CN107817238A - A kind of method that fluorescence based on carbon point recovers screening glutathione reductase inhibitor - Google Patents
A kind of method that fluorescence based on carbon point recovers screening glutathione reductase inhibitor Download PDFInfo
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- CN107817238A CN107817238A CN201711362481.0A CN201711362481A CN107817238A CN 107817238 A CN107817238 A CN 107817238A CN 201711362481 A CN201711362481 A CN 201711362481A CN 107817238 A CN107817238 A CN 107817238A
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
It is to use Ag the invention discloses a kind of method that fluorescence based on carbon point recovers screening glutathione reductase inhibitor+The yellow fluorescence carbon point that ion specific can be quenched is as fluorescence probe；Oxidized form of glutathione can be catalyzed and be reduced to reduced glutathione by glutathione reductase in the presence of coenzyme NADP 11, and reduced glutathione being capable of competition binding Ag+Ion and then the fluorescence for recovering carbon point；And the glutathione reductase that inactivation is handled through glutathione reductase inhibitor can not be catalyzed reduction-oxidation type glutathione, thus the fluorescence of carbon point can not be recovered, accordingly, the screening of glutathione reductase activity inhibitor can be realized by fluorescence recovery situation.The features such as method of present invention screening glutathione reductase inhibitor has high sensitivity, and selectivity is good, and cost is low, and toxicity is low, environment-friendly, and stability is strong, the related drugs suppressed based on glutathione reductase activity are screened significant.
Description
Technical field
The present invention relates to a kind of screening technique of glutathione reductase inhibitor, more particularly to it is a kind of based on the glimmering of carbon point
The method that light recovers screening glutathione reductase inhibitor, belongs to biochemical analysis and drug screening field.
Background technology
Malaria is one of most common infectious disease, easily causes Perenniporia martius country common people's disease and death.Because of malaria
The clinical case that disease triggers is up to several hundred million every year, causes people up to ten thousand dead, wherein most is infant.In recent years, because malaria is former
The resistance to the action of a drug enhancing of worm, the death rate constantly rise.Research finds, the increase meeting of reduced glutathione content in plasmodium falciparum
Strengthen its drug resistance to antimalarial, and the decline of reduced glutathione content can recover sensitivity of the drug resistance strain to antimalarial
Property.In fact, reduced glutathione can protect parasite from oxidative damage.Glutathione is mainly with reduced form gluathione
The form of peptide is present in into the cell, and intracellular higher reduced glutathione level depends on glutathione reductase
Catalysis reduction efficient to oxidized form of glutathione.It in addition, there will be research to find, in the strong tumour cell of some drug resistances
The glutathione reductase of overexpression can be observed.Therefore, the research of glutathione reductase inhibitor to anti-malarial and resists
Tumour medicine research and development are significant.
So far, a variety of glutathione reductases based on glutathione reductase activity detection have been developed to suppress
Agent screening technique.Wherein classical glutathione reductase activity assay method is based on detection NADPH and DTNB UV absorptions
Change and establish, although this method still in extensive use, the survey of the UV absorption of chaff interference to system UV absorption
Constant volume is also easy to produce interference.In addition, based on vulcanization Silver Electrode, high performance liquid chromatography, amperometric sensor, F19The methods of nuclear magnetic resonance
It has been successfully applied to glutathione reductase activity measure and its inhibitor screening.However, high equipment cost and cumbersome
Operating procedure can undoubtedly limit its extensive use.And with high sensitivity, high-spatial and temporal resolution, fluorescence probe skill easy to operate
Art, the analysis to glutathione reductase activity are significant.At present, existing researcher uses organic dyestuff fluorescence probe
Evaluated in glutathione reductase activity, but low quantum yield, easy photobleaching, and poor water solubility, complicated preparation and
Purification step etc. limits extensive use of such fluorescence probe in glutathione reductase activity evaluation.Also there is researcher's base
A variety of fluorescence nanos for being used to evaluate glutathione reductase activity are developed in metal nanometre cluster, quantum dot, DNA biologies point etc.
Probe.But material therefor has the shortcomings that bio-toxicity is high, fluorescence is unstable, cost is high.
Carbon point compared with organic molecule, metal nanometre cluster, quantum dot, has glimmering as a kind of novel fluorescence nano material
Quantum yield is high, optical property is stable, bio-toxicity is low, many advantages such as environment-friendly.Based on its photoluminescent property, carbon point is
It is widely used in chemical analysis and field of bioanalysis.
The content of the invention
The purpose of the present invention is a kind of method that fluorescence based on carbon point recovers screening glutathione reductase inhibitor.
The method of present invention screening glutathione reductase inhibitor, is with Ag+The carbon point that ion specific can be quenched is made
For fluorescence probe, Ag is first used+Its fluorescence is quenched in ion；Glutathione reductase will can aoxidize in the presence of coenzyme NADP 11
The catalysis of type glutathione is reduced to reduced glutathione, and reduced glutathione being capable of competition binding Ag+Ion and then recovery
The fluorescence of carbon point；And the glutathione reductase that inactivation is handled through glutathione reductase inhibitor can not be catalyzed reduction-oxidation type
Glutathione, thus the fluorescence of carbon point can not be recovered, therefore, glutathione reductase activity can be realized by fluorescence recovery situation
The screening of inhibitor.Its specific screening technique comprises the following steps：
（1）In cushioning liquid, by Ag+The carbon point that ion specific can be quenched with it is incubated, and obtains incubation product；
Wherein, Ag+The carbon point that ion specific can be quenched is yellow fluorescence carbon point, and its excitation wavelength is 380 ~ 420 nm（It is preferred that
400 nm）, launch wavelength is 520 ~ 600 nanometers（It is preferred that 552 nm）.In cushioning liquid, the concentration of yellow fluorescence carbon point for 10 ~
500 μg mL-1（It is preferred that 30 ~ 80 μ g mL-1）, Ag+The concentration of ion is 1 ~ 50 μM（It is preferred that 5 ~ 12 μM）；Incubation temperature 20 ~
40℃（It is preferred that 25 DEG C）, incubation time is 1 ~ 30 min（It is preferred that 2 min）.
（2）In cushioning liquid, under coenzyme NADP 11 effect, by the glutathione reductase crossed through drug-treated and oxidation
Type glutathione is incubated, and obtains incubation product ；
In cushioning liquid, the concentration of coenzyme NADP 11 is 1 ~ 100 μM（It is preferred that 10 ~ 20 μM）, the concentration of oxidized form of glutathione is
1~100 μM（It is preferred that 3 ~ 10 μM）, the concentration of inhibited dose of treated glutathione reductase is 1 ~ 20 mU mL–1（It is preferred that 2
~5 mU mL–1）；Incubation temperature is 20 ~ 40 DEG C（It is preferred that 25 DEG C）, incubation time is 1 ~ 30 min（It is preferred that 5 ~ 15 min）.
The technique of the inhibitor processing glutathione reductase：In cushioning liquid, by glutathione reductase and medicine
After mixing, 1 ~ 30 min is incubated in 20 ~ 40 DEG C；The concentration of medicine is 1 ~ 300 μM, and the concentration of glutathione reductase is 1 ~ 20
mU mL–1。
（3）Above-mentioned gained is incubated product, be incubated product Be incubated again after mixing, and determine incubation after mix it is molten
The fluorescence intensity of liquid, according to the recovery efficiency analysis medicine of fluorescence intensity to the inhibitory action of glutathione reductase activity, nationality
This filters out efficient glutathione reductase inhibitor.
It is incubated product, be incubated product Volume ratio be 1:0.01~1:100；Incubation temperature is 20 ~ 40 DEG C（It is preferred that 25
℃）, the time is 1 ~ 30 min（It is preferred that 0.5 ~ 5 min）.
In above steps, cushioning liquid is HEPES cushioning liquid, and concentration is 5 ~ 100 mM, and pH is 5.0 ~ 9.0（It is preferred that
7.2）.
High efficiency, the height of glutathione reductase inhibitor are screened to fluorescence restoring method of the present invention below by specific experiment
Selectivity, reliability illustrate.
1st, fluorescence restoring method measure glutathione reductase activity
Carbon point/Ag+The incubation of solion：In 50 μ L HEPES cushioning liquid（30 mM, pH=7.2）In, yellow carbon point is molten
Liquid（100 μg mL-1）And Ag+Solion（8.0 μM）It is vortexed and mixes 10 s, is placed in water-bath, 2 min are incubated at 25 DEG C；
In 50 μ L HEPES cushioning liquid（30 mM, pH=7.2）In, by glutathione reductase solution（0.6 U mL-1）With
Coenzyme NADP 11 solution（2.0 mM）With oxidized form of glutathione solution（1.0 mM）It is vortexed and mixes 10 s, is placed in water-bath, 25
10 min are incubated at DEG C；
40 μ L are taken to be transferred to above-mentioned carbon point/Ag+In solion, it is vortexed and mixes 15 s, after 1 min is incubated at 25 DEG C, measure
The fluorescence intensity of the mixed solution（It is respectively 400 nm and 552 nm to excite with launch wavelength）.
Fig. 1 is that fluorescence of the present invention recovers the feasibility analysis that measure medicine influences on glutathione reductase activity.Wherein,
A is carbon dots solution（50 μg mL–1）, B is carbon point/Ag+Solion（50 μg mL–1/8.0 μM）；Control group C is oxidized form
Glutathione and carbon point/Ag+Product after solion incubation, D are NADPH and carbon point/Ag+Product after solion incubation,
E is glutathione reductase and carbon point/Ag+Product after solion incubation, F oxidizeds form of glutathione are incubated with NADPH
Product again with carbon point/Ag+Product after solion incubation, G are that oxidized form of glutathione is incubated with glutathione reductase
Product again with carbon point/Ag+Solion be incubated after product, H be product that NADPH and glutathione reductase are incubated again with carbon
Point/Ag+Product after solion incubation；Experimental group I is that oxidized form of glutathione is incubated with NADPH and glutathione reductase
Products therefrom carbon point/Ag+Product after solion incubation.
It will be seen from figure 1 that under the same terms, the fluorescence intensity of control group is markedly less than experimental group.In other words, oxidized form
The incubation of one or both of glutathione, NADPH and glutathione reductase obtains product and fails to make the fluorescence of carbon point bright
Aobvious to recover, the product for being incubated to obtain when only oxidized form of glutathione, NADPH and glutathione reductase coexist can be effectively extensive
The fluorescence of carbon restoration point.As can be seen here, recover that the measure of glutathione reductase activity can be realized by fluorescence.
2nd, fluorescence recovers the relation between efficiency and glutathione reductase concentration, incubation time
In 50 μ L HEPES cushioning liquid（30 mM, pH 7.2）In, by the glutathione reductase solution of various concentrations（0.1~
0.6 U mL-1) and NADPH solution（2.0 mM）With oxidized form of glutathione solution（1.0 mM）It is vortexed and mixes 15 s, is placed in water
In bath, 1 ~ 15 min is incubated at 25 DEG C.Then, 40 μ L are taken to be transferred to carbon point/Ag+Solion（50 μg mL-1/8.0 μ
M）In, it is vortexed and mixes 15 s, after being incubated 1 min, determines the fluorescence intensity of the mixed solution（Excite and be respectively with launch wavelength
400 nm and 552 nm）.
Fig. 2 is that fluorescence recovers the graph of a relation that efficiency changes with glutathione reductase concentration and incubation time.Can by Fig. 2
To find out, as glutathione reductase concentration increases, fluorescence recovers the increase speed of efficiency in the catalytic reduction reaction short period
Degree is very fast, and fluorescence recovery efficiency is larger at the end of reaction.
3rd, fluorescence recovers the selectivity analysis of measure glutathione reductase activity
In 50 μ L HEPES cushioning liquid（30 mM, pH 7.2）In, by 0.5 U mL-1Protein or trypsase
（Try）, cromoci（Cyt）, bovine serum albumin（BSA）, avidin（Avi）, lysozyme（Lyz）, myoglobins（Myo）, core
Ribonuclease T.（Rib）, Chymetin（Chy）, fibrinogen（Fib）, hemoglobin（Hem）With NADPH solution（2.0
mM）With oxidized form of glutathione solution（1.0 mM）It is vortexed and mixes 15 s, is placed in water-bath, 10 min are incubated at 25 DEG C.So
Afterwards, 40 μ L are taken to be transferred to carbon point/Ag+Solion（50 μg mL–1/8.0 μM）In, it is vortexed and mixes 15 s, is incubated 1 min
Afterwards, the fluorescence intensity of the mixed solution is determined（It is respectively 400 nm and 552 nm to excite with launch wavelength）.
Fig. 3 is the selectivity analysis that fluorescence of the present invention recovers measure glutathione reductase activity.Can be with from Fig. 3
Find out, the glutathione reductase of any concentration（The mU mL of concentration 3.0–1It is approximately equal to 0.018 μ g mL–1）, nearly 1000 times
Protein or enzyme（10 μg mL–1）All fail to make carbon point fluorescence substantially recover, show the inventive method to glutathione reduction
Enzyme assay has high selectivity.
4th, double (2- chloroethyls) -1- nitroso ureas of the inhibitor 1,3- of various concentrations（BCNU）Glutathione reductase is lived
The suppression of property
With double (2- the chloroethyls) -1- nitroso ureas of 1,3-（BCNU）Exemplified by, illustrate the inhibitor of various concentrations to glutathione also
The inhibiting rate of original enzyme activity.In 30 μ L HEPES cushioning liquid（30 mM, pH 7.2）In, by glutathione reductase（0.3 U
mL-1）With double (2- the chloroethyls) -1- nitroso ureas of various concentrations 1,3-（BCNU, 3.3 ~ 25 mM）After 15 s that are vortexed are mixed, in water
10 min are incubated in bath at 25 DEG C, then, add 10 μ L NADPH solution（3.0 mM）It is molten with 10 μ L oxidizeds form of glutathione
Liquid（2.0 mM）, it is vortexed and mixes 15 s, and is incubated 10 min at 25 DEG C in a water bath.The 40 μ L mixed solutions are taken to be transferred to carbon
Point/Ag+Solion（50 μg mL-1/8.0 μM）In, it is vortexed and mixes 15 s, after being incubated 1 min, determines the mixed solution
Fluorescence intensity（It is respectively 400 nm and 552 nm to excite with launch wavelength）, and various concentrations inhibitor is calculated according to equation below
Inhibiting rates of the BCNU to glutathione reductase activity（I%）：
Wherein, △F 0Refer to the carbon point/Ag for not adding inhibitor+The fluorescence recovery value of ionic system, △FRefer to the carbon for adding inhibitor
Point/Ag+The fluorescence recovery value of ionic system.
Fig. 4 is the concentration and glutathione reduction of double (2- the chloroethyls) -1- nitroso ureas (BCNU) of measure inhibitor 1,3-
The graph of a relation of inhibition of enzyme activity rate.As seen from Figure 4, increase with BCNU concentration, the suppression of glutathione reductase activity
Rate processed gradually increases, and calculates the IC50 of gained（60 μM）It is suitable with literature value, show glutathione reductase inhibitor of the present invention
Screening technique there is good reliability.
In summary, the present invention has the advantage that compared with prior art：
1st, the present invention by oxidized form of glutathione by being reduced to reduced glutathione, and reduced glutathione can compete
With reference to the Ag on carbon point surface+Ion so that because of Ag+The carbon point that ion exists and fluorescent quenching occurs recovers its fluorescence, so as to realize
The activity rating of glutathione reductase, influence of the measure medicine to glutathione reductase activity is recovered by fluorescence, can
Reduce ambient interferences, high sensitivity, selectivity is good, simple and reliable, is a kind of inexpensive and efficient glutathione reductase
Inhibitor screening method；
2nd, the present invention is based on Ag+Ion regulation carbon point fluorescence builds fluorescence probe, low with toxicity, environment-friendly, fluorescent stabilization
The advantages that；The carbon point fluorescence probe transmitting fluorescence of structure is gold-tinted simultaneously, can effectively evade coenzyme NADP 11 autofluorescence（It is blue
Light）To the fluorimetric interference of system.
Brief description of the drawings
Fig. 1 is that fluorescence of the present invention recovers measure medicine to glutathione reductase activity influence.
Fig. 2 is that fluorescence of the present invention recovers the pass that efficiency changes with glutathione reductase concentration and incubation time
System's figure.
Fig. 3 is the selectivity analysis that fluorescence of the present invention recovers measure glutathione reductase activity.
Fig. 4 is to determine double (2- the chloroethyls) -1- nitroso ureas (BCNU) of inhibitor 1,3- based on the method for the invention
Concentration and the graph of a relation of glutathione reductase activity inhibiting rate.
Fig. 5 is the fluorescence point suppressed based on the method for the invention 10 kinds of medicines of measure to glutathione reductase activity
Analysis.
Embodiment
Below by specific embodiment to fluorescence of the present invention recover screening glutathione reductase inhibitor method make into
One step explanation.
10 kinds of medicines are selected, its suppression to glutathione reductase activity is determined by the inventive method and sieved
Choosing.
In 50 μ L HEPES cushioning liquid（30 mM, pH=7.2）In, by yellow carbon dots solution（50 μg mL-1）And Ag+
Solion（8.0 μM）It is vortexed and mixes 10 s, is placed in water-bath, 2 min are incubated at 25 DEG C, obtain incubation product--- carbon
Point/Ag+Solion（50 μg mL–1/8.0 μM）；
In 30 μ L HEPES cushioning liquid（30 mM, pH 7.2）In, by glutathione reductase（0.5 U mL–1）With difference
Medicine（50 mM）After 15 s that are vortexed are mixed, 10 min are incubated under the conditions of 25 DEG C in a water bath；Then it is molten that 10 μ L NADPH are added
Liquid（3.0 mM）With 10 μ L oxidized form of glutathione solution（2.0 mM）, it is vortexed and mixes 15 s, and 25 DEG C of conditions in a water bath
10 min of lower incubation, obtain incubation product ；
40 μ L are taken to be incubated product , it is transferred to incubation productIn, it is vortexed and mixes 15 s, after being incubated 1 min, it is molten determines the mixing
The fluorescence intensity of liquid（It is respectively 400 nm and 552 nm to excite with launch wavelength）.
Inhibiting rate according to the medicine to be measured that equation below calculating concentration is 150 μM to glutathione reductase activity
（I%）, rejection ability of the com-parison and analysis to glutathione reductase activity：
Wherein, △F 0Refer to the fluorescence recovery value for the system for not adding medicine, △FRefer to the fluorescence recovery value for the system for adding medicine.
Fig. 5 is the fluorescence analysis that above-mentioned 10 kinds of medicines suppress to glutathione reductase activity.Wherein, 1 is carbon dots solution；
2 be carbon point/Ag+ (50 μg mL–1/ 8.0 μM) solution；3 be to add oxidized form of glutathione and NADPH and glutathione also
Protoenzyme is incubated carbon point/Ag of product+Solion；4 ~ 8 are followed successively by medicine 1,1,3,3- tetramethyl -2- thiocarbamides, 1,5- diphenyl
Carbonohydrazides, N- diethylnitrosamines, urea, thiocarbamide；9 ~ 13 be followed successively by it has been reported that inhibitor 1- ethyl -1- nitroso ureas,
2- chloroethyl isocyanates, 1,3- double (2- chloroethyls) -1- nitroso ureas, cyclohexyl isocyanate, 1- (2- chloroethyls) -3-
(1- cyclohexyl) -1- nitroso ureas.As shown in Figure 5, it is stronger to add the fluorescence of the system of medicine 4 ~ 8, and adds concentration phase therewith
The fluorescence intensity of the system of same inhibitor 9 ~ 13 is extremely weak, shows 4 ~ 8 pairs of glutathione reductase activity unrestraints effects of medicine,
It is higher that system fluorescence recovers efficiency；The inhibitory action of 9 ~ 13 pairs of glutathione reductase activities of inhibitor is stronger, and system fluorescence is extensive
It is multiple extremely inefficient.
Table 1 is that the above method determines inhibiting rate of 10 kinds of medicines to glutathione reductase activity.4 ~ 8 pairs of paddy Guangs of medicine
The inhibiting rate of sweet fabk polypeptide activity is below 8%, and inhibitor is above 72% to the inhibiting rate of glutathione reductase activity.
The result of table 1 is consistent with Fig. 5 result, it is thereby achieved that the successful screening of glutathione reductase inhibitor.
Claims (9)
1. a kind of method that fluorescence based on carbon point recovers screening glutathione reductase inhibitor, it is characterised in that：It is with Ag+
The yellow carbon point that specific its fluorescence can be quenched in ion first uses Ag as fluorescence probe+Its fluorescence is quenched in ion；Glutathione is also
Oxidized form of glutathione can be catalyzed and be reduced to reduced glutathione, reduced form paddy by protoenzyme in the presence of coenzyme NADP 11
The sweet peptide of Guang being capable of competition binding Ag+Ion and then the fluorescence for recovering carbon point；And handle and inactivate through glutathione reductase inhibitor
Glutathione reductase can not be catalyzed reduction-oxidation type glutathione, thus the fluorescence of carbon point can not be recovered, therefore, can passed through
Fluorescence recovery situation realizes the screening of glutathione reductase activity inhibitor.
2. a kind of fluorescence based on carbon point recovers the method for screening glutathione reductase inhibitor as claimed in claim 1, its
It is characterised by：Comprise the following steps：
（1）In cushioning liquid, by Ag+The carbon point that ion specific can be quenched with it is incubated, and obtains incubation product；
（2）In cushioning liquid, under coenzyme NADP 11 effect, by inhibited dose of treated glutathione reductase and oxidized form
Glutathione is incubated, and obtains incubation product ；
（3）Above-mentioned gained is incubated product, be incubated product Be incubated again after mixing, and determine be incubated after mixed solution
Fluorescence intensity, according to the recovery efficiency analysis medicine of fluorescence intensity to the inhibitory action of glutathione reductase activity, take this to sieve
Select efficient glutathione reductase inhibitor.
3. a kind of fluorescence based on carbon point recovers the method for screening glutathione reductase inhibitor as claimed in claim 2, its
It is characterised by：Step（1）In, Ag+Ion can the carbon point that be quenched of specificity be yellow fluorescence carbon point, its excitation wavelength for 380 ~
420 nm, launch wavelength are 520 ~ 600 nanometers.
4. a kind of fluorescence based on carbon point recovers the method for screening glutathione reductase inhibitor as claimed in claim 2, its
It is characterised by：Step（1）Cushioning liquid in, the concentration of yellow fluorescence carbon point is 10 ~ 500 μ g mL-1, Ag+The concentration of ion
For 1 ~ 50 μM；20 ~ 40 DEG C of incubation temperature, incubation time are 1 ~ 30 min.
5. a kind of fluorescence based on carbon point recovers the method for screening glutathione reductase inhibitor as claimed in claim 2, its
It is characterised by：Step（2）Cushioning liquid in, the concentration of coenzyme NADP 11 is 1 ~ 100 μM, and the concentration of oxidized form of glutathione is
1 ~ 100 μM, the concentration of inhibited dose of treated glutathione reductase is 1 ~ 20 mU mL–1；Incubation temperature is 20 ~ 40 DEG C,
Incubation time is 1 ~ 30 min.
6. a kind of fluorescence based on carbon point recovers the method for screening glutathione reductase inhibitor as claimed in claim 2, its
It is characterised by：Step（3）In, it is incubated product, be incubated product Volume ratio be 1:0.01~1:100；Incubation temperature is 20 ~ 40
DEG C, the time is 1 ~ 30 min.
7. a kind of fluorescence based on carbon point recovers the method for screening glutathione reductase inhibitor as claimed in claim 2, its
It is characterised by：Step（2）In, the technique of the inhibitor processing glutathione reductase：In cushioning liquid, by glutathione also
After protoenzyme mixes with medicine, 1 ~ 30 min is incubated in 20 ~ 40 DEG C.
8. a kind of fluorescence based on carbon point recovers the method for screening glutathione reductase inhibitor as claimed in claim 7, its
It is characterised by：The concentration of inhibitor is 1 ~ 300 μM, and the concentration of glutathione reductase is 1 ~ 20 mU mL–1。
9. a kind of fluorescence based on carbon point recovers the method for screening glutathione reductase inhibitor as claimed in claim 1, its
It is characterised by：In above steps, cushioning liquid is HEPES cushioning liquid, and concentration is 5 ~ 100 mM, and pH is 5.0 ~ 9.0.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108220388A (en) * | 2018-01-23 | 2018-06-29 | 青岛海洋生物医药研究院股份有限公司 | A kind of screening technique of aminoglycoside dual-function modified enzyme AAC (6 ')-APH (2 ") inhibitor |
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| CN113740369A (en) * | 2020-05-29 | 2021-12-03 | 华东师范大学 | Reduction method for detecting heavy metal ions based on in-situ low-field nuclear magnetic resonance relaxation method |
| CN113740369B (en) * | 2020-05-29 | 2024-03-12 | 华东师范大学 | Reduction method for detecting heavy metal ions based on in-situ low-field nuclear magnetic resonance relaxation method |
| CN112063685A (en) * | 2020-09-04 | 2020-12-11 | 湖南中医药大学 | Screening method and application of glutathione S-transferase inhibitor |
| CN112063685B (en) * | 2020-09-04 | 2024-02-02 | 湖南中医药大学 | Screening method and application of glutathione S-transferase inhibitor |
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