixing, use ddH
2o is settled to 500ml;
B. prepare 1mol/L Cys (MW=121.16) and get the phosphate buffer of 0.12g+1ml above-mentioned (1) preparation;
C. prepare 20mol/L (0.5%) PLP (MW=265.1) and get the phosphate buffer of 0.50g+100ml above-mentioned (1) preparation;
(5) detection method experimental procedure of the present invention is as follows:
A. for tissue sample, be first cut into small pieces, then grind, for cell, histiocyte lysate capable (by force) carries out cracking (green skies biotechnology institute, Nantong, China);
B. histocyte pyrolysis product is through protein quantification;
C. histocyte pyrolysis product is added in the reaction tank of reaction bulb outer ring, add the PLP of 2mmol/L and the Cys of 10mmol/L and lysate simultaneously.This is the reaction system of reaction bulb;
D. absorption system is added in the absorption cell of inner ring, adds a kind of sulfuretted hydrogen fluorescence probe (20 μ mol/L) 100 μ l and reaction adjuvant Cetrimide (CTAB, 10 μ mol/L) 100 μ l, and is settled to 10ml with phosphate buffer (PBS).
E. under 37 ℃ of conditions, carry out airtight absorption reaction 60min;
F. fluorometric assay or fluorescence imaging.
2.H
2s fluorescence probe detects the application approach of sulfuretted hydrogen synthase activity
By H of the present invention
2s fluorescence probe is for the histiocytic detection of living things system sulfide hydrogen synzyme, biological tissue or Hemapoiesis H
2the analyzing and testing of the function of S and fluorescence imaging detect.
Compared with prior art, its beneficial effect is the new method that the present invention detects sulfuretted hydrogen synthase activity:
(1) histiocyte lysate capable cell lysis tissue, lysate itself does not affect the catalytic activity of enzyme, and its cracking degree is more thorough than multigelation method;
(2) probe good stability, can long preservation be used (20 ℃);
(3) easy and simple to handle, testing conditions is easily realized, favorable repeatability, visual detection;
(4) probe selectivity is high, can be specifically and H2S reaction, and it produces fluorescence signal has unique selectivity to H2S, not disturbed as halfcystine, glutathione by other biological mercaptan.
(5) probe and H
2before and after S reaction, change rapidly, quantum yield is higher, under 37 ℃ of conditions and H
2s reaction 1h can reach high fluorescent, is applicable to H in reaction system
2the Fast Measurement of S;
(6) probe detects and is limited to micromole (μ mol) in phosphate buffer solution, detects sensitive; But 10 micromolar H in blood plasma and cell can be detected
2s concentration, detects effect stability.
(7) probe autofluorescence faint (Φ=0.003), only after reaction, fluorescence strengthens (Φ=0.392), can not cause lower signal to noise ratio (S/N ratio), and can be used for the quantitative measurement of fluorescence during for fluorescence imaging;
H of the present invention
2s fluorescence probe detects sulfuretted hydrogen synthase activity and can be research H
2the signal path that S is relevant, and H
2the S meaning in diease occurrence process and diagnosis in some diseases or biosome provide good research method.
H of the present invention
2s fluorescence probe detects sulfuretted hydrogen synthase activity method can intuitively reflect that biological a certain histocyte produces H under specific physiology or pathology environment
2the ability of S, testing result can be used for fluorescence imaging, has good practicality in bioengineering industry.
Four, accompanying drawing explanation
Fig. 1: be reaction bulb figure, its volume is 50ml, and reaction system is added in the reaction tank of reaction bulb outer ring, and absorption system is added on the absorption cell of inner ring, and absorption cell volume is 2ml.
Fig. 2: novel absorption system is (containing the H of reaction adjuvant Cetrimide
2s fluorescence probe working fluid) detect the H that NaHS donor discharges
2s content, control group adds the PBS of certain volume to reaction tank, experimental group adds the NaHS donor of the variable concentrations of same volume to reaction tank, two groups all add identical absorption system in absorption cell, 37 ℃, jog 1h, by reaction bulb-fluorescence probe method fluorescence intensity, fluorescence intensity can reflect H in absorption liquid
2the content of S.
Fig. 3: different soak times, H
2s fluorescence probe detects the H that NaHS donor discharges
2s content, control group adds the PBS of certain volume to reaction tank, and experimental group adds the NaHS donor of the variable concentrations of same volume to reaction tank, and two groups all add identical absorption system (containing the H of reaction adjuvant Cetrimide in absorption cell
2s fluorescence probe working fluid), 37 ℃, jog different time, by reaction bulb-fluorescence probe method fluorescence intensity, fluorescence intensity can reflect H in absorption liquid
2the content of S.
Fig. 4: novel absorption system is (containing the H of reaction adjuvant Cetrimide
2s fluorescence probe working fluid) detect the H that NaHS donor discharges
2s content, prepares respectively the NaHS donor of variable concentrations, directly adds identical H
2s fluorescence probe, with the multi-functional readout instrument of full wavelength scanner (Varioskan Flash3001) (maximum excitation wavelength X ex (max)=476nm, maximum emission wavelength λ em (max)=513nm).
Fig. 5: detect H
2the cancellation situation of S fluorescence probe, control group adds the PBS of certain volume to reaction tank, and experimental group adds 600 μ mol/LNa of same volume to reaction tank
2s donor, two groups all add identical absorption system (containing the H of reaction adjuvant Cetrimide in absorption cell
2s fluorescence probe working fluid), 37 ℃, jog 1h by reaction bulb-fluorescence probe method fluorescence intensity, observes its fluorescent quenching situation under fluorescence.
Fig. 6: HaCaT cell through the dextrorotation-glucose (D-Glu) of variable concentrations and osmotic pressure contrast left-handed-glucose (L-Glu) processes after 72h, improve original multigelation method, the histiocyte lysate capable (by force) providing by green skies biotechnology carries out cracking.With reaction bulb-methylene blue development process of improvement, detect the H that high sugar is processed HaCaT cell
2the activity of S synzyme (CBS).
Fig. 7: by 50 μ mol/L S-NH
2s donor (Yz-2-54) adds in the HaCaT cell of cultivation processes 1h, uses H
2in S probe direct-detection nutrient culture media and cell in H
2the release conditions of S.
Five, embodiment:
Below by embodiment, specifically describe the present invention, but the present invention is not subject to the restriction of following embodiment.
Embodiment 1: novel absorption system detects the H that NaHS donor discharges
2s content
There is document to show, HS in NaHS solution
-/ H2S ratio is 3:1, so major part is HS in NaHS solution
-.Control group adds the PBS of certain volume to reaction tank, experimental group adds the variable concentrations NaHS donor of same volume to reaction tank, and two groups all add identical absorption system (containing the H of reaction adjuvant Cetrimide in absorption cell
2s fluorescence probe working fluid), 37 ℃, jog 1h, by reaction bulb-fluorescence probe method fluorescence intensity, fluorescence intensity can reflect H in absorption liquid
2the content of S.Result shows as Fig. 2, and the fluorescence intensity of high concentration group (600 μ mol/L) is greater than low concentration group (200 μ mol/L), and experimental group fluorescence intensity is obviously greater than control group.
Embodiment 2: different soak times, H
2s fluorescence probe detects the H that NaHS donor discharges
2s content
Control group adds the PBS of certain volume to reaction tank, experimental group adds the NaHS donor of the variable concentrations of same volume to reaction tank, and two groups all add identical absorption system (containing the H of reaction adjuvant Cetrimide in absorption cell
2s fluorescence probe working fluid), 37 ℃, jog different time, by reaction bulb-fluorescence probe method fluorescence intensity, fluorescence intensity can reflect H in absorption liquid
2the content of S.Result shows as Fig. 3, and the fluorescence intensity of 600 μ mol/L NaHS groups is greater than 200 μ mol/L NaHS groups, and experimental group fluorescence intensity is obviously greater than control group, in experimental group, and identical NaHS concentration, 1h group fluorescence intensity is slightly larger than 1/2h group,
*p < 0.01 compares with Con-1/2h group,
##p < 0.01 compares with Con-1h group.
Embodiment 3:H
2the H that S fluorescence probe direct-detection NaHS donor discharges
2s content
The NaHS donor of preparing respectively variable concentrations, adds identical H
2s fluorescence probe, with the multi-functional readout instrument of full wavelength scanner (Varioskan Flash3001) (maximum excitation wavelength X ex (max)=476nm, maximum emission wavelength λ em (max)=513nm).Result shows as Fig. 4, along with H
2the rising of S concentration, the corresponding rising of fluorescence intensity, is dose dependent.
*p < 0.01 compares with Blank group,
##p < 0.01 and 0 μ mol/L NaHS group are relatively.
Embodiment 4: detect H
2the cancellation situation of S fluorescence probe
Control group adds the PBS of certain volume to reaction tank, experimental group adds 600 μ mol/L NaHS donors of same volume to reaction tank, and two groups all add identical absorption system (containing the H of reaction adjuvant Cetrimide in absorption cell
2s fluorescence probe working fluid), 37 ℃, jog 1h by reaction bulb-fluorescence probe method fluorescence intensity, observes its fluorescent quenching situation under fluorescence, and result shows as Fig. 5, H
2s fluorescence probe is exposed to the more obvious cancellation of generation after 3min under fluorescence.
Embodiment 5: methylene blue detection method is measured the CBS activity that high sugar is processed HaCaT cell
HaCaT cell through the dextrorotation-glucose (D-Glu) of variable concentrations and osmotic pressure contrast left-handed-glucose (L-Glu) processes after 72h, on original reaction bulb-methylene blue development process, improve, the histiocyte lysate capable (by force) providing by green skies biotechnology carries out cracking.Detect H in born of the same parents
2the activity of S synzyme.Result shows as Fig. 6, and high sugared processing can suppress the activity of CBS, and this effect and osmotic pressure are irrelevant.
*p < 0.05,
*p < 0.01 compares with 5mmol/L D-Glu group.And lysate itself does not affect the catalytic activity of enzyme, cracking degree is more thorough than original multigelation method, and test has good stability.
Embodiment 6:H
2the S-NH that S probe in detecting is novel
2s donor (Yz-2-54) discharges H
2the ability of S
S-NH
2s donor (Yz-2-54) molecular formula is as Fig. 7 (2) demonstration.50 μ mol/L Yz-2-54 are added in the HaCaT cell of cultivation and process 1h, finding all has H in nutrient culture media and in cell
2s generates.Result is as Fig. 7 (1) demonstration, and novel Yz-2-54 can discharge H
2s molecule, and by H
2s fluorescence probe detects.
Claims (6)
1. with the fluorescence probe of sulfuretted hydrogen, detect a new method for sulfuretted hydrogen synthase activity, it is characterized in that:
(1) detection method of the present invention is applicable to detect the activity of CBS, CSE and CL (need add in addition nitrite), experimental procedure is as follows: for tissue sample, first be cut into small pieces, grind again, for cell, histiocyte lysate capable (by force) carries out cracking (green skies biotechnology institute, Nantong, China); Product of cell lysis is after protein quantification, add in reaction bulb, reaction system is added in the reaction tank of outer ring, comprise 2mmol/L PLP 400 μ l, 10mmol/L Cys 40 μ l and histiocyte lysate capables, and be settled to 2ml with 100mmol/L phosphate buffer (PBS, PH=7.4).Absorption system is added in the absorption cell of inner ring, adds 20 μ mol/L sulfuretted hydrogen fluorescence probe working fluid 1ml, wherein containing responding adjuvant Cetrimide (CTAB, 10 μ mol/L).Absorption reaction 60min in the closed environment of 37 ℃, the solution of getting absorption cell can be used for fluorometric assay and fluorescence imaging;
(2) detect the test of sulfuretted hydrogen synthase activity, traditional histocyte cleavage method is: directly add damping fluid, through the cracking of-20 ℃ of room temperature multigelations.Cleavage method of the present invention is: the histiocyte lysate capable (by force) providing by green skies biotechnology carries out cracking, for 55mm Tissue Culture Dish, adds 100 μ l cell pyrolysis liquids.
(3) detect the test of sulfuretted hydrogen synthase activity, traditional reaction bulb absorption system is: get 0.5ml2% zinc acetate (regulating pH value to alkalescence) and add in central absorption cell, to conical flask, be filled with appropriate nitrogen, with plug, seal, conical flask is placed in to 37 ℃ of constant temperature ovens, and jog 120min, with after 50% trichloroacetic acid 0.5ml stopped reaction, continue reaction 60min, thoroughly absorbing hydrogen sulphide.Absorption system of the present invention is: containing the sulfuretted hydrogen fluorescence probe working fluid of adjuvant Cetrimide (CTAB, the 10 μ mol/L) that respond, absorption reaction 60min in the closed environment of 37 ℃;
(4) detect the test of sulfuretted hydrogen synthase activity, traditional detection method is methylene blue detection method: after the zinc acetate in absorption cell is mixed, draw 0.4ml (rifle head notes not contacting conical flask wall), add 7.2mol/L p-phenylenediamine (PPD) 40 μ l, 1.2mol/L ferric trichloride 40 μ l, after room temperature reaction 20min, with microplate reader (Molecular Devices, Sunnyvale, CA, USA) detect the absorbance at 670nm wavelength place.Draw H
2s typical curve can quantitative test.Detection method of the present invention is: get absorption cell solution, with AMG fluorescent microscope (Advanced Microscopy Group, USA) carry out fluorescence imaging, or carry out fluorometric assay (λ ex (max)=476nm with the multi-functional readout instrument of full wavelength scanner (Varioskan Flash3001), λ em (max)=513nm), draw H
2s typical curve can carry out quantitative test.
2. the compound method of reaction bulb absorption system according to claim 1, is characterized in that:
(1) preparation 100mmol/L phosphate buffer (PBS, PH=7.4)
A liquid: 1mol/L K
2hPO
43H
2o (MW=228.22) gets 9.13g+40ml ddH
2o;
B liquid: 1mol/L KH
2pO
4(MW=136.09) get 1.36g+10ml ddH
2o;
Get A liquid 40.1ml+B liquid 9.9ml, after mixing, use ddH
2o is settled to 500ml;
(2) preparation detects H
2the fluorescence probe liquid of S: 4mg fluorescence probe (MW=591.65) is dissolved in to 13.5ml acetonitrile, the fluorescence probe mother liquor of compound concentration 50mmol/L,-20 ℃ of long preservation, during use, first get 100mmol/L phosphate buffer (PBS, PH=7.4) 500 μ l, then add acetonitrile 500 μ l, after mixing, get fluorescence probe mother liquor 20 μ l and add wherein mixing with it, the fluorescence probe working fluid of compound concentration 20 μ mmol/L;
(3) preparation Cetrimide (CTAB): get 364mg CTAB (MW=364.45) and be dissolved in the Cetrimide mother liquor that is made into 10mmol/L in 100ml ethanol, normal temperature is preserved, during use, by 1: 1000 use 10mmol/L phosphate buffer (PBS, PH=7.4) dilute, be made into the Cetrimide working fluid of concentration 10 μ mol/L;
(4) preparation reaction bulb absorption system: the sulfuretted hydrogen probe working fluid 100 μ l that get concentration 20 μ mmol/L, add on this basis reaction adjuvant Cetrimide mother liquor (CTAB, 10mmol/L) 10 μ l, and be settled to 10ml with phosphate buffer (PBS), be that CTAB final concentration is 10 μ mol/L, get the reaction system 1ml preparing and add reaction bulb absorption cell, matching while using.
3. the compound method of reaction bulb reaction system according to claim 1, is characterized in that:
(1) preparation 100mmol/L phosphate buffer (PBS, PH=7.4)
A liquid: 1mol/L K
2hPO
43H
2o (MW=228.22) gets 9.13g+40ml ddH
2o;
B liquid: 1mol/L KH
2pO
4(MW=136.09) get 1.36g+10ml ddH
2o;
Get A liquid 40.1ml+B liquid 9.9ml, after mixing, use ddH
2o is settled to 500ml;
(2) preparation 1mol/L Cys (MW=121.16) is got the phosphate buffer of 0.12g+1ml above-mentioned (1) preparation;
(3) preparation 20mol/L (0.5%) PLP (MW=265.1) is got the phosphate buffer of 0.50g+100ml above-mentioned (1) preparation.
4. according to claim 1 with histiocyte lysate capable application in bioengineering industry in the new method of histocyte being carried out to cracking.
5. the application of the new method of the fluorescence probe detection sulfuretted hydrogen synthase activity of use sulfuretted hydrogen in bioengineering industry according to claim 1.
6. use according to claim 1 the new method of the fluorescence probe detection sulfuretted hydrogen synthase activity of sulfuretted hydrogen studying H
2the signal path that S is relevant and H
2meaning in the diease occurrence process of S in disease or biosome and the application of diagnosis.
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| CN104894277A (en) * | 2015-06-19 | 2015-09-09 | 许昌学院 | Method for preventing aerosol pollution caused by nucleic acid isothermal amplification product |
| CN104910060A (en) * | 2015-05-28 | 2015-09-16 | 中国科学院烟台海岸带研究所 | Fluorophore derivative and application thereof |
| CN105017128A (en) * | 2014-04-28 | 2015-11-04 | 中国科学院烟台海岸带研究所 | Cyanine compound and application thereof |
| CN110646417A (en) * | 2019-10-24 | 2020-01-03 | 福建医科大学 | Rapid pyridoxal phosphate determination method taking nanogold as chromogenic probe |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105017128A (en) * | 2014-04-28 | 2015-11-04 | 中国科学院烟台海岸带研究所 | Cyanine compound and application thereof |
| CN105017128B (en) * | 2014-04-28 | 2017-09-15 | 中国科学院烟台海岸带研究所 | A kind of cyanine compound and its application |
| CN104910060A (en) * | 2015-05-28 | 2015-09-16 | 中国科学院烟台海岸带研究所 | Fluorophore derivative and application thereof |
| CN104910060B (en) * | 2015-05-28 | 2017-03-29 | 中国科学院烟台海岸带研究所 | A kind of fluorophore derivatives and its application |
| CN104894277A (en) * | 2015-06-19 | 2015-09-09 | 许昌学院 | Method for preventing aerosol pollution caused by nucleic acid isothermal amplification product |
| CN104894277B (en) * | 2015-06-19 | 2020-09-11 | 许昌学院 | Method for preventing aerosol pollution of isothermal amplification product of nucleic acid |
| CN110646417A (en) * | 2019-10-24 | 2020-01-03 | 福建医科大学 | Rapid pyridoxal phosphate determination method taking nanogold as chromogenic probe |
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|---|---|
| CN103630518B (en) | 2017-11-07 |
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